Nippostrongylus brasiliensis and Heligmosomoides polygyrus activate different immunomodulatory pathways in murine macrophages in vitro

Nippostrongylus brasiliensis and Heligmosomoides polygyrus activate different immunomodulatory pathways in murine macrophages in vitro | 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 Nippostrongylus brasiliensis and Heligmosomoides polygyrus activate different immunomodulatory pathways in murine macrophages in vitro Covadonga Varea, Antonio J. Martín-Galiano, Sara Vazquez, Ana Montero-Calle, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8017434/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Hookworm infections affect hundreds of millions of people worldwide and rank among the most significant neglected tropical diseases in terms of morbidity. Due to the inverse correlation between hookworm infection and the incidence of immune-mediated inflammatory diseases, considerable research has focused on understanding how parasitic helminths modulate host inflammation. Heligmosomoides polygyrus and Nippostrongylus brasiliensis are two hookworm-like rodent models widely used to investigate fundamental aspects of immune regulation. However, no comparative study has yet analysed the distinct immunological pathways activated by these helminths in their hosts. In this study, we compared cytokine profiles and M1/M2 macrophage polarization markers induced by H. polygyrus and N. brasiliensis , alongside proteomic pathways involved in their activation, using the RAW 264.7 murine macrophage cell line. H. polygyrus downregulated proinflammatory cytokines such as TNF-α and MCP-1, whereas N. brasiliensis did not affect TNF-α expression. Both parasites upregulated Th2 and regulatory cytokines, including IL-4 and TGF-β. Furthermore, H. polygyrus predominantly polarized macrophages toward an M2 phenotype, while N. brasiliensis maintained a balanced M1/M2 profile. Proteomic analysis revealed that N. brasiliensis and H. polygyrus exert their anti-inflammatory effects through distinct mechanisms, with N. brasiliensis acting via peripheral pathways and H. polygyrus via central regulatory mechanisms. Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology Nippostrongylus brasiliensis Heligmosomoides polygyrus RAW264.7 macrophages nematoda immunomodulation Figures Figure 1 Figure 2 Figure 3 Figure 4 1 INTRODUCTION Infections caused by parasitic helminths have a high prevalence worldwide, in particular in non-industrialised countries from tropical and subtropical regions [ 1 , 2 ]. According to the WHO, in 2018, 25% of the global population was infected with a helminth species, with nematodes being the most predominant group [ 3 ]. More importantly, these infections are associated with significant morbidity and mortality, particularly in children [ 4 ]. Species from the genera Ascaris , Ancylostoma , Necator , Strongyloides , and Trichuris , all known to parasitize their hosts through soil contact, infect approximately one billion people worldwide, significantly contributing to disability-adjusted life years (DALYs) particularly in impoverished and resource-limited settings. [ 2 ]. Despite the negative effects associated with parasites, there is evidence that their complete eradication can have adverse consequences for human health. Indeed, it is well known that limited exposure to pathogens leads to an underdeveloped regulatory immune system, ultimately increasing the prevalence of immune-related disorders [ 5 , 6 ]. Nematodes, aiming to survive within their host, must evade or modulate the immune response in their favour, strongly down-regulating host inflammatory responses [ 6 ]. This immunomodulatory ability affects the immune response in various ways, such as inhibiting protective mechanisms like mast cell degranulation, impairing the induction of immune responses, and promoting regulatory T cell induction [ 7 ]. These mechanisms are primarily mediated by bioactive molecules excreted and/or secreted by the parasites known as excretory/secretory products (ESPs) [ 6 , 8 ]. Hookworms have been described as one of the groups of nematodes with the highest immunomodulatory potential [ 9 ], and some of their secreted molecules ameliorate the symptoms of inflammatory diseases such as asthma and inflammatory bowel disease [ 6 , 10 ]. Due to the difficulty in reproducing human hookworms’ life cycle in laboratory settings, different hookworm-like rodent nematodes such as Nippostrongylus brasiliensis and Heligmosomoides polygyrus have been widely used for the study of hookworm biological features. Indeed, recent proteomic and transcriptomic studies have shown evidence of the high similarity between these rodent models and their human counterparts, making them highly suitable for the study of the immunobiology of gastrointestinal nematode infections [ 11 ]. N. brasiliensis belongs to the phylum Nematoda and the superfamily Trichostrongyloidea, with rats as its natural hosts, though it can also infect other rodent species like mice. Primary infection elicits pulmonary protection against reinfection, which depends on CD4 + T cells and alternatively activated macrophages (M2). These M2 macrophages may also contribute to tissue repair following the lung phase of the parasite’s life cycle [ 12 ]. In mice, infection with this parasite triggers a dominant Th2 response characterized by increased IL-4, IL-5, and IL-13 cytokines and elevated IgE antibody production [ 13 ]. Numerous studies highlight N. brasiliensis as a potent immunomodulator, with ESPs playing a significant role in this process [ 14 , 15 ]. Among these ESPs, proteins belonging to the SCP/TAPS family stand out as key mediators in evasion of the host's immune response [ 15 ]. The gastrointestinal nematode Heligmosomoides polygyrus belongs to the phylum Nematoda, superfamily Trichostrongyloidea, and family Heligmosomidae , with wild house mice ( Mus musculus ) as its natural host. Despite the similarities at a proteomic and transcriptomic level with human hookworms, H. polygyrus has a slightly different life cycle. In the case of H. polygyrus , definitive hosts acquire infection by oral ingestion of infective larvae, which then invade the duodenal mucosa, penetrating the muscular layer to reside beneath the serosal membrane, and returning to the intestinal lumen as adult worms about eight days later [ 16 ]. Due to its manageable life cycle in the laboratory, H. polygyrus has been widely used to study immunomodulation [ 17 ]. Infection is accompanied by regulatory T cell populations, dendritic cells, macrophages, B cell hyperstimulation, and localized changes in the intestinal environment [ 17 ]. H. polygyrus ESPs include Hp ARI ( H. polygyrus Alarmin Release Inhibitor), which suppresses the release of alarmins like IL-25 and IL-33 produced by epithelial cells [ 18 , 19 ]. More recently, a TGF-β mimic molecule secreted by H. polygyrus has been identified to activate TGF-β signalling and inducing the proliferation of mouse and human Foxp3 + Treg cells [ 20 ]. Additionally, H. polygyrus secretions contain apyrases that degrade ATP, reducing inflammatory DAMP signals and inhibiting damage detection responses [ 21 ]. Despite recent efforts in studying the bioactive molecules from N. brasiliensis and H. polygyrus ESPs, to our knowledge, no comparative study analysing the different pathways activated by both helminths in their hosts have been performed. Thus, the main objective of this work is to identify the key signalling pathways activated by both helminths in murine macrophages. This study provides insights into the similarities and differences in host-pathogen interactions mediated by two of the most widely used helminths in immunomodulatory studies. 2 RESULTS 2.1 H. polygyrus ESPs exert a more potent anti-inflammatory effect on murine macrophages in vitro compared to N. brasiliensis ESPs following LPS stimulation. To comparatively assess the immunomodulatory properties of helminth-derived ESPs, we investigated the ability of H. polygyrus ( Hp ESP) and N. brasiliensis ( Nb ESP) to modulate the inflammatory response of murine macrophages following LPS stimulation. Cytometric analyses enabled the quantification of five cytokines in total. Standard curves were generated for each cytokine, yielding coefficients of determination (R²) ≥ 0.99. Upon interpolation of the signal obtained for each cytokine on the corresponding standard curve, the resulting concentrations are presented in Figure 1, which also highlights the statistically significant differences identified through subsequent statistical analyses. Hp ESP was able to reverse the previously LPS-induced stimulation of TNF-α (Figure 1A). Significant differences were observed in the groups treated with LPS in combination with the anti-inflammatory corticosteroid dexamethasone (LPS + Dx) ( p < 0.005) or Hp ESP ( p < 0.001), compared to LPS-treated control. However, no statistically significant changes were detected in the Nb ESP + LPS group (Figure 1A). These findings were further validated by ELISA, confirming the results obtained by CBA. As observed in the CBA assay, treatment with LPS + Dx significantly reduced TNF-α levels ( p < 0.01) compared to the LPS-stimulated group (Supplementary Figure 1). Similarly, a significant reduction ( p < 0.01) was observed in the Hp ESP + LPS group. No statistically significant differences were detected in the Nb ESP + LPS group. Interestingly, in the case of the monocyte chemoattractant protein-1 (MCP-1), a statistically significant reduction was observed for Hp ESP + LPS ( p < 0.001), Nb ESP + LPS ( p < 0.05), and LPS + Dx ( p < 0.05) (Figure 1B). Regarding the concentration levels of IL-6, significant differences were observed after treatment with either Dx, Hp ESP and Nb ESP compared to the LPS-only stimulated group (p < 0.01) (Figure 1C). In this case, expression of IL-6 was reduced by 68.3%, 79.8%, and 92.7% following treatment with LPS + Dx, Nb ESP + LPS, and Hp ESP + LPS, respectively (Figure 1C). On the contrary, no significant differences were observed across experimental groups for IL-12p70 (Figure 1D) and IL-10 (Figure 1E). The expression of IL-4 and TGF-b genes was measured by qPCR. Interestingly the expression in both cases was significantly increased in RAW264.7 macrophages after treatment with Nb ESP+LPS and Hp ESP+LPS, although Nb ESP seemed to have a significantly higher effect on the expression of TGF-b (Figure 2A, B). In the case of IL-4, treatment with Nb ESP+LPS and Hp ESP+LPS increased its expression 77.1 and 124.7 times, respectively, compared to the control (Figure 2A). Similarly, the expression of TGF-b was increased 2,612 and 1,791.5 times after treatment with Nb ESP+LPS and Hp ESP+LPS, respectively (Figure 2B). 2.2 H. polygyrus polarises macrophages mainly towards a M2 phenotype, while N. brasiliensis maintains a M1/M2 balance Genes associated with macrophage polarization towards classical and alternative activation were analysed by qPCR. The expression of Ym1 and ARG, two genes positively implicated in the polarization towards a M2 phenotype of macrophages, was analysed. Treatment with Nb ESP + LPS and Hp ESP + LPS (as well as with the positive control LPS + Dx) significantly induced the expression of Ym1 (Figure 2C). In the case of Nb ESP + LPS the expression was increased 317.1 times ( p < 0.0001), while 76.1 times ( p < 0.005) when cells were treated with Hp ESP + LPS (Figure 2C). Similarly, the expression of ARG was significantly induced 207.2 (p < 0.0001) and 65.3 times ( p < 0.0005) after treatment with Nb ESP + LPS and Hp ESP + LPS, respectively (Figure 2D). In the case of iNOS, an enzyme implicated in polarization towards a M1 phenotype, treatment of cells with Nb ESP + LPS and Hp ESP + LPS significantly induced the expression of this gene compared to controls (Figure 2E; p < 0.05). Interestingly, the expression of iNOS increased 180.5 times after treatment with Nb ESP + LPS, while only 20.1-fold when cells were treated with Hp ESP + LPS (Figure 2E). 2.3 Hp ESP and Nb ESP induce different proteomic expression profiles in content and amount of differentially expressed proteins For a more comprehensive analysis of the immunomodulatory mechanisms used by H. polygyrus and N. brasiliensis , RAW264.7 cells incubated with Hp ESP and Nb ESP were analyzed by LC-MS/MS. A total of 64,843 spectra were acquired and used to assign 49,121 unique peptides, leading to the identification of 4,348 proteins (4,281 protein groups). After removing N. brasiliensis , H. polygyrus and contaminant proteins, a total of 4,245 protein groups were kept for further analyses. The quantification analysis was performed using Spectronaut and only proteins with a q value 0.58 or <−0.58 (for upregulated and downregulated proteins, respectively) were taken into consideration for further analysis. After removing potential contaminants and parasite proteins, a total of 990 and 2,222 proteins had significantly dysregulated expression profiles in RAW264.7 cells after treatment with Hp ESP and Nb ESP, respectively (Supplementary Table 1). PCA was conducted to reduce data dimensionality and visualize sample clustering. The resulting PCA plot shows that the first two principal components, PC1 and PC2, explain 35.7% and 21.3% of the total variance, respectively. The samples cluster differently according to their treatment groups (Figure 3A), supporting that exposure to Nb ESP and Hp ESP potentially induces distinct proteomic profiles in RAW264.7 cells. This was further supported by the amount of differentially expressed proteins (DEPs) with respect to controls (Figure 3B, C). 