CHAC2-mediated Glutathione Metabolic Reprogramming Drives N1 Polarization of Bone Marrow Neutrophils and Exacerbates Inflammatory Co-morbidities

CHAC2-mediated Glutathione Metabolic Reprogramming Drives N1 Polarization of Bone Marrow Neutrophils and Exacerbates Inflammatory Co-morbidities | 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 CHAC2-mediated Glutathione Metabolic Reprogramming Drives N1 Polarization of Bone Marrow Neutrophils and Exacerbates Inflammatory Co-morbidities Xuliang Deng, Ying Huang, Yuting Niu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7792660/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 13 You are reading this latest preprint version Abstract The proinflammatory (N1) polarization of bone marrow (BM) neutrophils, driven by central immune remodeling in response to peripheral inflammation, plays a critical role in propagating localized inflammatory conditions, such as periodontitis, to systemic levels. Although this process involves metabolic reprogramming, the specific underlying metabolic mechanisms of neutrophil N1 polarization within the periodontitis-modified BM niche remain poorly defined. Integrated transcriptomic and metabolomic analyses in this study revealed that periodontitis reprograms intracellular glutathione (GSH) metabolism in BM neutrophils, facilitating their N1 polarization. Central to this mechanism is the upregulation of Chac2, an enzyme that promotes GSH accumulation. This enhancement is accompanied by elevated GSH redox cycling, which supports sustained ROS production and NET formation, thereby amplifying inflammatory responses. We further identified type I interferon (IFN-I) signaling as a key upstream regulator that induces Chac2 expression and drives metabolic reprogramming. Importantly, the intraosseous delivery of AAV-delivered Chac2 shRNA in db/db mice with periodontitis markedly reduced neutrophil-aggravated systemic inflammatory co-morbidity symptoms and improved glycemic control, underscoring the functional relevance of this pathway in diabetic co-morbidity. Together, these findings thus delineate the IFN-I–Chac2–GSH axis as a core signaling mechanism regulating neutrophil N1 polarization in the BM niche, providing new insights into how periodontal inflammation reprograms immune functions at the systemic level. This study thus broadens the conceptual framework of neutrophil immunometabolism and proposes targeting the Chac2–GSH axis as a potential therapeutic strategy for systemic comorbidities associated with periodontitis. Health sciences/Diseases/Oral diseases/Periodontitis Health sciences/Diseases/Endocrine system and metabolic diseases/Diabetes Neutrophil N1 polarization Glutathione metabolic reprograming Chac2 IFN-I signaling Central immune remodeling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Local chronic inflammation not only extends beyond a pathological state localized within specific tissues/organs, but also profoundly reshape systemic immunity through peripheral inflammatory signaling 1-4 . In this process, the bone marrow (BM), as the central tissue/organ of hematopoiesis, undergoes marked alterations in hematopoietic dynamics, manifested by skewed myeloid differentiation and increased neutrophil production 5 . Our previous study had revealed that this quantitative expansion is accompanied by the functional activation of neutrophils, characterized by enhanced release of reactive oxygen species (ROS), increased formation of neutrophil extracellular traps (NETs), and elevated secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) 6 . Collectively, these functional changes are consistent with neutrophil polarization toward the N1 proinflammatory phenotype 7 , underscoring the propensity of neutrophils to undergo such polarization within the BM microenvironment remodeled by chronic inflammation. It must be noted that immune cell polarization rarely occurs in isolation but is intricately linked to underlying metabolic reprogramming, which serves as a critical determinant of cellular phenotype and effector activity 8-10 . The intrinsic connection between immune cell polarization and metabolic reprogramming has emerged as a central theme in immunometabolism research, offering critical insights into the regulatory mechanisms of immune responses. Indeed, substantial evidence have demonstrated that specific metabolic reprogramming directly regulates immune cell function, providing new perspectives and potential targets for the metabolic modulation of immune-mediated diseases. This principle has been validated across multiple immune cell types. For example, glycolytic metabolism supports macrophage M1 polarization 11-14 , lipid metabolism governs T cell differentiation 15-18 , and mitochondrial dynamics influence dendritic cell activation 19-22 . Building on the accumulated scientific data on these broadly conserved mechanisms, growing research attention is now directed towards the metabolic adaptations of neutrophils 23-25 , particularly how these immune cells remodel their metabolism in response to specific pathological microenvironments. In the tumor microenvironment, neutrophils have been shown to undergo glycolytic reprogramming in response to inflammatory cytokines, driving their polarization toward an N1 phenotype that exerts anti-tumor functions 26,27 . However, it is still unclear whether neutrophils experience comparable metabolic adaptations within the BM microenvironment remodeled by chronic inflammation (such as periodontitis). More importantly, how such metabolic remodeling directly governs the process of N1 polarization in this context is still unresolved, representing a critical knowledge gap on how metabolic regulation is linked to neutrophil inflammatory phenotypes. Among the various diverse pathways of immunometabolic regulation, the metabolism of glutathione (GSH), a key endogenous antioxidant, play key roles in maintaining the redox homeostasis, survival, and effector functions of immune cells 28,29 . Its intracellular levels are precisely regulated by a metabolic network that includes the synthetic enzyme Gss 30 , the degradation enzyme Chac1, and the degradation regulator Chac2, which work together to sustain GSH metabolic balance 29,31 . While the role of GSH biosynthesis in immune regulation has been extensively studied 32,33 , the role of its degradation pathway—particularly the regulatory functions of Chac2 under pathological conditions—remains poorly understood. In particular, it is necessary to investigate whether neutrophils can undergo Chac2-mediated GSH metabolic reprogramming within the BM niche remodeled by chronic inflammation, which could drive their polarization toward the N1 state. In this study, a murine periodontitis model was used to investigate how local chronic inflammation triggers neutrophil proinflammatory N1 polarization within the BM niche. By integrating transcriptomic and untargeted metabolomic analyses of BM neutrophils, we revealed a pivotal role for Chac2-driven GSH metabolic reprogramming in skewing neutrophils toward the N1 phenotype, which is orchestrated by upstream IFN-I signaling. By further investigating the systemic impact of this polarization in diabetic mice and evaluating the effects of Chac2 inhibition, we provide mechanistic insights into the underlying metabolic mechanisms of neutrophil N1 polarization, which opens up a new therapeutic avenue for targeting BM neutrophil-driven co-morbidities. Results Periodontitis promotes N1-type polarization of BM neutrophils through enhanced GSH metabolism To investigate the impact of periodontitis on the polarization state of BM neutrophils, we analyzed RNA-seq data from BM neutrophils of the control (Con) and ligature-induced periodontitis (Lig) mice obtained in our previous study (Figure 1A). Gene Ontology (GO) analysis of differentially expressed genes revealed enrichment of pathways related to neutrophil activation and inflammation, including “innate immune response,” “cellular response to LPS,” and “regulation of chemokine production” (Figure 1B). Notably, Lig-derived neutrophils exhibited upregulated expression of representative N1-type markers such as S100a9, Ifitm1, Ifitm2, and Cxcr1 (Figure 1C). Flow cytometry confirmed elevated surface expression of CD11b and CD66a (Figure 1D-E), along with heightened functional responses, including increased ROS production upon LPS stimulation (Figure 1G), enhanced secretion of IL-1ra, IL-16, and TNF-α (Figure 1H), and greater NET formation (Figure 1I). These results thus indicate that periodontitis drives BM neutrophil polarization towards a proinflammatory N1 phenotype at the transcriptional, phenotypic, and functional levels. Given the critical role of metabolism in shaping immune cell function, we next examined whether N1 polarization was accompanied by alterations in metabolic pathways. Untargeted metabolomic profiling of sorted BM neutrophils revealed a distinct separation between the Con and Lig groups by PCA (Figure 2A–B), with 122 metabolites significantly altered (Figure S1A), among which amino acid derivatives were most enriched (Figure 2C). Both KEGG and MSEA pathway analyses identified glutathione metabolism as the top-enriched pathway (Figures 2D–E). Notably, intracellular GSH levels were elevated nearly fourfold in Lig-derived neutrophils (Figure 2F–G), which was further validated by ELISA quantification (Figure 2H). To functionally assess the role of GSH in driving neutrophil polarization, we utilized the HL60 cell line differentiated into neutrophil-like cells. Cells were treated with either glutathione diethyl ester (GDE) to enhance intracellular GSH or L-buthionine sulfoximine (L-BSO) to inhibit GSH synthesis. GSH levels were confirmed by flow cytometry using a GSH-sensitive probe (Figure S1B). GDE-treated cells exhibited upregulation of N1 markers, along with increased NET formation, ROS production, and proinflammatory cytokine secretion, all of which were significantly suppressed by L-BSO treatment (Figure 2J-M). Together, these results thus demonstrate that periodontitis promotes N1-type polarization of BM neutrophils and is closely associated with enhanced GSH metabolic activity, implicating GSH metabolism as a key regulatory axis in inflammation-driven neutrophil reprogramming. Periodontitis induces glutathione metabolic reprogramming in BM neutrophils To investigate how periodontitis alters GSH metabolism in BM neutrophils, we conducted an integrated analysis of transcriptomic and metabolomic datasets from Con and Lig mice. GO enrichment analysis of genes upregulated in Lig mice identified significant activation of the glutathione metabolic process (Figure 3A). Differential gene expression profiling revealed notable changes in key glutathione-related genes (Figure 3B). Subsequently, an integrated GSH metabolic network map based on both datasets was constructed, which revealed a distinct reprogramming pattern in the Lig group. Despite elevated intracellular GSH levels and increased redox cycling activity, the transcriptomic data indicated downregulation of Gss (glutathione synthetase), Gclc (glutamate cysteine ligase catalytic), and Chac1 (a canonical GSH-degrading enzyme), alongside significant upregulation of Chac2, a less characterized homolog involved in glutathione regulation (Figure 3C). Given that previous studies have shown that Chac2 can antagonize Chac1-mediated GSH degradation, we hypothesized that Chac2 upregulation may suppress GSH catabolism and promote GSH accumulation. This hypothesis was supported by altered total GSH concentration, GSH/GSSG ratio, and RT-qPCR validation of gene expression patterns in Con and Lig neutrophils (Figure 3D-E). Western blot analysis further confirmed a ~3.5-fold increase in CHAC2 protein expression in Lig samples compared to controls (Figure 3F). The expression of GSR and GPX4 increased at transcriptional level, and the enzymatic activity of neutrophils in the Lig group was enhanced (Figure S1D), suggesting that the accumulation of GSH within cells was accompanied by an increase in the redox cycle. Collectively, these findings suggest that periodontitis induces a distinct reprogramming of GSH metabolism in BM neutrophils, characterized by CHAC2-mediated suppression of GSH degradation (Figure 3G), which may play a pivotal role in modulating neutrophil polarization. Chac2-mediated GSH metabolic reprogramming drives N1-type polarization of neutrophils To determine whether Chac2 is functionally involved in GSH-driven N1-type polarization, we performed both loss- and gain-of-function experiments using HL60-derived neutrophil-like