NAMPT/NAD+ signaling drives neutrophil dysfunction and enhances bacterial infection susceptibility in cancer patients

NAMPT/NAD+ signaling drives neutrophil dysfunction and enhances bacterial infection susceptibility in cancer patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article NAMPT/NAD+ signaling drives neutrophil dysfunction and enhances bacterial infection susceptibility in cancer patients Jadwiga Jablonska, Ekaterina Pylaeva, Lea Tollrian, Jana Riedesel, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6098692/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Despite advancements in cancer therapies, bacterial complications remain a major challenge, delaying treatment and worsening outcomes. While immunosuppressive therapies and prolonged hospitalizations contribute, they do not fully explain the elevated infection risk in cancer patients. We observed that elevated G-CSF production by tumors was associated with the persistence of Gram-negative pathogens in head and neck squamous cell carcinoma patients. The presence of Gram-negative pathogens in oral rinse positively correlated with poor prognosis of such patients. As neutrophils are the main cells controlling bacterial infections, we aimed to identify the molecular mechanisms underlying tumor-induced suppression of antibacterial activity of these cells. We identified tumor-driven activation of the NAMPT/NAD+ signaling axis in neutrophils (already at the progenitor level), which induced long-lasting cytoskeletal alterations, impairing phagocytosis and NETosis. Moreover, NAMPT/NAD+ activity accelerated neutrophil senescence and promoted accumulation of tissue-toxic CD62Llow subpopulations, ultimately disrupting lung tissue integrity and facilitating bacterial persistence in tumor-bearing hosts. Targeting the G-CSF/NAMPT signaling effectively reduced the generation of dysfunctional neutrophils and improved bacterial clearance in vivo. These findings reveal tumor-induced, NAMPT-dependent neutrophil reprogramming as a central mechanism driving impaired antimicrobial defenses. Implementing immune-modulating strategies, such as targeting G-CSF/NAMPT signaling, could improve infection control and enhance survival in cancer patients. Health sciences/Oncology/Cancer/Head and neck cancer Health sciences/Medical research/Translational research Biological sciences/Cancer/Tumour immunology Biological sciences/Immunology/Antimicrobial responses Cancer neutrophils antibacterial responses G-CSF/NAMPT signaling granulopoiesis Gram-negative pathogens Pseudomonas aeruginosa head and neck cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background In patients with cancer, infectious complications significantly impact clinical outcomes, delaying treatment and increasing morbidity and mortality. Among these, infections caused by Pseudomonas aeruginosa represent a particularly serious threat, with cancer patients experiencing a 50-fold higher risk of bacteremia compared to the general population [ 1 ]. Alongside other Gram-negative pathogens such as Klebsiella pneumoniae, Escherichia coli , and Haemophilus influenzae , P. aeruginosa remains a high-priority target in the 2024 Bacterial Priority Pathogens List due to its high rates of antibiotic resistance and global prevalence, especially in healthcare settings. The World Health Organization underscores the urgent need for innovative prevention and control measures, as well as alternative therapeutic strategies, to combat these pathogens effectively [ 2 ]. While traditional risk factors, including prolonged hospitalization and immunosuppressive treatments, contribute to the increased infection burden in cancer patients, they do not fully explain the vulnerability observed even in untreated cases. Emerging evidence suggests that tumors themselves may drive an intrinsic reprogramming of immune functionality, significantly impairing the body’s ability to resist bacterial infections [ 3 , 4 ]. This raises critical questions about the mechanisms of immune dysfunction in cancer and their implications for both infection control and therapeutic intervention. Neutrophils, central to antibacterial immunity, have historically been viewed as short-lived innate effector cells with limited adaptability [ 5 , 6 ]. However, recent discoveries in the field of trained immunity challenge this perspective, demonstrating that innate immune cells, including neutrophils, can acquire a form of memory through epigenetic modulation following specific stimuli [ 7 ]. While trained immunity enhances responses to subsequent infections [ 8 ] or even malignancies [ 9 ], the tumor microenvironment appears to induce a distinct phenomenon: immune reprogramming. Tumor-derived factors drive this reprogramming, potentially resulting in maladaptive immune responses that compromise the host's ability to clear bacterial infections [ 10 , 11 ]. Despite its clinical significance, the long-term impact of tumor-driven innate immune reprogramming on neutrophil function remains poorly understood. This study seeks to address this critical gap by investigating the effects of chronic exposure to tumor-derived factors on neutrophil development and function. Specifically, we focus on the role of G-CSF in driving neutrophil reprogramming, its contribution to impaired antibacterial defenses, and the elevated risk of infections in cancer patients. Furthermore, we explore whether targeting the G-CSF signaling pathway can reverse these changes and restore effective immunity. By elucidating the mechanisms of tumor-induced immune dysfunction, this work aims to inform the development of therapeutic approaches targeting patients' immune antibacterial defenses to mitigate infection-related complications and improve outcomes in patients with cancer. Results Increased susceptibility of cancer patients to bacterial infections is associated with high G-CSF release from tumor tissue Cancer patients suffer from recurring bacterial infections. To assess the abundance of clinically relevant Gram-negative pathogens in such patients ( P. aeruginosa, E. coli, K. pneumonia, H. influenza ) [ 12 , 13 ], we performed microbiological analysis of oral rinse of clinically asymptomatic HNSCC patients and compared it to healthy individuals (Table 1 ). Of note, the frequency of Gram-negative pathogens was significantly higher in such patients (Fig. 1 A). Table 1 Clinicopathological characterization of the study participants Healthy n = 28 HNC n = 45 Male, % 52% 78% Mean age, years 61 64 Active smokers, % 35% 65% Mean pack-years 14.8 35.5 HPV-positive, % - 38% UICC stage I - 13 II - 12 III - 3 IV - 17 Tumor localization Oral, oropharynx - 28 Hypopharynx - 6 Larynx - 10 Other - 1 Moreover, in a 2-years prospective study, we demonstrated that the presence of Gram-negative pathogens in the oral cavity indicated the poor 2 years outcome (Fig. 1 B), associated with significantly increased risk of bacterial complications (Fig. 1 C, clinical characteristics in the Table 2 ). Table 2 Infectious complications in participated patients with HNC. Group Mean age (min-max) at admission, years Mean time interval (min-max), months Source of infection Infectious agent Gram-neg bacteria at admission No infection (n = 10) 62.2 (49–76) 24.5 (7–36) - - 60% (no), 0% (yes), 40% (NA) Later infection (n = 8) 54.7 (40–77) 4 (1–12) See below See below 12.5% (no), 62.5% (yes), 25% (NA) ID19 61 1 Tracheal secret, bronchial secret E. cloacae yes ID25 76 1 Throat E. coli yes ID26 60 1 Breast A. lwoffi yes ID30 77 4 Blood (pneumogenic) NA no ID32 32 1 Blood B. cereus , S. oralis, E. coli , K. pneumoniae yes ID40 40 8 Odontogenic NA NA ID42 42 12 Pneumonia E. coli NA ID50 50 4 Demerskatheter, BAL S. epidermidis, Candida yes Given that bacterial infections are mainly controlled by neutrophil granulocytes and their anti-bacterial functions are modulated by G-CSF, we assessed blood and tumor levels of G-CSF in cancer patients, and observed a significant increase in tumor tissue (Fig. 1 D). Moreover, G-CSF levels were also increased in the oral cavity of patients, compared to healthy individuals (Fig. 1 E). Next, we evaluated if G-CSF levels in the oral cavity correlate with patient susceptibility to bacterial infections. Using a received operating characteristic (ROC) analysis, we determined the cut-off G-CSF value in oral rinse as 392 pg/ml, with the highest sensitivity (75%) and specificity (82.3%), indicating the presence of Gram-negative pathogens in the oral cavity (Fig. 1 F). Indeed, we observed higher frequencies of Gram-negative pathogens in patients with high G-CSF levels (Fig. 1 G), with positive likelihood ratio 4.25 (95% CI 1.34–11.96) (Fig. 1 H). Of note, in the general population (both, healthy and HNSCC) increased G-CSF levels in oral rinse (400 pg/ml or higher) predict the presence of Gram-negative pathogens with a specificity of 91% and a positive likelihood ratio of 8.4 (95% CI 2.64–26.71) (Figure S1 ). G-CSF chronically released by the tumor tissue promotes bacterial infections Short-term exposure to G-CSF is reported to have anti-bacterial and neutrophil-stimulating properties [ 14 ]. In agreement, treatment of isolated human blood neutrophils with G-CSF results in elevated antibacterial responses of these cells (elevated ROS and NETs production) (Figure S2). However, tumor presence is associated rather with long-lasting G-CSF release, leading us to hypothesis that it may have detrimental effect on neutrophil properties. To test this, we modified a murine oropharyngeal carcinoma (MOPC [ 15 ]) cell line to overexpress G-CSF ( G MOPC) at levels comparable to those measured in patients’ tumors (Fig. 2 A). To evaluate whether chronically elevated G-CSF impacts susceptibility to lower respiratory tract bacterial infections, we infected tumor-bearing mice (MOPC versus G MOPC) intratracheally ( i.t. ) with P. aeruginosa , and followed the course of infection. Indeed, similarly to patients, elevated chronic G-CSF availability ( G MOPC) increased the susceptibility of tumor-bearing animals to infection (Fig. 2 B). This was accompanied by the elevated lung tissue damage and loss of aerated area (Fig. 2 C,D) in these mice. Moreover, higher levels of TNFα as a marker of tissue damage (Fig. 2 E) and augmented severity of the disease (Fig. 2 F) were observed. Chronic exposure to tumor-derived G-CSF impairs neutrophil antibacterial functions and facilitates their tissue toxicity G-CSF chronically released by the tumor tissue stimulates granulopoiesis and neutrophil release from the bone marrow, resulting in elevated neutrophil accumulation in different compartments, such as tumor, lung (Fig. 2 G, gating strategy Figure S3), as well as in bone marrow and blood (Figure S4). Similar phenomenon is observed in HNSCC patients, where a higher accumulation of neutrophils in blood, tumor or oral rinse positively correlates with elevated G-CSF levels (Figure S5). Notably, the predominant neutrophil subpopulations associated with elevated G-CSF levels, were CD62L low CD11b dim senescent subsets (Fig. 2 H). To investigate the molecular mechanism behind possibly dysregulated antibacterial activity of neutrophils chronically exposed to tumor-derived G-CSF, we compared proteome of lung neutrophils isolated from mice bearing G MOPC (further referred as “ G neutrophils”) versus MOPC tumors (“neutrophils”). By quantitative proteomics we identified 115 differentially expressed proteins (63 up-regulated versus 52 down-regulated proteins) in G neutrophils (Table S2). Unsupervised hierarchical clustering analysis showed a unique proteomic signature of neutrophils under chronic exposure to G-CSF (Fig. 2 H, I). One of the molecules significantly downregulated in G neutrophils is CD11b (integrin αM), which plays a major role in neutrophil antibacterial activity. We validated the robustness of our label-free quantitative proteomic analysis and confirmed the downregulation of CD11b under chronic G-CSF exposure in G neutrophils using flow cytometry (Fig. 2 H, Figure S6). Next, we performed gene ontology (GO) enrichment analysis of differentially expressed proteins to gain insights into the biological processes (Fig. 2 J), cellular compartments (Fig. 2 K) and molecular functions (Fig. 2 L) that are affected in neutrophils by the long-term G-CSF exposure. Proteins up-regulated in such G neutrophils were significantly enriched in top 5 GO categories linking to neutrophil transcriptional activity, suggesting a possible dysregulation in their maturation, whereas proteins down-regulated in G neutrophils were associated with cytoskeleton reorganization (Fig. 2 J-L, supplemental files 1–7). G Neutrophils show impaired phagocytosis and NET formation due to defects in cytoskeleton polymerization In agreement with altered phenotype, we observed a significant downregulation of pathways involved in actin polymerization and phagocytosis in G neutrophils (Fig. 3 A, blue). While total expression of actin was higher (Figure S7A), actin-regulatory proteins responsible for actin dynamics (polymerization, depolymerization and branching), such as Arpc4 (Actin Related Protein 2/3 Complex Subunit 4) or Cap1 (Cyclase Associated Actin Cytoskeleton Regulatory Protein 1), were downregulated (Figure S7B,C). Apparently, cytoskeleton-dependent functions of G neutrophils are impaired due to chronic G-CSF exposure. To determine wherever the notable alterations of the cytoskeleton influence antibacterial functions of neutrophils, we challenge these cells with P. aeruginosa. We observed significantly reduced phagocytosis (Fig. 3 B) and NET formation (Fig. 3 C,E, Figure S7D,E) by G neutrophils, but no changes in total actin polarization (Fig. 3 D). The expression of other anti-bacterial proteins, such as Padi4 (Protein-arginine deiminase type-4) and non-oxidative branch of pentose-phosphate pathway (Transaldolase 1 Taldo, Transketolase Tkt) that are involved in NET formation, as well as Cybb (cytochrome b-245, beta chain), involved in phagolysosome pathogen killing, were also decreased upon G-CSF exposure (Figure S7F-I). This possibly contributes to the impaired bactericidal activity of these cells. Importantly, NET formation by G neutrophils in response to P. aeruginosa was decreased as compared to neutrophils transiently stimulated by G-CSF (Figure S2A-C), once again demonstrating different mechanisms involved in chronic versus acute G-CSF stimulation. Chronic exposure to G-CSF boosts tissue toxicity of neutrophils during bacterial infection G Neutrophils show upregulated Response to reactive oxygen species pathway (Fig. 3 A, orange). In agreement, we observed an increased expression of proteins involved in ROS production, such as Mpo (Myeloperoxidase, Fig. 3 F). As Mpo is a granule protein, high Mpo gene expression indicates high de novo synthesis of this protein, rather than decreased degranulation (Fig. 3 G). Besides non-mitochondrial ROS, we observe elevated mitochondrial activity in G neutrophils (Fig. 3 H-I, Figure S8A). At the same time, downregulated expression of the components of ROS detoxication machinery, such as Gpx1 (Glutathione peroxidase 1, Figure S8B) is observed. Such an elevated ROS production, both in steady state and during infection (Fig. 3 J-L), can be responsible for the observed lung tissue damage. Another potential cause of lung tissue damage could be an increased release of enzymes involved in matrix remodeling. Indeed, we observed significantly elevated levels of MMP9 in lung homogenates of infected animals (Fig. 3 M). Consistently, isolated G neutrophils release higher amounts of MMP9 in response to P. aeruginosa challenge in vitro (Fig. 3 N), and also show elevated Mmp9 gene levels in steady state (Fig. 3 O). G Neutrophils show a distinct aged phenotype G Neutrophil proteome analyses revealed dysregulation of neutrophil ageing and apoptosis (Fig. 4 A). In agreement, we observe significantly decelerated apoptosis of lung G neutrophils (Fig. 4 B), accompanied by significantly downregulated Caspase 3 expression (Fig. 4 C). At the same time, we observed accumulation of aged CD62L low G neutrophils in lungs (Fig. 4 D) and to a lesser extend also in bone marrow and blood (Figure S4) of mice bearing G-CSF producing tumors. Such aged CD62L low G neutrophils are strongly cytotoxic, with low (degranulation-associated) SSC expression (Fig. 4 E), elevated Mmp9 production (Fig. 4 F) and enhanced cytotoxicity in vitro (Fig. 4 G). Moreover, these cells distinct exhausted phenotype, with suppressed antibacterial activity, including phagocytosis (Fig. 4 H), impaired NET formation (Fig. 4 I-K) and inability to produce ROS in response to bacteria (Figure S9). Importantly, a similar trend can be observed in cancer patients: high production and release of G-CSF by the tumor microenvironment is associated with the accumulation of CD62L low aged neutrophils in tissues (Fig. 4 L,M). Such CD62L low neutrophils show high cytotoxic properties, with spontaneous ROS production (Fig. 4 N). Moreover, these cells show an exhausted phenotype with diminished antibacterial activity in response to P. aeruginosa , reduced NET production (Fig. 4 O) and decreased phagocytosis (Fig. 4 P). Neutrophils differentiated in the presence of G-CSF phenocopy G neutrophils Next, we were interested if the long-term exposure to tumor-derived G-CSF impacts neutrophil development and maturation, in addition to observed modulation of mature neutrophil phenotype. Therefore, we developed the system of in vitro maturation of bone marrow-derived progenitors for mechanistic studies (modified from our previous studies [ 16 ]) (scheme of the experiment Fig. 5 A). Progenitors maturated in the long-term presence of tumor-conditioned medium containing high levels of G-CSF ( G MOPC, Fig. 2 A) showed accelerated differentiation into mature Ly6G + neutrophils (Fig. 5 B,C). Such neutrophils phenocopied tumor-induced G neutrophils, showing decreased granularity (Fig. 5 D), elevated aged CD62L low population (Fig. 5 E), decreased CD11b expression (Fig. 5 F), higher spontaneous ROS production (Fig. 5 G) and decreased phagocytic capacity (Fig. 5 H). To prove the essential role of tumor-secreted G-CSF in the suppression of neutrophil bactericidal activity, we block G-CSF receptor during the entire neutrophil maturation using monoclonal antibodies. In line with our hypothesis, we observed impaired accumulation of aged CD62L low cells and suppressed ROS production. Similarly, after blocking downstream G-CSF signaling using STAT3 inhibitor LLL12, we observed partial restoration of neutrophil properties, namely inhibited neutrophil degranulation and lower accumulation of CD62L low cells, confirming the key role of G-CSF/ G-CSFR axis in the tumor-induced dysregulation of neutrophil maturation and functions (Figure S10). To exclude that observed changes are simply the consequence of delayed apoptosis in G neutrophils, we inhibited Caspase 3 using QVD-OPh and assessed neutrophil activity. Importantly, the treatment did not induce any changes observed after prolonged G-CSF exposure (Figure S11). Long term exposure to G-CSF persistently reprograms neutrophil progenitors Development of neutrophils from hematopoietic stem cells in the bone marrow takes approximately 14 days [ 17 ], therefore we hypothesized that the clinical consequence of cancer would be tumor-associated modulation of granulopoiesis and impaired neutrophil functionality, even after surgical removal of the tumor. To test this, we performed in vitro granulopoiesis assay, using bone marrow progenitors and compared neutrophil development in the presence and absence of tumor-derived factors (scheme