2.4 N. brasiliensis and H. polygyrus ESPs activate different inflammatory-related pathways in murine macrophages To better understand the pathways involved in immune modulation by N. brasiliensis and H. polygyrus , proteins with the GO term “Immune system response” (GO:0002376) as a parent term were further analysed. A protein-protein interaction network was generated using StringDB data and clusters were manually curated (Figure 4A). The network revealed a central cluster of 109 proteins primarily associated with common immune responses to helminths, including activation of IL-5 (Th2 response) and other cytokine signalling pathways. In addition to this central node, several smaller clusters (comprising 2–4 proteins each, e.g., Progranulin, Prosaposin, Protein unc-93 homolog B1, Toll-like receptor, ATPase family, Evolutionarily conserved signalling intermediate in Toll pathway, subunits of the AP-3 complex, Vesicle-associated membrane protein 7 (VAMP-7), MAP kinase-activated protein and Rab-27A) were identified and found to be directly involved in anti-inflammatory responses. Interestingly, most of the proteins involved in these “anti-inflammatory pathways” were directly regulated by N. brasiliensis . However, two proteins related to lysosome metabolism (e.g. Progranulin, Prosaposin) and a protein involved in innate and adaptive immunity (Protein unc-93 homolog B1) were regulated by H. polygyrus . Other clusters were enriched in proteins involved in Th1-related biological processes such as nucleotide metabolism (e.g. lmpdh1 and ctps1), mRNA regulation (RNA demethylase, YTH domain-containing family protein 2 ) proteasome-mediated antigen processing (Proteasome activator complex subunits), and T-cell receptor signalling (several subunits of the translational initiation factor 2B (elF2B). To further investigate the functional relevance of these proteins, enrichment analyses were performed using STRINGdb. WikiPathways analysis (Figure 4B) revealed significant involvement in the “Microglia pathogen phagocytosis” ( e.g. Trem2) and “IL-5 signalling” ( e.g. Stat3) pathways, along with additional cytokine-related pathways including IL-3, IL-6, IL-2, and IL-7 ( e.g. NF-kappa-B). Reactome analysis (Figure 4C) highlighted broader immune-related pathways such as “Neutrophil degranulation,” “Signalling by the BCR,” “Toll-like receptor cascades,” and “C-type lectin receptor signalling” ( e.g. Rela, NF-kappa-B), supporting the role of these proteins in both innate and adaptive immune responses. 3 DISCUSSION Of all known species, only a dozen nematode helminths frequently parasitise humans, yet they infect approximately 2 billion people, nearly a third of the global population. This high prevalence highlights their ability to evade host defences and underscores their potential as immunomodulatory agents of the human immune system [22]. Evidence suggests that the release of ESPs, including soluble proteins, lipids, carbohydrates, and extracellular vesicles, during nematode helminth infections plays a key role in suppressing or regulating inflammatory responses and eosinophilia. This helps create a regulatory or suppressive immune environment that supports the parasite’s long-term survival [23,24]. Despite efforts to identify bioactive molecules, the targets of these molecules and the immunological pathways that helminths exploit to regulate immune responses remain largely unknown. Therefore, elucidating how these ESPs modulate host immune responses may enhance our understanding of host-parasite interactions and support the development of helminth-derived therapeutics for autoimmune and inflammatory diseases. Here, we used murine macrophages from the RAW 264.7 cell line as a model to investigate the immune response to ESPs from two popular rodent hookworm-like nematodes following stimulation with LPS. TNF-α levels significantly decreased following treatment with dexamethasone and Hp ESP, as validated by two techniques, while reduction of TNF-levels by Nb ESPs was not significant. ESPs released by N. brasiliensis L3 larvae have been shown to modulate TNF-α levels rat models of LPS-induced pulmonary inflammation [25,26]. Similarly, N. brasiliensis adult ESPs can significantly reduce TNF-α levels in bone marrow-derived macrophages (BMDM) from LPS-treated C57BL/6 mice [27]. While using isolated cells like the RAW 264.7 cell line has inherent limitations compared to studying immune responses in whole organisms, this model is widely used to investigate the immunomodulatory potential of helminth ESPs due to its key role in immune activation. Furthermore, it is well known that N. brasiliensis secretes different proteins throughout its life cycle, including during the L3 and adult stages [15], which can influence their immunomodulatory effects and, consequently, the in vitro responses. A decrease in other pro-inflammatory cytokines (IL-6 and MCP-1) was also observed after treatment with Hp ESP and Nb ESPs. Experiments performed in BMDMs showed that EVs present in the Hp ESP could suppress the secretion of IL-6 and TNF-a after LPS stimulation [28]. In this sense, previous studies showed that IL-6 -/- mice infected with nematodes had a higher parasite burden, indicating that a decrease in IL-6 expression at the macrophage level is considered beneficial for the parasite [29]. Production of anti-inflammatory and regulatory cytokines was also evaluated. IL-4 (Figure 2A) showed a significant increase in the LPS + Dx group, while ESP-treated groups exhibited only a slight, non-significant trend. In contrast, TGF-β (Figure 2B) levels were significantly elevated in both the LPS + Dx and Nb ESP + LPS groups. The differential cytokine responses observed between the Nb ESP + LPS and Hp ESP + LPS groups may reflect differences in the molecular composition of their respective ESPs. Regarding the results for IL-10 (Figure 1E), no significant differences were observed in any of the cases. This finding is consistent with previous studies that noted that the extracellular vesicles of N. brasiliensis ( Nb -EVs) promoted higher levels of IL-10 secretion compared to ESPs depleted of EVs [30]. Alternatively, it is possible that the release of this cytokine occurs at a later time point, and thus its measurement at 6 hours post-stimulation may not accurately capture its entire expression dynamics. Further studies are warranted to clarify this temporal pattern. In addition to pro- and anti-inflammatory cytokines, Th1/Th2 markers were also analyzed to further characterize the type of macrophage response. Treatment of RAW264.7 macrophages with H. polygyrus ESPs induced a polarization mainly towards Th2, while treatment with N. brasiliensis ESPs presented a Th1/Th2 balance. In the case of genes involved in M2 polarization, a higher expression of Ym1 and ARG1 levels was observed, correlating with a decrease in pro-inflammatory cytokine levels, such as IL-6, MCP-1, TNF-α, and iNOS. These results suggest that H. polygyrus ESPs promote alternative activation of macrophages by shifting the M1/M2 ratio. This coincides with studies by other authors who observed a Th2 polarization induced by this intestinal parasite [31]. In the case of N. brasiliensis , an increase in iNOS gene expression was observed, an enzyme involved in macrophage polarization towards the pro-inflammatory M1 profile, while no significant changes were found in TNF-α levels quantified by ELISA or in IL-12p70 gene expression. This may also be associated with a polarization towards inflammatory responses. However, it is important to note that an increase in Ym1 gene expression, typically expressed in M2 cells, was observed, and a decrease in IL-6 was noted. Previous studies have demonstrated alternative activation of macrophages in the lungs of mice following infection with N. brasiliensis , accompanied by increased expression of YM-1, ARG, and other Th2-associated markers. However, it has been speculated that such responses may only be sustained in the presence of functional T cells [32], which could partially explain why we did not observe a purely Th2-skewed response. These findings support the notion that N. brasiliensis , similar to other intestinal nematodes such as Trichinella spiralis , elicits a mixed Th1/Th2 immune response in macrophages [33,34]. These slight differences in M1/M2 polarization and cytokine production of RAW 264.7 cells after treatment with N. brasiliensis and H. polygyrus ESPs, were also reflected in the proteomic analysis. The PCA plot revealed distinct clustering of the three groups, with minimal overlap between N. brasiliensis -treated, H. polygyrus -treated, and control samples. This clear separation suggests that each helminth species induces a unique proteomic signature in macrophages, reflecting species-specific modulation of host immune responses. These findings support the hypothesis that helminth-derived ESPs differentially influence macrophage function, potentially through distinct molecular pathways, in agreement with the cytokine profiles and M1/M2 marker expression also observed in this study. Our study revealed a marked innate immune response by murine macrophages against both N. brasiliensis and H. polygyrus , as indicated by the central cluster in Figure 4A, which is consistent with previous studies on immune response against helminths [35,36]. Interestingly, while both nematodes exhibited a significant anti-inflammatory effect on murine macrophages, N. brasiliensis appeared to act more specifically on proteins involved in anti-inflammatory responses, whereas H. polygyrus affected only three of these proteins directly. This suggests that H. polygyrus and N. brasiliensis may employ different pathways to exert its immunomodulatory effects. For instance, it has been previously shown that H. polygyrus secretes a TGF-β mimic ( Hp- TGM) that activate mouse and human Foxp3 + Treg cells and can expand the host Treg population [20]. Furthermore, two additional TGF-β mimics from H. polygyrus have been shown to be functionally active [37]. However, to the best of our knowledge, no similar molecules have yet been identified in N. brasiliensis . Based on these findings, it is tempting to speculate that H. polygyrus may exert a more direct influence on cytokine release, particularly TGF-β, whereas N. brasiliensis appears to act more indirectly, affecting proteins involved in anti-inflammatory responses, innate immune signalling, neutrophil degranulation, and Toll-like receptor pathways. Furthermore, it is worth noting that N. brasiliensis ESPs have an effect on proteins involved in the “purine metabolism” pathway ( e.g ., CTP synthase 1, Inosine-5'-monophosphate dehydrogenase, AMP deaminase 3, Adenosine deaminase and Purine nucleoside phosphorylase). While this might indicate the activation of macrophage proliferation, some of these proteins have been shown to act as a positive regulator of T-cell coactivation and to play a crucial role in the proliferation of activated lymphocytes in humans [38,39]. In conclusion, the results obtained using murine macrophages suggest that ESPs from N. brasiliensis and H. polygyrus exert their anti-inflammatory effects through distinct mechanisms. While both nematodes stimulate the production of anti-inflammatory and regulatory cytokines, they appear to activate different pathways involved in immune modulation. This important divergence should be considered when interpreting results in the context of human helminth infections. 4. MATERIALS AND METHODS 4.1 Ethical statement All animals were maintained in the animal facility at the National Center for Microbiology in conformity with the Directive on the protection of animals used for scientific purposes (Directive 2010/63/UE, Decision 2020/569/UE and RD 1386/2018). All animal procedures were conducted in accordance with relevant institutional and national guidelines and regulations. The experimental protocols involving animal experiments were approved by the Ethical Animal Experimentation Committee of the Instituto de Salud Carlos III and Comunidad de Madrid (PROEX 124/19 and PROEX 119.3/20). All methods are reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org/) for the reporting of animal research. 