cells. Knockdown of Chac2 (Sh-Chac2) via lentiviral transduction significantly reduced its expression at both the mRNA and protein levels, as confirmed by RT-qPCR and Western blot (Figures 4A-B). Chac2 silencing led to a marked decrease in intracellular GSH content, accompanied by significantly reduced levels of proinflammatory cytokines, ROS production, NET formation, and suppressed expression of N1 signature genes (Figure 4C-G, S2A). These results thus indicate that Chac2 knockdown impairs GSH accumulation and attenuates N1-type inflammatory polarization. Conversely, the overexpression of Chac2 (OE-Chac2) in HL60 cells (Figure 4H-I) resulted in a robust increase in GSH levels, validating its role in maintaining GSH homeostasis. Functionally, Chac2 overexpression led to enhanced secretion of IL-6, TNF-α, and IL-1β, elevated ROS production, increased NET formation, and significant upregulation of N1-associated genes (Figure 4J-N, S2B), collectively indicating a shift towards a pro-inflammatory N1 phenotype. Together, these results thus demonstrate that Chac2 serves as a key metabolic regulator linking glutathione homeostasis to neutrophil functional polarization. Specifically, Chac2 promotes GSH accumulation and drives N1-type polarization, thus contributing to the inflammatory potential of neutrophils in the context of periodontitis. IFN-I signaling promotes Chac2-mediated GSH reprogramming and N1-type polarization of neutrophils To identify upstream regulatory pathways responsible for the observed GSH metabolic reprogramming in BM neutrophils during periodontitis, we performed gene set enrichment analysis (GSEA) of neutrophil transcriptomes from Lig and Con mice. GSEA analysis revealed significant enrichment of IFN-I signaling pathways in the Lig group (Figure 5A), while the corresponding heatmap analysis confirmed upregulation of IFN-I–responsive genes in Lig-derived neutrophils (Figure 5B). Consistently, the IFN-I levels in BM supernatants were markedly elevated in periodontitis mice (Figure 5C), indicating robust activation of IFN-I signaling during periodontitis. To functionally evaluate the role of IFN-I signaling, we subjected both WT and Ifnar1 ⁻/⁻ mice (lacking the type I interferon receptor) to ligature-induced periodontitis. In Ifnar1 ⁻/⁻ mice, periodontitis failed to induce the metabolic and phenotypic changes observed in WT animals. Specifically, intracellular GSH levels (Figure 5D) and Chac2 expression (Figure 5E) remained unchanged in Lig-Ifnar1 ⁻/⁻ neutrophils compared to controls. Additionally, the key pro-inflammatory features of N1 polarization—including ROS generation (Figure 5F) and proinflammatory cytokine secretion (Figure S2C)—were significantly attenuated. These findings thus suggest that IFN-I signaling is required for Chac2 induction and GSH accumulation, as well as for driving the N1-type pro-inflammatory programming of neutrophils in periodontitis. To further delineate the IFN-I–CHAC2 signaling axis in neutrophil polarization, we treated DMSO-differentiated HL60 cells with recombinant IFN-I in the presence or absence of Chac2 knockdown. In Sh-NC cells, IFN-I stimulation induced a robust increase in intracellular GSH levels, upregulated Chac2 expression, and enhanced transcription of N1-type signature genes. Functionally, IFN-I also promoted NET formation, ROS production, and pro-inflammatory cytokine release. However, in Sh-Chac2 cells, these IFN-I–induced effects were markedly ablated—demonstrating that Chac2 is a key mediator linking IFN-I signaling to GSH metabolic reprogramming and N1-type polarization (Figure 5G-L, S2D-F). Hence, these results validated that IFN-I signaling promotes proinflammatory N1-type polarization of neutrophils by inducing Chac2 expression and modulating GSH metabolism. AAV-shChac2 delivery alleviated systemic inflammation and improved metabolic outcomes in diabetic mice To evaluate the systemic impact of N1-polarized neutrophils under diabetic conditions, BM neutrophils isolated from Con and Lig mice were adoptively transferred into db/db diabetic recipient mice (Figure S4A). Mice receiving Lig-Neus displayed worsened metabolic parameters, including elevated random blood glucose levels, increased glycated serum protein levels, impaired glucose tolerance, and reduced insulin sensitivity (Figure S4B-C). Histological analyses further revealed disrupted islet function and enhanced neutrophil infiltration in the pancreas (Figure S4D). These results thus suggest that BM N1-type neutrophils induced by periodontitis exacerbate pancreatic dysfunction in diabetes. To directly assess the role of GSH-driven neutrophil N1 polarization in systemic metabolic dysfunction, we adoptively transferred HL60-derived neutrophil-like cells pre-treated with either GDE (GDE-HL60) or L-BSO (L-BSO-HL60) into db/db diabetic mice (Figure 6A). Mice receiving GDE-treated HL60 cells, characterized by elevated intracellular GSH and N1-type polarization, exhibited significantly increased random blood glucose and glycated serum protein levels compared to controls (Figure 6B-C). Line charts of the intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) revealed more severe dysfunction in the GDE-HL60 group versus the L-BSO-HL60 group (Figure 6D). Additionally, immunofluorescence staining of pancreatic islets revealed marked insulin depletion and enhanced glucagon expression, indicating disrupted endocrine function (Figure 6E). Conversely, L-BSO-HL60 cells with suppressed GSH synthesis and attenuated inflammatory polarization failed to induce these metabolic abnormalities. Mice in this group showed improved glycemic control and preserved islet architecture, with restored insulin expression and limited neutrophil infiltration. These results thus validated that the core mechanism by which pro-inflammatory neutrophils exacerbate pancreatic dysfunction in diabetes is intracellular GSH accumulation within the neutrophils. To explore whether targeted intervention of Chac2 in neutrophils could alleviate the inflammatory co-morbidities of periodontitis, we constructed AAV9 viruses targeting neutrophils to specifically knock down Chac2 (AAV-shCHAC2) and a control virus with no payload (AAV-NC), and administered them via intraosseous injection into a mouse model with periodontitis and diabetes comorbidities (Figure 6F). During three weeks of modeling, the random blood glucose and glycated serum protein levels of the AAV-shCHAC2 group were significantly lower than those of the control group (Figure 6G-H), and islet dysfunction, as indicated by IPGTT, ITT, and insulin/glucagon fluorescence staining of pancreatic tissue, was alleviated (Figure 6I-J). Double immunofluorescence staining for detection of MPO/Chac2 in pancreatic tissue demonstrated the effective targeting and knockdown of neutrophils by AAV treatment (Figure 6K-L). These results thus suggest that Chac2-regulated N1 polarization of BM neutrophils plays a key role in the pathological process by which periodontitis exacerbates diabetic co-morbidity.. Together, these results functionally demonstrated that intracellular GSH elevation promotes neutrophil N1 polarization, which in turn contributes to the exacerbation of diabetic co-morbidity in the context of periodontitis. Correspondingly, targeted knockdown of Chac2 in BM neutrophils can reverse N1 polarization and effectively alleviate the aggravating effects of local chronic inflammation on distal co-morbidities. This IFN-I–CHAC2–GSH signaling axis thus represents a critical upstream regulatory pathway linking local chronic inflammation to systemic neutrophil activation and immune-metabolic dysfunction. Discussion There is emerging evidence that local chronic inflammation, such as periodontitis, is a key contributor to systemic immune dysregulation and multi-organ co-morbidities 34,35 . To delineate the role of BM neutrophils in this process, we employed integrated metabolomic and transcriptomic profiling and demonstrated that periodontitis triggers their polarization toward a pro-inflammatory N1 phenotype via GSH metabolic reprogramming. This reprogramming is driven by IFN-I signaling, which upregulates Chac2 expression at the transcriptional level. In contrast to its canonical role in GSH degradation, we found that in the context of inflammation, Chac2 upregulation led to a pronounced intracellular accumulation of GSH, which in turn enhanced neutrophil N1 effector functions. These insights thus broaden the conceptual framework of neutrophil immunometabolism and suggest that targeting the IFN-I–Chac2–GSH signaling axis may provide novel strategies for mitigating inflammation-driven co-morbidities. Neutrophils are capable of undergoing N1 polarization across diverse pathological microenvironments 26,36,37 , but their functional properties, metabolic basis, and regulatory mechanisms exhibit fundamental divergences and nuances that vary according to the disease condition and state. For instance, within the tumor microenvironment, N1-polarized neutrophils exert anti-tumor protective effects by generating ROS, NETs, and proinflammatory cytokines, which directly kill tumor cells and activate adaptive immunity 27,38,39 . In contrast, within the context of systemic inflammation—such as the BM microenvironment being remodeled by periodontitis—N1 polarization amplifies inflammatory cascades, disrupts tissue homeostasis, and drives multi-organ dysfunction 40,41 . Our study revealed that this functional divergence originates from distinct metabolic reprogramming processes. For example, tumor-associated N1 polarization is primarily driven by local cytokines such as TGF-β and IFN-γ 39 , and is often intertwined with hypoxia-induced glycolysis and the dynamics of N2 polarization 42,43 , but lacks well-defined metabolic signatures. By comparison, in the BM microenvironment affected by peripheral inflammation, N1 polarization is orchestrated by systemic IFN-I signaling and is precisely regulated through a newly identified IFN-I–Chac2–GSH signaling axis. This study specifically uncovers a non-canonical role of GSH metabolism within the BM microenvironment remodeled by peripheral inflammation. Traditionally, GSH has been regarded as a key antioxidant maintaining redox homeostasis, primarily functioning through the GSH redox cycle to scavenge ROS and preserve cell survival 28,29 . However, in the periodontitis model, we observed a fundamental reprogramming of GSH metabolism in BM neutrophil, with IFN-I signaling markedly upregulating the degradation-regulatory protein Chac2, which can in turn repressed the catalytic activity of Chac1 44-47 , resulting in substantial intracellular GSH accumulation. This accumulation was accompanied by enhanced expression of Gpx4 and Gsr, thereby strengthening redox cycling efficiency and providing essential metabolic support for sustained ROS generation and NETs formation. We demonstrated that Chac2-mediated GSH metabolic remodeling constitutes the core metabolic basis for the pro-inflammatory functions of N1-polarized neutrophils. By preventing collapse from oxidative stress while simultaneously fueling inflammatory effector activity, this pathway enables neutrophils to maintain persistent pro-inflammatory outputs. These findings thus challenge the conventional view of GSH metabolism as a passive redox buffer and instead establish it as an active driver of inflammatory processes. From a mechanistic perspective, the rate of GSH consumption in neutrophils under inflammatory stress greatly exceeds its rate of synthesis 48 , with Chac2-mediated expansion of the intracellular GSH pool emerging as a critical compensatory adaptation. This study not only uncovers the central role of Chac2 in immunometabolism reprogramming for the first time, but also provides a new perspective for understanding how periodontal inflammation alters neutrophil functional states to influence systemic disease progression. More importantly, we demonstrated that within the BM microenvironment remodeled by periodontitis, neutrophils undergo N1 polarization through an IFN-I–Chac2–GSH signaling axis, thereby exacerbating co-morbidities such as diabetes. This finding thus underscores that periodontal inflammation is not merely an isolated oral condition but a potent driver of distal tissue damage and systemic co-morbidity progression through immunometabolic reprogramming. Therefore, controlling periodontal inflammation is not only critical for maintaining oral health but also essential for preventing systemic dissemination of inflammation and reducing the risks of co-morbidities 35,49,50 . Hence, targeting the Chac2–GSH signaling axis offers a