of the experiment Fig. 5 A). Indeed, such neutrophils exposed to G MOPC -conditioned medium show decreased apoptosis (Fig. 5 I), elevated degranulation (Fig. 5 J), lower CD11b expression (Fig. 5 K), elevated spontaneous production of ROS (Fig. 5 L) and downregulated phagocytic capacity (Fig. 5 M), as compared to neutrophils that mature in control conditions. Next, to assess the effect of G-CSF on human neutrophil progenitors, we analyzed Gene Expression Omnibus databases GSE11247 [ 18 ], Figure S12), in which gene expression profiles of CD133 + circulating stem cells mobilized by CXCR4 inhibitor AMD3100 alone, or in combination with G-CSF, were investigated, and compared to our proteomics data. Among the pathways significantly upregulated by G-CSF were those responsible for defense response and leukocyte activation (Figure S12A), including beta-actin (Actb) commonly regulated (Figure S12B). Downregulated pathways include those involved in cell adhesion and migration, regulation of cell death and response to oxygen-containing compound (Figure S12A), with CD11b (Itgam) and cytochrome b (Cybb) commonly downregulated (Figure S12C). This indicates that the changes induced by G-CSF in stem cells are persistent during their development and present also at the mature neutrophil level. These data support the hypothesis that the changes observed in G neutrophils are induced already at the neutrophil progenitor level, and could be persistent in neutrophils even after the removal of G-CSF and be responsible for a prolonged susceptibility for bacterial infections. Therapeutic targeting of G-CSF signaling rescues neutrophil antibacterial functionality G-CSF signaling pathway seems to be involved in the inhibition of neutrophil antibacterial properties, therefore we hypothesized that targeting this pathway would have beneficial effects on their functionality. Previously, we observed than Nampt/NAD + are essential for the activity of G-CSF signaling. Although we could not measure any alteration of Nampt expression in G neutrophils on gene and protein level (Figure S13), we confirmed significant accumulation of NAD + in G MOPC bearing mice (Fig. 6 A), suggesting elevated activity of Nampt. To mechanistically assess the role of Nampt/NAD + axis in neutrophil hyperactivation and tissue toxicity, we used Nampt inhibitor (FK866) as described before [ 19 ]. Indeed, inhibition of G-CSF downstream signaling reduced spontaneous ROS production by neutrophils maturated in the presence of tumor-derived factors (Fig. 6 B). Next, we assessed the effect of blocked G-CSF on the susceptibility for infection in vivo . In agreement with our hypothesis, treatment of G MOPC-bearing mice with FK866 decreased lung infiltration with CD62L low neutrophils (Fig. 6 C,D). Moreover, such neutrophils show a distinct pro-apoptotic phenotype (Fig. 6 E-G), lower tissue-toxic potential with decreased spontaneous ROS production (Fig. 6 H) and MMP9 release (Fig. 6 I), but no changes in phagocytosis (Fig. 6 J). In line with this, mice treated with FK866 demonstrated improved bacterial clearance (Fig. 6 K) and clinical performance (Fig. 6 L), in comparison to untreated mice. This implies the importance of therapeutic approaches aiming at the normalization of G-CSF axis in cancer individuals to prevent neutrophil tissue toxicity, but at the same time to support antibacterial properties of such neutrophils to minimize the susceptibility of patients to bacterial infections. To sum up, chronic exposure to tumor-derived G-CSF not only enhances granulopoiesis and the release of harmful cytotoxic neutrophils from the bone marrow, but also results in the local retention of senescent exhausted neutrophils with diminished antibacterial properties in the lung. This leads to the significant lung tissue damage and enhanced lung colonization by bacteria. Discussion Cancer patients often suffer from recurrent bacterial infections that have a fatal impact on their morbidity and mortality. One of the reasons responsible for this phenomenon could be G-CSF that is chronically released by the growing tumor. Long-term exposure to G-CSF, in contrast to short-term treatment, induces a dysfunctional, exhausted state of neutrophils, with elevated cytotoxic activity and diminished antibacterial responses. This leads to tissue damage and impaired bacterial clearance, and thus to prolonged infections. Neutrophils are the key players orchestrating antibacterial immunity [ 5 ], and are reported to be significantly affected by cancer-released factors [ 10 ]. One of such factors is granulocyte colony-stimulating factor (G-CSF) [ 20 ], which exerts ambivalent effects on neutrophil antibacterial activity. Importantly, while short-term G-CSF treatment stimulates antibacterial activity of neutrophils [ 14 ], the evidence from the clinical trials reports the lack of protective effect [ 21 , 22 ] or even immuno-inhibitory properties [ 23 , 24 ] of long-term G-CSF treatment. Short-term treatment with G-CSF was previously shown to induce mobilization of neutrophils and to stimulate antibacterial potential of circulating neutrophils [ 14 ]. Therefore, the role of G-CSF treatment for chemotherapy-induced febrile neutropenia and prevention of sepsis is hard to underestimate [ 25 ]. Under physiological conditions, G-CSF is primarily cleared by neutrophils and neutrophil precursors, meaning that clearance from the circulation is a self-regulating process. After binding of G-CSF to its receptor (G-CSFR), G-CSF/G-CSFR complex is internalized and degraded [ 26 ]. In the context of cancer, tumor tissue constantly releases G-CSF, therefore self-regulation of G-CSF concentration is not possible, resulting in aberrant neutrophil functionality. The impact of G-CSF on neutrophil activity apparently depends on the dose, duration and underlying disease. In the context of hematopoietic stem cell mobilization by G-CSF and their further transplantation, impaired chemotaxis was observed; both in donor and in recipient neutrophils [ 23 , 27 , 28 ]. At the same time, reported changes of neutrophil functions are controversial: increased functionality in healthy donors [ 28 ], decreased ROS and phagocytosis in transplant recipients [ 29 ], or no changes in ROS and phagocytosis in both [ 23 ]. Our experiments show diminished neutrophil functionality after long-lasting exposure to tumor-derived G-CSF in non-neutropenic conditions, associated with impaired antibacterial responses (NET formation and phagocytosis, due to impaired actin cytoskeleton reorganization). Moreover, our data demonstrate clear morphological cause (lack of proteins regulating actin polymerization) responsible for the impairment of G neutrophil motility and associated functions, including phagocytosis and NET formation, in addition to functional exhaustion [ 23 ]. Neutrophils represent the first line defenders in acute inflammatory responses [ 30 ]. Aged/exhausted CD62L low neutrophils have been shown to contribute to sterile vascular injury and thrombosis in the model of fungal infection, despite impaired actin cytoskeleton [ 31 ]. We demonstrated that tumor-derived G-CSF not only prolonged survival, but also reprogrammed neutrophils during granulopoiesis, which resulted in their aged, exhausted phenotype, with impaired antimicrobial activity and elevated tissue toxicity. Typical marker of such aged neutrophils is downregulated CD62L (Sell low ) surface expression. CD62L is highly expressed in young neutrophils, but decreased on aged or activated neutrophils due to reduced gene expression or shedding [ 32 ]. G-CSF was shown to support shedding of CD62L [ 33 ], therefore prolonged exposure to G-CSF in cancer can be responsible for CD62L low phenotype of neutrophils. Accumulation of such tissue toxic neutrophils in organs, driven by tumor-derived G-CSF, can be responsible for acute respiratory distress syndrome, which is characteristic for G-CSF treatment [ 34 ]. Several therapeutic strategies were already tested to neutralize the adverse effects of G-CSF in neutrophils. Neutralizing anti-G-CSFR antibodies were shown to block G-CSF-induced neutrophilia, without inducing neutropenia, in non-human primates [ 35 ]. Moreover, anti-G-CSFR antibodies reduced neutrophilic inflammation during pneumococcal or influenza respiratory infections, without compromising bacterial clearance [ 36 ] and increased neutrophil CD62L expression, reverting them to active antibacterial phenotype [ 37 ]. Neutrophil-released ROS is one of the most powerful cytotoxic agents triggering tissue damage. In addition, it supports formation of biofilms by various bacterial species, including P. aeruginosa , by inducing overproduction of capsule-like exopolysaccharide alginate (so called mucoid conversion) [ 38 ]. High ROS production can be a result of upregulated downstream G-CSF signaling, which supports salvage NAD + synthesis through upregulation of nicotinamide phosphoribosyltransferase (NAMPT) [ 39 ]. Elevated NAD + production coincides with the engagement of oxidative phosphorylation, as a result of increased oxygen availability [ 40 ]. Previously, NAMPT was demonstrated to mitigate colitis severity by supporting redox-sensitive activation of phagocytosis in inflammatory macrophages [ 41 ], nevertheless, we demonstrated that in particular cases (high G-CSF production by tumor and elevated tissue toxicity of neutrophils) inhibition of NAMPT can have beneficial effects for the host. We hypothesized here that reprogramming of neutrophils by tumor-derived G-CSF occurs already at the progenitor stage. In agreement, elimination of tumor-derived G-CSF from the system after prolonged neutrophil exposure to tumor-conditioned medium, failed to fully restore their phenotype and properties (ROS, phagocytosis), demonstrating the crucial impact of the early education of neutrophil progenitors for their functionality. Observations from transplantology, where the persistence of aberrant G-CSF-stimulated neutrophils was observed in recipients in certain cases up to several weeks after stem cell transplantation [ 23 , 29 ], confirm the long-lasting effect of neutrophil progenitor reprogramming. Hence, tumor-induced reprogramming of myeloid progenitors in hematopoietic organs can be responsible for their prolonged suppressed bactericidal activity. It is especially important, because clinically, the majority of bacterial complications occur within 30 days after surgery, with almost half of cases after hospital discharge [ 42 ]. Thus, developing ways to monitor neutrophil functionality in patients after oncologic surgery should have high priority to identify patients at risk. Early non-cancer mortality, usually defined as 90-day mortality after diagnosis or treatment initiation, is a main driver of overall mortality in the HNSCC population. Prevalence of early non-cancer death has been shown to be around 5% in several cohorts with about 30% of cases being caused by pulmonary infection or blood stream infection [ 43 ] affecting patients treated with primary surgery [ 44 ] or (chemo-)radiation [ 45 ] equally. Given that a significant proportion of these patients are expected to be cured from their tumor, non-cancer mortality is an unacceptable obstacle to achieving good patient outcomes. Unfavorable changes in neutrophil functionality due to prolonged G-CSF exposition may worsen the prognosis of cancer patients. Prognostic role of G-CSF expression in tumor tissue is also already known [ 19 ]. Here, we observed worse prognosis of HNSCC patients with elevated G-CSF levels, which was associated with the presence of Gram-negative pathogens and increased risk of bacterial complications. The aerodigestive tract, especially the oral cavity, serves as a reservoir of bacteria, which can then spread with saliva. The incidence of P. aeruginosa is twice higher in people with untreated head and neck cancer, in comparison to healthy individuals [ 4 ], indicating that cancer-derived factors influence immune antibacterial responses, independently of the treatment. Further anticancer therapies due to their cytostatic and thus immunosuppressive effects, as well as due to the disrupting mucosal barriers, may allow spreading of persisting pathogens. Gram-negative pathogens, including Pseudomonas , Escherichia or Klebsiella spp , represent the major reason for local and systemic infectious complications after surgery for multiple tumor entities, including HNSCC [ 42 ]. Therefore, prevention of bacterial complications and treatment remains the great challenge in cancer patients. Additionally, infectious complications might lead to cancer progression if they interfere with indicated standard of care treatment such as in the case of adjuvant radiation therapy. Delays in postoperative treatment have been repeatedly associated with worse oncologic outcome [ 46 ], but the reasons for extended lags between surgery and the initiation of postoperative radiotherapy are unclear at this point. Even though these reasons will be plentiful and variable, it is rational to assume that infections, their treatment, and time required to recuperate, are responsible for a significant subset of delays. This highlights the need of early identification and treatment of patients at risk of infectious complications. Conclusion Here we demonstrate that one of the reasons for bacterial persistence and spread in tumor-bearing hosts are severely impaired antibacterial properties of neutrophils, accompanied by their eminent tissue toxicity that is caused by tumor-derived G-CSF. Therefore, treatment modalities should be considered to neutralize the impact of cancer-related G-CSF stimulation of neutrophils in order to prevent tissue damage and bacterial persistence in damaged tissue. Prediction and early diagnostics of patient predisposition to bacterial complications would decrease possible therapy delay and thus improve patient survival. Abbreviations BAL bronchoalveolar lavage Casp3 caspase 3 CFUs colony–forming units G CSF–granulocyte colony stimulating factor GO gene ontology HNSCC head and neck squamous cell carcinoma MMP9 matrix metalloproteinase 9 MOPC murine oropharyngeal carcinoma MPO myeloperoxidase NETs neutrophil extracellular traps ROC received operating characteristic ROS reactive oxygen species SSC side scatter TNFa tumor necrosis factor alpha Materials And Methods Cell lines The murine oropharyngeal carcinoma cell line MOPC (C57BL/6-derived, HPV16 E6/E7 - ) was obtained from Dr. William Chad Spanos and John H. Lee (Sanford Research/University of South Dakota, Sioux Falls, SD, US) [47]. Employing CRISPR/Cas9-mediated targeted knock-in technology, we engineered murine HNSCC cell lines expressing low and high G-CSF levels. Elevated production of G-CSF by tumor cells ( G MOPC cell line) in cell culture conditioned medium was evaluated with ELISA according to the manufacturer's protocol. Cells were cultivated in a special medium (67% DMEM, 22% Hams F12 nutrient mix, 10% Fetal Bovine Serum, 1% penicillin-streptomycin, 0.5 µg/ml Hydrocortisone, 8.4 ng/ml Cholera Toxin, 5µg/ml Transferrin, 5 µg/ml Insulin, 1.36 ng/ml Tri-Iodo-Thyronine, 5 µg/ml E.G.F.). During cultivation, cell lines were regularly tested for mycoplasma contamination with negative results. Cells were grown in a monolayer at 37 °C in a humidified incubator with 5% CO 2 . Animals. C57BL/6JCrl mouse strain from own breeding (University Hospital Essen), originally a JAX strain bred by Charles River Laboratory, was used for experiments. For the experiments, female littermates between 8-12 weeks were used. Mice were housed and bred under specific pathogen-free conditions housed in cages of up to 5 mice per cage, 12 h light/dark cycle at animal facility of the University Hospital Essen. All animal experiments have been approved by the regulatory authorities LANUV (Das Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany). Our animal care and used protocols adhere to the regulations of German law according to das Deutsche Tierschutzgesetz (TierSchG) and follow the recommendations of Federation of European Laboratory Animal Science Associations (FELASA). The MOPC and G MOPC cells were injected subcutaneously (s.c. 1 x 10 6 in 100 µl PBS) into the flank of C57BL/6 mice, as described previously [16]. Tumor-free animals from the same strain were used as control animals. Mice treatment with the NAMPT inhibitor FK866 was performed per i.p . injection of 25 mg/kg at day 0 and further at every second day. Bacteria . P. aeruginosa strains that were used in this study: PA14 parental strain . Bacteria have been cultured in Luria-Bertani (LB) broth for 3 hours to reach the early exponential phase, washed twice in PBS, the optical density of 100 µl suspension was measured in 96 well flat-bottom cell culture plates (Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany) at 600 nm using a microplate reader Synergy 2 (BioTek Instruments, Inc., Vermont, U.S.). OD 0.4 corresponds to a bacterial density of 5x10 9 /ml, as determined by serial dilutions and colony-forming unit (CFU) assays. Bacteria concentration was adjusted to the desired values and verified by plating on 2% LB agar plates. Lower respiratory tract infection in mice . For intratracheal inoculation of P. aeruginosa, mice were anesthetized with Ketamin (bela-pharm GmbH & Co, Vechta, Germany) 100 mg/kg and Xylazin (Ceva Tiergesundheit GmbH, Düsseldorf, Germany) 10 mg/kg in 0.9% NaCl solution, intubated and 2x10 6 CFUs of P. aeruginosa in sterile PBS (50 µl) were administrated using the Minivent Mouse Ventilator type 845 (Harvard Apparatus, Massachusetts, U.S.) with stroke volume 150 µl and frequency 150 breaths/ min. The control of distribution of liquid in both lungs during intratracheal administration was performed prior to the experiments using Trypan blue (Sigma-Aldrich/Merck, Darmstadt, Germany). The adapted intratracheal method demonstrated accurate delivery and retention of P. aeruginosa in lungs. Animals were monitored post-operatively in a heated box until ambulant and clinically normal. Mice were transferred to a clean box with food and water ad libitum and monitored for 20 hours. To evaluate the clinical status of the mice a severity scoring was performed according to the experiment specific score sheet approved in our animal permission based on the guidelines of the Deutsche Tierschutzgesetz. After 20 hours, mice were sacrificed. Heparinized blood was collected via heart puncture, plasma was prepared after centrifugation. Broncho-alveolar lavage (BAL) was collected after bronchial perfusion trough the trachea with 1 ml of sterile PBS. BAL was plated in serial dilutions to estimate CFUs on 2% LB agar and examined after 24 hours incubation. Histology. For histological examination of lungs mice were infected i.t. with P. aeruginosa. At the certain time point mice were sacrificed, lungs perfused with Tissue-Tek O.C.T. Compound (Sakura Finetek, Japan) containing 5% paraformaldehyde, the lumen of the trachea was fixed with ligature; lungs were dissected and snap frozen at -80 o C. 7-μm cryosections were fixed with ice-cold acetone, stained with hematoxylin-eosin, dried and mounted with Neo-Mount (Merck, Darmstadt, Germany). Microscopy. Microscopy was performed using Zeiss AxioObserver.Z1 Inverted Microscope with ApoTome Optical Sectioning equipped with filters for: DAPI, FITC, Alexa Fluor 488, GFP, DsRed, Cy3 or Olympus BX51 upright epifluorescence microscope. Images were processed with ZEN Blue 2012 software or CellSens Dimension software (Olympus), respectively, and analyzed with ImageJ. Assessment of neutrophil infiltration in lungs. Lungs were collected as described