4.2 Parasite material and isolation of ESPs Nippostrongylus brasiliensis was maintained in C57BL/6 mice ( M. musculus) purchased from Janvier Labs (Le Genest-Saint-Isle, France). A total of sixty (60) mice were infected subcutaneously with 500 infective larvae (L3) per mouse. Six days post-infection, the mice were euthanized using CO₂, and both the small and large intestines were harvested. Adult worms were manually extracted from the small intestine, washed three times with PBS and twice with RPMI 1640 at 37 °C, and then incubated in adult culture medium (RPMI 1640 supplemented with glucose [4.5 g/mL], glutamine [2 mM], and antibiotic/antimycotic [2x]) at 37 °C with 5% CO₂. The final worm concentration in the medium was adjusted to 500 worms per millilitre. The media obtained during the first 24 h after parasite culturing was discarded. Subsequently, excretory-secretory products (ESPs) were collected every 48 hours over a 12-day period and subjected to sequential differential centrifugation at 500, 2,000, and 4,000 g for 30 min each to remove eggs and parasite debris. Finally, media was concentrated using a 3 kDa spin concentrator (Merck Millipore, Billerica, MA, USA) and stored at 1.0 mg/ml in PBS at −80°C until used. H. polygyrus was maintained in NMRI mice ( M. musculus ) purchased from Charles River Laboratories (MA, USA). A total of 10 mice was inoculated orally with 150 ± 15 third-stage larvae (L3) suspended in 0.3–0.4 mL of distilled water. The larval concentration was standardized prior to inoculation using a McMaster counting chamber. Infective L3 larvae were obtained as described previously with minor modifications [40]. Briefly, faecal pellets were collected from day 14 onwards, softened, and homogenized in a mortar with distilled water. The homogenate was centrifuged at 300 rpm for 3 minutes three times, retaining the pellet each time. The pellet was finally resuspended in distilled water and filtered through a mesh strainer and gauze. The filtrate was then centrifuged at 600 rpm for 6 minutes, and the pellet was placed onto the central area of an inverted watch glass covered with pre-moistened filter paper. This assembly was set in a 15 cm diameter Petri dish, with the edges of the filter paper kept in contact with distilled water to maintain humidity. The cultures were incubated at room temperature, in darkness and under high humidity conditions, for 8 days. The liquid phase of the coproculture was collected and the larvae were allowed to sediment for approximately 45 minutes and finally stored in distilled water at 4°C until use. H. polygyrus adult worms were collected at day 14 days post infection and cultivated as described previously [41]. Briefly, adult worms were manually extracted and cultured in the same medium used for N. brasiliensis worms, as described above. 4.3 Cell culture conditions and treatments Murine macrophage RAW 264.7 cells (ATCC® TIB-71™) purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) were propagated in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine at 37°C in a humidified atmosphere with 5% CO₂. The culture medium, pre-warmed to 37°C, was refreshed every 48 hours, with a total volume of 10 mL per T75 flask. RAW 264.7 cells were cultured in 6-well plates at the same density and divided into the following eight groups: i) Control group: Cells cultured in supplemented DMEM only; ii) LPS-treated group: Cells stimulated with 5 ng/mL of LPS; iii) Dexamethasone control group: Cells treated with 1 μM dexamethasone; iv) Dexamethasone + LPS intervention group: Cells treated with 1 μM dexamethasone and stimulated with 5 ng/mL of LPS; v) Nb ESP-treated group: Cells treated with 10 μg/mL of Nb ESP; vi) Nb ESP + LPS intervention group: Cells treated with 10 μg/mL Nb ESP and stimulated with 5 ng/mL of LPS; vii) Hp ESP-treated group: Cells treated with 10 μg/mL of Hp ESP; and viii) Hp ESP + LPS intervention group: Cells treated with 10 μg/mL Hp ESP and stimulated with 5 ng/mL of LPS. At time 0, groups were stimulated with LPS. One hour later, dexamethasone, Nb ESP or Hp ESP were added to the wells. Supernatants were collected 6 hours post-LPS stimulation, and cells from each well were harvested by adding cold PBS and centrifuging at 1,000 rpm for 5 minutes. Both supernatants and cell pellets were stored at −20°C until further analysis. 4.4 Cytokine quantification by cytometric bead array and ELISA The cytokines present in the cell culture supernatants were quantified using the BD Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Briefly, the beads coated with six specific capture antibodies were mixed for each assay. Subsequently, 50 μL of the mixed capture beads, 50 μL of the sample or standard dilution, and 50 μL of the PE detection reagent were added to each test tube. The tubes were, then, incubated for 2 hours at room temperature (RT) in the dark. After incubation, the samples were washed with 1 mL of wash buffer and centrifuged at 200 g for 5 minutes. The resulting microbead pellet was resuspended in 300 μL of buffer following the removal of the supernatant. Samples were finally analyzed using the BD Accuri C6 Plus flow cytometer (BD Biosciences) and data were processed with FlowJo software (BD Biosciences). The TNF- a cytokine levels were further confirmed using the Mouse TNF alpha Uncoated ELISA kit (Invitrogen) following the manufacturer’s instructions. Briefly, plates were coated with 100 μL/well of capture antibody diluted 1:250 in coating buffer overnight at 4°C. The following day, plates were washed x3 in washing buffer and blocked with 200 μL of ELISA/ELISPOT Diluent for 1 h at RT. Plates were washed again and incubated with 100 μL of standards or samples for 2 h at RT. After washing again x3 with washing buffer, 100 μL/well of Detection Antibody diluted 1:250 was added and plates were incubated for 1 h at room temperature. A wash was performed as described above and finally, 100 μL/well of diluted Streptavidin-HRP was added to each well and plates were developed after a final wash using 100 μl of 3,3’,5,5’-tetramethylbenzidine (TMB) for 15 min, followed by 100 μl of stop solution. Plates were read at a wavelength of 450 nm on a Heales mod. MB580 (Shenzhen Huisong Technology Development Co., Ltd. Shenzhen , China) microplate reader. 4.5 RNA extraction, cDNA synthesis and qPCR Total RNA from cells treated with excretory-secretory products (ESPs) was extracted using the Quick-RNA™ Miniprep Kit (Zymo), following the manufacturer’s instructions. The extracted RNA was quantified using a NanoDrop One/One spectrophotometer (ThermoFisher) by measuring absorbance at 260 nm. The purity of the RNA was assessed through absorbance ratios at 260/280 and 260/230 nm. Complementary DNA (cDNA) synthesis was performed using the SuperScript III kit (Thermo Fisher Scientific). The reaction was initiated by mixing 3 µL of RNA from each sample, 1 µL of oligo(dT) (50 µM), 1 µL of dNTP Mix (10 mM), and distilled water to a final volume of 13 µL. This mixture was incubated in a GeneAmp PCR System 2700 (Applied Biosystems) thermocycler for 5 minutes at 65°C and then for 1 minute at 4°C. Subsequently, 4 µL of 5X First-Strand Buffer, 1 µL of DTT (0.1 M), and 1 µL of RNaseOUT (RNase inhibitor, 40 U/µL) were added to reach a final reaction volume of 20 µL. The reaction was incubated for 60 minutes at 50°C, followed by 15 minutes at 70°C to inactivate the enzyme. The synthesized cDNA was quantified using the NanoDrop One/One spectrophotometer (Thermo Fisher Scientific) by measuring absorbance at 260 nm, with purity assessed via 260/280 and 260/230 ratios. The cDNA was stored at −20°C until use. cDNA amplification was performed using quantitative real-time PCR (qPCR) with the SYBR-Green system. The actin gene was used as an endogenous control, and the expression of IL-4 , TGFβ , iNOS , Ym1 , and ARG genes was analyzed. Amplifications were conducted in the presence of SYBR-Green PCR Master Mix (Applied Biosystems) using 2.5 µg of template cDNA and 1 µL of each primer (forward and reverse; Table 1) in a final reaction volume of 20 µL. qPCR reactions were performed on a Rotor-Gene Q (Qiagen) using the following conditions: initial denaturation at 95°C for 10 seconds (1 cycle) followed by 40 cycles of denaturation (95°C for 10 sec), annealing (55°C or 65°C for 15 sec) and extension (72°C for 20 sec). Gene expression was normalised to the housekeeping gene as described before [42,43] and relative expression levels were calculated using the 2 − ΔΔ Ct method using Control (untreated cells) as a reference group [44]. 4.6 Cell processing and protein extraction Three biological replicates of cells treated with Nb ESP or Hp ESP were lysed using 100 µL of RIPA buffer (Invitrogen) following the manufacturer’s instructions. Protein extracts (20 µg in 100 µL of RIPA) were reduced by adding 10 µL of 100 mM tris(2-carboxyethyl)phosphine (TCEP) and incubated at 37°C with 600 rpm for 45 minutes. Alkylation was performed by adding 11 µL of 400 mM chloroacetamide, followed by a 30-minute incubation at RT in the dark with 600 rpm shaking. Subsequently, 100 µL of SeraMag magnetic beads mix (GE Healthcare, Chicago, IL, USA; 50% hydrophilic beads and 50% hydrophobic beads) and 200 µL of acetonitrile (ACN) were added to facilitate protein binding to the beads. The mixture was incubated at RT with 600 rpm for 35 minutes. Supernatants were removed, and magnetic beads were washed twice with 70% ethanol and once with ACN. Proteins were then digested overnight at 37°C with 0.5 µg of porcine trypsin (Thermo Fisher Scientific) in 100 µL of 200 mM HEPES (pH 8.0) with 600 rpm. The following day, samples were sonicated twice, and the resulting supernatants were dried under vacuum in preparation for desalting. The Pierce C18 Spin Tips (Thermo Fisher Scientific) were used for peptide purification. The dried peptides were resuspended in 50 µL of 0.1% trifluoroacetic acid (TFA) and loaded onto the C18 Spin Tip, followed by centrifugation at 1,000–2,000 g to allow peptides to bind to the hydrophobic matrix. To remove contaminants, 20 µL of 0.1% TFA was added for washing. Peptides were eluted using 20 µL of 0.1% TFA in 80% ACN, with centrifugation at 1,000 g for 1 minute. The eluted peptides were dried under vacuum and stored at -80°C until analysis by LC-MS/MS. 4.7 Mass spectrometry and protein identification For LC-MS/MS, peptides were analyzed in an Orbitrap Astral mass spectrometer coupled to a Vanquish Neo UHPLC System (Thermo Fisher Scientific). Peptide samples were loaded into the precolumn PepMap Trap Catridge 5 µm, 300 µm x 5 mm (Thermo Fisher Scientific) and eluted in an Easy-Spray PepMap RSLC C18 2 µm, 50 µm x 15 cm (Thermo Fisher Scientific) heated at 50°C. The mobile phase flow rate was 300 nL/min and 0.1% formic acid (FA) in H 2 O mq and 0.1% FA in 80% acetonitrile (ACN) were used as elution buffers A and B, respectively. The 15 min elution gradient was: 4%-10% buffer B for 2 min, 10%-40% buffer B for 11 min, 40%-99% buffer B for 0.5 min, and 99% buffer B for 1.5 min. Prior to injection, samples were re-suspended in 10 µL of buffer A, and 1 µL of each sample were injected, and analyzed in data independent acquisition (DIA) mode. For ionization, 1900 V of liquid junction voltage and 280°C capillary temperature was used. The full scan method employed a m/z 380-980 mass selection, an Orbitrap resolution of 240000 (at m/z 200), an automatic gain control (AGC) value of 500%, and maximum injection time (IT) 5 ms. The MS/MS was performed with the Astral mass analyzer, using an AGC of 500%, an IT of 3 ms, and a normalized collision energy (NCE) of 25 for fragmentation of precursors. The scan range was set from 380 to 980 m/z, with an isolation window of 2 m/z, and window placement optimization was enabled. Thus, a total of 299 windows were analyzed in each cycle. A directDIA search was performed using Spectronaut v19.4 against a concatenated target/decoy database consisting of the mouse proteome (17,216 reviewed proteins from UP000000589 accessed in March 2024) and common contaminants (246 proteins from cRAP database accessed in October 2024) as well as proteomes from N. brasiliensis (PRJNA994163; 29.473 proteins) and H. polygyrus (PRJEB15396; 25.212 proteins) downloaded from WormBaseParasite in November 2024 (WBPS19). Enzyme was set to Trypsin/P, fixed modifications to Carbamidomethyl (C) and variable modifications to Acetyl (Protein N-term) and Oxidation (M). Precursor qvalue and PEP cutoffs were set to 0,01 and 0,2, respectively, while protein qvalue cutoff was set to 0,01. Peptide and protein FDR were set to 0,01. Single hit proteins were excluded from the analysis. Mass spectrometry data along with the identification results and search parameters have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository [45] with the dataset identifier PXD062974. 4.8 Gene Ontology and PPI analyses The GO information for each protein was retrieved from Uniprot database (https://www.uniprot.org/). Furthermore, an enrichment analysis was performed using STRINGdb using the default settings except for the minimum required interaction score, which was set to “high confidence” (0.7). The interactome graph involving “immune system response” proteins was depicted using the python NetworkX library (https://networkx.org/). Reactome and Wikipathways terms were grouped by similarity ≥ 0.8 and sorted by signal. 4.9 Statistical and bioinformatic analyses For the CBA, ELISA and qPCR analyses, statistical significance was calculated using Student’s t-test by comparing the LPS-treated group to each treatment or control. **** = p < 0.0001; *** = p < 0.001; ** = p < 0.01; * = p 0.05. For the proteomics data, a DIA quantitative analysis was performed following protein identification using Spectronaut® v19.4. Quantitative results were normalised by cross-run normalization of medians. An unpaired t-test was performed to compare the proteins with a significant regulation between two groups after log2 transformation. Fold-changes were considered as significant when ≥0.58 and adjusted p -value ≤ 0.05. Volcano and Principal Component Analysis (PCA) plots were performed using the ggplot2 R package using customised scripts. All scripts are available and can be downloaded from https://github.com/JavierSotillo/Scripts. Declarations Author contributions Covadonga Varea: Investigation . Antonio J. Martín-Galiano: Data Curation, Writing . Sara Vázquez-Ávila: Investigation, Methodology . Ana Montero-Calle: Investigation, Methodology, Software . Julia Cesteros: Investigation. María Jesús Perteguer: Investigation . Alberto Peláez-García: Investigation, Methodology, Software. Rodrigo Barderas: Investigation, Methodology, Software . Juan José García: Supervision, resources, Funding acquisition, Writing . Javier Sotillo: Supervision, resources, Funding acquisition, Writing . Data availability Mass spectrometry data along with the identification results and search parameters have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD062974. Reviewer can access the dataset by logging in to the PRIDE website using the following account details: Username: [email protected] Password: Tm9dBo09W50t Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. Funding This research was supported by grant PID2022-137661OB-I00 (MPY 341/23) funded by MCIN/AEI/10.13039/501100011033/, by ISCIII grant MPY337/24 and by FEDER A way to make Europe. References Hotez, P. J. et al. Helminth infections: the great neglected tropical diseases. J. Clin. Invest. 