promising strategy for precise intervention in neutrophil pathogenic activation, thereby providing a novel therapeutic avenue for mitigating periodontitis-associated systemic inflammatory responses. From the perspective of immunometabolism, this study further underscores the importance of periodontal disease management in maintaining systemic health, offering a theoretical foundation for clinical practice in which controlling local oral inflammation may slow the progression of systemic co-morbidities. Limitations Several limitations of this study should be acknowledged. First, while we identified IFN-I signaling as a key upstream inducer of Chac2 expression, the precise molecular details by which IFN-I regulates Chac2 transcription and activity require further investigation. Second, our in vivo experiments were conducted in a murine model of periodontitis with diabetic co-morbidity, and the extent to which these findings are applicable to human pathophysiology warrants further validation in clinical samples. Finally, while our results suggest promising therapeutic potential for targeting Chac2, innovative drug delivery strategies to achieve non-invasive and precise modulation of the neutrophil IFN-I–Chac2–GSH signaling axis in clinical settings remain to be developed. Addressing these questions will thus refine our understanding of the IFN-I–Chac2–GSH signaling axis and strengthen its potential as a therapeutic target in inflammation-driven comorbidities. Conclusion Within the BM microenvironment remodeled by periodontitis, neutrophils acquire pro-inflammatory functions by reprogramming their metabolic networks, particularly GSH metabolism. In this context, GSH metabolism is no longer merely a biosynthetic substrate but serves as a central regulatory switch that directly governs neutrophil functional states. The large pool of neutrophils generated through hematopoietic skewing after being reprogrammed by GSH metabolism plays a pivotal role in propagating systemic inflammation. These findings thus provide mechanistic insights into how local periodontal inflammation exacerbates systemic co-morbidities and underscore the critical importance of managing local inflammatory diseases. Materials and Methods Mice C57BL/6 wild-type mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Ifnar1 -/- mice were obtained from Cyagen Bioscience Inc. (Guangzhou, China). Db/db mice were purchased from GemPharmatech Co., Ltd (Jiangsu, China). Six-week-old male mice were used for all experiments. Mice were housed in specific pathogen-free conditions with a 12 : 12 h light/dark cycle with a temperature of 24 ± 0.5 °C and a relative humidity of 40-70%. Food and water were provided ad libitum during the experimental period. Animal experiments were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center (approved number: DLASBD0673). Ligature-induced periodontitis To investigate the impact of experimental periodontitis on BM neutrophils, LIP was performed in mice as previously described. In brief, bilateral maxillary and mandibular second molars were both tied with 5-0 silk ligatures for 14 days to induce periodontitis. Control mice did not undergo ligature placement on their teeth. Cell preparations and sample collection For BM neutrophil isolation, femurs and tibiae from C57BL/6 mice were flushed with ice-cold RPMI 1640 medium (ThermoFisher). The cells were then passed through a 70 µm nylon mesh to obtain a single-cell suspension for subsequent flow cytometric analysis and FACS cell sorting. To collect BM extracellular fluid, femurs and tibiae were flushed with 1 mL of ice-cold PBS, and the supernatant was collected after centrifugation at 500 x g for 5 min at 4 °C. Whole-blood samples were obtained through retrobulbar bleeding. Flow cytometry and sorting Flow cytometric analysis was performed by FACS Aria II cytometer (BD Biosciences), and all flow cytometry data were analyzed using the FlowJo Software (Tree Star Inc.). For cell surface phenotype analysis, anti-CD11b (clone M1/70), anti-Ly6G (clone 1A8), and anti-CD66a (clone MAb-CC1) antibodies were utilized. The BM neutrophils were gated as CD45 + CD11b + Ly6G + , and cell sorting was performed using a FACS Aria Sorp sorter (BD Biosciences). Cytokine assay The sorted BM neutrophils were cultured in RPMI 1640 medium (ThermoFisher) supplemented with 10% (v/v) FBS for 30 min. The neutrophils were then seeded into 12-well plates (1 x 10 6 cells/well) and stimulated with 150 ng/mL E. coli O111 : B4 LPS (InVivogen) for 17 h. Subsequently, the cell culture supernatants were collected for measurements of IL-1β, IL-6, and TNF-α concentrations using a mouse ELISA kit (Absin), following the manufacturer's instructions. The Mouse IFN-beta Quantikine ELISA Kit (R&D Systems) and Mouse IFN-alpha All Subtype Quantikine ELISA Kit (R&D Systems) were utilized to determine the concentrations of IFNα and IFNβ in the BM extracellular fluid, respectively, following the manufacturer's instructions. Histology and immunostaining Mouse pancreases were fixed with 4% (w/v) paraformaldehyde, dehydrated in a graded series of alcohol, paraffin-embedded, and tissue sectioned to 5 µm for histological evaluation. For immunofluorescence staining of pancreas, the slides were deparaffinized and subjected to antigen retrieval, then permeabilized, blocked, and incubated with various antibodies, including anti-glucagon (ab92517, Abcam), anti-insulin (ab181547, Abcam), anti-chac2 (16304-1-AP, Proteintech), and anti-MPO (AF3667, R&D systems). The images were then scanned with ZEISS AXIOSCAN 7 and analyzed with the ImageJ software. Immunofluorescence cell staining The cells were gently rinsed with PBS and fixed in 4% (w/v) paraformaldehyde for 15 min. Permeabilization was performed using 0.5% Triton X-100 (Sigma) for 5 min at room temperature. The cells were then blocked with 3% BSA (Solarbio) for 30 minutes before overnight incubation at 4°C with primary antibodies, including anti-MPO (AF3667, R&D Systems) and anti-CitH3 (ab5103, Abcam) antibodies. Following primary antibody incubation, cells were treated with goat anti-rabbit IgG H&L Alexa Fluor 488 (ab150077, Abcam) and DAPI (Invitrogen; 1:1000) for 1 hour in the dark at room temperature. The images were acquired using a TCS-SP8 STED 3X microscope and analyzed with Fiji software (v 2.0.0). Adoptive transfer of neutrophils BM neutrophils from donor mice (Con or Lig) were sorted by FACS. HL60-neutrophils were treated with GDE and L-BSO for 24h after being stimulated with 1.25% (v/v) DMSO for 5 days. Each recipient db/db mouse received tail vein injections of 5 x 10 6 neutrophils. Three weeks after adoptive transfer, the pancreatic tissues and whole blood were collected for tissue section staining and detection of glycated serum proteins to assess the impact of polarized N1 neutrophils on diabetes in recipient mice. RT-qPCR Total RNA was isolated from the BM neutrophils or HL60 cells using SteadyPure Quick RNA Extraction Kit (AG21023 , Accurate Biotechnology) according to the manufacturer’s instructions. cDNA was synthesized using the Evo M-MLV RT Premix (AG11706, Accurate Biotechnology), according to the manufacturer’s instructions. RT-qPCR was conducted using the Hieff UNICON ® Universal Blue qPCR SYBR Green Master Mix (11184ES08, Yeasen) together with specific primers, and analyzed by real-time fluorescence quantitative PCR instrument (QuantStudio 3, Thermo Fisher). The gene expression levels were normalized to Gapdh mRNA levels. The primer sequences are listed in Supplementary Table 1. PCR array The pro-inflammatory N1 polarization of neutrophils was assessed using the Inflammatory Cytokines & Receptors PCR Array (WC-MRNA0266, WcGene Biotech), which analyzed a total of 90 genes. GAPDH, 18S, β-Actin, and HPRT1 were utilized as internal control genes. Each plate included two blank wells as negative controls. Chac2 knockdown and overexpression To knock down the expression of CHAC2 in HL60 neutrophils, we utilized specific shRNA sequences as follows: 5'-GCTACAGAACCACAACAGTCA-3'. A non-targeting shRNA sequence was used as a control. The HL60 cells were transfected with CHAC-targeting shRNA lentiviral particles (Sh-CHAC2) or control shRNA lentiviral particles (Sh-NC) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. After 48 hours of transfection, the cells were cultured in complete medium supplemented with 2 µg/mL puromycin for 72 hours to select the successfully transfected cells. The efficiency of gene knockdown was confirmed by RT-PCR and WB. These selected cells were then used for downstream experimental analyses to ensure the purity and reliability of the results. The full-length CHAC2 coding sequence was cloned into a lentiviral expression vector (pRRLSIN-cPPT-SFFV-MCS-3FLAG-E2A-EGFP-SV40-puro). Lentivirus was produced in HEK293T cells by co-transfection with packaging plasmids and subsequently used to transfect HL-60 cells. The transduced cells were then selected with puromycin (1 μg/mL) for 5–7 days to establish stable lines. Overexpression efficiency was confirmed by qRT-PCR and Western blot analysis. The empty vector-transduced HL-60 cells served as controls. AAV production for Chac2 silencing To construct AAV vectors for the specific silencing of Chac2 in mouse BM neutrophils, we first selected target sequences for mouse Chac2 mRNA using the application program from Dharmacon siDESIGN center (http://www.dharmacon.com). The specific shRNA sequence 5’-CTACAGAACTACGACAGTCAT-3’ was designed. A non-specific control shRNA sequence (shNC) was also designed to serve as the negative control, ensuring no significant sequence similarity to any known mouse genes. These sequences were cloned under the control of the Ly6G promoter, which is highly active in neutrophils, and a U6 promoter was inserted into an AAV vector backbone to drive shRNA expression. For virus production, the recombinant plasmids were co-transfected with helper plasmids into HEK293 cells using Lipofectamine 3000 transfection reagent (L3000150, Invitrogen). The helper plasmids provided necessary AAV rep and cap genes, as well as adenoviral helper functions. The virus-containing medium was harvested at 72 hours post-transfection, and the virus particles were purified through gradient ultracentrifugation. The titer of the AAV particles was determined by the AAV Quantitation Titer Kit (Cell Biolabs, San Diego, CA, USA). AAV-shCHAC2 intraosseous injection of db/db mice with periodontitis Following 14 days of silk ligation-induced periodontitis, db/db mice were anesthetized with isoflurane, and both knees were flexed with support behind each knee. Hair was shaved around the joint area, and 70% (v/v) alcohol and iodine were used to clean the area. A 1 ml syringe with a 25 (5/8 length) gauge needle was inserted into the intrafemoral space by gentle twisting and application of pressure between the condyles at the top of the femur between the tibia and femur joint. The 25 (5/8 length) gauge needle and cap were left in place, while the 1ml syringe was gently removed. A (25μl 1702 RN) Hamilton syringe with a 32G needle (7803-04, 32 Gauge RN 2” point size 4, referred to as bone marrow needles) was inserted into the plastic cap opening and threaded through the needle opening of the 25 (5/8 length) gauge needle. The 32 G bone marrow needle was marked at 3.5 cm from the tip to indicate the length at which to discontinue insertion. Five microliters of solution (AAV-GFP (4.5×10 13 CFU/ml, AAV-shCHAC2 (1×10 13 TU/ml) was slowly injected by free hand into the shaft of the femur using the 25μl Hamilton syringe and slowly removed to limit backflow. The 25 (5/8 length) gauge needle was then gently removed, and mice were monitored and allowed to recover. RNA sequencing analysis Previously published RNA sequencing data were retrieved as raw files from the GEO database (GSE236477). These files were processed and transformed into Seurat-compatible objects for further analysis. All downstream bioinformatic analyses were performed using Omicsmart, a dynamic, real-time interactive online platform for data analysis (http://www.omicsmart.com). MESA enrichment analysis of metabolic pathways Metabolite Set Enrichment Analysis (MSEA) was conducted using the MetaboAnalyst software (v6.0). The pathway-associated metabolite set was used as the metabolite library, with all compounds included. Pathways showing a Holm-adjusted p -value < 0.05 were considered statistically significant in pairwise comparisons across different time points. Glucose and insulin tolerance tests The blood glucose levels of mice were assessed using an electronic dehydrogenase blood glucose