above; organs from non-infected animals were used as a control. Lung tissue was digested using dispase 0.2µg/ml, collagenase A 0.2µg/ml, DNase I 100µg/ml (all Sigma-Aldrich/Merck, Darmstadt, Germany) solution in DMEM (Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.) containing 10% FCS and 1% penicillin-streptomycin). Cells were meshed through 50µm filters (Cell Trics, Partec, Sysmex Europe GmbH, Goerlitz, Germany) and erythrocytes lysed in ACK buffer containing NH 4 Cl 150 mM, KHCO 3 10 mM, Na 2 EDTA 0.1 mM. Single-cell suspensions were stained with antibodies and reagents listed below. Isolation of bone marrow neutrophils. Neutrophils were isolated from bone marrow of non-infected mice. Bone marrow cells were collected via perfusion of femoral bones from each animal under aseptic conditions. Cells were meshed through 50µm filters (Cell Trics, Partec, Sysmex Europe GmbH, Goerlitz, Germany) and erythrocytes lysed in ACK buffer containing NH 4 Cl 150 mM, KHCO 3 10 mM, Na 2 EDTA 0.1 mM. Single-cell suspensions were stained with antibodies listed below, Ly6G + viable neutrophils were sorted using a FACS Aria cell sorter (BD Biosciences, BD, New Jersey, U.S.), and the purity of cells was assessed (≥95%), all neutrophils are CD11b + . After sorting cells were resuspended in DMEM containing 10% FCS. Blood was collected after sacrificing via heart puncture in heparinized tubes. Plasma was collected after centrifugation at 2000g, frozen at -80C until further analysis. Alternatively, white blood cells were collected from blood after threefold lysis of red blood cells with ACK buffer containing NH 4 Cl 150 mM, KHCO 3 10 mM, Na 2 EDTA 0.1 mM. Visualization of mitochondria . Staining for nuclei, cell membrane and mitochondria with Hoerst, PKH and Mitospy, respectively, was performed according to the manufacturer’s protocols, samples were evaluated with flow cytometry and on cytospins microscopically. Reactive oxygen species . Cells were washed and resuspended in DMEM containing 10% FCS, P. aeruginosa PA14 WT MOI 10 was added. Sterile medium was used as negative control. ROS production by Ly6G + viable neutrophils was estimated after 60 min of exposure to P. aeruginosa using Dihydrorhodamine 123 (Sigma-Aldrich/Merck, Darmstadt, Germany) with flow cytometry. Phagocytosis of CFSE-labelled P. aeruginosa . Lung tissue was harvested from non-infected animals under aseptic conditions; single cell suspension was prepared and stained with antibodies. Cells were then washed and resuspended in DMEM containing 10% FCS and DNase (to prevent binding of non-phagocyted bacteria in NETs and false-positive results), P. aeruginosa PA14 WT labeled with CFSE (MOI 10) added. Phagocytosis of CFSE-labeled bacteria by Ly6G + neutrophils was estimated after 60 min using flow cytometry. Phagocytosis of FITC-labelled beads (Caymann) according to manufacturer’s protocol. Isolation of lung neutrophils. For estimation of neutrophil functions, neutrophils were isolated from lungs of non-infected mice. Lung tissue was harvested from each animal under aseptic conditions; single cell suspension was prepared as described above. Single-cell suspensions were stained with antibodies listed below, Ly6G + viable neutrophils, as well as subpopulations Ly6G + CD62L high , Ly6g + CD62L low were sorted using a FACS Aria cell sorter (BD Biosciences, BD, New Jersey, U.S.), and the purity of cells was assessed (≥95%), all neutrophils are CD11b + . After sorting cells were used in pellet for proteomics or resuspended in DMEM containing 10% FCS for functional assays. Proteomics of isolated lung neutrophils Sample preparation. 80.000 mouse lung neutrophils per sample were dissolved in 70 µl of lysis buffer (50 mM Tris-HCl (pH 7.8) 150 mM NaCl, and 1% SDS supplemented with complete mini-EDTA free protease inhibitor, Roche, Penzberg). The proteins were reduced for 30 min at 37°C in 10 mM DTT and alkylated in 30 mM IAA for 30 min at RT in the dark. After that, the proteins were precipitated with nine volumes of Ethanol for 1h at -80°C and centrifuged for 30 min at 20,000g. The supernatant was removed, and the pellet was dried and dissolved first in 1 µL of 6M GuHCl and then in 29 µL of 50 mM ammonium bicarbonate buffer, pH 7.8 containing 2 mM CaCl 2 and 50 ng of Trypsin (sequencing grade, Promega) and incubated for 18 h at 37°C. The enzymatic digestion was stopped by acidifying the sample to pH<2.5 with TFA. High pH fractionation . 8 high pH reversed phase fractions were created for spectral library generation using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific). For fractionation, equal amounts of each analyzed sample were combined to a total 50 µg peptides. Peptides were vacuum dried and dissolved in 0.1% TFA according to the instructions and fractionation was performed following the manual. Fractionated and vacuum dried peptide samples were dissolved in 10 µL 0.1% FA for LC-MS/MS measurement. LC-MS/MS acquisition. For liquid-chromatography-coupled tandem mass spectrometry (LC-MS/MS) measurements, 2 µL tryptic peptides were injected for individual samples. Spectral library fractions were injected with 2 µL (fraction 1) and 4 µL (fraction 2-8). Measurements were performed on a quadrupole-ion-trap-orbitrap MS (Orbitrap Fusion, Thermo Fisher) coupled to a nano-UPLC (Dionex Ultimate 3000 UPLC system, Thermo Fisher). Chromatographic separation of peptides was achieved with a two-buffer system (buffer A: 0.1% FA in water, buffer B: 0.1% FA in ACN). Attached to the UPLC was a peptide trap (100 μm × 200 mm, 100 Å pore size, 5 μm particle size, C18, Thermo Fisher Scientific) for online desalting and purification followed by a 25 cm C18 reversed-phase column (75 μm × 250 mm, 130 Å pore size, 1.7 μm particle size, Peptide BEH C18, Waters). Peptides were separated using an 80-min method with linearly increasing ACN concentration from 2% to 30% ACN in 60 minutes. Eluting peptides were ionized using a nano-electrospray ionization source (nano-ESI) with a spray voltage of 1800, transferred into the MS, and analyzed in data-dependent acquisition (DDA) mode. For each MS1 scan, ions were accumulated for a maximum of 120 milliseconds or until a charge density of 2x10 5 ions (AGC Target) was reached. Fourier-transformation-based mass analysis of the data from the orbitrap mass analyzer was performed covering a mass range of 400-1200 m/z with a resolution of 120000 at m/z = 200. Peptides with charge states between 2+ - 5+ above an intensity threshold of 1000 were isolated within a 1.6 m/z isolation window in Top Speed mode for 3 seconds from each precursor scan and fragmented with a normalized collision energy of 30% using higher energy collisional dissociation (HCD). MS2 scanning was performed, using an ion trap mass analyzer at a rapid scan rate, covering a mass range starting at m/z 120, and accumulated for 60ms or to an AGC target of 1x10 5 . Already fragmented peptides were excluded for 30 seconds. Raw data processing and normalization. LC-MS/MS from DDA were searched with the Sequest algorithm integrated into the Proteome Discoverer software (v 2.4.1.15), Thermo Fisher Scientific) against a reviewed murine Swissprot database, obtained in October 2020, containing 17053 entries. Carbamidomethylation was set as fixed modification for cysteine residues and the oxidation of methionine, and pyro-glutamate formation at glutamine residues at the peptide N-terminus, as well as acetylation of the protein N-terminus were allowed as variable modifications. A maximum number of 2 missing tryptic cleavages was set. Peptides between 6 and 144 amino acids were considered. A strict cutoff (FDR<0.01) was set for peptide and protein identification. Quantification was performed using the Minora Algorithm, implemented in Proteome Discoverer. LC-MS/MS from pH fractions were handled in a separate processing step within the software. A multi-consensus workflow was applied to sample and library mgf files to generate a combined output and increase the protein-identification rate for individual samples trough feature mapper. Protein abundances for individual samples were exported and submitted to subsequent statistical analysis. Protein abundances for library fractions were discarded prior to normalization. Lowess algorithm was applied for data normalization [48]. Statistics Significant regulation was considered for the proteins and transcripts with Log2 FC>±2 and p-Value<0.05. Subsequently, the p-values were adjusted for the false discovery rate (FDR) with Benjamini-Hochberg. The signal-to-noise (SNR) value was calculated using SNR=log2x1-x2d1+d2 with d1 and d2 representing the respective standard deviation. Gene ontology (GO) enrichment analysis of the generated datasets of differentially expressed proteins was performed using open access ShinyGO platform (Version 0.78, http://bioinformatics.sdstate.edu/go/, [49]). The hypergeometric test after the Benjamini-Hochberg false discovery rate (FDR) correction was used to assess statistical significance. Enriched GO terms with FDR-corrected P < 0.05 were considered statistically significant. In addition to the use of functional annotation tools, we also searched PubMed manually to gain insights into the functions of the identified differentially expressed proteins. NETs release. Isolated neutrophils 15.000/well were incubated with P. aeruginosa (MOI 10) in glass-bottom 96-well plate (MatTek Corporation, Massachusetts, U.S.) pre-coated with poly-D-lysine 1 mg/ml (Sigma-Aldrich/Merck, Darmstadt, Germany) for 4 hours at 37 o C, 5% CO 2 , sterile medium was used as a negative control. Samples were fixed with paraformaldehyde (Thermo Fisher Scientific, Massachusetts, U.S.) to the final concentration 4%, permeabilized with Triton X-100 (Sigma-Aldrich/Merck, Darmstadt, Germany) 0.2% containing buffer. Since the visualization of NETs using DNA-intercalating dyes alone has the risk of detection of necrotic cells or the generation of artificial results based on dye-blocking peptides associated with NETs, antibody-based techniques are required to visualize NETs. Anti-histone 1 antibodies (Merck Millipore, Darmstadt, Germany) were used to detect all NETs. Donkey-anti-mouse-AF564 (Invitrogen, Thermo Fisher Scientific, Massachusetts, U.S.) were used as secondary antibodies. Stainings were mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen, Thermo Fisher Scientific, Massachusetts, U.S.). Percent of NET-producing cells and NETs length and area were estimated by microscopy followed by analysis with ImageJ Fiji software. Isolation of bone marrow progenitors and in vitro maturation assay (adapted from Siakaeva et al, REF). Murine BM progenitor cells were negative selected by depletion of CD3e + CD45R + NK1.1 + CD11b + Ter119 + BM cells using the Streptavidin MicroBeads and LD Columns from Miltenyi Biotec (Bergisch Gladbach, Germany) according to the manufactuer’s protocols. For maturation isolated cells were cultured in 24-well plates at the concentration of 0,3x10 6 cells/ml in tumor cell line-condenced media (MOPC, G-MOPC or control M-medium) with addtition of mrSCF and mrIL3 (all from Peprotec, Hamburg, Germany; end concenration 50 ng/ml) 7 days long at 37°C and 5% CO 2 , medium was changed into fresh at day 4. aG-CSFR in concentration (5 µg/ml), LLL12 (1µM), FK866 (100 nM) were used. At day 4 tumor-condenced medium was exchanged into sterile M-medium. At day 7 the composition of the cultivated cells in all conditions was analyzed with microscopy and flow cytometry. Phenotype (viability, Ly6G, CD11b, CD62L expression) and functions (ROS, phagocytosis) of Ly6G + cells were evaluated with flow cytometry as described previously. For morphological analysis , cytospin preparations of maturated bone marrow cells on SuperfrostTM slides (Gibco, Thermo Fisher Scientific, Waltham, MA, US) were fixed with pure methanol, stained by Giemsa (Sigma-Aldrich, Merck KGaA, St. Louis, MO, US) and nuclear morphology was assessed using light microscope Olympus BX5 (Olympus, Tokyo, Japan). At least 10 fields of view were counted and percentages of immature, band and segmented nuclei were calculated. NAD assay in plasma was performed according to manufacturer’s protocol Human cohort. Patients with head and neck cancer and healthy individuals participated in the study (clinical characteristics in the Table 1). Peripheral blood obtained from healthy donors, was drawn into 3.8% sodium citrate anticoagulant monovettes (Sarstedt, Nuembrecht, Germany) and mixed 1:1 with PBS (Gibco, Thermo Fisher Scientific, Waltham, MA, US) before separation by density gradient centrifugation (Pancoll density 1,077 g/ml). The mononuclear cell fraction was discarded and neutrophils (purity ≥95%) were isolated by sedimentation over 1% polyvinyl alcohol, followed by hypotonic lysis (0.2% NaCl) of erythrocytes and reconstitution of osmolarity with 1.2% NaCl. Isolated neutrophils (1 mln/ml in RPMI medium containing 10% FCS) alone or with P. aeruginosa MOI 10 were incubated for 1 hour, afterwards analysis of ROS, phagocytosis (as described above) and NET formation (using Sytox Green reagent in combination with anti-MPO according to manufacturer’s protocol) was performed. Oral rinse with 15 ml of sterile saline was collected from n=28 healthy controls and n=45 patients with HNSCC directly after awaking prior to oral hygiene and food/drink consumption, for 1 min. The absolute amount of cells in the rinse was evaluated, the proportion of viable CD66b + neutrophils and their activation (CD62L expression) were estimated with flow cytometry. Soluble fraction of oral rinse was collected and centrifuged at 3000g. After discarding the supernatant, the bacteria were resuspended in a total of 0.4 ml of sterile saline, and 10 µl of the resuspended bacteria were inoculated onto Columia blood agar and Chocolate blood agar as universal media, and onto Mac Conkey agar (all media from Oxoid, Wesel, Germany), a selective medium for the growth of Gram-negative rods. Bacteria were cultured at 36°C under aerobic conditions with 5% CO 2 and growth was assessed after 24 and 48 hours. Bacteria were identified using VITEK2 TM (bioMérieux, Marcy-l’Étoile, France), MicroScan WalkAway (Beckman Coulter, Brea, US), VITEK MS (bioMérieux) or MALDI Biotyper (Bruker, Billerica, US). Tumor samples were cut into 1 mm 3 pieces with sterile instruments and incubated in RPMI medium containing 10% FCS, 1% Pen/Strep and 0.2% of Fungizon in proportion 0.02 g/ 0.6 ml for 4 hours, afterward supernatant was collected. In parallel, single cell suspension of the tumor was derived how is explained above, the content and activation (expression of CD62L) of viable tissue CD66b + neutrophils was evaluated with flow cytometry. ELISA. G-CSF, MMP9 and TNF-a in murine plasma samples and lung supernatants and neutrophi-conditioned medium, G-CSF in human tumor supernatants and oral rinse were analyzed with ELISA (R&D Systems, Minnesota, U.S.) according to manufacturer protocols. RT-qPCR. The RNA was isolated using Qia Shredder and RNeasy Mini Kit (Qiagen, Hilden, Germany) and the cDNA was produced using the Superscript II Reverse Transcriptase Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, US). qRT-PCR was performed at 60 °C annealing temperature using primers listed below. As housekeeping gene, Rps9 was used. The mRNA expression was measured using the Luna Universal qPCR Master Mix (New England BioLabs, Ipswich, MA, US). Relative gene expressions were calculated by 2^-ΔCt formulations. Real-time RT-PCR was performed using primers listed in the supplemental file 9. ­ List of reagents, antibodies and consumables in the supplemental file 9. ­Analysis of the data deposited in the Gene Expression Omnibus databases. We used previously published microarray data deposited in the Gene Expression Omnibus databases GSE11247 (GSM283955, GSM283956, GSM283957, GSM283958, GSM283959, GSM283960, GSM283961, GSM283962) Statistics Statistical analyses were performed using Kruskal-Wallis ANOVA for multiple comparisons with the Bonferroni correction, and Mann-Whitney U-test for two independent samples, and Whilcoxon test for dependent samples; correlations were analyzed with Spearman R test. Sensitivity, specificity of the diagnostic test, relative risk were calculated using MedCalc’s calculators (available online https://www.medcalc.org/calc/). To assess the accuracy of model predictions, receiver operating characteristic (ROC) analysis was performed. P<0.05 was considered significant. Declarations Ethics approval and consent to participate The animal experiments have been approved by the regulatory authorities LANUV (Das Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen), Germany. Our animal care and use protocols adhere to the regulations of das Deutsche Tierschutzgesetz (TierSchG) and follow FELASA recommendations. Research involving human material, or human data was performed in accordance with the Declaration of Helsinki and was approved by the ethics committee of the University Hospital Essen, Germany (19-8599-BO, 16-7135-BO). Informed consent to participate in the study was obtained from participants Consent for publication Not applicable. Availability of data and materials The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [50] partner repository with the dataset identifier PXD052631. Other materials are available upon request to interested researchers. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding The study is supported by grants from the Deutsche Forschungsgemeinschaft (DFG/ JA 2461/2-1, DFG/ JA 2461/7-1, TR332 A5) and Deutsche Krebshilfe (111647). BS and HS received funding from the INST 337/15-1, INST 337/16-1, INST 152/837-1 and INST 152/947-1 FUGG. DRE received funding from the Deutsche Forschungsgemeinschaft FOR5427 SP4 (DRE); EN984/15-1, 16-1 and 18-1 (DRE); TR296 P09 (DRE); TR332 A3 and Z1 (DRE), and INST 20876/486-1. OS received funding from the Deutsche Forschungsgemeinschaft: FOR5427 SP1. Authors' contributions Conceptualization, E.P. and J.J.; Methodology, E.P., O.S., H.H., C.K., J.K., D.R.E. and J.J.; Software, O.S., B.S., H.S.; Validation, E.P. and J.J., Formal Analysis, E.P., L.T., J.R., O.S. and A.S.; Investigation, E.P., L.T., J.R., O.S., I.T., J.A., I.O., B.S., H.S. and C.H.; Resources, O.S., B.S., H.S., H.H., S.M., J.K., D.R.E., S.L. and J.J.; Data Curation, E.P., O.S., B.S., H.S. and J.J.; Writing – Original Draft Preparation, E.P., O.S. and C.K.; Writing – Review & Editing, H.H., S.M., J.K., D.R.E., S.L. and J.J. ; Visualization, E.P., O.S., B.S. and H.S.; Supervision, S.L. and J.J., Project Administration, J.J.; Funding Acquisition, O.S., D.R.E., S.L. and J.J. Acknowledgements We acknowledge support by the Open Access Publication Fund of the University of Duisburg-Essen, the Imaging Center Essen (Alexandra Brenzel and Dr. Anthony Squire) and the Immunoproteomics group (Stephanie Tautges-Schaefer, Stephanie Thiebes and Jenny Dick). 