118 , 1311–1321 (2008). Pullan, R. L., Smith, J. L., Jasrasaria, R. & Brooker, S. J. Global numbers of infection and disease burden of soil transmitted helminth infections in 2010. Parasit. Vectors 7 , 37 (2014). WHO. Soil-transmitted helminth infections (Fact sheet). https://www.who.int/news-room/fact-sheets/detail/soil-transmitted-helminth-infections. 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Anti-Inflammatory Responses Produced with Nippostrongylus brasiliensis -Derived Uridine via the Mitochondrial ATP-Sensitive Potassium Channel and Its Anti-Atherosclerosis Effect in an Apolipoprotein E Gene Knockout Mouse Model. Biomolecules 14 , 672 (2024). Coakley, G. et al. Extracellular Vesicles from a Helminth Parasite Suppress Macrophage Activation and Constitute an Effective Vaccine for Protective Immunity. Cell Reports 19 , 1545–1557 (2017). Muhsin, M. et al. IL-6 is required for protective immune responses against early filarial infection. Int. J. Parasitol. 48 , 925–935 (2018). Eichenberger, R. M. et al. Hookworm Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice. Front. Immunol. 9 , 850 (2018). Su, C. W. et al. Helminth infection protects against high fat diet-induced obesity via induction of alternatively activated macrophages. Sci. Rep. 8 , 4607 (2018). Reece, J. J., Siracusa, M. C. & Scott, A. L. Innate Immune Responses to Lung-Stage Helminth Infection Induce Alternatively Activated Alveolar Macrophages. Infect. Immun. 74 , 4970–4981 (2006). Ilic, N. et al. Trichinella spiralis antigens prime mixed Th1/Th2 response but do not induce de novo generation of Foxp3 + T cells in vitro . Parasite Immunol. 33 , 572–582 (2011). Song, Y. et al. Regulation of host immune cells and cytokine production induced by Trichinella spiralis infection. Parasite 26 , 74 (2019). Lechner, A., Bohnacker, S. & Esser-von Bieren, J. Macrophage regulation & function in helminth infection. Semin Immunol. 53 , 101526 (2021). Kreider, T., Anthony, R. M., Urban, J. F. & Gause, W. C. Alternatively activated macrophages in helminth infections. Curr. Opin. Immunol. 19 , 448–453 (2007). Smyth, D. J. et al. TGF-β mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus. Int. J. Parasitol. 48 , 379–385 (2018). Martinez-Navio, J. M. et al. Adenosine deaminase potentiates the generation of effector, memory, and regulatory CD4+ T cells. J. Leukoc. Biol. 89 , 127–136 (2010). Han, G.-S. et al. Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A. J. Biol. Chem. 280 , 38328–38336 (2005). Burren, C. H. A method for obtaining large numbers of clean infective larvae of Nematospiroides dubius . Z. Parasitenkd. 62 , 111–112 (1980). Johnston, C. J. C. et al. Cultivation of Heligmosomoides polygyrus : An Immunomodulatory Nematode Parasite and its Secreted Products. JoVE 52412 (2015) doi:10.3791/52412. Chung, J., Choi, M. J., Jeong, S. Y., Oh, J. S. & Kim, H. K. Chemokines Gene Expression of RAW 264.7 Cells by Actinobacillus actinomycetemcomitans Lipopolysaccharide Using Microarray and RT-PCR Analysis. Mol. Cells 27 , 257–261 (2009). Zhou, C. et al. Anti-inflammatory Mechanism of Action of Benzoylmesaconine in Lipopolysaccharide-Stimulated RAW264.7 Cells. eCAM 2022 , 1–12 (2022). Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 , 402–408 (2001). Vizcaíno, J. A. et al. The Proteomics Identifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. 41 , D1063–D1069 (2012). Table Table 1: Oligonucleotides Used for Gene Expression Analysis via SYBR-Green qPCR GENE PRIMER FORWARD PRIMER REVERSE ACT 5´-CGGTTCCGATGCCCTGAGGCTCTT-3´ 5´-CGTCACACTTCATGATGGAATTGA-3´ IL-4 5´-CCCCCAGCTAGTTGTCATCC-3´ 5´-AGGACGTTTGGCACATCCAT-3´ iNOS 5´-CAGCTGGGCTGTACAAACCTT-3´ 5´-CATTGGAAGTGAAGCGTTTCG-3´ TGF β 5´-TTGCTTCAGCTCCACAGAGA-3´ 5´-TGGTTGTAGAGGGCAAGGAC-3´ Ym1 5´-TTTCTCCAGTGTAGCCATCCTT-3´ 5´-TCTGGGTACAAGATCCCTGAA-3´ ARG 5´-TTTTTCCAGCAGACCAGCTT-3´ 5´-AGAGATTATCGGAGCGCCTT-3´ Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.tif Supplementary Figure 1. TNF-a levels in the supernatant of RAW264.7 murine macrophages measured by ELISA. RAW264.7 cells were stimulated with LPS and treated with excretory/secretory products from Nippostrongylus brasiliensis (LPS + Nb ESP) or Heligmosomoides polygyrus (LPS + Hp ESP) or with dexamethasone as positive control (LPS + Dx). SupplementaryTable1.xlsx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 06 Apr, 2026 Reviews received at journal 03 Apr, 2026 Reviews received at journal 02 Apr, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers agreed at journal 13 Mar, 2026 Reviewers invited by journal 11 Nov, 2025 Editor assigned by journal 11 Nov, 2025 Submission checks completed at journal 06 Nov, 2025 First submitted to journal 06 Nov, 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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05:41:28","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":123272,"visible":true,"origin":"","legend":"","description":"","filename":"ee9f129e34bd4a9781a5021eb5fb77361structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8017434/v1/aaa8ac8a924c05847879c071.xml"},{"id":96436386,"identity":"d350c542-afa5-42aa-b0a7-41582c0c7820","added_by":"auto","created_at":"2025-11-21 05:41:29","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137426,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8017434/v1/639d3abc90c1b042c23fba25.html"},{"id":96436361,"identity":"9de281fb-f086-40fb-a3cb-fac27c9bcf61","added_by":"auto","created_at":"2025-11-21 05:41:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1003193,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine levels in the supernatant of RAW264.7 murine macrophages measured by cytometric bead array (CBA). RAW264.7 cells were stimulated with LPS and treated with excretory/secretory products from \u003cem\u003eNippostrongylus brasiliensis\u003c/em\u003e (LPS + \u003cem\u003eNb\u003c/em\u003eESP) or \u003cem\u003eHeligmosomoides polygyrus\u003c/em\u003e (LPS + \u003cem\u003eHp\u003c/em\u003eESP) or with dexamethasone as positive control (LPS + Dx). The levels of TNF-a (A), MCP-1 (B), IL-6 (C), IL-12p (D) and IL-10 (E) were measure with the CBA Mouse Inflammation Kit.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8017434/v1/7066c17c0932b5d5e817b880.png"},{"id":96436362,"identity":"5d32ccde-fc2c-47aa-a896-99682655e2ca","added_by":"auto","created_at":"2025-11-21 05:41:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":947543,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression measured by qPCR in RAW264.7 murine macrophages following stimulation with LPS and treatment with excretory/secretory products from \u003cem\u003eNippostrongylus brasiliensis\u003c/em\u003e (LPS + \u003cem\u003eNb\u003c/em\u003eESP) or \u003cem\u003eHeligmosomoides polygyrus\u003c/em\u003e (LPS + \u003cem\u003eHp\u003c/em\u003eESP) or with dexamethasone as positive control (LPS + Dx). Gene expression of IL-4 (A), TGF-b (B), YM-1 (C), Arginase (D) and iNOS (E) were measure by qPCR and analysed following the 2-\u003csup\u003eΔΔCt\u003c/sup\u003e method.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8017434/v1/a17cec87d0ed8f219594f217.png"},{"id":96455523,"identity":"848e1afb-14c1-41fa-982b-97d04ae18f18","added_by":"auto","created_at":"2025-11-21 10:04:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3051268,"visible":true,"origin":"","legend":"\u003cp\u003eProteomics analysis of RAW264.7 cells after incubation with \u003cem\u003eNippostrongylus brasiliensis\u003c/em\u003e (\u003cem\u003eNb\u003c/em\u003eESP) or \u003cem\u003eHeligmosomoides polygyrus \u003c/em\u003e(\u003cem\u003eHp\u003c/em\u003eESP) excretory/secretory products. (A) Principal component analysis of each of the three replicates of RAW264.7 cells following treatment with \u003cem\u003eNb\u003c/em\u003eESP and \u003cem\u003eHp\u003c/em\u003eESP. Volcano plot representing all proteins identified in RAW264.7 cells after incubation with \u003cem\u003eHp\u003c/em\u003eESP (B) and \u003cem\u003eNb\u003c/em\u003eESP (C). Purple and cyan nodes represent upregulated and downregulated proteins, respectively.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8017434/v1/b829169b61bbab65213b7360.png"},{"id":96455128,"identity":"3fe3d409-a559-4570-afce-5efad6bd737b","added_by":"auto","created_at":"2025-11-21 10:03:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4378887,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of immune-related proteins regulated by \u003cem\u003eN. brasiliensis\u003c/em\u003e and \u003cem\u003eH. polygyrus\u003c/em\u003e. (A) Protein-protein interaction network showing clusters grouped by biological processes. WikiPathways (B) and Reactome (C) enrichment analysis of proteins annotated under the GO term “Immune system”.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8017434/v1/17f6f5d69092bb8c24a5ed2f.png"},{"id":97135356,"identity":"5be716de-907c-4de0-95e4-4521f40b2c07","added_by":"auto","created_at":"2025-12-01 09:37:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10065092,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8017434/v1/13556709-aa88-4b7b-8892-d6bde0579033.pdf"},{"id":96436369,"identity":"e870a49d-c2c4-475c-8e73-530ffb341c08","added_by":"auto","created_at":"2025-11-21 05:41:28","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":505756,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e. TNF-a levels in the supernatant of RAW264.7 murine macrophages measured by ELISA. RAW264.7 cells were stimulated with LPS and treated with excretory/secretory products from \u003cem\u003eNippostrongylus brasiliensis\u003c/em\u003e (LPS + \u003cem\u003eNb\u003c/em\u003eESP) or \u003cem\u003eHeligmosomoides polygyrus\u003c/em\u003e (LPS + \u003cem\u003eHp\u003c/em\u003eESP) or with dexamethasone as positive control (LPS + Dx).\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8017434/v1/0583f0a667c8686416464e58.tif"},{"id":96455494,"identity":"24bb394c-84fa-4494-81c2-418400cc68db","added_by":"auto","created_at":"2025-11-21 10:04:10","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1994449,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8017434/v1/6cb1d8d81baa8789a8d7fa06.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nippostrongylus brasiliensis and Heligmosomoides polygyrus activate different immunomodulatory pathways in murine macrophages in vitro","fulltext":[{"header":"1 INTRODUCTION","content":"\u003cp\u003eInfections caused by parasitic helminths have a high prevalence worldwide, in particular in non-industrialised countries from tropical and subtropical regions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to the WHO, in 2018, 25% of the global population was infected with a helminth species, with nematodes being the most predominant group [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. More importantly, these infections are associated with significant morbidity and mortality, particularly in children [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Species from the genera \u003cem\u003eAscaris\u003c/em\u003e, \u003cem\u003eAncylostoma\u003c/em\u003e, \u003cem\u003eNecator\u003c/em\u003e, \u003cem\u003eStrongyloides\u003c/em\u003e, and \u003cem\u003eTrichuris\u003c/em\u003e, all known to parasitize their hosts through soil contact, infect approximately one billion people worldwide, significantly contributing to disability-adjusted life years (DALYs) particularly in impoverished and resource-limited settings. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite the negative effects associated with parasites, there is evidence that their complete eradication can have adverse consequences for human health. Indeed, it is well known that limited exposure to pathogens leads to an underdeveloped regulatory immune system, ultimately increasing the prevalence of immune-related disorders [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Nematodes, aiming to survive within their host, must evade or modulate the immune response in their favour, strongly down-regulating host inflammatory responses [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This immunomodulatory ability affects the immune response in various ways, such as inhibiting protective mechanisms like mast cell degranulation, impairing the induction of immune responses, and promoting regulatory T cell induction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These mechanisms are primarily mediated by bioactive molecules excreted and/or secreted by the parasites known as excretory/secretory products (ESPs) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHookworms have been described as one of the groups of nematodes with the highest immunomodulatory potential [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and some of their secreted molecules ameliorate the symptoms of inflammatory diseases such as asthma and inflammatory bowel disease [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Due to the difficulty in reproducing human hookworms\u0026rsquo; life cycle in laboratory settings, different hookworm-like rodent nematodes such as \u003cem\u003eNippostrongylus brasiliensis\u003c/em\u003e and \u003cem\u003eHeligmosomoides polygyrus\u003c/em\u003e have been widely used for the study of hookworm biological features. Indeed, recent proteomic and transcriptomic studies have shown evidence of the high similarity between these rodent models and their human counterparts, making them highly suitable for the study of the immunobiology of gastrointestinal nematode infections [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eN. brasiliensis\u003c/em\u003e belongs to the phylum Nematoda and the superfamily Trichostrongyloidea, with rats as its natural hosts, though it can also infect other rodent species like mice. Primary infection elicits pulmonary protection against reinfection, which depends on CD4\u003csup\u003e+\u003c/sup\u003e T cells and alternatively activated macrophages (M2). These M2 macrophages may also contribute to tissue repair following the lung phase of the parasite\u0026rsquo;s life cycle [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In mice, infection with this parasite triggers a dominant Th2 response characterized by increased IL-4, IL-5, and IL-13 cytokines and elevated IgE antibody production [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Numerous studies highlight \u003cem\u003eN. brasiliensis\u003c/em\u003e as a potent immunomodulator, with ESPs playing a significant role in this process [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among these ESPs, proteins belonging to the SCP/TAPS family stand out as key mediators in evasion of the host's immune response [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe gastrointestinal nematode \u003cem\u003eHeligmosomoides polygyrus\u003c/em\u003e belongs to the phylum Nematoda, superfamily Trichostrongyloidea, and family \u003cem\u003eHeligmosomidae\u003c/em\u003e, with wild house mice (\u003cem\u003eMus musculus\u003c/em\u003e) as its natural host. Despite the similarities at a proteomic and transcriptomic level with human hookworms, \u003cem\u003eH. polygyrus\u003c/em\u003e has a slightly different life cycle. In the case of \u003cem\u003eH. polygyrus\u003c/em\u003e, definitive hosts acquire infection by oral ingestion of infective larvae, which then invade the duodenal mucosa, penetrating the muscular layer to reside beneath the serosal membrane, and returning to the intestinal lumen as adult worms about eight days later [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Due to its manageable life cycle in the laboratory, \u003cem\u003eH. polygyrus\u003c/em\u003e has been widely used to study immunomodulation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Infection is accompanied by regulatory T cell populations, dendritic cells, macrophages, B cell hyperstimulation, and localized changes in the intestinal environment [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cem\u003eH. polygyrus\u003c/em\u003e ESPs include \u003cem\u003eHp\u003c/em\u003eARI (\u003cem\u003eH. polygyrus\u003c/em\u003e Alarmin Release Inhibitor), which suppresses the release of alarmins like IL-25 and IL-33 produced by epithelial cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. More recently, a TGF-β mimic molecule secreted by \u003cem\u003eH. polygyrus\u003c/em\u003e has been identified to activate TGF-β signalling and inducing the proliferation of mouse and human Foxp3\u003csup\u003e+\u003c/sup\u003e Treg cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, \u003cem\u003eH. polygyrus\u003c/em\u003e secretions contain apyrases that degrade ATP, reducing inflammatory DAMP signals and inhibiting damage detection responses [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite recent efforts in studying the bioactive molecules from \u003cem\u003eN. brasiliensis\u003c/em\u003e and \u003cem\u003eH. polygyrus\u003c/em\u003e ESPs, to our knowledge, no comparative study analysing the different pathways activated by both helminths in their hosts have been performed. Thus, the main objective of this work is to identify the key signalling pathways activated by both helminths in murine macrophages. This study provides insights into the similarities and differences in host-pathogen interactions mediated by two of the most widely used helminths in immunomodulatory studies.\u003c/p\u003e"},{"header":"2 RESULTS","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.1 H. polygyrus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;ESPs exert a more potent anti-inflammatory effect on murine macrophages \u003cem\u003ein vitro\u003c/em\u003e compared to \u003ci\u003eN. brasiliensis\u003c/i\u003e ESPs following LPS stimulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo comparatively assess the immunomodulatory properties of helminth-derived ESPs, we investigated the ability of \u003cem\u003eH. polygyrus\u003c/em\u003e (\u003cem\u003eHp\u003c/em\u003eESP) and \u003cem\u003eN. brasiliensis\u003c/em\u003e (\u003cem\u003eNb\u003c/em\u003eESP) to modulate the inflammatory response of murine macrophages following LPS stimulation. Cytometric analyses enabled the quantification of five cytokines in total. Standard curves were generated for each cytokine, yielding coefficients of determination (R\u0026sup2;) \u0026ge; 0.99. Upon interpolation of the signal obtained for each cytokine on the corresponding standard curve, the resulting concentrations are presented in Figure 1, which also highlights the statistically significant differences identified through subsequent statistical analyses.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHp\u003c/em\u003eESP was able to reverse the previously LPS-induced stimulation of TNF-\u0026alpha; (Figure 1A). Significant differences were observed in the groups treated with LPS in combination with the anti-inflammatory corticosteroid dexamethasone (LPS + Dx) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005) or \u003cem\u003eHp\u003c/em\u003eESP (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), compared to LPS-treated control. However, no statistically significant changes were detected in the \u003cem\u003eNb\u003c/em\u003eESP + LPS group (Figure 1A). These findings were further validated by ELISA, confirming the results obtained by CBA. As observed in the CBA assay, treatment with LPS + Dx significantly reduced TNF-\u0026alpha; levels (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) compared to the LPS-stimulated group (Supplementary Figure 1). Similarly, a significant reduction (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) was observed in the \u003cem\u003eHp\u003c/em\u003eESP + LPS group. No statistically significant differences were detected in the \u003cem\u003eNb\u003c/em\u003eESP + LPS group.\u003c/p\u003e\n\u003cp\u003eInterestingly, in the case of the monocyte chemoattractant protein-1 (MCP-1), a statistically significant reduction was observed for\u0026nbsp;\u003cem\u003eHp\u003c/em\u003eESP + LPS (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001),\u0026nbsp;\u003cem\u003eNb\u003c/em\u003eESP + LPS\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), and\u0026nbsp;LPS + Dx\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05)\u0026nbsp;(Figure 1B). Regarding the concentration levels of IL-6, significant differences were observed after treatment with either Dx, \u003cem\u003eHp\u003c/em\u003eESP and\u0026nbsp;\u003cem\u003eNb\u003c/em\u003eESP\u0026nbsp;compared to the LPS-only stimulated group (p \u0026lt; 0.01)\u0026nbsp;(Figure 1C). In this case,\u0026nbsp;expression of\u0026nbsp;IL-6\u0026nbsp;was reduced by 68.3%, 79.8%, and 92.7% following treatment with LPS + Dx, \u003cem\u003eNb\u003c/em\u003eESP + LPS, and \u003cem\u003eHp\u003c/em\u003eESP + LPS, respectively\u0026nbsp;(Figure 1C).\u0026nbsp;On the\u0026nbsp;contrary, no significant differences were observed across experimental groups for IL-12p70\u0026nbsp;(Figure 1D)\u0026nbsp;and IL-10\u0026nbsp;(Figure 1E).\u003c/p\u003e\n\u003cp\u003eThe expression of IL-4 and TGF-b genes was measured by qPCR. Interestingly the expression in both cases was significantly increased in RAW264.7 macrophages after treatment with \u0026nbsp; \u003cem\u003eNb\u003c/em\u003eESP+LPS and \u003cem\u003eHp\u003c/em\u003eESP+LPS, although \u003cem\u003eNb\u003c/em\u003eESP seemed to have a significantly higher effect on the expression of TGF-b (Figure 2A, B). In the case of IL-4, treatment with \u003cem\u003eNb\u003c/em\u003eESP+LPS and \u003cem\u003eHp\u003c/em\u003eESP+LPS increased its expression 77.1 and 124.7 times, respectively, compared to the control (Figure 2A). Similarly, the expression of TGF-b was increased 2,612 and 1,791.5 times after treatment with \u003cem\u003eNb\u003c/em\u003eESP+LPS and \u003cem\u003eHp\u003c/em\u003eESP+LPS, respectively (Figure 2B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.2 H. polygyrus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003epolarises macrophages mainly towards a M2 phenotype, while \u003cem\u003eN. brasiliensis\u003c/em\u003e maintains a M1/M2 balance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenes associated with macrophage polarization towards classical and alternative activation were analysed by qPCR. The expression of Ym1 and ARG, two genes positively implicated in the polarization towards a M2 phenotype of macrophages, was analysed. Treatment with \u0026nbsp;\u003cem\u003eNb\u003c/em\u003eESP + LPS and \u003cem\u003eHp\u003c/em\u003eESP + LPS (as well as with the positive control LPS + Dx) significantly induced the expression of Ym1 (Figure 2C). In the case of \u003cem\u003eNb\u003c/em\u003eESP + LPS the expression was increased 317.1 times (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001), while 76.1 times (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005) when cells were treated with \u003cem\u003eHp\u003c/em\u003eESP + LPS (Figure 2C). Similarly, the expression of ARG was significantly induced 207.2 (p \u0026lt; 0.0001) and 65.3 times (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0005) after treatment with \u003cem\u003eNb\u003c/em\u003eESP + LPS and \u003cem\u003eHp\u003c/em\u003eESP + LPS, respectively (Figure 2D).\u003c/p\u003e\n\u003cp\u003eIn the case of iNOS, an enzyme implicated in polarization towards a M1 phenotype, treatment of cells with \u003cem\u003eNb\u003c/em\u003eESP + LPS and \u003cem\u003eHp\u003c/em\u003eESP + LPS significantly induced the expression of this gene compared to controls (Figure 2E; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Interestingly, the expression of iNOS increased 180.5 times after treatment with \u003cem\u003eNb\u003c/em\u003eESP + LPS, while only 20.1-fold when cells were treated with \u003cem\u003eHp\u003c/em\u003eESP + LPS (Figure 2E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.3 Hp\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eESP and \u003cem\u003eNb\u003c/em\u003eESP induce different proteomic expression profiles in content and amount of differentially expressed proteins\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor a more comprehensive analysis of the immunomodulatory mechanisms used by \u003cem\u003eH. polygyrus\u003c/em\u003e and \u003cem\u003eN. brasiliensis\u003c/em\u003e, RAW264.7 cells incubated with \u003cem\u003eHp\u003c/em\u003eESP and \u003cem\u003eNb\u003c/em\u003eESP were analyzed by LC-MS/MS. A total of 64,843 spectra were acquired and used to assign 49,121 unique peptides, leading to the identification of 4,348 proteins (4,281 protein groups). After removing \u003cem\u003eN. brasiliensis\u003c/em\u003e, \u003cem\u003eH. polygyrus\u003c/em\u003e and contaminant proteins, a total of 4,245 protein groups were kept for further analyses. The quantification analysis was performed using Spectronaut and only proteins with a \u003cem\u003eq\u003c/em\u003e value \u0026lt;0.05 and a log\u003csub\u003e2\u003c/sub\u003e fold-change \u0026gt;0.58 or \u0026lt;\u0026minus;0.58 (for upregulated and downregulated proteins, respectively) were taken into consideration for further analysis. After removing potential contaminants and parasite proteins, a total of 990 and 2,222 proteins had significantly dysregulated expression profiles in RAW264.7 cells after treatment with \u003cem\u003eHp\u003c/em\u003eESP and \u003cem\u003eNb\u003c/em\u003eESP, respectively (Supplementary Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCA was conducted to reduce data dimensionality and visualize sample clustering. The resulting PCA plot shows that the first two principal components, PC1 and PC2, explain 35.7% and 21.3% of the total variance, respectively. The samples cluster differently according to their treatment groups (Figure 3A), supporting that exposure to \u003cem\u003eNb\u003c/em\u003eESP and \u003cem\u003eHp\u003c/em\u003eESP potentially induces distinct proteomic profiles in RAW264.7 cells. This was further supported by the amount of differentially expressed proteins (DEPs) with respect to controls (Figure 3B, C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 \u003cem\u003eN. brasiliensis\u003c/em\u003e and \u003cem\u003eH. polygyrus\u003c/em\u003e ESPs activate different inflammatory-related pathways in murine macrophages\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo better understand the pathways involved in immune modulation by \u003cem\u003eN. brasiliensis\u003c/em\u003e and \u003cem\u003eH. polygyrus\u003c/em\u003e, proteins with the GO term \u0026ldquo;Immune system response\u0026rdquo; (GO:0002376) as a parent term were further analysed. A protein-protein interaction network was generated using StringDB data and clusters were manually curated (Figure 4A). The network revealed a central cluster of 109 proteins primarily associated with common immune responses to helminths, including activation of IL-5 (Th2 response) and other cytokine signalling pathways.