meter (Yuwell 921), determined from tail vein samples, with RBG measured at 3 weeks after ligature. Both the IPGTT and the ITT were conducted. These tests were performed once, at 30 days post-intervention, and were not carried out before the intervention. For IPGTT, the mice fasted for 14 h and received a 20% glucose injection at 2 g/kg, with glucose levels being measured at 0, 30, 60, and 120 min post-injection for IPGTT calculation. In the ITT, mice were fasted for 6h and were then injected with insulin at 0.5 IU/kg, with plasma glucose being measured at the same time point as IPGTT. Statistics The animals were randomly assigned to treatment or control groups. All experiments were performed in duplicates. The two-tailed Student’s t-test was utilized to compare data between the two independent groups. For data involving three or more groups with a single variable, the one-way ANOVA test was employed, followed by Tukey’s tests for multiple comparisons. Statistical analysis was conducted using the GraphPad Prism 9.0.2 software. All data are presented as mean ± SD, and statistical significance was defined as P < 0.05. Declarations Acknowledgements This work was supported by the Beijing Research Ward Excellence Program (BRWEP2024W194100100), the National Natural Science Foundation of China (No. 82501151), the Clinical Research Foundation of Peking University School and Hospital of Stomatology (PKUSS-2024CRFG02), the Beijing Natural Science Foundation (L242154). Conflict of interests The authors have declared that no competing interests exist. Author contributions Conceptualization, Y.N., Y.H. and X.D.; Investigation, Y.N, Y.L., S.S., C.D, Y.C., G.Y., R.L., and Z.H.; Formal Analysis, Y.N, Y.L., F.H., and Y.C.; Writing, Y.N., Y.L, Y.H., B.H., and X.D.; Visualization, Y.N, Y.L., S.S. and T.X.; Funding Acquisition, Y.L., Y.H., and X.D.; Supervision, Y.H. and X.D. Supplementary material Supplemental material includes 3 figures and 1 table. Data availability All original data used for this study are available from the corresponding authors upon reasonable request. References Hasturk, H. & Kantarci, A. 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Additional Declarations There is no conflict of interest Supplementary Files Supplementarymaterials.pdf Supplemental material Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 05 Jan, 2026 Review # 3 received at journal 04 Jan, 2026 Review # 4 received at journal 23 Dec, 2025 Reviewer # 4 agreed at journal 11 Dec, 2025 Review # 2 received at journal 07 Nov, 2025 Review # 1 received at journal 04 Nov, 2025 Reviewer # 3 agreed at journal 25 Oct, 2025 Reviewer # 2 agreed at journal 21 Oct, 2025 Reviewer # 1 agreed at journal 21 Oct, 2025 Reviewers invited by journal 21 Oct, 2025 Submission checks completed at journal 20 Oct, 2025 Editor assigned by journal 06 Oct, 2025 First submitted to journal 06 Oct, 2025 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. 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06:57:03","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1696968,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7792660/v1/df1c9edea3f2f52924cc7d98.pdf"},{"id":94985271,"identity":"efae3376-96de-4475-870b-ca4e53f1120e","added_by":"auto","created_at":"2025-11-03 06:57:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":818020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePeriodontitis induces N1-type polarization of BM neutrophils.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic illustration of RNA sequencing analyses of BM neutrophils from Con and Lig mice. \u003cstrong\u003e(B)\u003c/strong\u003eGO enrichment analysis for genes differentially upregulated in the Lig group compared to the Con group. \u003cstrong\u003e(C)\u003c/strong\u003e Heat map of the expression of N1 neutrophil-related genes. \u003cstrong\u003e(D-E)\u003c/strong\u003e Flow cytometric analysis of the expression of CD11b and CD66a in neutrophils obtained from the BM. \u003cstrong\u003e(F) \u003c/strong\u003eSchematic illustration of \u003cem\u003eex vivo\u003c/em\u003e challenge of neutrophils with LPS (100 ng/mL; 17 h). \u003cstrong\u003e(G)\u003c/strong\u003e Cytokine antibody array dot plot (left) and relative quantitative statistics (right) of differentially-expressed cytokines produced by neutrophils. \u003cstrong\u003e(H)\u003c/strong\u003e Overlay histogram of ROS production in neutrophils and the corresponding mean fluorescence intensity. \u003cstrong\u003e(I) \u003c/strong\u003eRepresentative images of ankle joint sections stained with citH3 (red), MPO (green), and DAPI (blue). NETs are visualized by co-localization of citH3 and DAPI staining (merged images) (scale bar = 50 µm). All data are presented as the mean ± SD from at least three independent replicate experiments.\u003cem\u003e P\u003c/em\u003e values were calculated using two-tailed Student's t test; *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ns indicates no significant difference.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792660/v1/3a07d59009f285f2ff7ea321.jpg"},{"id":94838829,"identity":"ade25f4f-3c7e-434f-94fe-16d239bb552d","added_by":"auto","created_at":"2025-10-31 08:58:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":871306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlutathione accumulation is associated with N1-type neutrophil activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic diagram for LC-MS Metabolomics analysis of BM neutrophils from Con and Lig mice.\u003cstrong\u003e (B) \u003c/strong\u003ePCA of normalized metabolite data from the Con group and Lig group (n = 6 per group). \u003cstrong\u003e(C)\u003c/strong\u003e Nightingale rose chart of classification of differential metabolites. \u003cstrong\u003e(D) \u003c/strong\u003eGO enrichment analysis of metabolites differentially upregulated in the Lig group versus the Con group. \u003cstrong\u003e(E)\u003c/strong\u003e MSEA enrichment analysis for metabolites differentially upregulated in the Lig group versus the Con group.\u003cstrong\u003e (F)\u003c/strong\u003e Volcano plot analysis identifying the most differentially expressed metabolite between the Lig and Con groups. Metabolites upregulated in the Lig group are highlighted in red, while downregulated metabolites are depicted in green. \u003cstrong\u003e(G) \u003c/strong\u003eViolin plot comparing the GSH quantities of the Con group versus the Lig group in the raw intensity of metabolomic data. \u003cstrong\u003e(H) \u003c/strong\u003eQuantification of total GSH concentrations in BM neutrophils from the Con and Lig mice, measured by ELISA. \u003cstrong\u003e(I)\u003c/strong\u003e Schematic illustration of the experimental design for the GDE and L-BSO treatments in HL60 neutrophils. \u003cstrong\u003e(J)\u003c/strong\u003e Heat map of gene expression related to N1-type neutrophil polarization. \u003cstrong\u003e(K)\u003c/strong\u003e Representative immunofluorescent staining of CitH3(green), MPO (red), and cell nuclei (DAPI, blue) of neutrophils. Scale bar=10 μm. \u003cstrong\u003e(L)\u003c/strong\u003e Overlay histogram of ROS production in neutrophils (left) and the corresponding mean fluorescence intensity (right). \u003cstrong\u003e(M)\u003c/strong\u003e Quantitative statistical analysis of reactive cytokines IL-6, IL-1β, and TNF-α produced by neutrophils in response to LPS stimulation. Data are presented as the mean ± SD from at least three independent replicate experiments.\u003cem\u003e P\u003c/em\u003e values were calculated using the two-tailed Student's t test; *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ns indicates no significant difference.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792660/v1/73030148435a4bd80229cc05.jpg"},{"id":94838827,"identity":"1b56f0a9-2b8d-4cf3-94dd-ed38b85f1f44","added_by":"auto","created_at":"2025-10-31 08:58:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":810040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlutathione metabolic reprogramming in BM neutrophils is driven by CHAC2-mediated inhibition of its degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e GO enrichment analysis of metabolites differentially upregulated in the Lig group versus the Con group. \u003cstrong\u003e(B)\u003c/strong\u003e Heat map of gene expression related to the GSH metabolic pathway. \u003cstrong\u003e(C)\u003c/strong\u003e Diagram showing the up-regulation (red) and down-regulation (blue) of key metabolites and key genes in the GSH metabolic pathway. \u003cstrong\u003e(D)\u003c/strong\u003e Quantitative statistics of the relative mRNA expression levels of \u003cem\u003eGss\u003c/em\u003e, \u003cem\u003eGsr\u003c/em\u003e, \u003cem\u003eChac1\u003c/em\u003e, and \u003cem\u003eChac2.\u003c/em\u003e \u003cstrong\u003e(E)\u003c/strong\u003e Western blotting and quantitative analyses of Chac2 protein expression levels in BM neutrophils from Con versus Lig mice. \u003cstrong\u003e(F)\u003c/strong\u003e Schematic diagram illustrating the regulatory role of CHAC2 in GSH degradation. Data are presented as the mean ± SD from at least three independent experiments. \u003cem\u003eP\u003c/em\u003e values were calculated using two-tailed Student's t-test; *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792660/v1/f419739c1ad031409097e98e.jpg"},{"id":94838837,"identity":"0de42605-bb08-498e-9671-7601be5a6015","added_by":"auto","created_at":"2025-10-31 08:58:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":814563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCHAC2 serves as a key metabolic regulator linking glutathione homeostasis to HL60 neutrophil polarization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Western blotting and quantitative analyses of CHAC2 and CHAC1 protein expression levels in HL60 neutrophils of Sh-NC and Sh-CHAC2 groups. \u003cstrong\u003e(B) \u003c/strong\u003eStatistical chart of relative changes in \u003cem\u003eChac2\u003c/em\u003e mRNA expression levels. \u003cstrong\u003e(C)\u003c/strong\u003e Quantitative statistical analyses of the expression levels of reactive cytokines IL-6, IL-1βand TNF-α produced by HL60 neutrophils in response to LPS stimulation.\u003cstrong\u003e (D)\u003c/strong\u003eOverlay histogram of GSH concentration in HL60 neutrophils (left) and the corresponding mean fluorescence intensity (right).\u003cstrong\u003e (E)\u003c/strong\u003e Overlay histogram of ROS production in HL60 neutrophils (left) and the corresponding mean fluorescence intensity (right). \u003cstrong\u003e(F) \u003c/strong\u003eRepresentative immunofluorescent staining of CitH3(red), MPO (green), and cell nuclei (DAPI, blue) of HL60 neutrophils. Scale bar=10 μm.\u003cstrong\u003e(G) \u003c/strong\u003eHeat map of PCR array-based gene expression analysis related to N1-type polarization of Sh-NC and Sh-Chac2 neutrophils.\u003cstrong\u003e (H)\u003c/strong\u003e Western blotting and quantitative analyses of CHAC2 and CHAC1 protein expression levels in HL60 neutrophils of OE-NC and OE-CHAC2 groups.\u003cstrong\u003e (I)\u003c/strong\u003eStatistical chart of relative changes in \u003cem\u003eCHAC2\u003c/em\u003e mRNA expression levels. \u003cstrong\u003e(J)\u003c/strong\u003eQuantitative statistical analyses of the relative expression levels of reactive cytokines IL-6, IL-1β, and TNF-α produced by HL60 neutrophils in response to LPS stimulation.\u003cstrong\u003e (K)\u003c/strong\u003e Overlay histogram of GSH concentration in HL60 neutrophils (left) and the corresponding mean fluorescence intensity (right).\u003cstrong\u003e (L)\u003c/strong\u003e Overlay histogram of ROS production in HL60 neutrophils (left) and the corresponding mean fluorescence intensity (right).\u003cstrong\u003e (M)\u003c/strong\u003e Representative immunofluorescent staining of CitH3(red), MPO (green), and cell nuclei (DAPI, blue) of HL60 neutrophils. Scale bar=10 μm.\u003cstrong\u003e(N)\u003c/strong\u003e Heat map of PCR array-based gene expression analysis related to N1-type polarization of OE-NC and OE-Chac2 neutrophils. Data are presented as the mean ± SD from at least three independent experiments.\u003cem\u003e \u003c/em\u003eThe\u003cem\u003e P\u003c/em\u003e values were calculated using one-way ANOVA; *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ns indicates no significant difference.