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Supplementary Files supplementalfigureswithlegends.docx supplemental figures GA.png Graphical abstract Revieweraccessdetails.docx ProteomeXchange accession code for the mass spectrometry raw data Cite Share Download PDF Status: Published Journal Publication published 12 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6098692","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":423276062,"identity":"8ba7b5a6-99bb-42b8-a248-eabc4c18fffd","order_by":0,"name":"Jadwiga 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rinse with 50 ml of sterile saline was collected in n=28 healthy controls and n=45 patients with HNSCC directly after awaking prior to oral hygiene and food/drink consumption. Frequencies of Gram-negative pathogens (E. coli, K. pneumoniae, H. influenza, P. aeruginosa) were evaluated by culture using universal and selective agar media, G-CSF levels were investigated with ELISA. The 2 years follow-up information was collected. (A) Frequency of clinically significant Gram-negative pathogens detected in oral rinse is higher in untreated patients with HNSCC in comparison to healthy volunteers. (C) In HNSCC, presence of Gram-negative pathogens in oral rinse is associated with poor 2-years outcome. (B) In HNSCC, presence of Gram-negative pathogens in oral rinse is associated with increased risk of bacterial infections in a 2 years follow-up. (D) HNSCC tumors produce high levels of G-CSF (E) G-CSF levels in oral rinse are elevated in patients with HNSCC. (F) A receiver operating characteristic (ROC) curve of G-CSF in oral rinse as a predictor for presence of Gram-negative pathogens in oral cavity in HNSCC. (G) Concentration of G-CSF in oral rinse higher than 392 pg/ml is a predictor for presence of Gram-negative pathogens in oral cavity in HNSCC. (H) The risk of detection of Gram-negative pathogens in oral cavity is 4.25 times higher in patients with HNSCC with concentration of G-CSF in oral rinse higher than 392 pg/ml. H - healthy, HNSCC - head and neck cancer, G-CSF - granulocyte colony-stimulating factor, AUC - area under the ROC curve. Mean values are shown, dots represent individual values. Chi-square test (A,C,G), Kruskal-Wallis test for comparison of multiple groups and Mann-Whitney test for two groups comparison (D,E), comparison of Kaplan-Meier survival curves using Log-rank (Mantel-Cox) test (B). * p \u0026lt; 0.05, # p=0.05.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6098692/v1/6f4338a1ffa4486635edd10b.png"},{"id":79128865,"identity":"f3d990ba-d6e8-4d26-b997-e2839548e19b","added_by":"auto","created_at":"2025-03-24 18:07:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":963688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMurine model of HNSCC with high production of G-CSF (G-MOPC) is characterized by impaired neutrophilic bactericidal responses.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e(A) G-CSF levels in tumor supernatants from patients with HNSCC and in murine model of oropharyngeal carcinoma with low and high production of G-CSF. (B-F) Mice were injected s.c. with PBS (Co group), MOPC or \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC (n=8 in each roup), at day 14 10\u003c/em\u003e\u003csup\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e P.aeruginosa were inoculated intratracheally, after 18 hours animals were sacrificed, bronchoalveolar lavage and lung tissue were collected. (B) Decreased clearance of P. aeruginosa from lower respiratory tract of mice bearing \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC.(C) Lung tissue damage, representative image, HE, scale bar 30 µm.(D) Decreased aerated area in lungs of mice bearing \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC.(E) Elevated expression of TNFa in lungs of mice bearing \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC.(F)Worse general clinical performance of mice bearing \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC. (G-H) Mice were injected s.c. with PBS (Co group), MOPC or \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC, at day 14 animals were sacrificed, lung tissue was collected, alive Ly6G+ lung neutrophils were isolated. (G) Mice bearing \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC are characterized with prominent lung infiltration of neutrophils in steady state. (H) Predominance of CD62Llow CD11bdim neutrophil subsets in lungs of \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-bearing mice. (I-M) Mice were injected s.c. with PBS (Co group), MOPC or \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC, at day 14 animals were sacrificed, lung tissue was collected, alive Ly6G+ lung neutrophils were isolated, proteome of isolated neutrophils was assessed. (I-J). Lung \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils have distinct proteome signature of \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. (K-M). GO enrichment analysis for the pathways up- (orange) and down-regulated (blue) in lung \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils (K. biological processes,\u0026nbsp; L. cellular components, M. molecular functions). Co - control group of tumor-free mice, MOPC - mice bearing G-CSF-not-producing tumors, \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC - mice bearing G-CSF producing tumors, BAL – bronchoalveolar lavage, Tnfa – tumor necrosis factor alpha, CFUs - colony-forming units. Mean values are shown, dots represent individual values. Chi-square test (F), Kruskal-Wallis test for comparison of multiple groups and Mann-Whitney test for two groups comparison (A,B,D,E,G). \u0026nbsp;* p \u0026lt; 0.05, # p=0.05.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6098692/v1/bfaa24e8363c1f628ef0e05c.png"},{"id":79128874,"identity":"8c00d6f3-a59a-4820-9b7e-3a81d75c6340","added_by":"auto","created_at":"2025-03-24 18:07:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":981363,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChronic exposure to tumor-derived G-CSF (\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eneutrophils) impairs bactericidal activity and elevates tissue toxicity of neutrophils\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. (A) Mice were injected s.c. with MOPC or \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC, at day 14, animals were sacrificed, lung tissue was collected, viable Ly6G+ lung neutrophils were isolated and analyzed by LC-MS/MS based proteomics. Volcano plot depicts downregulated proteins involved in actin polymerization and phagocytosis (blue) and upregulated proteins involved in response to reactive oxygen species (orange) in \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. (B-L, O) Mice were injected s.c. with PBS (Co group), MOPC or \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC, at day 14, animals were sacrificed, lung tissue was collected, viable Ly6G+ lung neutrophils were isolated, properties and functions were evaluated in vitro in absence/presence of P. aeruginosa MOI 10. (B) Decreased phagocytic capacity of \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils towards CFSE-labelled P. aeruginosa. (C) Decreased NET release by \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils towards P. aeruginosa. (D) Decreased actin polimerization in \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils in presence of lipopolysaccharide, estimated by intracellular staining of F-actin with phalloidin-FITC. (E) Representative image of decreased NET release by \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils towards P. aeruginosa. Samples were fixed, permeabilized,\u0026nbsp; stained and evaluated microscopically, scale bar 65 µm, orange Histone 1, blue DAPI. (F) Elevated expression of Mpo protein in \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. (G) \u0026nbsp;Elevated expression of Mpo gene in \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils\u0026nbsp; \u0026nbsp;(H) Increased mitochondrial count in \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. (I) Representative image of increased mitochondrial count in \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. Isolated neutrophils were stained with MitoSpy, PKH26 and Hoechst, evaluated microscopically, scale bar 10 µm, red membrane dye PKH 26, MitoSpy green, Hoechst blue. (J) Elevated ROS production of unstimulated \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils, assessed with 123-DHR. (K) Elevated ROS production of P.aeruginosa-stimulated \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils, assessed with 123-DHR. \u0026nbsp;(L) Representative flow cytometry histogram for ROS, light green – unstimulated neutrophils, dark green – P. aeruginosa stimulated neutrophils, light red – unstimulated \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils, dark red – P. aeruginosa stimulated \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils.\u0026nbsp; \u0026nbsp;(M,N) Mmp9 levels were estimated with ELISA. (M) Elevated expression of Mmp9 in plasma of \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC bearing mice. (N) Elevated expression of Mmp9 in supernatant after 24 hour cultivation of \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils.\u0026nbsp; \u0026nbsp;(O) Elevated expression of Mmp9 gene in \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. Co - control group of tumor-free mice, MOPC - mice bearing G-CSF-not-producing tumors, \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC - mice bearing G-CSF producing tumors, NETs - neutrophil extracellular traps, Mpo - myeloperoxidase, ROS - reactive oxygen species, Mmp9 - matrix metalloproteinase 9, CFUs - colony-forming units. Mean values are shown, dots represent individual values. Kruskal-Wallis test for comparison of multiple groups and Mann-Whitney test for two groups comparison. \u0026nbsp;* p \u0026lt; 0.05, # p=0.05.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6098692/v1/0a493f15ea8fbe957d2dd042.png"},{"id":79128866,"identity":"591a162d-4fa1-41a8-a07b-c7c6040fddf0","added_by":"auto","created_at":"2025-03-24 18:07:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":761568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChronic exposure to tumor-derived G-CSF (\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eneutrophils) impairs ageing and accumulation of tissue toxic CD62L\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003elow\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e subpopulation of neutrophils.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u0026nbsp;(A) Volcano plot illustrating downregulated proteins involved in regulation of neutrophil ageing and apoptosis in \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. Mice were injected s.c. with MOPC or \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC, at day 14 animals were sacrificed, lung tissue was collected, viable Ly6G+ lung neutrophils were isolated, proteome of isolated neutrophils was assessed. \u0026nbsp;(B-K) Mice were injected s.c. with PBS (Co group), MOPC or \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC. On day 14, animals were sacrificed, lung tissue was collected. The properties and functions of viable Ly6G+ lung \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils were then evaluated in vitro in absence/presence of P. aeruginosa at the multiplicity of infection (MOI) of 10, depending on their CD62L surface expression. (B) Decreased apoptosis of \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. \u0026nbsp;(C) Decreased Casp3 gene expression in \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. \u0026nbsp;(D). Accumulation of CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils in lungs. \u0026nbsp;(E) Elevated degranulation of CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils assessed by SSC. (F) Elevated release of Mmp9 by CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. (G) Elevated \u0026nbsp;killing of epithelial tumor cells by CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. (H) Decreased phagocytic capacity of CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils towards CFSE-labelled P. aeruginosa. (I) Representative image of decreased NET release by CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils towards P. aeruginosa, Samples were fixed, permeabilized, stained and evaluated microscopically, scale bar 100 µm, orange Histone 1, blue DAPI. (J) Decreased percentage of NET-producing cells from CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. (K) Decreased length of released NETs by CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. (L) High release of G-CSF by tumor tissue analyzed in tumor-condensed medium, \u0026nbsp;is associated with deceased CD62L expression on tumor neutrophils. \u0026nbsp;(M) In HNSCC patients (n=45), concentration of G-CSF oral rinse was analyzed and correlated with expression of CD62L on CD66b+ viable neutrophils. High levels of G-CSF in saliva are associated with deceased CD62L expression on salivary neutrophils. \u0026nbsp;(N-P) Circulating neutrophils were isolated from peripheral venous blood of healthy volunteers with density gradient, functions were determined in relation to the surface expression of CD62L. (N) Elevated spontaneous ROS production of CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow \u003c/em\u003e\u003c/sup\u003e\u003cem\u003ehuman neutrophils. (O) Decreased capacity to produce NETs towards P. aeruginosa MOI 10\u0026nbsp; by CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e human neutrophils, estimated by staining with Sytox Green. (P) Decreased phagocytic capacity of CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e human neutrophils towards CFSE-labelled P. aeruginosa. G-CSF - granulocyte colony stimulating factor, Co - control group of tumor-free mice, MOPC - mice bearing G-CSF-not-producing tumors, G-MOPC - mice bearing G-CSF producing tumors, Casp3 - caspase 3, SSC - side scatter, Mmp9 - matrix metalloproteinase 9, NETs - neutrophil extracellular traps, ROS - reactive oxygen species, Mmp9 - matrix metalloproteinase 9, CFUs - colony-forming units. Mean values are shown, dots represent individual values. Kruskal-Wallis test for comparison of multiple groups and Mann-Whitney test for two groups comparison (B-L), Spearman (M), paired Student T test (N-P) was used\u0026nbsp; * p \u0026lt; 0.05, # p=0.05.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6098692/v1/bf6e0214e72e0e0b64a5fb14.png"},{"id":79130317,"identity":"d1323e47-f89b-4dd7-a99a-6c2748a91623","added_by":"auto","created_at":"2025-03-24 18:31:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":649106,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTumor-derived G-CSF induces persistent changes in neutrophil progenitors in vitro.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u0026nbsp;(A) Scheme of the experiment. Bone marrow progenitors CD3e-CD45R-NK1.1-CD11b-Ly6G-Ter119-we prepared by immunomagnetic isolation by negative selection as described previously ([17], with modifications). Isolated progenitors were exposed to MOPC or \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium for 6 days (A, left) or 3 days followed by 3 days of exposure to medium (absence of stimulation) (A, right), phenotype and functions of de novo differentiated Ly6G+ neutrophils were analyzed at day 6. \u0026nbsp;(B-H) Isolated bone marrow progenitors were exposed to MOPC or \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium for 6 days, analysis of morphology (B) and phenotype and functions of de novo differentiated Ly6G+ neutrophils with flow cytometry (C-H) was performed. (B) Representative image of Giemsa-stained samples, scale bar 20 µm. (C) \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium induced differentiation of progenitors into of Ly6G+ neutrophils. (D) \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium promoted degranulation. (E) \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium promoted accumulation of CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Ly6G+ neutrophils. (F) \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium decreased expression of CD11b. (G) \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium elevated unstimulated ROS production. (H) \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium non-significantly decreased phagocytosis of Ly6G+ neutrophils towards CFSE-labelled P. aeruginosa. (I-M) Isolated progenitors were exposed to MOPC or \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium for 3 days followed by 3 days of exposure to medium (absence of stimulation), analysis of phenotype and functions of de novo differentiated Ly6G+ neutrophils with flow cytometry was performed. (I) Initial exposure to \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium promoted degranulation. (J) Initial exposure to \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium increased apoptosis. (K) Initial exposure to \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium decreased expression of CD11b. (L) Initial exposure to \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium elevated unstimulated ROS production. (M) Initial exposure to \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium decreased phagocytosis of Ly6G+ neutrophils towards CFSE-labelled P. aeruginosa. Co - control (initial) state, MOPC – cells incubated with MOPC-condenced medium, \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC – cells incubated with \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-condenced medium, SSC - side scatter, ROS - reactive oxygen species. Mean values are shown, dots represent individual values\u003c/em\u003e \u003cem\u003eKruskal-Wallis test for comparison of multiple groups and Mann-Whitney test for two groups comparison. \u0026nbsp;\u0026nbsp;* p \u0026lt; 0.05, # p=0.05.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6098692/v1/7bd4865716146c4654abbc59.png"},{"id":79128869,"identity":"d503ae9b-147e-4037-bf31-fa93bb4c5263","added_by":"auto","created_at":"2025-03-24 18:07:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1184769,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eInhibition of G-CSF receptor downstream pathway abrogates tissue toxicity of \u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eneutrophils and improves bacterial clearance\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. (A). Mice were injected s.c. with PBS (Co group), MOPC or \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC, at day 14 animals were sacrificed, blood was collected.\u0026nbsp; Elevated NAD levels in plasma \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-bearing mice. \u0026nbsp;(B). Isolated bone marrow progenitors were exposed \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC-conditioned medium for 6 days in absence or presence of NAMPT inhibitor FK866. NAMPT inhibition normalized unstimulated ROS production in de novo maturated \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. \u0026nbsp;(C-L). Mice bearing \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC were treated i.p. with NAMPT inhibitor FK866 every second day, at the day 14, mice were sacrificed, neutrophil phenotype and functionality were analyzed in lung single cell suspension. \u0026nbsp;(C) NAMPT inhibition decreased infiltration of lungs with \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. \u0026nbsp;(D) NAMPT inhibition prevented accumulation of CD62L\u003c/em\u003e\u003csup\u003e\u003cem\u003elow\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. (E-G) NAMPT inhibition enhanced \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophil cell death. (H) NAMPT inhibition normalized unstimulated ROS production by \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. \u0026nbsp;(I) NAMPT inhibition normalized P. aeruginosa-stimulated Mmp9 release by \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils, \u0026nbsp;(J) NAMPT inhibition did not influence phagocytosis of CFSE-labeled P. aeruginosa by \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eneutrophils. (K-L) Mice bearing G-MOPC were treated i.p. with NAMPT inhibitor FK866 every second day, at the day 14 days mice were infected with P. aeruginosa intratracheally, and sacrificed after 18 hours. \u0026nbsp;(K) NAMPT inhibition promoted bacterial clearance in the lower respiratory tract, assessed by bacterial culture of bronchoalveolar lavage on universal and selective agar media \u0026nbsp;ß. \u0026nbsp;(L) NAMPT inhibition improved clinical performance of infected animals. \u0026nbsp;Co - control group of tumor-free mice, MOPC - mice bearing G-CSF-not-producing tumors, \u003c/em\u003e\u003csup\u003e\u003cem\u003eG\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eMOPC - mice bearing G-CSF producing tumors, NAD - nicotinamide adenine dinucleotide, ROS - reactive oxygen species, Mmp9 - matrix metalloproteinase 9, CFUs - colony-forming units. . Mean values are shown, dots represent individual values. Kruskal-Wallis test for comparison of multiple groups and Mann-Whitney test for two groups comparison (A-K) and Chi-square test (M) was used * p \u0026lt; 0.05, # p=0.05.