\u003c/p\u003e\n\u003cp\u003eIn addition to this central node, several smaller clusters (comprising 2\u0026ndash;4 proteins each, \u003cem\u003ee.g.,\u003c/em\u003e Progranulin, Prosaposin, Protein unc-93 homolog B1, Toll-like receptor, ATPase family, Evolutionarily conserved signalling intermediate in Toll pathway, subunits of the AP-3 complex, Vesicle-associated membrane protein 7 (VAMP-7), MAP kinase-activated protein and Rab-27A) were identified and found to be directly involved in anti-inflammatory responses. Interestingly, most of the proteins involved in these \u0026ldquo;anti-inflammatory pathways\u0026rdquo; were directly regulated by \u003cem\u003eN. brasiliensis\u003c/em\u003e. However, two proteins related to lysosome metabolism (e.g. Progranulin, Prosaposin) and a protein involved in innate and adaptive immunity (Protein unc-93 homolog B1) were regulated by \u003cem\u003eH. polygyrus\u003c/em\u003e. Other clusters were enriched in proteins involved in Th1-related biological processes such as nucleotide metabolism (e.g. lmpdh1 and ctps1), mRNA regulation (RNA demethylase, YTH domain-containing family protein 2\u003cstrong\u003e)\u003c/strong\u003e proteasome-mediated antigen processing (Proteasome activator complex subunits), and T-cell receptor signalling (several subunits of the translational initiation factor 2B (elF2B).\u003c/p\u003e\n\u003cp\u003eTo further investigate the functional relevance of these proteins, enrichment analyses were performed using STRINGdb. WikiPathways analysis (Figure 4B) revealed significant involvement in the \u0026ldquo;Microglia pathogen phagocytosis\u0026rdquo; (\u003cem\u003ee.g.\u003c/em\u003e Trem2) and \u0026ldquo;IL-5 signalling\u0026rdquo; (\u003cem\u003ee.g.\u003c/em\u003e Stat3) pathways, along with additional cytokine-related pathways including IL-3, IL-6, IL-2, and IL-7 (\u003cem\u003ee.g.\u003c/em\u003e NF-kappa-B). Reactome analysis (Figure 4C) highlighted broader immune-related pathways such as \u0026ldquo;Neutrophil degranulation,\u0026rdquo; \u0026ldquo;Signalling by the BCR,\u0026rdquo; \u0026ldquo;Toll-like receptor cascades,\u0026rdquo; and \u0026ldquo;C-type lectin receptor signalling\u0026rdquo; (\u003cem\u003ee.g.\u003c/em\u003e Rela, NF-kappa-B), supporting the role of these proteins in both innate and adaptive immune responses.\u003c/p\u003e"},{"header":"3 DISCUSSION","content":"\u003cp\u003eOf all known species, only a dozen nematode helminths frequently parasitise humans, yet they infect approximately 2 billion people, nearly a third of the global population. This high prevalence highlights their ability to evade host defences and underscores their potential as immunomodulatory agents of the human immune system [22]. Evidence suggests that the release of ESPs, including soluble proteins, lipids, carbohydrates, and extracellular vesicles, during nematode helminth infections plays a key role in suppressing or regulating inflammatory responses and eosinophilia. This helps create a regulatory or suppressive immune environment that supports the parasite\u0026rsquo;s long-term survival [23,24]. Despite efforts to identify bioactive molecules, the targets of these molecules and the immunological pathways that helminths exploit to regulate immune responses remain largely unknown. Therefore, elucidating how these ESPs modulate host immune responses may enhance our understanding of host-parasite interactions and support the development of helminth-derived therapeutics for autoimmune and inflammatory diseases.\u003c/p\u003e\n\u003cp\u003eHere, we used murine macrophages from the RAW 264.7 cell line as a model to investigate the immune response to ESPs from two popular rodent hookworm-like nematodes following stimulation with LPS. TNF-\u0026alpha; levels significantly decreased following treatment with dexamethasone and \u003cem\u003eHp\u003c/em\u003eESP, as validated by two techniques, while reduction of TNF-levels by \u003cem\u003eNb\u003c/em\u003eESPs was not significant. ESPs released by \u003cem\u003eN. brasiliensis\u003c/em\u003e L3 larvae have been shown to modulate TNF-\u0026alpha; levels rat models of LPS-induced pulmonary inflammation [25,26]. Similarly, \u003cem\u003eN. brasiliensis\u003c/em\u003e adult ESPs can significantly reduce TNF-\u0026alpha; levels in bone marrow-derived macrophages (BMDM) from LPS-treated C57BL/6 mice [27]. While using isolated cells like the RAW 264.7 cell line has inherent limitations compared to studying immune responses in whole organisms, this model is widely used to investigate the immunomodulatory potential of helminth ESPs due to its key role in immune activation. Furthermore, it is well known that \u003cem\u003eN. brasiliensis\u003c/em\u003e secretes different proteins throughout its life cycle, including during the L3 and adult stages [15], which can influence their immunomodulatory effects and, consequently, the \u003cem\u003ein vitro\u003c/em\u003e responses. A decrease in other pro-inflammatory cytokines (IL-6 and MCP-1) was also observed after treatment with \u003cem\u003eHp\u003c/em\u003eESP and \u003cem\u003eNb\u003c/em\u003eESPs. Experiments performed in BMDMs showed that EVs present in the \u003cem\u003eHp\u003c/em\u003eESP could suppress the secretion of IL-6 and TNF-a\u0026nbsp;after LPS stimulation\u0026nbsp;[28]. In this sense, previous studies showed that IL-6\u003csup\u003e-/-\u003c/sup\u003e mice infected with nematodes had a higher parasite burden, indicating that a decrease in IL-6 expression at the macrophage level is considered beneficial for the parasite [29].\u003c/p\u003e\n\u003cp\u003eProduction of anti-inflammatory and regulatory cytokines was also evaluated. IL-4 (Figure 2A) showed a significant increase in the LPS + Dx group, while ESP-treated groups exhibited only a slight, non-significant trend. In contrast, TGF-\u0026beta; (Figure 2B) levels were significantly elevated in both the LPS + Dx and \u003cem\u003eNb\u003c/em\u003eESP + LPS groups. The differential cytokine responses observed between the \u003cem\u003eNb\u003c/em\u003eESP + LPS and \u003cem\u003eHp\u003c/em\u003eESP + LPS groups may reflect differences in the molecular composition of their respective ESPs. Regarding the results for IL-10 (Figure 1E), no significant differences were observed in any of the cases. This finding is consistent with previous studies that noted that the extracellular vesicles of \u003cem\u003eN. brasiliensis\u003c/em\u003e (\u003cem\u003eNb\u003c/em\u003e-EVs) promoted higher levels of IL-10 secretion compared to ESPs depleted of EVs [30]. Alternatively, it is possible that the release of this cytokine occurs at a later time point, and thus its measurement at 6 hours post-stimulation may not accurately capture its entire expression dynamics. Further studies are warranted to clarify this temporal pattern.\u003c/p\u003e\n\u003cp\u003eIn addition to pro- and anti-inflammatory cytokines, Th1/Th2 markers were also analyzed to further characterize the type of macrophage response. Treatment of RAW264.7 macrophages with \u003cem\u003eH. polygyrus\u003c/em\u003e ESPs induced a polarization mainly towards Th2, while treatment with \u003cem\u003eN. brasiliensis\u003c/em\u003e ESPs presented a Th1/Th2 balance. In the case of genes involved in M2 polarization, a higher expression of Ym1 and ARG1 levels was observed, correlating with a decrease in pro-inflammatory cytokine levels, such as IL-6, MCP-1, TNF-\u0026alpha;, and iNOS. These results suggest that \u003cem\u003eH. polygyrus\u003c/em\u003e ESPs promote alternative activation of macrophages by shifting the M1/M2 ratio. This coincides with studies by other authors who observed a Th2 polarization induced by this intestinal parasite [31]. In the case of \u003cem\u003eN. brasiliensis\u003c/em\u003e, an increase in iNOS gene expression was observed, an enzyme involved in macrophage polarization towards the pro-inflammatory M1 profile, while no significant changes were found in TNF-\u0026alpha; levels quantified by ELISA or in IL-12p70 gene expression. This may also be associated with a polarization towards inflammatory responses. However, it is important to note that an increase in Ym1 gene expression, typically expressed in M2 cells, was observed, and a decrease in IL-6 was noted. Previous studies have demonstrated alternative activation of macrophages in the lungs of mice following infection with \u003cem\u003eN. brasiliensis\u003c/em\u003e, accompanied by increased expression of YM-1, ARG, and other Th2-associated markers. However, it has been speculated that such responses may only be sustained in the presence of functional T cells \u0026nbsp;[32], which could partially explain why we did not observe a purely Th2-skewed response. These findings support the notion that \u003cem\u003eN. brasiliensis\u003c/em\u003e, similar to other intestinal nematodes such as \u003cem\u003eTrichinella spiralis\u003c/em\u003e, elicits a mixed Th1/Th2 immune response in macrophages [33,34].\u003c/p\u003e\n\u003cp\u003eThese slight differences in M1/M2 polarization and cytokine production of RAW 264.7 cells after treatment with \u003cem\u003eN. brasiliensis\u003c/em\u003e and \u003cem\u003eH. polygyrus\u003c/em\u003e ESPs, were also reflected in the proteomic analysis. The PCA plot revealed distinct clustering of the three groups, with minimal overlap between \u003cem\u003eN. brasiliensis\u003c/em\u003e-treated, \u003cem\u003eH. polygyrus\u003c/em\u003e-treated, and control samples. This clear separation suggests that each helminth species induces a unique proteomic signature in macrophages, reflecting species-specific modulation of host immune responses. These findings support the hypothesis that helminth-derived ESPs differentially influence macrophage function, potentially through distinct molecular pathways, in agreement with the cytokine profiles and M1/M2 marker expression also observed in this study. Our study revealed a marked innate immune response by murine macrophages against both \u003cem\u003eN. brasiliensis\u003c/em\u003e and \u003cem\u003eH. polygyrus\u003c/em\u003e, as indicated by the central cluster in Figure 4A, which is consistent with previous studies on immune response against helminths [35,36].\u003c/p\u003e\n\u003cp\u003eInterestingly, while both nematodes exhibited a significant anti-inflammatory effect on murine macrophages, \u003cem\u003eN. brasiliensis\u003c/em\u003e appeared to act more specifically on proteins involved in anti-inflammatory responses, whereas \u003cem\u003eH. polygyrus\u003c/em\u003e affected only three of these proteins directly. This suggests that \u003cem\u003eH. polygyrus\u003c/em\u003e and \u003cem\u003eN. brasiliensis\u003c/em\u003e may employ different pathways to exert its immunomodulatory effects. For instance, it has been previously shown that \u003cem\u003eH. polygyrus\u003c/em\u003e secretes a TGF-\u0026beta; mimic (\u003cem\u003eHp-\u003c/em\u003eTGM) that activate mouse and human Foxp3\u003csup\u003e+\u003c/sup\u003e Treg cells and can expand the host Treg population [20]. Furthermore, two additional TGF-\u0026beta; mimics from \u003cem\u003eH. polygyrus\u003c/em\u003e have been shown to be functionally active [37]. However, to the best of our knowledge, no similar molecules have yet been identified in \u003cem\u003eN. brasiliensis\u003c/em\u003e. Based on these findings, it is tempting to speculate that \u003cem\u003eH. polygyrus\u003c/em\u003e may exert a more direct influence on cytokine release, particularly TGF-\u0026beta;, whereas \u003cem\u003eN. brasiliensis\u003c/em\u003e appears to act more indirectly, affecting proteins involved in anti-inflammatory responses, innate immune signalling, neutrophil degranulation, and Toll-like receptor pathways.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, it is worth noting that \u003cem\u003eN. brasiliensis\u003c/em\u003e ESPs have an effect on proteins involved in the \u0026ldquo;purine metabolism\u0026rdquo; pathway (\u003cem\u003ee.g\u003c/em\u003e., CTP synthase 1, Inosine-5\u0026apos;-monophosphate dehydrogenase, AMP deaminase 3, Adenosine deaminase and Purine nucleoside phosphorylase). While this might indicate the activation of macrophage proliferation, some of these proteins have been shown to act as a positive regulator of T-cell coactivation and to play a crucial role in the proliferation of activated lymphocytes in humans [38,39].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, the results obtained using murine macrophages suggest that ESPs from \u003cem\u003eN. brasiliensis\u003c/em\u003e and \u003cem\u003eH. polygyrus\u003c/em\u003e exert their anti-inflammatory effects through distinct mechanisms. While both nematodes stimulate the production of anti-inflammatory and regulatory cytokines, they appear to activate different pathways involved in immune modulation. This important divergence should be considered when interpreting results in the context of human helminth infections.