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792660/v1/b1c4ff57f5c1582e2815fd35.jpg"},{"id":94984899,"identity":"fce712a7-b1ba-44d7-b1f8-c90944fdf44c","added_by":"auto","created_at":"2025-11-03 06:56:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":855801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFN-I signaling promotes proinflammatory N1-type neutrophil polarization by inducing Chac2 expression and modulating GSH metabolism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e GSEA analysis of upregulated genes expressed in the RNA-seq data of neutrophils. \u003cstrong\u003e(B) \u003c/strong\u003eHeatmap of the expression of IFN-I response-related gene sets in the RNAseq data. \u003cstrong\u003e(C)\u003c/strong\u003e Quantification of IFN-α and IFN-β levels in the BM supernatant of Con and Lig mice. \u003cstrong\u003e(D)\u003c/strong\u003e Overlay histogram of ROS production in neutrophils (left) and the corresponding mean fluorescence intensity (right). \u003cstrong\u003e(E)\u003c/strong\u003e Quantitative analysis of the relative expression levels of reactive cytokines IL-6, IL-1β, and TNF-α produced by neutrophils in response to LPS stimulation. \u003cstrong\u003e(F)\u003c/strong\u003e Overlay histogram of GSH concentration in neutrophils. \u003cstrong\u003e(G)\u003c/strong\u003e Overlay histogram of GSH concentrations in Sh-NC and Sh-CHAC2 neutrophils of the blank or IFN-I treatment groups. \u003cstrong\u003e(H)\u003c/strong\u003e Statistical chart of relative changes in CHAC2 mRNA expression levels. \u003cstrong\u003e(I)\u003c/strong\u003e Heat map of gene expression related to N1-type neutrophil polarization. \u003cstrong\u003e(J)\u003c/strong\u003e Representative immunofluorescent staining of CitH3(green), MPO (red), and cell nuclei (DAPI, blue) of neutrophils. Scale bar=10 μm. \u003cstrong\u003e(K)\u003c/strong\u003e Overlay histogram of ROS production in Sh-NC and Sh-CHAC2 neutrophils of the blank or IFN-I treatment groups. \u003cstrong\u003e(L)\u003c/strong\u003e Quantitative analysis of the expression levels of the reactive cytokines IL-6, IL-1β, and TNF-α produced by neutrophils in response to LPS stimulation. Data are presented as the mean ± SD from at least three independent experiments.\u003cem\u003e P\u003c/em\u003evalues were calculated using one-way ANOVA; *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ns indicates no significant difference.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792660/v1/4321c37c9aae24b97b6dc8c6.jpg"},{"id":94838843,"identity":"06699f0a-c3c5-4760-ba10-2de17b36c1e8","added_by":"auto","created_at":"2025-10-31 08:58:50","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1128142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAAV-shChac2 delivery alleviates N1 neutrophil-mediated systemic inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic illustration of the experimental design for the transfer of GDE or L-BSO-treated BM neutrophils to db/db mice.\u003cstrong\u003e (B) \u003c/strong\u003eStatistical curves of random blood glucose measurements in the GDE-Neus and LBSO-Neus groups.\u003cstrong\u003e (C) \u003c/strong\u003eQuantitative analysis of serum glycated protein content. \u003cstrong\u003e(D) \u003c/strong\u003eStatistical curves of blood glucose measurements during IPGTT and ITT. \u003cstrong\u003e(E)\u003c/strong\u003e Representative immunofluorescent staining of glucagon (green), insulin (red), and cell nuclei (DAPI, blue), together with immunohistochemical staining of MPO in pancreas tissue sections. Scale bar = 100 μm. \u003cstrong\u003e(F)\u003c/strong\u003e Schematic illustration of the experimental design for intraosseous injection of AAV-NC or AAV-shCHAC2 into db/db mice.\u003cstrong\u003e (G) \u003c/strong\u003eStatistical curves of random blood glucose measurements in the AAV-NC or AAV-shCHAC2 groups. \u003cstrong\u003e(H) \u003c/strong\u003eQuantitative analysis of serum glycated protein content. \u003cstrong\u003e(I)\u003c/strong\u003e Statistical curves of blood glucose measurements during IPGTT and ITT. \u003cstrong\u003e(J)\u003c/strong\u003e Representative immunofluorescent staining of Chac2 (green), MPO (red), and cell nuclei (DAPI, blue) in pancreatic tissue sections. Scale bar = 100 μm.\u003cstrong\u003e (K)\u003c/strong\u003e Representative immunofluorescent staining of glucagon (green), insulin (red), and cell nuclei (DAPI, blue). Scale bar = 200 μm. \u003cstrong\u003e(L)\u003c/strong\u003e Quantification of fluorescence intensities of Chac2 and MPO in pancreatic tissue sections. Data are presented as the mean ± SD from at least three independent replicate experiments. The \u003cem\u003eP\u003c/em\u003e values were calculated using two-tailed Student's t test; *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ns indicates no significant difference.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7792660/v1/45415d8a3d40a5d7544c2919.jpg"},{"id":94990331,"identity":"fda98988-dff0-46d6-b0f2-7d8951a91a3d","added_by":"auto","created_at":"2025-11-03 07:16:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6617534,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7792660/v1/2c2dbe7a-0b5b-479e-8342-8cf59fc224f7.pdf"},{"id":94838831,"identity":"111ca302-2d8f-4515-82df-7c3fb1a91a31","added_by":"auto","created_at":"2025-10-31 08:58:50","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1696968,"visible":true,"origin":"","legend":"Supplemental material","description":"","filename":"Supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7792660/v1/9b008389722e407f8df02e57.pdf"}],"financialInterests":"There is no conflict of interest","formattedTitle":"CHAC2-mediated Glutathione Metabolic Reprogramming Drives N1 Polarization of Bone Marrow Neutrophils and Exacerbates Inflammatory Co-morbidities","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLocal chronic inflammation not only extends beyond a pathological state localized within specific tissues/organs, but also profoundly reshape systemic immunity through peripheral inflammatory signaling\u003csup\u003e1-4\u003c/sup\u003e. In this process, the bone marrow (BM), as the central tissue/organ of hematopoiesis, undergoes marked alterations in hematopoietic dynamics, manifested by skewed myeloid differentiation and increased neutrophil production\u003csup\u003e5\u003c/sup\u003e. Our previous study had revealed that this quantitative expansion is accompanied by the functional activation of neutrophils, characterized by enhanced release of reactive oxygen species (ROS), increased formation of neutrophil extracellular traps (NETs), and elevated secretion of pro-inflammatory cytokines such as tumor necrosis factor-α\u0026nbsp;(TNF-α)\u003csup\u003e6\u003c/sup\u003e. Collectively, these functional changes are consistent with neutrophil polarization toward the N1 proinflammatory phenotype\u003csup\u003e7\u003c/sup\u003e, underscoring the propensity of neutrophils to undergo such polarization within the BM microenvironment remodeled by chronic inflammation. It must be noted that immune cell polarization rarely occurs in isolation but is intricately linked to underlying metabolic reprogramming, which serves as a critical determinant of cellular phenotype and effector activity\u003csup\u003e8-10\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe intrinsic connection between immune cell polarization and metabolic reprogramming has emerged as a central theme in immunometabolism research, offering critical insights into the regulatory mechanisms of immune responses. Indeed, substantial evidence have demonstrated that specific metabolic reprogramming directly regulates immune cell function, providing new perspectives and potential targets for the metabolic modulation of immune-mediated diseases. This principle has been validated across multiple immune cell types. For example, glycolytic metabolism supports macrophage M1 polarization\u003csup\u003e11-14\u003c/sup\u003e, lipid metabolism governs T cell differentiation\u003csup\u003e15-18\u003c/sup\u003e, and mitochondrial dynamics influence dendritic cell activation\u003csup\u003e19-22\u003c/sup\u003e. Building on the accumulated scientific data on these broadly conserved mechanisms, growing research attention is now directed towards the metabolic adaptations of neutrophils\u003csup\u003e23-25\u003c/sup\u003e, particularly how these immune cells remodel their metabolism in response to specific pathological microenvironments.\u003c/p\u003e\n\u003cp\u003eIn the tumor microenvironment, neutrophils have been shown to undergo glycolytic reprogramming in response to inflammatory cytokines, driving their polarization toward an N1 phenotype that exerts anti-tumor functions\u003csup\u003e26,27\u003c/sup\u003e. However, it is still unclear whether neutrophils experience comparable metabolic adaptations within the BM microenvironment remodeled by chronic inflammation (such as periodontitis). More importantly, how such metabolic remodeling directly governs the process of N1 polarization in this context is still unresolved, representing a critical knowledge gap on how metabolic regulation is linked to neutrophil inflammatory phenotypes.\u003c/p\u003e\n\u003cp\u003eAmong the various diverse pathways of immunometabolic regulation, the metabolism of glutathione (GSH), a key endogenous antioxidant, play key roles in maintaining the redox homeostasis, survival, and effector functions of immune cells\u003csup\u003e28,29\u003c/sup\u003e. Its intracellular levels are precisely regulated by a metabolic network that includes the synthetic enzyme Gss\u003csup\u003e30\u003c/sup\u003e, the degradation enzyme Chac1, and the degradation regulator Chac2, which work together to sustain GSH metabolic balance\u003csup\u003e29,31\u003c/sup\u003e. While the role of GSH biosynthesis in immune regulation has been extensively studied\u003csup\u003e32,33\u003c/sup\u003e, the role of its degradation pathway—particularly the regulatory functions of Chac2 under pathological conditions—remains poorly understood. In particular, it is necessary to investigate whether neutrophils can undergo Chac2-mediated GSH metabolic reprogramming within the BM niche remodeled by chronic inflammation, which could drive their polarization toward the N1 state.\u003c/p\u003e\n\u003cp\u003eIn this study, a murine periodontitis model was used to investigate how local chronic inflammation triggers neutrophil proinflammatory N1 polarization within the BM niche. By integrating transcriptomic and untargeted metabolomic analyses of BM neutrophils, we revealed a pivotal role for Chac2-driven GSH metabolic reprogramming in skewing neutrophils toward the N1 phenotype, which is orchestrated by upstream IFN-I signaling. By further investigating the systemic impact of this polarization in diabetic mice and evaluating the effects of Chac2 inhibition, we provide mechanistic insights into the underlying metabolic mechanisms of neutrophil N1 polarization, which opens up a new therapeutic avenue for targeting BM neutrophil-driven co-morbidities.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePeriodontitis promotes N1-type polarization of BM neutrophils through enhanced GSH metabolism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the impact of periodontitis on the polarization state of BM neutrophils, we analyzed RNA-seq data from BM neutrophils of the control (Con) and ligature-induced periodontitis (Lig) mice obtained in our previous study (Figure 1A). Gene Ontology (GO) analysis of differentially expressed genes revealed enrichment of pathways related to neutrophil activation and inflammation, including\u0026nbsp;“innate immune response,”\u0026nbsp;“cellular response to LPS,”\u0026nbsp;and\u0026nbsp;“regulation of chemokine production”\u0026nbsp;(Figure 1B). Notably, Lig-derived neutrophils exhibited upregulated expression of representative N1-type markers such as S100a9, Ifitm1, Ifitm2, and Cxcr1 (Figure 1C). Flow cytometry confirmed elevated surface expression of CD11b and CD66a (Figure 1D-E), along with heightened functional responses, including increased ROS production upon LPS stimulation (Figure 1G), enhanced secretion of IL-1ra, IL-16, and TNF-α\u0026nbsp;(Figure 1H), and greater NET formation (Figure 1I). These results thus indicate that periodontitis drives BM neutrophil polarization towards a proinflammatory N1 phenotype at the transcriptional, phenotypic, and functional levels.\u003c/p\u003e\n\u003cp\u003eGiven the critical role of metabolism in shaping immune cell function, we next examined whether N1 polarization was accompanied by alterations in metabolic pathways. Untargeted metabolomic profiling of sorted BM neutrophils revealed a distinct separation between the Con and Lig groups by PCA (Figure 2A–B), with 122 metabolites significantly altered (Figure S1A), among which amino acid derivatives were most enriched (Figure 2C). Both KEGG and MSEA pathway analyses identified glutathione metabolism as the top-enriched pathway (Figures 2D–E). Notably, intracellular GSH levels were elevated nearly fourfold in Lig-derived neutrophils (Figure 2F–G), which was further validated by ELISA quantification (Figure 2H).