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6098692/v1/3d87327c1e00d27ebfe3c849.png"},{"id":98290932,"identity":"2f94c1a7-e18a-4367-b23b-c83e9d708e22","added_by":"auto","created_at":"2025-12-16 08:11:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7283770,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6098692/v1/e79e8c4f-8a68-4736-8b21-65d47969b04d.pdf"},{"id":79128861,"identity":"c8e296cd-f6dd-4d0b-ab22-e779ef24b04e","added_by":"auto","created_at":"2025-03-24 18:07:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1975260,"visible":true,"origin":"","legend":"supplemental figures","description":"","filename":"supplementalfigureswithlegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6098692/v1/355b24af8fb6e542987c754e.docx"},{"id":79130043,"identity":"d9c8c178-241a-407c-a10f-9a583dbc9df7","added_by":"auto","created_at":"2025-03-24 18:23:53","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":494650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-6098692/v1/58e610b0f560f3157d17a97a.png"},{"id":79129582,"identity":"5b612e23-0910-4a11-98f3-0bf67499086f","added_by":"auto","created_at":"2025-03-24 18:15:53","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13594,"visible":true,"origin":"","legend":"ProteomeXchange accession code for the mass spectrometry raw data","description":"","filename":"Revieweraccessdetails.docx","url":"https://assets-eu.researchsquare.com/files/rs-6098692/v1/dfeae4a49a4739c3e8f1c4bc.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"NAMPT/NAD+ signaling drives neutrophil dysfunction and enhances bacterial infection susceptibility in cancer patients","fulltext":[{"header":"Background","content":"\u003cp\u003eIn patients with cancer, infectious complications significantly impact clinical outcomes, delaying treatment and increasing morbidity and mortality. Among these, infections caused by \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e represent a particularly serious threat, with cancer patients experiencing a 50-fold higher risk of bacteremia compared to the general population [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Alongside other Gram-negative pathogens such as \u003cem\u003eKlebsiella pneumoniae, Escherichia coli\u003c/em\u003e, and \u003cem\u003eHaemophilus influenzae\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e remains a high-priority target in the 2024 Bacterial Priority Pathogens List due to its high rates of antibiotic resistance and global prevalence, especially in healthcare settings. The World Health Organization underscores the urgent need for innovative prevention and control measures, as well as alternative therapeutic strategies, to combat these pathogens effectively [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile traditional risk factors, including prolonged hospitalization and immunosuppressive treatments, contribute to the increased infection burden in cancer patients, they do not fully explain the vulnerability observed even in untreated cases. Emerging evidence suggests that tumors themselves may drive an intrinsic reprogramming of immune functionality, significantly impairing the body\u0026rsquo;s ability to resist bacterial infections [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This raises critical questions about the mechanisms of immune dysfunction in cancer and their implications for both infection control and therapeutic intervention.\u003c/p\u003e \u003cp\u003eNeutrophils, central to antibacterial immunity, have historically been viewed as short-lived innate effector cells with limited adaptability [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, recent discoveries in the field of trained immunity challenge this perspective, demonstrating that innate immune cells, including neutrophils, can acquire a form of memory through epigenetic modulation following specific stimuli [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. While trained immunity enhances responses to subsequent infections [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] or even malignancies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], the tumor microenvironment appears to induce a distinct phenomenon: immune reprogramming. Tumor-derived factors drive this reprogramming, potentially resulting in maladaptive immune responses that compromise the host's ability to clear bacterial infections [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Despite its clinical significance, the long-term impact of tumor-driven innate immune reprogramming on neutrophil function remains poorly understood.\u003c/p\u003e \u003cp\u003eThis study seeks to address this critical gap by investigating the effects of chronic exposure to tumor-derived factors on neutrophil development and function. Specifically, we focus on the role of G-CSF in driving neutrophil reprogramming, its contribution to impaired antibacterial defenses, and the elevated risk of infections in cancer patients. Furthermore, we explore whether targeting the G-CSF signaling pathway can reverse these changes and restore effective immunity. By elucidating the mechanisms of tumor-induced immune dysfunction, this work aims to inform the development of therapeutic approaches targeting patients' immune antibacterial defenses to mitigate infection-related complications and improve outcomes in patients with cancer.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eIncreased susceptibility of cancer patients to bacterial infections is associated with high G-CSF release from tumor tissue\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCancer patients suffer from recurring bacterial infections. To assess the abundance of clinically relevant Gram-negative pathogens in such patients (\u003cem\u003eP. aeruginosa, E. coli, K. pneumonia, H. influenza\u003c/em\u003e) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], we performed microbiological analysis of oral rinse of clinically asymptomatic HNSCC patients and compared it to healthy individuals (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of note, the frequency of Gram-negative pathogens was significantly higher in such patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinicopathological characterization of the study participants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHealthy\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;28\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHNC\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;45\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eMale, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eMean age, years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eActive smokers, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eMean pack-years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHPV-positive, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUICC stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor localization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOral, oropharynx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHypopharynx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarynx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMoreover, in a 2-years prospective study, we demonstrated that the presence of Gram-negative pathogens in the oral cavity indicated the poor 2 years outcome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), associated with significantly increased risk of bacterial complications (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, clinical characteristics in the Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInfectious complications in participated patients with HNC.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean age (min-max)\u003c/p\u003e \u003cp\u003eat admission, years\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean time interval\u003c/p\u003e \u003cp\u003e(min-max), months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSource of infection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInfectious agent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGram-neg bacteria at admission\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo infection (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.2 (49\u0026ndash;76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.5 (7\u0026ndash;36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60% (no), 0% (yes), 40% (NA)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLater infection (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54.7 (40\u0026ndash;77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (1\u0026ndash;12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSee below\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSee below\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.5% (no), 62.5% (yes), 25% (NA)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTracheal secret, bronchial secret\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eE. cloacae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThroat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBreast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eA. lwoffi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBlood (pneumogenic)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eNA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBlood\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e,\u003c/p\u003e \u003cp\u003e\u003cem\u003eS. oralis, E. coli\u003c/em\u003e,\u003c/p\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOdontogenic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eNA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePneumonia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDemerskatheter, BAL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eS. epidermidis, Candida\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGiven that bacterial infections are mainly controlled by neutrophil granulocytes and their anti-bacterial functions are modulated by G-CSF, we assessed blood and tumor levels of G-CSF in cancer patients, and observed a significant increase in tumor tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Moreover, G-CSF levels were also increased in the oral cavity of patients, compared to healthy individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eNext, we evaluated if G-CSF levels in the oral cavity correlate with patient susceptibility to bacterial infections. Using a received operating characteristic (ROC) analysis, we determined the cut-off G-CSF value in oral rinse as 392 pg/ml, with the highest sensitivity (75%) and specificity (82.3%), indicating the presence of Gram-negative pathogens in the oral cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Indeed, we observed higher frequencies of Gram-negative pathogens in patients with high G-CSF levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), with positive likelihood ratio 4.25 (95% CI 1.34\u0026ndash;11.96) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Of note, in the general population (both, healthy and HNSCC) increased G-CSF levels in oral rinse (400 pg/ml or higher) predict the presence of Gram-negative pathogens with a specificity of 91% and a positive likelihood ratio of 8.4 (95% CI 2.64\u0026ndash;26.71) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eG-CSF chronically released by the tumor tissue promotes bacterial infections\u003c/h2\u003e \u003cp\u003eShort-term exposure to G-CSF is reported to have anti-bacterial and neutrophil-stimulating properties [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In agreement, treatment of isolated human blood neutrophils with G-CSF results in elevated antibacterial responses of these cells (elevated ROS and NETs production) (Figure S2).\u003c/p\u003e \u003cp\u003eHowever, tumor presence is associated rather with long-lasting G-CSF release, leading us to hypothesis that it may have detrimental effect on neutrophil properties. To test this, we modified a murine oropharyngeal carcinoma (MOPC [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]) cell line to overexpress G-CSF (\u003csup\u003eG\u003c/sup\u003eMOPC) at levels comparable to those measured in patients\u0026rsquo; tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTo evaluate whether chronically elevated G-CSF impacts susceptibility to lower respiratory tract bacterial infections, we infected tumor-bearing mice (MOPC versus \u003csup\u003eG\u003c/sup\u003eMOPC) intratracheally (\u003cem\u003ei.t.\u003c/em\u003e) with \u003cem\u003eP. aeruginosa\u003c/em\u003e, and followed the course of infection. Indeed, similarly to patients, elevated chronic G-CSF availability (\u003csup\u003eG\u003c/sup\u003eMOPC) increased the susceptibility of tumor-bearing animals to infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This was accompanied by the elevated lung tissue damage and loss of aerated area (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,D) in these mice. Moreover, higher levels of TNFα as a marker of tissue damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) and augmented severity of the disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) were observed.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChronic exposure to tumor-derived G-CSF impairs neutrophil antibacterial functions and facilitates their tissue toxicity\u003c/h3\u003e\n\u003cp\u003eG-CSF chronically released by the tumor tissue stimulates granulopoiesis and neutrophil release from the bone marrow, resulting in elevated neutrophil accumulation in different compartments, such as tumor, lung (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, gating strategy Figure S3), as well as in bone marrow and blood (Figure S4). Similar phenomenon is observed in HNSCC patients, where a higher accumulation of neutrophils in blood, tumor or oral rinse positively correlates with elevated G-CSF levels (Figure S5). Notably, the predominant neutrophil subpopulations associated with elevated G-CSF levels, were CD62L\u003csup\u003elow\u003c/sup\u003e CD11b\u003csup\u003edim\u003c/sup\u003e senescent subsets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eTo investigate the molecular mechanism behind possibly dysregulated antibacterial activity of neutrophils chronically exposed to tumor-derived G-CSF, we compared proteome of lung neutrophils isolated from mice bearing \u003csup\u003eG\u003c/sup\u003eMOPC (further referred as \u0026ldquo;\u003csup\u003eG\u003c/sup\u003eneutrophils\u0026rdquo;) versus MOPC tumors (\u0026ldquo;neutrophils\u0026rdquo;). By quantitative proteomics we identified 115 differentially expressed proteins (63 up-regulated versus 52 down-regulated proteins) in \u003csup\u003eG\u003c/sup\u003eneutrophils (Table S2). Unsupervised hierarchical clustering analysis showed a unique proteomic signature of neutrophils under chronic exposure to G-CSF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, I).\u003c/p\u003e \u003cp\u003eOne of the molecules significantly downregulated in \u003csup\u003eG\u003c/sup\u003eneutrophils is CD11b (integrin αM), which plays a major role in neutrophil antibacterial activity. We validated the robustness of our label-free quantitative proteomic analysis and confirmed the downregulation of CD11b under chronic G-CSF exposure in \u003csup\u003eG\u003c/sup\u003eneutrophils using flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, Figure S6).\u003c/p\u003e \u003cp\u003eNext, we performed gene ontology (GO) enrichment analysis of differentially expressed proteins to gain insights into the biological processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ), cellular compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK) and molecular functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL) that are affected in neutrophils by the long-term G-CSF exposure. Proteins up-regulated in such \u003csup\u003eG\u003c/sup\u003eneutrophils were significantly enriched in top 5 GO categories linking to neutrophil transcriptional activity, suggesting a possible dysregulation in their maturation, whereas proteins down-regulated in \u003csup\u003eG\u003c/sup\u003eneutrophils were associated with cytoskeleton reorganization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-L, supplemental files 1\u0026ndash;7).\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003eG\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eNeutrophils show impaired phagocytosis and NET formation due to defects in cytoskeleton polymerization\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIn agreement with altered phenotype, we observed a significant downregulation of pathways involved in \u003cem\u003eactin polymerization and phagocytosis\u003c/em\u003e in \u003csup\u003eG\u003c/sup\u003eneutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, blue). While total expression of actin was higher (Figure S7A), actin-regulatory proteins responsible for actin dynamics (polymerization, depolymerization and branching), such as Arpc4 (Actin Related Protein 2/3 Complex Subunit 4) or Cap1 (Cyclase Associated Actin Cytoskeleton Regulatory Protein 1), were downregulated (Figure S7B,C). Apparently, cytoskeleton-dependent functions of \u003csup\u003eG\u003c/sup\u003eneutrophils are impaired due to chronic G-CSF exposure.\u003c/p\u003e \u003cp\u003eTo determine wherever the notable alterations of the cytoskeleton influence antibacterial functions of neutrophils, we challenge these cells with \u003cem\u003eP. aeruginosa.\u003c/em\u003e We observed significantly reduced phagocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and NET formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC,E, Figure S7D,E) by \u003csup\u003eG\u003c/sup\u003eneutrophils, but no changes in total actin polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe expression of other anti-bacterial proteins, such as Padi4 (Protein-arginine deiminase type-4) and non-oxidative branch of pentose-phosphate pathway (Transaldolase 1 Taldo, Transketolase Tkt) that are involved in NET formation, as well as Cybb (cytochrome b-245, beta chain), involved in phagolysosome pathogen killing, were also decreased upon G-CSF exposure (Figure S7F-I). This possibly contributes to the impaired bactericidal activity of these cells. Importantly, NET formation by \u003csup\u003eG\u003c/sup\u003eneutrophils in response to \u003cem\u003eP. aeruginosa\u003c/em\u003e was decreased as compared to neutrophils transiently stimulated by G-CSF (Figure S2A-C), once again demonstrating different mechanisms involved in chronic versus acute G-CSF stimulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eChronic exposure to G-CSF boosts tissue toxicity of neutrophils during bacterial infection\u003c/h3\u003e\n\u003cp\u003e \u003csup\u003eG\u003c/sup\u003eNeutrophils show upregulated \u003cem\u003eResponse to reactive oxygen species\u003c/em\u003e pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, orange). In agreement, we observed an increased expression of proteins involved in ROS production, such as Mpo (Myeloperoxidase, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). As Mpo is a granule protein, high \u003cem\u003eMpo\u003c/em\u003e gene expression indicates high \u003cem\u003ede novo\u003c/em\u003e synthesis of this protein, rather than decreased degranulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Besides non-mitochondrial ROS, we observe elevated mitochondrial activity in \u003csup\u003eG\u003c/sup\u003eneutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-I, Figure S8A). At the same time, downregulated expression of the components of ROS detoxication machinery, such as Gpx1 (Glutathione peroxidase 1, Figure S8B) is observed. Such an elevated ROS production, both in steady state and during infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-L), can be responsible for the observed lung tissue damage.