\u003c/p\u003e"},{"header":"4. MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e4.1 Ethical statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animals were maintained in the animal facility at the National Center for Microbiology in conformity with the Directive on the protection of animals used for scientific purposes (Directive 2010/63/UE, Decision 2020/569/UE and RD 1386/2018). All animal procedures were conducted in accordance with relevant institutional and national guidelines and regulations. The experimental protocols involving animal experiments were approved by the Ethical Animal Experimentation Committee of the Instituto de Salud Carlos III and Comunidad de Madrid (PROEX 124/19 and PROEX 119.3/20). All methods are reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org/) for the reporting of animal research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 Parasite material and isolation of ESPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNippostrongylus brasiliensis\u003c/em\u003e was maintained in C57BL/6 mice (\u003cem\u003eM. musculus)\u0026nbsp;\u003c/em\u003epurchased from\u003cem\u003e\u0026nbsp;\u003c/em\u003eJanvier Labs (Le Genest-Saint-Isle, France). \u0026nbsp;A total of sixty (60) mice were infected subcutaneously with 500 infective larvae (L3) per mouse. Six days post-infection, the mice were euthanized using CO₂, and both the small and large intestines were harvested. Adult worms were manually extracted from the small intestine, washed three times with PBS and twice with RPMI 1640 at 37 \u0026deg;C, and then incubated in adult culture medium (RPMI 1640 supplemented with glucose [4.5 g/mL], glutamine [2 mM], and antibiotic/antimycotic [2x]) at 37 \u0026deg;C with 5% CO₂. The final worm concentration in the medium was adjusted to 500 worms per millilitre. The media obtained during the first 24 h after parasite culturing was discarded. Subsequently, excretory-secretory products (ESPs) were collected every 48 hours over a 12-day period and subjected to sequential differential centrifugation at 500, 2,000, and 4,000 g for 30 min each to remove eggs and parasite debris. Finally, media was concentrated using a 3 kDa spin concentrator (Merck Millipore, Billerica, MA, USA) and stored at 1.0 mg/ml in PBS at \u0026minus;80\u0026deg;C until used.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eH. polygyrus\u003c/em\u003e was maintained in NMRI mice (\u003cem\u003eM. musculus\u003c/em\u003e) purchased from\u003cem\u003e\u0026nbsp;\u003c/em\u003eCharles River Laboratories (MA, USA). A total of 10 mice was inoculated orally with 150 \u0026plusmn; 15 third-stage larvae (L3) suspended in 0.3\u0026ndash;0.4 mL of distilled water. The larval concentration was standardized prior to inoculation using a McMaster counting chamber. Infective L3 larvae were obtained as described previously with minor modifications [40]. Briefly, faecal pellets were collected from day 14 onwards, softened, and homogenized in a mortar with distilled water. The homogenate was centrifuged at 300 rpm for 3 minutes three times, retaining the pellet each time. The pellet was finally resuspended in distilled water and filtered through a mesh strainer and gauze. The filtrate was then centrifuged at 600 rpm for 6 minutes, and the pellet was placed onto the central area of an inverted watch glass covered with pre-moistened filter paper. This assembly was set in a 15 cm diameter Petri dish, with the edges of the filter paper kept in contact with distilled water to maintain humidity. The cultures were incubated at room temperature, in darkness and under high humidity conditions, for 8 days. The liquid phase of the coproculture was collected and the larvae were allowed to sediment for approximately 45 minutes and finally stored in distilled water at 4\u0026deg;C until use. \u003cem\u003eH. polygyrus\u003c/em\u003e adult worms were collected at day 14 days post infection and cultivated as described previously [41]. Briefly, adult worms were manually extracted and cultured in the same medium used for \u003cem\u003eN. brasiliensis\u003c/em\u003e worms, as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 Cell culture conditions and treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMurine macrophage RAW 264.7 cells (ATCC\u0026reg; TIB-71\u0026trade;) purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) were propagated in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 \u0026mu;g/mL streptomycin, and 2 mM glutamine at 37\u0026deg;C in a humidified atmosphere with 5% CO₂. The culture medium, pre-warmed to 37\u0026deg;C, was refreshed every 48 hours, with a total volume of 10 mL per T75 flask.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRAW 264.7 cells were cultured in 6-well plates at the same density and divided into the following eight groups: i) Control group: Cells cultured in supplemented DMEM only; ii) LPS-treated group: Cells stimulated with 5 ng/mL of LPS; iii) Dexamethasone control group: Cells treated with 1 \u0026mu;M dexamethasone; iv) Dexamethasone + LPS intervention group: Cells treated with 1 \u0026mu;M dexamethasone and stimulated with 5 ng/mL of LPS; v) \u003cem\u003eNb\u003c/em\u003eESP-treated group: Cells treated with 10 \u0026mu;g/mL of \u003cem\u003eNb\u003c/em\u003eESP; vi) \u003cem\u003eNb\u003c/em\u003eESP + LPS intervention group: Cells treated with 10 \u0026mu;g/mL \u003cem\u003eNb\u003c/em\u003eESP and stimulated with 5 ng/mL of LPS; vii) \u003cem\u003eHp\u003c/em\u003eESP-treated group: Cells treated with 10 \u0026mu;g/mL of \u003cem\u003eHp\u003c/em\u003eESP; and viii) \u003cem\u003eHp\u003c/em\u003eESP + LPS intervention group: Cells treated with 10 \u0026mu;g/mL \u003cem\u003eHp\u003c/em\u003eESP and stimulated with 5 ng/mL of LPS. At time 0, groups were stimulated with LPS. One hour later, dexamethasone, \u003cem\u003eNb\u003c/em\u003eESP or \u003cem\u003eHp\u003c/em\u003eESP were added to the wells. Supernatants were collected 6 hours post-LPS stimulation, and cells from each well were harvested by adding cold PBS and centrifuging at 1,000 rpm for 5 minutes. Both supernatants and cell pellets were stored at \u0026minus;20\u0026deg;C until further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4 Cytokine quantification by cytometric bead array and ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cytokines present in the cell culture supernatants were quantified using the BD Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Biosciences, San Jose, CA, USA) following the manufacturer\u0026apos;s instructions. Briefly, the beads coated with six specific capture antibodies were mixed for each assay. Subsequently, 50 \u0026mu;L of the mixed capture beads, 50 \u0026mu;L of the sample or standard dilution, and 50 \u0026mu;L of the PE detection reagent were added to each test tube. The tubes were, then, incubated for 2 hours at room temperature (RT) in the dark. After incubation, the samples were washed with 1 mL of wash buffer and centrifuged at 200 g for 5 minutes. The resulting microbead pellet was resuspended in 300 \u0026mu;L of buffer following the removal of the supernatant. Samples were finally analyzed using the BD Accuri C6 Plus flow cytometer (BD Biosciences) and data were processed with FlowJo software (BD Biosciences).\u003c/p\u003e\n\u003cp\u003eThe TNF-\u003cem\u003ea\u003c/em\u003e cytokine levels were further confirmed using the Mouse TNF alpha Uncoated ELISA kit (Invitrogen) following the manufacturer\u0026rsquo;s instructions. Briefly, plates were coated with 100 \u0026mu;L/well of capture antibody diluted 1:250 in coating buffer overnight at 4\u0026deg;C. The following day, plates were washed x3 in washing buffer and blocked with 200 \u0026mu;L of ELISA/ELISPOT Diluent for 1 h at RT. Plates were washed again and incubated with 100 \u0026mu;L of standards or samples for 2 h at RT. After washing again x3 with washing buffer, 100 \u0026mu;L/well of Detection Antibody diluted 1:250 was added and plates were incubated for 1 h at room temperature. A wash was performed as described above and finally, 100 \u0026mu;L/well of diluted Streptavidin-HRP was added to each well and plates were developed after a final wash using 100 \u0026mu;l of 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethylbenzidine (TMB) for 15 min, followed by 100 \u0026mu;l of stop solution. Plates were read at a wavelength of 450 nm on a Heales mod. MB580 (Shenzhen Huisong Technology Development Co., Ltd. Shenzhen , China) microplate reader.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5 RNA extraction, cDNA synthesis and qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA from cells treated with excretory-secretory products (ESPs) was extracted using the Quick-RNA\u0026trade; Miniprep Kit (Zymo), following the manufacturer\u0026rsquo;s instructions. The extracted RNA was quantified using a NanoDrop One/One spectrophotometer (ThermoFisher) by measuring absorbance at 260 nm. The purity of the RNA was assessed through absorbance ratios at 260/280 and 260/230 nm. Complementary DNA (cDNA) synthesis was performed using the SuperScript III kit (Thermo Fisher Scientific). The reaction was initiated by mixing 3 \u0026micro;L of RNA from each sample, 1 \u0026micro;L of oligo(dT) (50 \u0026micro;M), 1 \u0026micro;L of dNTP Mix (10 mM), and distilled water to a final volume of 13 \u0026micro;L. This mixture was incubated in a GeneAmp PCR System 2700 (Applied Biosystems) thermocycler for 5 minutes at 65\u0026deg;C and then for 1 minute at 4\u0026deg;C. Subsequently, 4 \u0026micro;L of 5X First-Strand Buffer, 1 \u0026micro;L of DTT (0.1 M), and 1 \u0026micro;L of RNaseOUT (RNase inhibitor, 40 U/\u0026micro;L) were added to reach a final reaction volume of 20 \u0026micro;L. The reaction was incubated for 60 minutes at 50\u0026deg;C, followed by 15 minutes at 70\u0026deg;C to inactivate the enzyme. The synthesized cDNA was quantified using the NanoDrop One/One spectrophotometer (Thermo Fisher Scientific) by measuring absorbance at 260 nm, with purity assessed via 260/280 and 260/230 ratios. The cDNA was stored at \u0026minus;20\u0026deg;C until use.\u003c/p\u003e\n\u003cp\u003ecDNA amplification was performed using quantitative real-time PCR (qPCR) with the SYBR-Green system. The actin gene was used as an endogenous control, and the expression of \u003cem\u003eIL-4\u003c/em\u003e, \u003cem\u003eTGF\u0026beta;\u003c/em\u003e, \u003cem\u003eiNOS\u003c/em\u003e, \u003cem\u003eYm1\u003c/em\u003e, and \u003cem\u003eARG\u003c/em\u003e genes was analyzed. Amplifications were conducted in the presence of SYBR-Green PCR Master Mix (Applied Biosystems) using 2.5 \u0026micro;g of template cDNA and 1 \u0026micro;L of each primer (forward and reverse; Table 1) in a final reaction volume of 20 \u0026micro;L. qPCR reactions were performed on a Rotor-Gene Q (Qiagen) using the following conditions: initial denaturation at 95\u0026deg;C for 10 seconds (1 cycle) followed by 40 cycles of denaturation (95\u0026deg;C for 10\u0026thinsp;sec), annealing (55\u0026deg;C or 65\u0026deg;C for 15\u0026thinsp;sec) and extension (72\u0026deg;C for 20 sec). Gene expression was normalised to the housekeeping gene as described before [42,43] and relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e\u0026Delta;\u0026Delta;\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e method using Control (untreated cells) as a reference group [44].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6 Cell processing and protein extraction\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree biological replicates of cells treated with \u003cem\u003eNb\u003c/em\u003eESP or \u003cem\u003eHp\u003c/em\u003eESP were lysed using 100 \u0026micro;L of RIPA buffer (Invitrogen) following the manufacturer\u0026rsquo;s instructions. Protein extracts (20 \u0026micro;g in 100 \u0026micro;L of RIPA) were reduced by adding 10 \u0026micro;L of 100 mM tris(2-carboxyethyl)phosphine (TCEP) and incubated at 37\u0026deg;C with 600 rpm for 45 minutes. Alkylation was performed by adding 11 \u0026micro;L of 400 mM chloroacetamide, followed by a 30-minute incubation at RT in the dark with 600 rpm shaking.\u003c/p\u003e\n\u003cp\u003eSubsequently, 100 \u0026micro;L of SeraMag magnetic beads mix (GE Healthcare, Chicago, IL, USA; 50% hydrophilic beads and 50% hydrophobic beads) and 200 \u0026micro;L of acetonitrile (ACN) were added to facilitate protein binding to the beads. The mixture was incubated at RT with 600 rpm for 35 minutes. Supernatants were removed, and magnetic beads were washed twice with 70% ethanol and once with ACN. Proteins were then digested overnight at 37\u0026deg;C with 0.5 \u0026micro;g of porcine trypsin (Thermo Fisher Scientific) in 100 \u0026micro;L of 200 mM HEPES (pH 8.0) with 600 rpm. The following day, samples were sonicated twice, and the resulting supernatants were dried under vacuum in preparation for desalting.\u003c/p\u003e\n\u003cp\u003eThe Pierce C18 Spin Tips (Thermo Fisher Scientific) were used for peptide purification. The dried peptides were resuspended in 50 \u0026micro;L of 0.1% trifluoroacetic acid (TFA) and loaded onto the C18 Spin Tip, followed by centrifugation at 1,000\u0026ndash;2,000 g to allow peptides to bind to the hydrophobic matrix. To remove contaminants, 20 \u0026micro;L of 0.1% TFA was added for washing. Peptides were eluted using 20 \u0026micro;L of 0.1% TFA in 80% ACN, with centrifugation at 1,000 g for 1 minute. The eluted peptides were dried under vacuum and stored at -80\u0026deg;C until analysis by LC-MS/MS.