\u003c/p\u003e\n\u003cp\u003eTo functionally assess the role of GSH in driving neutrophil polarization, we utilized the HL60 cell line differentiated into neutrophil-like cells. Cells were treated with either glutathione diethyl ester (GDE) to enhance intracellular GSH or L-buthionine sulfoximine (L-BSO) to inhibit GSH synthesis. GSH levels were confirmed by flow cytometry using a GSH-sensitive probe (Figure S1B). GDE-treated cells exhibited upregulation of N1 markers, along with increased NET formation, ROS production, and proinflammatory cytokine secretion, all of which were significantly suppressed by L-BSO treatment (Figure 2J-M).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, these results thus demonstrate that periodontitis promotes N1-type polarization of BM neutrophils and is closely associated with enhanced GSH metabolic activity, implicating GSH metabolism as a key regulatory axis in inflammation-driven neutrophil reprogramming.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeriodontitis induces glutathione metabolic reprogramming in BM neutrophils\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate how periodontitis alters GSH metabolism in BM neutrophils, we conducted an integrated analysis of transcriptomic and metabolomic datasets from Con and Lig mice. GO enrichment analysis of genes upregulated in Lig mice identified significant activation of the glutathione metabolic process (Figure 3A). Differential gene expression profiling revealed notable changes in key glutathione-related genes (Figure 3B).\u003c/p\u003e\n\u003cp\u003eSubsequently, an integrated GSH metabolic network map based on both datasets was constructed, which revealed a distinct reprogramming pattern in the Lig group. Despite elevated intracellular GSH levels and increased redox cycling activity, the transcriptomic data indicated downregulation of Gss (glutathione synthetase), Gclc (glutamate cysteine ligase catalytic), and Chac1 (a canonical GSH-degrading enzyme), alongside significant upregulation of Chac2, a less characterized homolog involved in glutathione regulation (Figure 3C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven that previous studies have shown that Chac2 can antagonize Chac1-mediated GSH degradation, we hypothesized that Chac2 upregulation may suppress GSH catabolism and promote GSH accumulation. This hypothesis was supported by altered total GSH concentration, GSH/GSSG ratio, and RT-qPCR validation of gene expression patterns in Con and Lig neutrophils (Figure 3D-E). Western blot analysis further confirmed a ~3.5-fold increase in CHAC2 protein expression in Lig samples compared to controls (Figure 3F). The expression of GSR and GPX4 increased at transcriptional level, and the enzymatic activity of neutrophils in the Lig group was enhanced (Figure S1D), suggesting that the accumulation of GSH within cells was accompanied by an increase in the redox cycle.\u003c/p\u003e\n\u003cp\u003eCollectively, these findings suggest that periodontitis induces a distinct reprogramming of GSH metabolism in BM neutrophils, characterized by CHAC2-mediated suppression of GSH degradation (Figure 3G), which may play a pivotal role in modulating neutrophil polarization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChac2-mediated GSH metabolic reprogramming drives N1-type polarization of neutrophils\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether Chac2 is functionally involved in GSH-driven N1-type polarization, we performed both loss- and gain-of-function experiments using HL60-derived neutrophil-like cells. Knockdown of Chac2 (Sh-Chac2) via lentiviral transduction significantly reduced its expression at both the mRNA and protein levels, as confirmed by RT-qPCR and Western blot (Figures 4A-B). Chac2 silencing led to a marked decrease in intracellular GSH content, accompanied by significantly reduced levels of proinflammatory cytokines, ROS production, NET formation, and suppressed expression of N1 signature genes (Figure 4C-G, S2A). These results thus indicate that Chac2 knockdown impairs GSH accumulation and attenuates N1-type inflammatory polarization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConversely, the overexpression of Chac2 (OE-Chac2) in HL60 cells (Figure 4H-I) resulted in a robust increase in GSH levels, validating its role in maintaining GSH homeostasis. Functionally, Chac2 overexpression led to enhanced secretion of IL-6, TNF-α, and IL-1β, elevated ROS production, increased NET formation, and significant upregulation of N1-associated genes (Figure 4J-N, S2B), collectively indicating a shift towards a pro-inflammatory N1 phenotype.\u003c/p\u003e\n\u003cp\u003eTogether, these results thus demonstrate that Chac2 serves as a key metabolic regulator linking glutathione homeostasis to neutrophil functional polarization. Specifically, Chac2 promotes GSH accumulation and drives N1-type polarization, thus contributing to the inflammatory potential of neutrophils in the context of periodontitis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIFN-I signaling promotes Chac2-mediated GSH reprogramming and N1-type polarization of neutrophils\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify upstream regulatory pathways responsible for the observed GSH metabolic reprogramming in BM neutrophils during periodontitis, we performed gene set enrichment analysis (GSEA) of neutrophil transcriptomes from Lig and Con mice. GSEA analysis revealed significant enrichment of IFN-I signaling pathways in the Lig group (Figure 5A), while the corresponding heatmap analysis confirmed upregulation of IFN-I–responsive genes in Lig-derived neutrophils (Figure 5B). Consistently, the IFN-I levels in BM supernatants were markedly elevated in periodontitis mice (Figure 5C), indicating robust activation of IFN-I signaling during periodontitis.\u003c/p\u003e\n\u003cp\u003eTo functionally evaluate the role of IFN-I signaling, we subjected both WT and Ifnar1\u003csup\u003e⁻/⁻\u003c/sup\u003e mice (lacking the type I interferon receptor) to ligature-induced periodontitis. In Ifnar1\u003csup\u003e⁻/⁻\u003c/sup\u003e mice, periodontitis failed to induce the metabolic and phenotypic changes observed in WT animals. Specifically, intracellular GSH levels (Figure 5D) and Chac2 expression (Figure 5E) remained unchanged in Lig-Ifnar1\u003csup\u003e⁻/⁻\u003c/sup\u003e neutrophils compared to controls. Additionally, the key pro-inflammatory features of N1 polarization—including ROS generation (Figure 5F) and proinflammatory cytokine secretion (Figure S2C)—were significantly attenuated. These findings thus suggest that IFN-I signaling is required for Chac2 induction and GSH accumulation, as well as for driving the N1-type pro-inflammatory programming of neutrophils in periodontitis.\u003c/p\u003e\n\u003cp\u003eTo further delineate the IFN-I–CHAC2 signaling axis in neutrophil polarization, we treated DMSO-differentiated HL60 cells with recombinant IFN-I in the presence or absence of Chac2 knockdown. In Sh-NC cells, IFN-I stimulation induced a robust increase in intracellular GSH levels, upregulated Chac2 expression, and enhanced transcription of N1-type signature genes. Functionally, IFN-I also promoted NET formation, ROS production, and pro-inflammatory cytokine release. However, in Sh-Chac2 cells, these IFN-I–induced effects were markedly ablated—demonstrating that Chac2 is a key mediator linking IFN-I signaling to GSH metabolic reprogramming and N1-type polarization (Figure 5G-L, S2D-F). Hence, these results validated that IFN-I signaling promotes proinflammatory N1-type polarization of neutrophils by inducing Chac2 expression and modulating GSH metabolism.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAAV-shChac2 delivery alleviated systemic inflammation and improved metabolic outcomes in diabetic mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the systemic impact of N1-polarized neutrophils under diabetic conditions, BM neutrophils isolated from Con and Lig mice were adoptively transferred into db/db diabetic recipient mice (Figure S4A). Mice receiving Lig-Neus displayed worsened metabolic parameters, including elevated random blood glucose levels, increased glycated serum protein levels, impaired glucose tolerance, and reduced insulin sensitivity (Figure S4B-C). Histological analyses further revealed disrupted islet function and enhanced neutrophil infiltration in the pancreas (Figure S4D). These results thus suggest that BM N1-type neutrophils induced by periodontitis exacerbate pancreatic dysfunction in diabetes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo directly assess the role of GSH-driven neutrophil N1 polarization in systemic metabolic dysfunction, we adoptively transferred HL60-derived neutrophil-like cells pre-treated with either GDE (GDE-HL60) or L-BSO (L-BSO-HL60) into db/db diabetic mice (Figure 6A). Mice receiving GDE-treated HL60 cells, characterized by elevated intracellular GSH and N1-type polarization, exhibited significantly increased random blood glucose and glycated serum protein levels compared to controls (Figure 6B-C). Line charts of the intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) revealed more severe dysfunction in the GDE-HL60 group versus the L-BSO-HL60 group (Figure 6D). Additionally, immunofluorescence staining of pancreatic islets revealed marked insulin depletion and enhanced glucagon expression, indicating disrupted endocrine function (Figure 6E). Conversely, L-BSO-HL60 cells with suppressed GSH synthesis and attenuated inflammatory polarization failed to induce these metabolic abnormalities. Mice in this group showed improved glycemic control and preserved islet architecture, with restored insulin expression and limited neutrophil infiltration. These results thus validated that the core mechanism by which pro-inflammatory neutrophils exacerbate pancreatic dysfunction in diabetes is intracellular GSH accumulation within the neutrophils.\u003c/p\u003e\n\u003cp\u003eTo explore whether targeted intervention of Chac2 in neutrophils could alleviate the inflammatory co-morbidities of periodontitis, we constructed AAV9 viruses targeting neutrophils to specifically knock down Chac2 (AAV-shCHAC2) and a control virus with no payload (AAV-NC), and administered them via intraosseous injection into a mouse model with periodontitis and diabetes comorbidities (Figure 6F). During three weeks of modeling, the random blood glucose and glycated serum protein levels of the AAV-shCHAC2 group were significantly lower than those of the control group (Figure 6G-H), and islet dysfunction, as indicated by IPGTT, ITT, and insulin/glucagon fluorescence staining of pancreatic tissue, was alleviated (Figure 6I-J). Double immunofluorescence staining for detection of MPO/Chac2 in pancreatic tissue demonstrated the effective targeting and knockdown of neutrophils by AAV treatment (Figure 6K-L). These results thus suggest that Chac2-regulated N1 polarization of BM neutrophils plays a key role in the pathological process by which periodontitis exacerbates diabetic co-morbidity..\u003c/p\u003e\n\u003cp\u003eTogether, these results functionally demonstrated that intracellular GSH elevation promotes neutrophil N1 polarization, which in turn contributes to the exacerbation of diabetic co-morbidity in the context of periodontitis. Correspondingly, targeted knockdown of Chac2 in BM neutrophils can reverse N1 polarization and effectively alleviate the aggravating effects of local chronic inflammation on distal co-morbidities. This IFN-I–CHAC2–GSH signaling axis thus represents a critical upstream regulatory pathway linking local chronic inflammation to systemic neutrophil activation and immune-metabolic dysfunction.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere is emerging evidence that local chronic inflammation, such as periodontitis, is a key contributor to systemic immune dysregulation and multi-organ co-morbidities\u003csup\u003e34,35\u003c/sup\u003e. To delineate the role of BM neutrophils in this process, we employed integrated metabolomic and transcriptomic profiling and demonstrated that periodontitis triggers their polarization toward a pro-inflammatory N1 phenotype via GSH metabolic reprogramming. This reprogramming is driven by IFN-I signaling, which upregulates Chac2 expression at the transcriptional level. In contrast to its canonical role in GSH degradation, we found that in the context of inflammation, Chac2 upregulation led to a pronounced intracellular accumulation of GSH, which in turn enhanced neutrophil N1 effector functions. These insights thus broaden the conceptual framework of neutrophil immunometabolism and suggest that targeting the IFN-I–Chac2–GSH signaling axis may provide novel strategies for mitigating inflammation-driven co-morbidities.