\u003c/p\u003e \u003cp\u003eAnother potential cause of lung tissue damage could be an increased release of enzymes involved in matrix remodeling. Indeed, we observed significantly elevated levels of MMP9 in lung homogenates of infected animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). Consistently, isolated \u003csup\u003eG\u003c/sup\u003eneutrophils release higher amounts of MMP9 in response to \u003cem\u003eP. aeruginosa\u003c/em\u003e challenge \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN), and also show elevated \u003cem\u003eMmp9\u003c/em\u003e gene levels in steady state (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO).\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003eG\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eNeutrophils show a distinct aged phenotype\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003eG\u003c/sup\u003eNeutrophil proteome analyses revealed dysregulation of neutrophil ageing and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In agreement, we observe significantly decelerated apoptosis of lung \u003csup\u003eG\u003c/sup\u003eneutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), accompanied by significantly downregulated Caspase 3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). At the same time, we observed accumulation of aged CD62L\u003csup\u003elow G\u003c/sup\u003eneutrophils in lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and to a lesser extend also in bone marrow and blood (Figure S4) of mice bearing G-CSF producing tumors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSuch aged CD62L\u003csup\u003elow G\u003c/sup\u003eneutrophils are strongly cytotoxic, with low (degranulation-associated) SSC expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), elevated Mmp9 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) and enhanced cytotoxicity \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Moreover, these cells distinct exhausted phenotype, with suppressed antibacterial activity, including phagocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), impaired NET formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-K) and inability to produce ROS in response to bacteria (Figure S9).\u003c/p\u003e \u003cp\u003eImportantly, a similar trend can be observed in cancer patients: high production and release of G-CSF by the tumor microenvironment is associated with the accumulation of CD62L\u003csup\u003elow\u003c/sup\u003e aged neutrophils in tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL,M). Such CD62L\u003csup\u003elow\u003c/sup\u003e neutrophils show high cytotoxic properties, with spontaneous ROS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN). Moreover, these cells show an exhausted phenotype with diminished antibacterial activity in response to \u003cem\u003eP. aeruginosa\u003c/em\u003e, reduced NET production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO) and decreased phagocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eP).\u003c/p\u003e \u003cp\u003e \u003cem\u003eNeutrophils differentiated in the presence of G-CSF phenocopy\u003c/em\u003e \u003csup\u003eG\u003c/sup\u003e\u003cem\u003eneutrophils\u003c/em\u003e\u003c/p\u003e \u003cp\u003eNext, we were interested if the long-term exposure to tumor-derived G-CSF impacts neutrophil development and maturation, in addition to observed modulation of mature neutrophil phenotype. Therefore, we developed the system of \u003cem\u003ein vitro\u003c/em\u003e maturation of bone marrow-derived progenitors for mechanistic studies (modified from our previous studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]) (scheme of the experiment Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eProgenitors maturated in the long-term presence of tumor-conditioned medium containing high levels of G-CSF (\u003csup\u003eG\u003c/sup\u003eMOPC, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) showed accelerated differentiation into mature Ly6G\u003csup\u003e+\u003c/sup\u003e neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,C). Such neutrophils phenocopied tumor-induced \u003csup\u003eG\u003c/sup\u003eneutrophils, showing decreased granularity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), elevated aged CD62L\u003csup\u003elow\u003c/sup\u003e population (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), decreased CD11b expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), higher spontaneous ROS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) and decreased phagocytic capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo prove the essential role of tumor-secreted G-CSF in the suppression of neutrophil bactericidal activity, we block G-CSF receptor during the entire neutrophil maturation using monoclonal antibodies. In line with our hypothesis, we observed impaired accumulation of aged CD62L\u003csup\u003elow\u003c/sup\u003e cells and suppressed ROS production. Similarly, after blocking downstream G-CSF signaling using STAT3 inhibitor LLL12, we observed partial restoration of neutrophil properties, namely inhibited neutrophil degranulation and lower accumulation of CD62L\u003csup\u003elow\u003c/sup\u003e cells, confirming the key role of G-CSF/ G-CSFR axis in the tumor-induced dysregulation of neutrophil maturation and functions (Figure S10).\u003c/p\u003e \u003cp\u003eTo exclude that observed changes are simply the consequence of delayed apoptosis in \u003csup\u003eG\u003c/sup\u003eneutrophils, we inhibited Caspase 3 using QVD-OPh and assessed neutrophil activity. Importantly, the treatment did not induce any changes observed after prolonged G-CSF exposure (Figure S11).\u003c/p\u003e\n\u003ch3\u003eLong term exposure to G-CSF persistently reprograms neutrophil progenitors\u003c/h3\u003e\n\u003cp\u003eDevelopment of neutrophils from hematopoietic stem cells in the bone marrow takes approximately 14 days [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], therefore we hypothesized that the clinical consequence of cancer would be tumor-associated modulation of granulopoiesis and impaired neutrophil functionality, even after surgical removal of the tumor. To test this, we performed \u003cem\u003ein vitro\u003c/em\u003e granulopoiesis assay, using bone marrow progenitors and compared neutrophil development in the presence and absence of tumor-derived factors (scheme of the experiment Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Indeed, such neutrophils exposed to \u003csup\u003eG\u003c/sup\u003eMOPC -conditioned medium show decreased apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI), elevated degranulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ), lower CD11b expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK), elevated spontaneous production of ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL) and downregulated phagocytic capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM), as compared to neutrophils that mature in control conditions.\u003c/p\u003e \u003cp\u003eNext, to assess the effect of G-CSF on human neutrophil progenitors, we analyzed Gene Expression Omnibus databases GSE11247 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], Figure S12), in which gene expression profiles of CD133\u003csup\u003e+\u003c/sup\u003e circulating stem cells mobilized by CXCR4 inhibitor AMD3100 alone, or in combination with G-CSF, were investigated, and compared to our proteomics data. Among the pathways significantly upregulated by G-CSF were those responsible for defense response and leukocyte activation (Figure S12A), including beta-actin (Actb) commonly regulated (Figure S12B). Downregulated pathways include those involved in cell adhesion and migration, regulation of cell death and response to oxygen-containing compound (Figure S12A), with CD11b (Itgam) and cytochrome b (Cybb) commonly downregulated (Figure S12C). This indicates that the changes induced by G-CSF in stem cells are persistent during their development and present also at the mature neutrophil level.\u003c/p\u003e \u003cp\u003eThese data support the hypothesis that the changes observed in \u003csup\u003eG\u003c/sup\u003eneutrophils are induced already at the neutrophil progenitor level, and could be persistent in neutrophils even after the removal of G-CSF and be responsible for a prolonged susceptibility for bacterial infections.\u003c/p\u003e\n\u003ch3\u003eTherapeutic targeting of G-CSF signaling rescues neutrophil antibacterial functionality\u003c/h3\u003e\n\u003cp\u003eG-CSF signaling pathway seems to be involved in the inhibition of neutrophil antibacterial properties, therefore we hypothesized that targeting this pathway would have beneficial effects on their functionality. Previously, we observed than Nampt/NAD\u003csup\u003e+\u003c/sup\u003e are essential for the activity of G-CSF signaling. Although we could not measure any alteration of Nampt expression in \u003csup\u003eG\u003c/sup\u003eneutrophils on gene and protein level (Figure S13), we confirmed significant accumulation of NAD\u003csup\u003e+\u003c/sup\u003e in \u003csup\u003eG\u003c/sup\u003eMOPC bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), suggesting elevated activity of Nampt.\u003c/p\u003e \u003cp\u003eTo mechanistically assess the role of Nampt/NAD\u003csup\u003e+\u003c/sup\u003e axis in neutrophil hyperactivation and tissue toxicity, we used Nampt inhibitor (FK866) as described before [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Indeed, inhibition of G-CSF downstream signaling reduced spontaneous ROS production by neutrophils maturated in the presence of tumor-derived factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we assessed the effect of blocked G-CSF on the susceptibility for infection \u003cem\u003ein vivo\u003c/em\u003e. In agreement with our hypothesis, treatment of \u003csup\u003eG\u003c/sup\u003eMOPC-bearing mice with FK866 decreased lung infiltration with CD62L\u003csup\u003elow\u003c/sup\u003e neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC,D). Moreover, such neutrophils show a distinct pro-apoptotic phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-G), lower tissue-toxic potential with decreased spontaneous ROS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH) and MMP9 release (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI), but no changes in phagocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). In line with this, mice treated with FK866 demonstrated improved bacterial clearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK) and clinical performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL), in comparison to untreated mice.\u003c/p\u003e \u003cp\u003eThis implies the importance of therapeutic approaches aiming at the normalization of G-CSF axis in cancer individuals to prevent neutrophil tissue toxicity, but at the same time to support antibacterial properties of such neutrophils to minimize the susceptibility of patients to bacterial infections.\u003c/p\u003e \u003cp\u003eTo sum up, chronic exposure to tumor-derived G-CSF not only enhances granulopoiesis and the release of harmful cytotoxic neutrophils from the bone marrow, but also results in the local retention of senescent exhausted neutrophils with diminished antibacterial properties in the lung. This leads to the significant lung tissue damage and enhanced lung colonization by bacteria.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCancer patients often suffer from recurrent bacterial infections that have a fatal impact on their morbidity and mortality. One of the reasons responsible for this phenomenon could be G-CSF that is chronically released by the growing tumor. Long-term exposure to G-CSF, in contrast to short-term treatment, induces a dysfunctional, exhausted state of neutrophils, with elevated cytotoxic activity and diminished antibacterial responses. This leads to tissue damage and impaired bacterial clearance, and thus to prolonged infections.\u003c/p\u003e \u003cp\u003eNeutrophils are the key players orchestrating antibacterial immunity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and are reported to be significantly affected by cancer-released factors [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. One of such factors is granulocyte colony-stimulating factor (G-CSF) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], which exerts ambivalent effects on neutrophil antibacterial activity. Importantly, while short-term G-CSF treatment stimulates antibacterial activity of neutrophils [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the evidence from the clinical trials reports the lack of protective effect [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] or even immuno-inhibitory properties [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] of long-term G-CSF treatment.\u003c/p\u003e \u003cp\u003eShort-term treatment with G-CSF was previously shown to induce mobilization of neutrophils and to stimulate antibacterial potential of circulating neutrophils [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, the role of G-CSF treatment for chemotherapy-induced febrile neutropenia and prevention of sepsis is hard to underestimate [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnder physiological conditions, G-CSF is primarily cleared by neutrophils and neutrophil precursors, meaning that clearance from the circulation is a self-regulating process. After binding of G-CSF to its receptor (G-CSFR), G-CSF/G-CSFR complex is internalized and degraded [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the context of cancer, tumor tissue constantly releases G-CSF, therefore self-regulation of G-CSF concentration is not possible, resulting in aberrant neutrophil functionality.\u003c/p\u003e \u003cp\u003eThe impact of G-CSF on neutrophil activity apparently depends on the dose, duration and underlying disease. In the context of hematopoietic stem cell mobilization by G-CSF and their further transplantation, impaired chemotaxis was observed; both in donor and in recipient neutrophils [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. At the same time, reported changes of neutrophil functions are controversial: increased functionality in healthy donors [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], decreased ROS and phagocytosis in transplant recipients [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], or no changes in ROS and phagocytosis in both [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Our experiments show diminished neutrophil functionality after long-lasting exposure to tumor-derived G-CSF in non-neutropenic conditions, associated with impaired antibacterial responses (NET formation and phagocytosis, due to impaired actin cytoskeleton reorganization). Moreover, our data demonstrate clear morphological cause (lack of proteins regulating actin polymerization) responsible for the impairment of \u003csup\u003eG\u003c/sup\u003eneutrophil motility and associated functions, including phagocytosis and NET formation, in addition to functional exhaustion [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNeutrophils represent the first line defenders in acute inflammatory responses [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Aged/exhausted CD62L\u003csup\u003elow\u003c/sup\u003e neutrophils have been shown to contribute to sterile vascular injury and thrombosis in the model of fungal infection, despite impaired actin cytoskeleton [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. We demonstrated that tumor-derived G-CSF not only prolonged survival, but also reprogrammed neutrophils during granulopoiesis, which resulted in their aged, exhausted phenotype, with impaired antimicrobial activity and elevated tissue toxicity. Typical marker of such aged neutrophils is downregulated CD62L (Sell\u003csup\u003elow\u003c/sup\u003e) surface expression. CD62L is highly expressed in young neutrophils, but decreased on aged or activated neutrophils due to reduced gene expression or shedding [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. G-CSF was shown to support shedding of CD62L [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], therefore prolonged exposure to G-CSF in cancer can be responsible for CD62L\u003csup\u003elow\u003c/sup\u003e phenotype of neutrophils. Accumulation of such tissue toxic neutrophils in organs, driven by tumor-derived G-CSF, can be responsible for acute respiratory distress syndrome, which is characteristic for G-CSF treatment [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral therapeutic strategies were already tested to neutralize the adverse effects of G-CSF in neutrophils. Neutralizing anti-G-CSFR antibodies were shown to block G-CSF-induced neutrophilia, without inducing neutropenia, in non-human primates [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Moreover, anti-G-CSFR antibodies reduced neutrophilic inflammation during pneumococcal or influenza respiratory infections, without compromising bacterial clearance [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and increased neutrophil CD62L expression, reverting them to active antibacterial phenotype [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNeutrophil-released ROS is one of the most powerful cytotoxic agents triggering tissue damage. In addition, it supports formation of biofilms by various bacterial species, including \u003cem\u003eP. aeruginosa\u003c/em\u003e, by inducing overproduction of capsule-like exopolysaccharide alginate (so called mucoid conversion) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. High ROS production can be a result of upregulated downstream G-CSF signaling, which supports salvage NAD\u003csup\u003e+\u003c/sup\u003e synthesis through upregulation of nicotinamide phosphoribosyltransferase (NAMPT) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Elevated NAD\u003csup\u003e+\u003c/sup\u003e production coincides with the engagement of oxidative phosphorylation, as a result of increased oxygen availability [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Previously, NAMPT was demonstrated to mitigate colitis severity by supporting redox-sensitive activation of phagocytosis in inflammatory macrophages [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], nevertheless, we demonstrated that in particular cases (high G-CSF production by tumor and elevated tissue toxicity of neutrophils) inhibition of NAMPT can have beneficial effects for the host.