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.7 Mass spectrometry and protein identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor LC-MS/MS, peptides were analyzed in an Orbitrap Astral mass spectrometer coupled to a Vanquish Neo UHPLC System (Thermo Fisher Scientific). Peptide samples were loaded into the precolumn PepMap Trap Catridge 5 \u0026micro;m, 300 \u0026micro;m x 5 mm (Thermo Fisher Scientific) and eluted in an Easy-Spray PepMap RSLC C18 2 \u0026micro;m, 50 \u0026micro;m x 15 cm (Thermo Fisher Scientific) heated at 50\u0026deg;C. The mobile phase flow rate was 300 nL/min and 0.1% formic acid (FA) in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003emq\u003c/sub\u003e and 0.1% FA in 80% acetonitrile (ACN) were used as elution buffers A and B, respectively. The 15 min elution gradient was: 4%-10% buffer B for 2 min, 10%-40% buffer B for 11 min, 40%-99% buffer B for 0.5 min, and 99% buffer B for 1.5 min. Prior to injection, samples were re-suspended in 10 \u0026micro;L of buffer A, and 1 \u0026micro;L of each sample were injected, and analyzed in data independent acquisition (DIA) mode. For ionization, 1900 V of liquid junction voltage and 280\u0026deg;C capillary temperature was used. The full scan method employed a m/z 380-980 mass selection, an Orbitrap resolution of 240000 (at m/z 200), an automatic gain control (AGC) value of 500%, and maximum injection time (IT) 5 ms. The MS/MS was performed with the Astral mass analyzer, using an AGC of 500%, an IT of 3 ms, and a normalized collision energy (NCE) of 25 for fragmentation of precursors. The scan range was set from 380 to 980 m/z, with an isolation window of 2 m/z, and window placement optimization was enabled. Thus, a total of 299 windows were analyzed in each cycle.\u003c/p\u003e\n\u003cp\u003eA directDIA search was performed using Spectronaut v19.4 against a concatenated target/decoy database consisting of the mouse proteome (17,216 reviewed proteins from UP000000589 accessed in March 2024) and common contaminants (246 proteins from cRAP database accessed in October 2024) as well as proteomes from \u003cem\u003eN. brasiliensis\u003c/em\u003e (PRJNA994163; 29.473 proteins) and \u003cem\u003eH. polygyrus\u0026nbsp;\u003c/em\u003e(PRJEB15396; 25.212 proteins) downloaded from WormBaseParasite in November 2024 (WBPS19). Enzyme was set to Trypsin/P, fixed modifications to Carbamidomethyl (C) and variable modifications to Acetyl (Protein N-term) and Oxidation (M). Precursor qvalue and PEP cutoffs were set to 0,01 and 0,2, respectively, while protein qvalue cutoff was set to 0,01. Peptide and protein FDR were set to 0,01. Single hit proteins were excluded from the analysis. Mass spectrometry data along with the identification results and search parameters have been deposited in the ProteomeXchange Consortium \u003cem\u003evia\u003c/em\u003e the PRIDE partner repository\u0026nbsp;[45]\u0026nbsp;with the dataset identifier PXD062974.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.8 Gene Ontology and PPI analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GO information for each protein was retrieved from Uniprot database (https://www.uniprot.org/). Furthermore, an enrichment analysis was performed using STRINGdb using the default settings except for the minimum required interaction score, which was set to \u0026ldquo;high confidence\u0026rdquo; (0.7). The interactome graph involving \u0026ldquo;immune system response\u0026rdquo; proteins was depicted using the python NetworkX library (https://networkx.org/). Reactome and Wikipathways terms were grouped by similarity \u0026ge; 0.8 and sorted by signal.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.9 Statistical and bioinformatic analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the CBA, ELISA and qPCR analyses, statistical significance was calculated using Student\u0026rsquo;s t-test by comparing the LPS-treated group to each treatment or control. **** = \u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.0001; *** = \u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.001; ** = \u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.01; * = \u003cem\u003ep \u0026lt;\u0026nbsp;\u003c/em\u003e0.05; no value = \u003cem\u003ep \u0026gt;\u0026nbsp;\u003c/em\u003e0.05. For the proteomics data, a DIA quantitative analysis was performed following protein identification using Spectronaut\u0026reg;\u0026nbsp;v19.4. Quantitative results were normalised by cross-run normalization of medians. An unpaired t-test was performed to compare the proteins with a significant regulation between two groups after log2 transformation. Fold-changes were considered as significant when \u0026ge;0.58 and adjusted \u003cem\u003ep\u003c/em\u003e-value \u0026le; 0.05. Volcano and Principal Component Analysis (PCA) plots were performed using the ggplot2 R package using customised scripts. All scripts are available and can be downloaded from https://github.com/JavierSotillo/Scripts.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCovadonga Varea: \u003cem\u003eInvestigation\u003c/em\u003e. Antonio J. Mart\u0026iacute;n-Galiano: \u003cem\u003eData Curation, Writing\u003c/em\u003e. Sara V\u0026aacute;zquez-\u0026Aacute;vila: \u003cem\u003eInvestigation, Methodology\u003c/em\u003e. Ana Montero-Calle: \u003cem\u003eInvestigation, Methodology, Software\u003c/em\u003e. Julia Cesteros: \u003cem\u003eInvestigation.\u003c/em\u003e Mar\u0026iacute;a Jes\u0026uacute;s Perteguer: \u003cem\u003eInvestigation\u003c/em\u003e. Alberto Pel\u0026aacute;ez-Garc\u0026iacute;a: \u003cem\u003eInvestigation, Methodology, Software.\u003c/em\u003e Rodrigo Barderas: \u003cem\u003eInvestigation, Methodology, Software\u003c/em\u003e. Juan Jos\u0026eacute; Garc\u0026iacute;a: \u003cem\u003eSupervision, resources, Funding acquisition, Writing\u003c/em\u003e. Javier Sotillo: \u003cem\u003eSupervision, resources, Funding acquisition, Writing\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMass spectrometry data along with the identification results and search parameters have been deposited in the ProteomeXchange Consortium \u003cem\u003evia\u003c/em\u003e the PRIDE partner repository with the dataset identifier PXD062974. Reviewer can access the dataset by logging in to the PRIDE website using the following account details:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eUsername:\u0026nbsp;\u003c/strong\[email protected]\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003ePassword:\u0026nbsp;\u003c/strong\u003eTm9dBo09W50t\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grant PID2022-137661OB-I00 (MPY 341/23) funded by MCIN/AEI/10.13039/501100011033/, by ISCIII grant MPY337/24 and by FEDER A way to make Europe.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHotez, P. 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C. \u003cem\u003eet al.\u003c/em\u003e Cultivation of \u003cem\u003eHeligmosomoides polygyrus\u003c/em\u003e: An Immunomodulatory Nematode Parasite and its Secreted Products. \u003cem\u003eJoVE\u003c/em\u003e 52412 (2015) doi:10.3791/52412.\u003c/li\u003e\n\u003cli\u003eChung, J., Choi, M. J., Jeong, S. Y., Oh, J. S. \u0026amp; Kim, H. K. Chemokines Gene Expression of RAW 264.7 Cells by \u003cem\u003eActinobacillus actinomycetemcomitans \u003c/em\u003eLipopolysaccharide Using Microarray and RT-PCR Analysis. \u003cem\u003eMol. Cells\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 257\u0026ndash;261 (2009).\u003c/li\u003e\n\u003cli\u003eZhou, C. \u003cem\u003eet al.\u003c/em\u003e Anti-inflammatory Mechanism of Action of Benzoylmesaconine in Lipopolysaccharide-Stimulated RAW264.7 Cells. \u003cem\u003eeCAM \u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 1\u0026ndash;12 (2022).\u003c/li\u003e\n\u003cli\u003eLivak, K. J. \u0026amp; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. \u003cem\u003eMethods\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 402\u0026ndash;408 (2001).\u003c/li\u003e\n\u003cli\u003eVizca\u0026iacute;no, J. A. \u003cem\u003eet al.\u003c/em\u003e The Proteomics Identifications (PRIDE) database and associated tools: status in 2013. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, D1063\u0026ndash;D1069 (2012).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Oligonucleotides Used for Gene Expression Analysis via SYBR-Green qPCR\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGENE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePRIMER FORWARD\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePRIMER REVERSE\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eACT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-CGGTTCCGATGCCCTGAGGCTCTT-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-CGTCACACTTCATGATGGAATTGA-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL-4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-CCCCCAGCTAGTTGTCATCC-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-AGGACGTTTGGCACATCCAT-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eiNOS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-CAGCTGGGCTGTACAAACCTT-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-CATTGGAAGTGAAGCGTTTCG-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTGF \u0026beta;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-TTGCTTCAGCTCCACAGAGA-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-TGGTTGTAGAGGGCAAGGAC-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eYm1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-TTTCTCCAGTGTAGCCATCCTT-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-TCTGGGTACAAGATCCCTGAA-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eARG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-TTTTTCCAGCAGACCAGCTT-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5\u0026acute;-AGAGATTATCGGAGCGCCTT-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Nippostrongylus brasiliensis, Heligmosomoides polygyrus, RAW264.7, macrophages, nematoda, immunomodulation","lastPublishedDoi":"10.21203/rs.3.rs-8017434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8017434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHookworm infections affect hundreds of millions of people worldwide and rank among the most significant neglected tropical diseases in terms of morbidity. Due to the inverse correlation between hookworm infection and the incidence of immune-mediated inflammatory diseases, considerable research has focused on understanding how parasitic helminths modulate host inflammation. \u003cem\u003eHeligmosomoides polygyrus\u003c/em\u003e and \u003cem\u003eNippostrongylus brasiliensis\u003c/em\u003e are two hookworm-like rodent models widely used to investigate fundamental aspects of immune regulation. However, no comparative study has yet analysed the distinct immunological pathways activated by these helminths in their hosts. In this study, we compared cytokine profiles and M1/M2 macrophage polarization markers induced by \u003cem\u003eH. polygyrus\u003c/em\u003e and \u003cem\u003eN. brasiliensis\u003c/em\u003e, alongside proteomic pathways involved in their activation, using the RAW 264.7 murine macrophage cell line. \u003cem\u003eH. polygyrus\u003c/em\u003e downregulated proinflammatory cytokines such as TNF-α and MCP-1, whereas \u003cem\u003eN. brasiliensis\u003c/em\u003e did not affect TNF-α expression. Both parasites upregulated Th2 and regulatory cytokines, including IL-4 and TGF-β. Furthermore, \u003cem\u003eH. polygyrus\u003c/em\u003e predominantly polarized macrophages toward an M2 phenotype, while \u003cem\u003eN. brasiliensis\u003c/em\u003e maintained a balanced M1/M2 profile. Proteomic analysis revealed that \u003cem\u003eN. brasiliensis\u003c/em\u003e and \u003cem\u003eH. polygyrus\u003c/em\u003e exert their anti-inflammatory effects through distinct mechanisms, with \u003cem\u003eN. brasiliensis\u003c/em\u003e acting via peripheral pathways and \u003cem\u003eH. polygyrus\u003c/em\u003e via central regulatory mechanisms.\u003c/p\u003e","manuscriptTitle":"Nippostrongylus brasiliensis and Heligmosomoides polygyrus activate different immunomodulatory pathways in murine macrophages in vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-21 05:41:23","doi":"10.21203/rs.3.rs-8017434/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-06T08:35:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-03T20:04:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T20:56:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156120792810696524858278236325144134807","date":"2026-03-16T15:37:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124614608292632992712272635740350970680","date":"2026-03-13T19:22:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-11T10:51:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-11T09:52:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-06T09:48:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-06T09:40:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6aa2ed18-601f-498f-9a0d-970850374053","owner":[],"postedDate":"November 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":58130884,"name":"Health sciences/Diseases"},{"id":58130885,"name":"Biological sciences/Immunology"},{"id":58130886,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-06T08:40:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-21 05:41:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8017434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8017434","identity":"rs-8017434","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}