\u003c/p\u003e\n\u003cp\u003eNeutrophils are capable of undergoing N1 polarization across diverse pathological microenvironments\u003csup\u003e26,36,37\u003c/sup\u003e, but their functional properties, metabolic basis, and regulatory mechanisms exhibit fundamental divergences and nuances that vary according to the disease condition and state. For instance, within the tumor microenvironment, N1-polarized neutrophils exert anti-tumor protective effects by generating ROS, NETs, and proinflammatory cytokines, which directly kill tumor cells and activate adaptive immunity\u003csup\u003e27,38,39\u003c/sup\u003e. In contrast, within the context of systemic inflammation—such as the BM microenvironment being remodeled by periodontitis—N1 polarization amplifies inflammatory cascades, disrupts tissue homeostasis, and drives multi-organ dysfunction\u003csup\u003e40,41\u003c/sup\u003e. Our study revealed that this functional divergence originates from distinct metabolic reprogramming processes. For example, tumor-associated N1 polarization is primarily driven by local cytokines such as TGF-β and IFN-γ\u003csup\u003e39\u003c/sup\u003e, and is often intertwined with hypoxia-induced glycolysis and the dynamics of N2 polarization\u003csup\u003e42,43\u003c/sup\u003e, but lacks well-defined metabolic signatures. By comparison, in the BM microenvironment affected by peripheral inflammation, N1 polarization is orchestrated by systemic IFN-I signaling and is precisely regulated through a newly identified IFN-I–Chac2–GSH signaling axis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study specifically uncovers a non-canonical role of GSH metabolism within the BM microenvironment remodeled by peripheral inflammation. Traditionally, GSH has been regarded as a key antioxidant maintaining redox homeostasis, primarily functioning through the GSH redox cycle to scavenge ROS and preserve cell survival\u003csup\u003e28,29\u003c/sup\u003e. However, in the periodontitis model, we observed a fundamental reprogramming of GSH metabolism in BM neutrophil, with IFN-I signaling markedly upregulating the degradation-regulatory protein Chac2, which can in turn repressed the catalytic activity of Chac1\u003csup\u003e44-47\u003c/sup\u003e, resulting in substantial intracellular GSH accumulation. This accumulation was accompanied by enhanced expression of Gpx4 and Gsr, thereby strengthening redox cycling efficiency and providing essential metabolic support for sustained ROS generation and NETs formation. We demonstrated that Chac2-mediated GSH metabolic remodeling constitutes the core metabolic basis for the pro-inflammatory functions of N1-polarized neutrophils. By preventing collapse from oxidative stress while simultaneously fueling inflammatory effector activity, this pathway enables neutrophils to maintain persistent pro-inflammatory outputs. These findings thus challenge the conventional view of GSH metabolism as a passive redox buffer and instead establish it as an active driver of inflammatory processes.\u003c/p\u003e\n\u003cp\u003eFrom a mechanistic perspective, the rate of GSH consumption in neutrophils under inflammatory stress greatly exceeds its rate of synthesis\u003csup\u003e48\u003c/sup\u003e, with Chac2-mediated expansion of the intracellular GSH pool emerging as a critical compensatory adaptation. This study not only uncovers the central role of Chac2 in immunometabolism reprogramming for the first time, but also provides a new perspective for understanding how periodontal inflammation alters neutrophil functional states to influence systemic disease progression. More importantly, we demonstrated that within the BM microenvironment remodeled by periodontitis, neutrophils undergo N1 polarization through an IFN-I–Chac2–GSH signaling axis, thereby exacerbating co-morbidities such as diabetes. This finding thus underscores that periodontal inflammation is not merely an isolated oral condition but a potent driver of distal tissue damage and systemic co-morbidity progression through immunometabolic reprogramming.\u003c/p\u003e\n\u003cp\u003eTherefore, controlling periodontal inflammation is not only critical for maintaining oral health but also essential for preventing systemic dissemination of inflammation and reducing the risks of co-morbidities\u003csup\u003e35,49,50\u003c/sup\u003e. Hence, targeting the Chac2–GSH signaling axis offers a promising strategy for precise intervention in neutrophil pathogenic activation, thereby providing a novel therapeutic avenue for mitigating periodontitis-associated systemic inflammatory responses. From the perspective of immunometabolism, this study further underscores the importance of periodontal disease management in maintaining systemic health, offering a theoretical foundation for clinical practice in which controlling local oral inflammation may slow the progression of systemic co-morbidities.\u003c/p\u003e"},{"header":"Limitations","content":"\u003cp\u003eSeveral limitations of this study should be acknowledged. First, while we identified IFN-I signaling as a key upstream inducer of Chac2 expression, the precise molecular details by which IFN-I regulates Chac2 transcription and activity require further investigation. Second, our \u003cem\u003ein vivo\u003c/em\u003e experiments were conducted in a murine model of periodontitis with diabetic co-morbidity, and the extent to which these findings are applicable to human pathophysiology warrants further validation in clinical samples. Finally, while our results suggest promising therapeutic potential for targeting Chac2, innovative drug delivery strategies to achieve non-invasive and precise modulation of the neutrophil IFN-I–Chac2–GSH signaling axis in clinical settings remain to be developed. Addressing these questions will thus refine our understanding of the IFN-I–Chac2–GSH signaling axis and strengthen its potential as a therapeutic target in inflammation-driven comorbidities.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWithin the BM microenvironment remodeled by periodontitis, neutrophils acquire pro-inflammatory functions by reprogramming their metabolic networks, particularly GSH metabolism. In this context, GSH metabolism is no longer merely a biosynthetic substrate but serves as a central regulatory switch that directly governs neutrophil functional states. The large pool of neutrophils generated through hematopoietic skewing after being reprogrammed by GSH metabolism plays a pivotal role in propagating systemic inflammation. These findings thus provide mechanistic insights into how local periodontal inflammation exacerbates systemic co-morbidities and underscore the critical importance of managing local inflammatory diseases.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eMice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6 wild-type mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Ifnar1\u003csup\u003e-/-\u003c/sup\u003e mice were obtained from Cyagen Bioscience Inc. (Guangzhou, China). Db/db mice were purchased from GemPharmatech Co., Ltd (Jiangsu, China). Six-week-old male mice were used for all experiments. Mice were housed in specific pathogen-free conditions with a 12 : 12 h light/dark cycle with a temperature of 24\u0026nbsp;±\u0026nbsp;0.5\u0026nbsp;°C and a relative humidity of 40-70%. Food and water were provided \u003cem\u003ead libitum\u003c/em\u003e during the experimental period. Animal experiments were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center (approved number: DLASBD0673). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLigature-induced periodontitis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the impact of experimental periodontitis on BM neutrophils, LIP was performed in mice as previously described. In brief, bilateral maxillary and mandibular second molars were both tied with 5-0 silk ligatures for 14 days to induce periodontitis. Control mice did not undergo ligature placement on their teeth.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell preparations and sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor BM neutrophil isolation, femurs and tibiae from C57BL/6 mice were flushed with ice-cold RPMI 1640 medium (ThermoFisher). The cells were then passed through a 70 µm nylon mesh to obtain a single-cell suspension for subsequent flow cytometric analysis and FACS cell sorting. To collect BM extracellular fluid, femurs and tibiae were flushed with 1 mL of ice-cold PBS, and the supernatant was collected after centrifugation at 500 x g for 5 min at 4 °C. Whole-blood samples were obtained through retrobulbar bleeding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry and sorting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometric analysis was performed by FACS Aria II cytometer (BD Biosciences), and all flow cytometry data were analyzed using the FlowJo Software (Tree Star Inc.). For cell surface phenotype analysis, anti-CD11b (clone M1/70), anti-Ly6G (clone 1A8), and anti-CD66a (clone MAb-CC1) antibodies were utilized. The BM neutrophils were gated as CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e, and cell sorting was performed using a FACS Aria Sorp sorter (BD Biosciences). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sorted BM neutrophils were cultured in RPMI 1640 medium (ThermoFisher) supplemented with 10% (v/v) FBS for 30 min. The neutrophils were then seeded into 12-well plates (1 x 10\u003csup\u003e6\u003c/sup\u003e cells/well) and stimulated with 150 ng/mL E. coli O111 : B4 LPS (InVivogen) for 17 h. Subsequently, the cell culture supernatants were collected for measurements of IL-1β, IL-6, and TNF-α concentrations using a mouse ELISA kit (Absin), following the manufacturer's instructions. The Mouse IFN-beta Quantikine ELISA Kit (R\u0026amp;D Systems) and Mouse IFN-alpha All Subtype Quantikine ELISA Kit (R\u0026amp;D Systems) were utilized to determine the concentrations of IFNα and IFNβ in the BM extracellular fluid, respectively, following the manufacturer's instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology and immunostaining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse pancreases were fixed with 4% (w/v) paraformaldehyde, dehydrated in a graded series of alcohol, paraffin-embedded, and tissue sectioned to 5 µm for histological evaluation. For immunofluorescence staining of pancreas, the slides were deparaffinized and subjected to antigen retrieval, then permeabilized, blocked, and incubated with various antibodies, including anti-glucagon (ab92517, Abcam), anti-insulin (ab181547, Abcam), anti-chac2 (16304-1-AP, Proteintech), and anti-MPO (AF3667, R\u0026amp;D systems).