\u003c/p\u003e \u003cp\u003eWe hypothesized here that reprogramming of neutrophils by tumor-derived G-CSF occurs already at the progenitor stage. In agreement, elimination of tumor-derived G-CSF from the system after prolonged neutrophil exposure to tumor-conditioned medium, failed to fully restore their phenotype and properties (ROS, phagocytosis), demonstrating the crucial impact of the early education of neutrophil progenitors for their functionality. Observations from transplantology, where the persistence of aberrant G-CSF-stimulated neutrophils was observed in recipients in certain cases up to several weeks after stem cell transplantation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], confirm the long-lasting effect of neutrophil progenitor reprogramming. Hence, tumor-induced reprogramming of myeloid progenitors in hematopoietic organs can be responsible for their prolonged suppressed bactericidal activity. It is especially important, because clinically, the majority of bacterial complications occur within 30 days after surgery, with almost half of cases after hospital discharge [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Thus, developing ways to monitor neutrophil functionality in patients after oncologic surgery should have high priority to identify patients at risk.\u003c/p\u003e \u003cp\u003eEarly non-cancer mortality, usually defined as 90-day mortality after diagnosis or treatment initiation, is a main driver of overall mortality in the HNSCC population. Prevalence of early non-cancer death has been shown to be around 5% in several cohorts with about 30% of cases being caused by pulmonary infection or blood stream infection [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] affecting patients treated with primary surgery [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] or (chemo-)radiation [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] equally. Given that a significant proportion of these patients are expected to be cured from their tumor, non-cancer mortality is an unacceptable obstacle to achieving good patient outcomes.\u003c/p\u003e \u003cp\u003eUnfavorable changes in neutrophil functionality due to prolonged G-CSF exposition may worsen the prognosis of cancer patients. Prognostic role of G-CSF expression in tumor tissue is also already known [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Here, we observed worse prognosis of HNSCC patients with elevated G-CSF levels, which was associated with the presence of Gram-negative pathogens and increased risk of bacterial complications. The aerodigestive tract, especially the oral cavity, serves as a reservoir of bacteria, which can then spread with saliva. The incidence of \u003cem\u003eP. aeruginosa\u003c/em\u003e is twice higher in people with untreated head and neck cancer, in comparison to healthy individuals [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], indicating that cancer-derived factors influence immune antibacterial responses, independently of the treatment. Further anticancer therapies due to their cytostatic and thus immunosuppressive effects, as well as due to the disrupting mucosal barriers, may allow spreading of persisting pathogens. Gram-negative pathogens, including \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eEscherichia\u003c/em\u003e or \u003cem\u003eKlebsiella spp\u003c/em\u003e, represent the major reason for local and systemic infectious complications after surgery for multiple tumor entities, including HNSCC [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Therefore, prevention of bacterial complications and treatment remains the great challenge in cancer patients. Additionally, infectious complications might lead to cancer progression if they interfere with indicated standard of care treatment such as in the case of adjuvant radiation therapy. Delays in postoperative treatment have been repeatedly associated with worse oncologic outcome [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], but the reasons for extended lags between surgery and the initiation of postoperative radiotherapy are unclear at this point. Even though these reasons will be plentiful and variable, it is rational to assume that infections, their treatment, and time required to recuperate, are responsible for a significant subset of delays. This highlights the need of early identification and treatment of patients at risk of infectious complications.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eHere we demonstrate that one of the reasons for bacterial persistence and spread in tumor-bearing hosts are severely impaired antibacterial properties of neutrophils, accompanied by their eminent tissue toxicity that is caused by tumor-derived G-CSF. Therefore, treatment modalities should be considered to neutralize the impact of cancer-related G-CSF stimulation of neutrophils in order to prevent tissue damage and bacterial persistence in damaged tissue. Prediction and early diagnostics of patient predisposition to bacterial complications would decrease possible therapy delay and thus improve patient survival.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBAL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebronchoalveolar lavage\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCasp3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecaspase 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCFUs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecolony\u0026ndash;forming units\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCSF\u0026ndash;granulocyte colony stimulating factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egene ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHNSCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehead and neck squamous cell carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMP9\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ematrix metalloproteinase 9\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMOPC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emurine oropharyngeal carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMPO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emyeloperoxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNETs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneutrophil extracellular traps\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereceived operating characteristic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eside scatter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNFa\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor necrosis factor alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe murine oropharyngeal carcinoma cell line MOPC (C57BL/6-derived, HPV16 E6/E7\u003csup\u003e-\u003c/sup\u003e) was obtained from Dr. William Chad Spanos and John H. Lee (Sanford Research/University of South Dakota, Sioux Falls, SD, US) [47]. Employing CRISPR/Cas9-mediated targeted knock-in technology, we engineered murine HNSCC cell lines expressing low and high G-CSF levels. \u0026nbsp;Elevated production of G-CSF by tumor cells (\u003csup\u003eG\u003c/sup\u003eMOPC cell line) in cell culture conditioned medium was evaluated with ELISA according to the manufacturer\u0026apos;s protocol. Cells were cultivated in a special medium (67% DMEM, 22% Hams F12 nutrient mix, 10% Fetal Bovine Serum, 1% penicillin-streptomycin, 0.5 \u0026micro;g/ml Hydrocortisone, 8.4 ng/ml Cholera Toxin, 5\u0026micro;g/ml Transferrin, 5 \u0026micro;g/ml Insulin, 1.36 ng/ml Tri-Iodo-Thyronine, 5 \u0026micro;g/ml E.G.F.). During cultivation, cell lines were regularly tested for mycoplasma contamination with negative results. Cells were grown in a monolayer at 37 \u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals.\u0026nbsp;\u003c/strong\u003eC57BL/6JCrl mouse strain from own breeding (University Hospital Essen), originally a JAX strain bred by Charles River Laboratory, was used for experiments. For the experiments, female littermates between 8-12 weeks were used. Mice were housed and bred under specific pathogen-free conditions housed in cages of up to 5 mice per cage, 12 h light/dark cycle at animal facility of the University Hospital Essen. All animal experiments have been approved by the regulatory authorities LANUV (Das Landesamt f\u0026uuml;r Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany). Our animal care and used protocols adhere to the regulations of German law according to das Deutsche Tierschutzgesetz (TierSchG) and follow the recommendations of Federation of European Laboratory Animal Science Associations (FELASA).\u003c/p\u003e\n\u003cp\u003eThe MOPC and \u003csup\u003eG\u003c/sup\u003eMOPC cells were injected subcutaneously (s.c. 1 x 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ein 100 \u0026micro;l PBS) into the flank of C57BL/6 mice, as described previously [16]. Tumor-free animals from the same strain were used as control animals. Mice treatment with the\u003cem\u003e\u0026nbsp;\u003c/em\u003eNAMPT inhibitor FK866 was performed per \u003cem\u003ei.p\u003c/em\u003e. injection of 25 mg/kg at day 0 and further at every second day.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacteria\u003c/strong\u003e. \u003cem\u003eP. aeruginosa\u003c/em\u003e strains that were used in this study: PA14 parental strain\u003cem\u003e.\u0026nbsp;\u003c/em\u003eBacteria have been cultured in Luria-Bertani (LB) broth for 3 hours to reach the early exponential phase, washed twice in PBS, the optical density of 100 \u0026micro;l suspension was measured in 96 well flat-bottom cell culture plates (Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany) at 600\u0026nbsp;nm using a microplate reader Synergy 2 (BioTek Instruments, Inc., Vermont, U.S.). OD 0.4 corresponds to a bacterial density of 5x10\u003csup\u003e9\u003c/sup\u003e/ml, as determined by serial dilutions and colony-forming unit (CFU) assays. Bacteria concentration was adjusted to the desired values and verified by plating on 2% LB agar plates. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLower respiratory tract infection in mice\u003c/strong\u003e. For intratracheal inoculation of \u003cem\u003eP. aeruginosa,\u003c/em\u003e mice were anesthetized with Ketamin (bela-pharm GmbH \u0026amp; Co, Vechta, Germany) 100 mg/kg and Xylazin (Ceva Tiergesundheit GmbH, D\u0026uuml;sseldorf, Germany) 10 mg/kg in 0.9% NaCl solution, intubated and 2x10\u003csup\u003e6\u003c/sup\u003e CFUs of \u003cem\u003eP. aeruginosa\u003c/em\u003e in sterile PBS (50 \u0026micro;l) were administrated using the Minivent Mouse Ventilator type 845 (Harvard Apparatus, Massachusetts, U.S.) with stroke volume 150 \u0026micro;l and frequency 150 breaths/ min. The control of distribution of liquid in both lungs during intratracheal administration was performed prior to the experiments using Trypan blue (Sigma-Aldrich/Merck, Darmstadt, Germany). The adapted intratracheal method demonstrated accurate delivery and retention of \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003ein lungs. Animals were monitored post-operatively in a heated box until ambulant and clinically normal. Mice were transferred to a clean box with food and water ad libitum and monitored for 20 hours. To evaluate the clinical status of the mice a severity scoring was performed according to the experiment specific score sheet approved in our animal permission based on the guidelines of the Deutsche Tierschutzgesetz. After 20 hours, mice were sacrificed. Heparinized blood was collected via heart puncture, plasma was prepared after centrifugation. Broncho-alveolar lavage (BAL) was collected after bronchial perfusion trough the trachea with 1 ml of sterile PBS. BAL was plated in serial dilutions to estimate CFUs on 2% LB agar and examined after 24 hours incubation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology.\u0026nbsp;\u003c/strong\u003eFor histological examination of lungs\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice were infected \u003cem\u003ei.t.\u003c/em\u003e with \u003cem\u003eP. aeruginosa.\u0026nbsp;\u003c/em\u003eAt the certain time point mice were sacrificed, lungs perfused with Tissue-Tek O.C.T. Compound (Sakura Finetek, Japan) containing 5% paraformaldehyde, the lumen of the trachea was fixed with ligature; lungs were dissected and snap frozen at -80\u003csup\u003eo\u003c/sup\u003eC. 7-\u0026mu;m cryosections were fixed with ice-cold acetone, stained with hematoxylin-eosin, dried and mounted with Neo-Mount (Merck, Darmstadt, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopy.\u0026nbsp;\u003c/strong\u003eMicroscopy was performed using Zeiss AxioObserver.Z1 Inverted Microscope with ApoTome Optical Sectioning equipped with filters for: DAPI, FITC, Alexa Fluor 488, GFP, DsRed, Cy3 or Olympus BX51 upright epifluorescence microscope. Images were processed with ZEN Blue 2012 software or CellSens Dimension software (Olympus), respectively, and analyzed with ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of neutrophil infiltration in lungs.\u0026nbsp;\u003c/strong\u003eLungs were collected as described above; organs from non-infected animals were used as a control. Lung tissue was digested using dispase 0.2\u0026micro;g/ml, collagenase A 0.2\u0026micro;g/ml, DNase I 100\u0026micro;g/ml (all Sigma-Aldrich/Merck, Darmstadt, Germany) solution in DMEM (Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.) containing 10% FCS and 1% penicillin-streptomycin). Cells were meshed through 50\u0026micro;m filters (Cell Trics, Partec, Sysmex Europe GmbH, Goerlitz, Germany) and erythrocytes lysed in ACK buffer containing NH\u003csub\u003e4\u003c/sub\u003eCl 150 mM, KHCO\u003csub\u003e3\u003c/sub\u003e 10 mM, Na\u003csub\u003e2\u003c/sub\u003eEDTA 0.1 mM. \u0026nbsp;Single-cell suspensions were stained with antibodies and reagents listed below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of bone marrow neutrophils.\u0026nbsp;\u003c/strong\u003eNeutrophils were isolated from bone marrow of non-infected mice. Bone marrow cells were collected via perfusion of femoral bones\u0026nbsp;from each animal under aseptic conditions. Cells were meshed through 50\u0026micro;m filters (Cell Trics, Partec, Sysmex Europe GmbH, Goerlitz, Germany) and erythrocytes lysed in ACK buffer containing NH\u003csub\u003e4\u003c/sub\u003eCl 150 mM, KHCO\u003csub\u003e3\u003c/sub\u003e 10 mM, Na\u003csub\u003e2\u003c/sub\u003eEDTA 0.1 mM. Single-cell suspensions were stained with antibodies listed below, Ly6G\u003csup\u003e+\u003c/sup\u003e viable neutrophils were sorted using a FACS Aria cell sorter (BD Biosciences, BD, New Jersey, U.S.), and the purity of cells was assessed (\u0026ge;95%), all neutrophils are CD11b\u003csup\u003e+\u003c/sup\u003e . After sorting cells were resuspended in DMEM containing 10% FCS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood\u0026nbsp;\u003c/strong\u003ewas collected after sacrificing via heart puncture in heparinized tubes. Plasma was collected after centrifugation at 2000g, frozen at -80C until further analysis. Alternatively, white blood cells were collected from blood after threefold lysis of red blood cells with\u0026nbsp;ACK buffer containing NH\u003csub\u003e4\u003c/sub\u003eCl 150 mM, KHCO\u003csub\u003e3\u003c/sub\u003e 10 mM, Na\u003csub\u003e2\u003c/sub\u003eEDTA 0.1 mM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization of mitochondria\u003c/strong\u003e. Staining for nuclei, cell membrane and mitochondria with Hoerst, PKH and Mitospy, respectively, was performed according to the manufacturer\u0026rsquo;s protocols, samples were evaluated with flow cytometry and on cytospins microscopically.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReactive oxygen species\u003c/strong\u003e.\u0026nbsp;Cells were washed and resuspended in DMEM containing 10% FCS, \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003ePA14 WT MOI 10 was added. Sterile medium was used as negative control. ROS production by Ly6G\u003csup\u003e+\u003c/sup\u003e viable neutrophils was estimated after 60 min of exposure to \u003cem\u003eP. aeruginosa\u003c/em\u003e using Dihydrorhodamine 123 (Sigma-Aldrich/Merck, Darmstadt, Germany) with flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhagocytosis of CFSE-labelled \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/strong\u003e Lung tissue was harvested from non-infected\u003csup\u003e\u0026nbsp;\u003c/sup\u003eanimals under aseptic conditions; single cell suspension was prepared and stained with antibodies. Cells were then washed and resuspended in DMEM containing 10% FCS and DNase (to prevent binding of non-phagocyted bacteria in NETs and false-positive results), \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003ePA14 WT labeled with CFSE (MOI 10) added. Phagocytosis of CFSE-labeled bacteria by Ly6G\u003csup\u003e+\u003c/sup\u003e neutrophils was estimated after 60 min using flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhagocytosis of FITC-labelled beads\u0026nbsp;\u003c/strong\u003e(Caymann) according to manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of lung neutrophils.\u0026nbsp;\u003c/strong\u003eFor estimation of neutrophil functions, neutrophils were isolated from\u0026nbsp;lungs of non-infected mice. Lung tissue was harvested from each animal under aseptic conditions; single cell suspension was prepared as described above. Single-cell suspensions were stained with antibodies listed below, Ly6G\u003csup\u003e+\u003c/sup\u003e viable neutrophils, as well as subpopulations Ly6G\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003ehigh\u003c/sup\u003e, Ly6g\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003e were sorted using a FACS Aria cell sorter (BD Biosciences, BD, New Jersey, U.S.), and the purity of cells was assessed (\u0026ge;95%), all neutrophils are CD11b\u003csup\u003e+\u003c/sup\u003e . After sorting cells were used in pellet for proteomics or resuspended in DMEM containing 10% FCS for functional assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteomics of isolated lung neutrophils\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSample preparation.\u0026nbsp;\u003c/em\u003e80.000 mouse lung neutrophils per sample were dissolved in 70 \u0026micro;l of lysis buffer (50 mM Tris-HCl (pH 7.8) 150 mM NaCl, and 1% SDS supplemented with complete mini-EDTA free protease inhibitor, Roche, Penzberg). The proteins were reduced for 30 min at 37\u0026deg;C in 10 mM DTT and alkylated in 30 mM IAA for 30 min at RT in the dark. \u0026nbsp; After that, the proteins were precipitated with nine volumes of Ethanol for 1h at -80\u0026deg;C and centrifuged for 30 min at 20,000g. The supernatant was removed, and the pellet was dried and dissolved first in 1 \u0026micro;L of 6M GuHCl and then in 29 \u0026micro;L of 50 mM ammonium bicarbonate buffer, pH 7.8 containing 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e and 50 ng of Trypsin (sequencing grade, Promega) and incubated for 18 h at 37\u0026deg;C. The enzymatic digestion was stopped by acidifying the sample to pH\u0026lt;2.5 with TFA. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHigh pH fractionation\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e8 high pH reversed phase fractions were created for spectral library generation using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific).\u0026nbsp;For fractionation, equal amounts of each analyzed sample were combined to a total 50 \u0026micro;g peptides. Peptides were vacuum dried and dissolved in 0.1% TFA according to the instructions and fractionation was performed following the manual. Fractionated and vacuum dried peptide samples were dissolved in 10 \u0026micro;L 0.1% FA for LC-MS/MS measurement.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLC-MS/MS acquisition.\u0026nbsp;\u003c/em\u003eFor liquid-chromatography-coupled tandem mass spectrometry (LC-MS/MS) measurements, 2\u0026nbsp;\u0026micro;L tryptic peptides were injected for individual samples. Spectral library fractions were injected with 2 \u0026micro;L (fraction 1) and 4 \u0026micro;L (fraction 2-8). Measurements were performed on a quadrupole-ion-trap-orbitrap MS (Orbitrap Fusion, Thermo Fisher) coupled to a nano-UPLC (Dionex Ultimate 3000 UPLC system, Thermo Fisher). Chromatographic separation of peptides was achieved with a two-buffer system (buffer A: 0.1% FA in water, buffer B: 0.1% FA in ACN). Attached to the UPLC was a peptide trap (100 \u0026mu;m \u0026times; 200 mm, 100 Å pore size, 5 \u0026mu;m particle size, C18, Thermo Fisher Scientific) for online desalting and purification followed by a 25 cm C18 reversed-phase column (75 \u0026mu;m \u0026times; 250 mm, 130 Å pore size, 1.7 \u0026mu;m particle size, Peptide BEH C18, Waters). Peptides were separated using an 80-min method with linearly increasing ACN concentration from 2% to 30% ACN in 60 minutes. Eluting peptides were ionized using a nano-electrospray ionization source (nano-ESI) with a spray voltage of 1800, transferred into the MS, and analyzed in data-dependent acquisition (DDA) mode. For each MS1 scan, ions were accumulated for a maximum of 120 milliseconds or until a charge density of 2x10\u003csup\u003e5\u003c/sup\u003e ions (AGC Target) was reached. Fourier-transformation-based mass analysis of the data from the orbitrap mass analyzer was performed covering a mass range of 400-1200 m/z with a resolution of 120000 at m/z = 200. Peptides with charge states between 2+ - 5+ above an intensity threshold of 1000 were isolated within a 1.6 m/z isolation window in Top Speed mode for 3 seconds from each precursor scan and fragmented with a normalized collision energy of 30% using higher energy collisional dissociation (HCD). MS2 scanning was performed, using an ion trap mass analyzer at a rapid scan rate, covering a mass range starting at m/z 120, and accumulated for 60ms or to an AGC target of 1x10\u003csup\u003e5\u003c/sup\u003e. Already fragmented peptides were excluded for 30 seconds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRaw data processing and normalization.\u0026nbsp;\u003c/em\u003eLC-MS/MS from DDA were searched with the Sequest algorithm integrated into the Proteome Discoverer software (v 2.4.1.15), Thermo Fisher Scientific) against a reviewed murine Swissprot database, obtained in October 2020, containing 17053 entries. Carbamidomethylation was set as fixed modification for cysteine residues and the oxidation of methionine, and pyro-glutamate formation at glutamine residues at the peptide N-terminus, as well as acetylation of the protein N-terminus were allowed as variable modifications. A maximum number of 2 missing tryptic cleavages was set. Peptides between 6 and 144 amino acids were considered. A strict cutoff (FDR\u0026lt;0.01) was set for peptide and protein identification. Quantification was performed using the Minora Algorithm, implemented in Proteome Discoverer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLC-MS/MS from pH fractions were handled in a separate processing step within the software. A multi-consensus workflow was applied to sample and library mgf files to generate a combined output and increase the protein-identification rate for individual samples trough feature mapper. Protein abundances for individual samples were exported and submitted to subsequent statistical analysis. Protein abundances for library fractions were discarded prior to normalization. Lowess algorithm was applied for data normalization [48].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistics\u0026nbsp;\u003c/em\u003eSignificant regulation was considered for the proteins and transcripts with Log2 FC\u0026gt;\u0026plusmn;2 and p-Value\u0026lt;0.05. Subsequently, the p-values were adjusted for the false discovery rate (FDR) with Benjamini-Hochberg. The signal-to-noise (SNR) value was calculated using SNR=log2x1-x2d1+d2 with d1 and d2 representing the respective standard deviation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGene ontology (GO) enrichment analysis of the generated datasets of differentially expressed proteins was performed using open access ShinyGO platform (Version 0.78, http://bioinformatics.sdstate.edu/go/, [49]). The hypergeometric test after the Benjamini-Hochberg false discovery rate (FDR) correction was used to assess statistical significance. Enriched GO terms with FDR-corrected P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. In addition to the use of functional annotation tools, we also searched PubMed manually to gain insights into the functions of the identified differentially expressed proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNETs release.\u0026nbsp;\u003c/strong\u003eIsolated neutrophils 15.000/well were incubated with \u003cem\u003eP. aeruginosa\u003c/em\u003e (MOI 10) in glass-bottom 96-well plate (MatTek Corporation, Massachusetts, U.S.) pre-coated with poly-D-lysine 1 mg/ml (Sigma-Aldrich/Merck, Darmstadt, Germany) for 4 hours at 37\u003csup\u003eo\u003c/sup\u003eC, 5% CO\u003csub\u003e2\u003c/sub\u003e, sterile medium was used as a negative control. \u0026nbsp; Samples were fixed with paraformaldehyde (Thermo Fisher Scientific, Massachusetts, U.S.) to the final concentration 4%, permeabilized with Triton X-100 (Sigma-Aldrich/Merck, Darmstadt, Germany) 0.2% containing buffer. Since the visualization of NETs using DNA-intercalating dyes alone has the risk of detection of necrotic cells or the generation of artificial results based on dye-blocking peptides associated with NETs, antibody-based techniques are required to visualize NETs. \u0026nbsp;Anti-histone 1 antibodies (Merck Millipore, Darmstadt, Germany) were used to detect all NETs. Donkey-anti-mouse-AF564 (Invitrogen, Thermo Fisher Scientific, Massachusetts, U.S.) were used as secondary antibodies. Stainings were mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen, Thermo Fisher Scientific, Massachusetts, U.S.). Percent of NET-producing cells and NETs length and area were estimated by microscopy followed by analysis with ImageJ Fiji software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of bone marrow progenitors and in vitro maturation assay\u003c/strong\u003e (adapted from Siakaeva et al, REF). Murine BM progenitor cells were negative selected by depletion of CD3e\u003csup\u003e+\u003c/sup\u003eCD45R\u003csup\u003e+\u003c/sup\u003eNK1.1\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eTer119\u003csup\u003e+\u003c/sup\u003e BM cells using the Streptavidin MicroBeads and LD Columns from Miltenyi Biotec (Bergisch Gladbach, Germany) according to the manufactuer\u0026rsquo;s protocols. For maturation isolated cells were cultured in 24-well plates at the concentration of 0,3x10\u003csup\u003e6\u003c/sup\u003e cells/ml in tumor cell line-condenced media (MOPC, G-MOPC or control M-medium) with addtition of mrSCF and mrIL3 (all from Peprotec, Hamburg, Germany; end concenration 50 ng/ml) 7 days long at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e, medium was changed into fresh at day 4. aG-CSFR in concentration (5 \u0026micro;g/ml), LLL12 (1\u0026micro;M), FK866 (100 nM) were used. At day 4 tumor-condenced medium was exchanged into sterile M-medium. At day 7 the composition of the cultivated cells in all conditions was analyzed with microscopy and flow cytometry. Phenotype (viability, Ly6G, CD11b, CD62L expression) and functions (ROS, phagocytosis) of Ly6G\u003csup\u003e+\u003c/sup\u003e cells were evaluated with flow cytometry as described previously.\u003c/p\u003e\n\u003cp\u003eFor \u003cstrong\u003emorphological analysis\u003c/strong\u003e, cytospin preparations of maturated bone marrow cells on SuperfrostTM slides (Gibco, Thermo Fisher Scientific, Waltham, MA, US) were fixed with pure methanol, stained by Giemsa (Sigma-Aldrich, Merck KGaA, St. Louis, MO, US) and nuclear morphology was assessed using light microscope Olympus BX5 (Olympus, Tokyo, Japan). At least 10 fields of view were counted and percentages of immature, band and segmented nuclei were calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNAD assay\u003c/strong\u003e in plasma was performed according to manufacturer\u0026rsquo;s protocol\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman cohort.\u003c/strong\u003e Patients with head and neck cancer and healthy individuals participated in the study (clinical characteristics in the Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeripheral blood\u003c/strong\u003e obtained from healthy donors, was drawn into 3.8% sodium citrate anticoagulant monovettes (Sarstedt, Nuembrecht, Germany) and mixed 1:1 with PBS (Gibco, Thermo Fisher Scientific, Waltham, MA, US) before separation by density gradient centrifugation (Pancoll density 1,077 g/ml). The mononuclear cell fraction was discarded and neutrophils (purity \u0026ge;95%) were isolated by sedimentation over 1% polyvinyl alcohol, followed by hypotonic lysis (0.2% NaCl) of erythrocytes and reconstitution of osmolarity with 1.2% NaCl. Isolated neutrophils (1 mln/ml in RPMI medium containing 10% FCS) alone or with \u003cem\u003eP. aeruginosa\u003c/em\u003e MOI 10 were incubated for 1 hour, afterwards analysis of ROS, phagocytosis (as described above) and NET formation (using Sytox Green reagent in combination with anti-MPO according to manufacturer\u0026rsquo;s protocol) was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOral rinse\u003c/strong\u003e with 15 ml of sterile saline was collected from n=28 healthy controls and n=45 patients with HNSCC directly after awaking prior to oral hygiene and food/drink consumption, for 1 min. The absolute amount of cells in the rinse was evaluated, the proportion of viable CD66b\u003csup\u003e+\u003c/sup\u003e neutrophils and their activation (CD62L expression) were estimated with flow cytometry. Soluble fraction of oral rinse was collected and centrifuged at 3000g. After discarding the supernatant, the bacteria were resuspended in a total of 0.4 ml of sterile saline, and 10 \u0026micro;l of the resuspended bacteria were inoculated onto Columia blood agar and Chocolate blood agar as universal media, and onto Mac Conkey agar (all media from Oxoid, Wesel, Germany), a selective medium for the growth of Gram-negative rods. Bacteria were cultured at 36\u0026deg;C under aerobic conditions with 5% CO\u003csub\u003e2\u003c/sub\u003e and growth was assessed after 24 and 48 hours. Bacteria were identified using VITEK2\u003csup\u003eTM\u003c/sup\u003e (bioM\u0026eacute;rieux, Marcy-l\u0026rsquo;\u0026Eacute;toile, France), MicroScan WalkAway (Beckman Coulter, Brea, US), VITEK MS (bioM\u0026eacute;rieux) or MALDI Biotyper (Bruker, Billerica, US).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumor samples\u003c/strong\u003e were cut into 1 mm\u003csup\u003e3\u003c/sup\u003e pieces with sterile instruments and incubated in RPMI medium containing 10% FCS, 1% Pen/Strep and 0.2% of Fungizon in proportion 0.02 g/ 0.6 ml for 4 hours, afterward supernatant was collected. In parallel, single cell suspension of the tumor was derived how is explained above, the content and activation (expression of CD62L) of viable tissue CD66b\u003csup\u003e+\u003c/sup\u003e neutrophils was evaluated with flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA.\u0026nbsp;\u003c/strong\u003eG-CSF, MMP9 and TNF-a in murine plasma samples and lung supernatants and neutrophi-conditioned medium, G-CSF in human tumor supernatants and oral rinse were analyzed with ELISA (R\u0026amp;D Systems, Minnesota, U.S.) according to manufacturer protocols.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-qPCR.\u0026nbsp;\u003c/strong\u003eThe RNA was isolated using Qia Shredder and RNeasy Mini Kit (Qiagen, Hilden, Germany) and the cDNA was produced using the Superscript II Reverse Transcriptase Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, US). qRT-PCR was performed at 60 \u0026deg;C annealing temperature using primers listed below. As housekeeping gene, \u003cem\u003eRps9\u003c/em\u003e was used. The mRNA expression was measured using the Luna Universal qPCR Master Mix (New England BioLabs, Ipswich, MA, US). Relative gene expressions were calculated by 2^-\u0026Delta;Ct \u0026nbsp;formulations. Real-time RT-PCR was performed using primers listed in the supplemental file 9.\u003c/p\u003e\n\u003cp\u003e\u0026shy;\u003cstrong\u003eList of reagents, antibodies and consumables\u0026nbsp;\u003c/strong\u003ein the supplemental file 9.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026shy;Analysis of the data deposited in the Gene Expression Omnibus databases.\u0026nbsp;\u003c/strong\u003eWe used previously published microarray data deposited in the Gene Expression Omnibus databases GSE11247 (GSM283955, GSM283956, GSM283957, GSM283958, GSM283959, GSM283960, GSM283961, GSM283962)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using Kruskal-Wallis ANOVA for multiple comparisons with the Bonferroni correction, and Mann-Whitney U-test for two independent samples, and Whilcoxon test for dependent samples; correlations were analyzed with Spearman R test. Sensitivity, specificity of the diagnostic test, relative risk were calculated using MedCalc\u0026rsquo;s calculators (available online https://www.medcalc.org/calc/). To assess the accuracy of model predictions, receiver operating characteristic (ROC) analysis was performed. P\u0026lt;0.05 was considered significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments have been approved by the regulatory authorities LANUV (Das Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen), Germany. Our animal care and use protocols adhere to the regulations of das Deutsche Tierschutzgesetz (TierSchG) and follow FELASA recommendations.\u003c/p\u003e\n\u003cp\u003eResearch involving human material, or human data was performed in accordance with the Declaration of Helsinki and was approved by the ethics committee of the University Hospital Essen, Germany (19-8599-BO, 16-7135-BO). Informed consent to participate in the study was obtained from participants\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [50] partner repository with the dataset identifier\u0026nbsp;PXD052631. Other materials are available upon request to interested researchers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study is supported by grants from the Deutsche Forschungsgemeinschaft (DFG/ JA 2461/2-1, DFG/ JA 2461/7-1, TR332 A5) and Deutsche Krebshilfe (111647). BS and HS received funding from the INST 337/15-1, INST 337/16-1, INST 152/837-1 and INST 152/947-1 FUGG. \u0026nbsp;DRE received funding from the Deutsche Forschungsgemeinschaft FOR5427 SP4 (DRE); EN984/15-1, 16-1 and 18-1 (DRE); TR296 P09 (DRE); TR332 A3 and Z1 (DRE), and INST 20876/486-1. OS received funding from the Deutsche Forschungsgemeinschaft: FOR5427 SP1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, E.P. and J.J.; Methodology, E.P., O.S., H.H., C.K., J.K., D.R.E. and J.J.; Software, O.S., B.S., H.S.; Validation, E.P. and J.J., Formal Analysis, E.P., L.T., J.R., O.S. and A.S.; Investigation, E.P., L.T., J.R., O.S., I.T., J.A., I.O., B.S., H.S. and C.H.; Resources, O.S., B.S., H.S., H.H., S.M., J.K., D.R.E., S.L. and J.J.; Data Curation, E.P., O.S., B.S., H.S. and J.J.; Writing – Original Draft Preparation, E.P., O.S. and C.K.; Writing – Review \u0026amp; Editing, H.H., S.M., J.K., D.R.E., S.L. and J.J. ; Visualization, E.P., O.S., B.S. and H.S.; Supervision, S.L. and J.J., Project Administration, J.J.; Funding Acquisition, O.S., D.R.E., S.L. and J.J.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge support by the Open Access Publication Fund of the University of Duisburg-Essen, the Imaging Center Essen (Alexandra Brenzel and Dr. Anthony Squire) and the Immunoproteomics group (Stephanie Tautges-Schaefer, Stephanie Thiebes and Jenny Dick).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eParkins MD, Gregson DB, Pitout JD, Ross T, Laupland KB. 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PMID: 34723319; PMCID: PMC8728295.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cancer, neutrophils, antibacterial responses, G-CSF/NAMPT signaling, granulopoiesis, Gram-negative pathogens, Pseudomonas aeruginosa, head and neck cancer","lastPublishedDoi":"10.21203/rs.3.rs-6098692/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6098692/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Despite advancements in cancer therapies, bacterial complications remain a major challenge, delaying treatment and worsening outcomes. While immunosuppressive therapies and prolonged hospitalizations contribute, they do not fully explain the elevated infection risk in cancer patients. We observed that elevated G-CSF production by tumors was associated with the persistence of Gram-negative pathogens in head and neck squamous cell carcinoma patients. The presence of Gram-negative pathogens in oral rinse positively correlated with poor prognosis of such patients. As neutrophils are the main cells controlling bacterial infections, we aimed to identify the molecular mechanisms underlying tumor-induced suppression of antibacterial activity of these cells. We identified tumor-driven activation of the NAMPT/NAD+ signaling axis in neutrophils (already at the progenitor level), which induced long-lasting cytoskeletal alterations, impairing phagocytosis and NETosis. Moreover, NAMPT/NAD+ activity accelerated neutrophil senescence and promoted accumulation of tissue-toxic CD62Llow subpopulations, ultimately disrupting lung tissue integrity and facilitating bacterial persistence in tumor-bearing hosts. Targeting the G-CSF/NAMPT signaling effectively reduced the generation of dysfunctional neutrophils and improved bacterial clearance in vivo. These findings reveal tumor-induced, NAMPT-dependent neutrophil reprogramming as a central mechanism driving impaired antimicrobial defenses. Implementing immune-modulating strategies, such as targeting G-CSF/NAMPT signaling, could improve infection control and enhance survival in cancer patients.","manuscriptTitle":"NAMPT/NAD+ signaling drives neutrophil dysfunction and enhances bacterial infection susceptibility in cancer patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 18:07:48","doi":"10.21203/rs.3.rs-6098692/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b1feab44-0de1-4784-8898-3e302ff018bf","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45107331,"name":"Health sciences/Oncology/Cancer/Head and neck cancer"},{"id":45107332,"name":"Health sciences/Medical research/Translational research"},{"id":45107333,"name":"Biological sciences/Cancer/Tumour immunology"},{"id":45107334,"name":"Biological sciences/Immunology/Antimicrobial responses"}],"tags":[],"updatedAt":"2025-12-16T08:10:36+00:00","versionOfRecord":{"articleIdentity":"rs-6098692","link":"https://doi.org/10.1038/s41467-025-67471-4","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-12 05:00:00","publishedOnDateReadable":"December 12th, 2025"},"versionCreatedAt":"2025-03-24 18:07:48","video":"","vorDoi":"10.1038/s41467-025-67471-4","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67471-4","workflowStages":[]},"version":"v1","identity":"rs-6098692","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6098692","identity":"rs-6098692","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}