\u0026nbsp;The images were then scanned with ZEISS AXIOSCAN 7 and analyzed with the ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence cell staining\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe cells were gently rinsed with PBS and fixed in 4% (w/v) paraformaldehyde for 15 min. Permeabilization was performed using 0.5% Triton X-100 (Sigma) for 5 min at room temperature. The cells were then blocked with 3% BSA (Solarbio) for 30 minutes before overnight incubation at 4°C with primary antibodies, including anti-MPO (AF3667, R\u0026amp;D Systems) and anti-CitH3 (ab5103, Abcam) antibodies. Following primary antibody incubation, cells were treated with goat anti-rabbit IgG H\u0026amp;L Alexa Fluor 488 (ab150077, Abcam) and DAPI (Invitrogen; 1:1000) for 1 hour in the dark at room temperature. The images were acquired using a\u0026nbsp;TCS-SP8 STED 3X microscope and analyzed with Fiji software (v 2.0.0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdoptive transfer of neutrophils\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBM neutrophils from donor mice (Con or Lig) were sorted by FACS. HL60-neutrophils were treated with GDE and L-BSO for 24h after being stimulated with 1.25% (v/v) DMSO for 5 days. Each recipient db/db mouse received tail vein injections of 5 x 10\u003csup\u003e6\u003c/sup\u003e neutrophils. Three weeks after adoptive transfer, the pancreatic tissues and whole blood were collected for tissue section staining and detection of glycated serum proteins to assess the impact of polarized N1 neutrophils on diabetes in recipient mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from the BM neutrophils or HL60 cells using SteadyPure Quick RNA Extraction Kit (AG21023 , Accurate Biotechnology) according to the manufacturer’s instructions. cDNA was synthesized using the Evo M-MLV RT Premix (AG11706, Accurate Biotechnology), according to the manufacturer’s instructions. RT-qPCR was conducted using the Hieff UNICON\u003csup\u003e®\u003c/sup\u003e Universal Blue qPCR SYBR Green Master Mix (11184ES08, Yeasen) together with specific primers, and analyzed by real-time fluorescence quantitative PCR instrument (QuantStudio 3, Thermo Fisher). The gene expression levels were normalized to \u003cem\u003eGapdh\u003c/em\u003e mRNA levels. The primer sequences are listed in Supplementary Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR array\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pro-inflammatory N1 polarization of neutrophils was assessed using the Inflammatory Cytokines \u0026amp; Receptors PCR Array (WC-MRNA0266, WcGene Biotech), which analyzed a total of 90 genes. GAPDH, 18S,\u0026nbsp;β-Actin, and HPRT1 were utilized as internal control genes. Each plate included two blank wells as negative controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChac2 knockdown and overexpression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo knock down the expression of \u003cem\u003eCHAC2\u003c/em\u003e in HL60 neutrophils, we utilized specific shRNA sequences as follows: 5'-GCTACAGAACCACAACAGTCA-3'. A non-targeting shRNA sequence was used as a control. The HL60 cells were transfected with CHAC-targeting shRNA lentiviral particles (Sh-CHAC2) or control shRNA lentiviral particles (Sh-NC) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. After 48 hours of transfection, the cells were cultured in complete medium supplemented with 2 µg/mL puromycin for 72 hours to select the successfully transfected cells. The efficiency of gene knockdown was confirmed by RT-PCR and WB. These selected cells were then used for downstream experimental analyses to ensure the purity and reliability of the results. The full-length \u003cem\u003eCHAC2\u003c/em\u003e coding sequence was cloned into a lentiviral expression vector (pRRLSIN-cPPT-SFFV-MCS-3FLAG-E2A-EGFP-SV40-puro). Lentivirus was produced in HEK293T cells by co-transfection with packaging plasmids and subsequently used to transfect HL-60 cells. The transduced cells were then selected with puromycin (1 μg/mL) for 5–7 days to establish stable lines. Overexpression efficiency was confirmed by qRT-PCR and Western blot analysis. The empty vector-transduced HL-60 cells served as controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAAV production for Chac2 silencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo construct AAV vectors for the specific silencing of Chac2 in mouse BM neutrophils, we first selected target sequences for mouse Chac2 mRNA using the application program from Dharmacon siDESIGN center (http://www.dharmacon.com). The specific shRNA sequence 5’-CTACAGAACTACGACAGTCAT-3’ was designed. A non-specific control shRNA sequence (shNC) was also designed to serve as the negative control, ensuring no significant sequence similarity to any known mouse genes. These sequences were cloned under the control of the Ly6G promoter, which is highly active in neutrophils, and a U6 promoter was inserted into an AAV vector backbone to drive shRNA expression. For virus production, the recombinant plasmids were co-transfected with helper plasmids into HEK293 cells using Lipofectamine 3000 transfection reagent (L3000150, Invitrogen). The helper plasmids provided necessary AAV rep and cap genes, as well as adenoviral helper functions. The virus-containing medium was harvested at 72 hours post-transfection, and the virus particles were purified through gradient ultracentrifugation. The titer of the AAV particles was determined by the AAV Quantitation Titer Kit (Cell Biolabs, San Diego, CA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAAV-shCHAC2 intraosseous injection of db/db mice with periodontitis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing 14 days of silk ligation-induced periodontitis, db/db mice were anesthetized with isoflurane, and both knees were flexed with support behind each knee. Hair was shaved around the joint area, and 70% (v/v) alcohol and iodine were used to clean the area. A 1 ml syringe with a 25 (5/8 length) gauge needle was inserted into the intrafemoral space by gentle twisting and application of pressure between the condyles at the top of the femur between the tibia and femur joint. The 25 (5/8 length) gauge needle and cap were left in place, while the 1ml syringe was gently removed. A (25μl 1702 RN) Hamilton syringe with a 32G needle (7803-04, 32 Gauge RN 2”\u0026nbsp;point size 4, referred to as bone marrow needles) was inserted into the plastic cap opening and threaded through the needle opening of the 25 (5/8 length) gauge needle. The 32 G bone marrow needle was marked at 3.5 cm from the tip to indicate the length at which to discontinue insertion. Five microliters of solution (AAV-GFP (4.5×10\u003csup\u003e13\u003c/sup\u003e CFU/ml, AAV-shCHAC2 (1×10\u003csup\u003e13\u003c/sup\u003e TU/ml) was slowly injected by free hand into the shaft of the femur using the 25μl Hamilton syringe and slowly removed to limit backflow. The 25 (5/8 length) gauge needle was then gently removed, and mice were monitored and allowed to recover.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreviously published RNA sequencing data were retrieved as raw files from the GEO database (GSE236477). These files were processed and transformed into Seurat-compatible objects for further analysis. All downstream bioinformatic analyses were performed using Omicsmart, a dynamic, real-time interactive online platform for data analysis (http://www.omicsmart.com).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMESA enrichment analysis of metabolic pathways\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetabolite Set Enrichment Analysis (MSEA) was conducted using the MetaboAnalyst software (v6.0). The pathway-associated metabolite set was used as the metabolite library, with all compounds included. Pathways showing a Holm-adjusted \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 were considered statistically significant in pairwise comparisons across different time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlucose and insulin tolerance tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe blood glucose levels of mice were assessed using an electronic dehydrogenase blood glucose meter (Yuwell 921), determined from tail vein samples, with RBG measured at 3 weeks after ligature. Both the IPGTT and the ITT were conducted. These tests were performed once, at 30 days post-intervention, and were not carried out before the intervention. For IPGTT, the mice fasted for 14 h and received a 20% glucose injection at 2 g/kg, with glucose levels being measured at 0, 30, 60, and 120 min post-injection for IPGTT calculation. In the ITT, mice were fasted for 6h and were then injected with insulin at 0.5 IU/kg, with plasma glucose being measured at the same time point as IPGTT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animals were randomly assigned to treatment or control groups. All experiments were performed in duplicates. The two-tailed Student’s t-test was utilized to compare data between the two independent groups. For data involving three or more groups with a single variable, the one-way ANOVA test was employed, followed by Tukey’s tests for multiple comparisons. Statistical analysis was conducted using the GraphPad Prism 9.0.2 software. All data are presented as mean ± SD, and statistical significance was defined as \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Beijing Research Ward Excellence Program\u0026nbsp;(BRWEP2024W194100100), the\u0026nbsp;National Natural Science Foundation of China (No.\u0026nbsp;82501151), the\u0026nbsp;Clinical Research Foundation of Peking University School and Hospital of Stomatology (PKUSS-2024CRFG02), the\u0026nbsp;Beijing Natural Science Foundation (L242154).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Y.N., Y.H. and X.D.; Investigation, Y.N, Y.L., S.S., C.D, Y.C., G.Y., R.L., and Z.H.; Formal Analysis, Y.N, Y.L., F.H., and Y.C.; Writing, Y.N., Y.L, Y.H., B.H., and X.D.; Visualization, Y.N, Y.L., S.S. and T.X.; Funding Acquisition, Y.L., Y.H., and X.D.; Supervision, Y.H. and X.D.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplemental material includes \u003cstrong\u003e3\u003c/strong\u003e figures and \u003cstrong\u003e1\u0026nbsp;\u003c/strong\u003etable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll original data used for this study are available from the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHasturk, H. \u0026amp; Kantarci, A. 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Although this process involves metabolic reprogramming, the specific underlying metabolic mechanisms of neutrophil N1 polarization within the periodontitis-modified BM niche remain poorly defined. Integrated transcriptomic and metabolomic analyses in this study revealed that periodontitis reprograms intracellular glutathione (GSH) metabolism in BM neutrophils, facilitating their N1 polarization. Central to this mechanism is the upregulation of Chac2, an enzyme that promotes GSH accumulation. This enhancement is accompanied by elevated GSH redox cycling, which supports sustained ROS production and NET formation, thereby amplifying inflammatory responses. We further identified type I interferon (IFN-I) signaling as a key upstream regulator that induces Chac2 expression and drives metabolic reprogramming. Importantly, the intraosseous delivery of AAV-delivered Chac2 shRNA in db/db mice with periodontitis markedly reduced neutrophil-aggravated systemic inflammatory co-morbidity symptoms and improved glycemic control, underscoring the functional relevance of this pathway in diabetic co-morbidity. Together, these findings thus delineate the IFN-I–Chac2–GSH axis as a core signaling mechanism regulating neutrophil N1 polarization in the BM niche, providing new insights into how periodontal inflammation reprograms immune functions at the systemic level. This study thus broadens the conceptual framework of neutrophil immunometabolism and proposes targeting the Chac2–GSH axis as a potential therapeutic strategy for systemic comorbidities associated with periodontitis.","manuscriptTitle":"CHAC2-mediated Glutathione Metabolic Reprogramming Drives N1 Polarization of Bone Marrow Neutrophils and Exacerbates Inflammatory Co-morbidities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 08:58:45","doi":"10.21203/rs.3.rs-7792660/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-01-05T10:02:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-04T15:58:59+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-12-23T14:03:26+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-12-11T08:56:16+00:00","index":4,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-07T06:47:57+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-04T13:17:26+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-25T04:37:34+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-21T13:53:17+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-21T12:24:00+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-10-21T08:00:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-21T03:43:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-06T15:30:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Oral Science","date":"2025-10-06T15:30:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-oral-science","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ijos2","sideBox":"Learn more about [International Journal of Oral Science](http://www.nature.com/ijos/)","snPcode":"41368","submissionUrl":"https://mts-ijos.nature.com/cgi-bin/main.plex","title":"International Journal of Oral Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3981986b-31de-4c40-961c-a363f43fcc8d","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":56622997,"name":"Health sciences/Diseases/Oral diseases/Periodontitis"},{"id":56622998,"name":"Health sciences/Diseases/Endocrine system and metabolic diseases/Diabetes"}],"tags":[],"updatedAt":"2026-05-09T09:30:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 08:58:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7792660","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7792660","identity":"rs-7792660","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}