Semaphorin 3A reduces the severity of aGVHD by promoting M2 macrophage polarization

Semaphorin 3A reduces the severity of aGVHD by promoting M2 macrophage polarization | 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 Semaphorin 3A reduces the severity of aGVHD by promoting M2 macrophage polarization Jiali Li, Bing Wang, Xixi Xiang, Hongyun Liu, Song Dong, Hanshan Yang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7904342/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Acute graft-versus-host disease (aGVHD) remains a serious complication allogeneic hematopoietic stem cell transplantation (allo-HSCT) and significantly impacts nonrelapse mortality (NRM). While Semaphorin 3A (Sema3A) is implicated in immune regulation during inflammatory conditions, its role in aGVHD pathogenesis was unclear. Using an aGVHD mouse model, intravenous recombinant Sema3A administration demonstrated therapeutic effects, significantly reducing tissue damage in target organs observed via histopathological analysis. Flow cytometry and immunofluorescence staining revealed an increased proportion of M2 macrophages (CD206⁺) in vivo. In vitro treatment of RAW264.7 murine macrophages with Sema3A promoted polarization towards the M2 phenotype, characterized by elevated expression of Arg1 and IL-10, and inhibited T cell function. Transcriptome sequencing of treated macrophages identified suppressed IL-17 signaling, which was validated by qPCR and western blot showing significant decreases in mRNA and protein levels of both IL-17A and its receptor IL-17RA. These findings indicate that Sema3A mitigates aGVHD by driving M2 macrophage polarization through inhibition of the IL-17 signaling pathway, thereby suppressing pathogenic T cell responses, highlighting its potential as a therapeutic target. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Immunology Biological sciences/Molecular biology aGVHD Sema3A Macrophage IL17 signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) [ 1 , 2 ]. Moreover, aGVHD is a significant cause of morbidity and mortality among allogeneic HCT recipients [ 3 ]. Owing to the complex mechanisms underlying aGVHD, existing therapeutic approaches are inadequate for its complete prevention. Semaphorin 3A (Sema3A) is a secreted protein that belongs to the class 3 semaphorin family. Over the past decade, Sema3A has emerged as one of the most active semaphorins (SEMAs) involved in regulating inflammatory responses. The increased expression levels of Sema3A in differentiated giant cells and activated T cells suggest that it plays a pivotal role in immune-mediated diseases [ 4 – 6 ]. In our previous study, we showed that Sema3A secreted by human dermal mesenchymal stromal cells decreases the expression of downstream factors, such as cyclin D1 and CDK4, thereby inhibiting T cell proliferation [ 7 ]. However, the role of Sema3A in aGVHD and its specific regulatory mechanisms remain unexplored. In the present study, we established aGVHD model mice and treated them with Sema3A. Moreover, we integrated histopathological assessment, macrophage polarization profiling (in vivo and in vitro), and transcriptome pathway analyses and identified IL-17 signaling as a key component in the mechanism by which Sema3A mediates aGVHD pathogenesis. Material and methods Mouse model of GVHD Preparation of mouse bone marrow cells and splenocytes: Male C57BL/6J donor mice (8–12 weeks old) were purchased from Charles River Laboratory (Beijing, China). The femora and tibiae were washed 2–3 times with PBS containing 2% FBS, and the ends of the bones were removed using ophthalmic scissors. A 10 mL syringe needle was then inserted into the bone marrow cavity, which was flushed with PBS containing 2% FBS. The resulting solution was collected in a 60 mm cell culture dish and passed through a 200-mesh cell strainer after gentle grinding. The cell suspension was transferred to a 50 mL centrifuge tube and centrifuged at 1500 rpm at 4℃ for 5 minutes. The supernatant was discarded, and the pellet was resuspended in PBS containing 2% FBS and filtered through a cell strainer. The bone marrow cell suspension was thus successfully prepared. For splenocyte preparation, the spleens were washed 2–3 times with PBS containing 2% FBS, carefully ground using a syringe plunger, and suspended in PBS containing 2% FBS. The splenocyte suspension was prepared as described above. Establishment of the mouse model: A mouse model of MHC-mismatched HSCT was generated by transplanting bone marrow (BM) cells from C57BL/6J mice into male BALB/c mice previously irradiated with X-rays at a dose of 4.0 Gy on the day prior to transplantation and irradiated with X-rays at a dose of 3.5 Gy on the day of transplantation [ 8 ]. The recipient male BALB/c mice were reconstituted with 5 × 10⁶ BM cells from the donor C57BL/6 mice, either with or without 3 × 10⁶ donor spleen cells. To investigate the effect of Sema3A (Sino Biological Inc., Beijing) on aGVHD, intravenous administration of Sema3A (0.5 mg/kg) was initiated concurrently with splenocyte and BM transplantation [ 9 ], followed by repeated dosing every 3 days through 9 days posttransplantation. The experiment comprised the following 5 groups: the control group, which included 6 normal mice; the X-ray group, which included 10 irradiated mice; the BM group, in which 8 mice received 3 × 10⁶ BM cells only; the aGVHD group, in which 19 mice received a mixture of 5 × 10⁶ BM cells and 3 × 10⁶ spleen cells from donor C57BL/6 mice; and the Sema3A-treated aGVHD (S-aGVHD) group, in which 18 mice received a mixture of 5 × 10⁶ BM cells and 3 × 10⁶ spleen cells from donor C57BL/6 mice plus Sema3A (0.5 mg/kg). Body weights and clinical scores were observed and recorded every two days up to 14 days posttransplantation, while survival rates were assessed up to 20 days after transplantation. At 14 days posttransplantation, the severity of clinical aGVHD was evaluated using a scoring system [ 10 ]. Histopathological changes in aGVHD target organs (liver, lung, skin, colon, and small intestine) were observed using HE staining, and histopathological scores for acute GVHD were determined following previously published protocols [ 11 ]. The euthanasia of mice is carried out by intraperitoneal injection of excessive phetobarbital sodium (St. Louis, MO, USA). According to the body weight of the mice, phenobarbital sodium solution was intraperitoneally injected at a dose of 150 mg/kg. The mice were observed at room temperature for 5–8 minutes to determine death. Cervical dislocation was then performed as a secondary measure to ensure death. All animal experiments were approved by the Institutional Laboratory Animal Care and Use Committee of Xinqiao Hospital. Cell lines and Cell culture: RAW264.7 and CTLL-2 cells were obtained from the Procell Cell Bank (Wuhan, China). RAW264.7 cells were cultured in DMEM (GIBCO, USA) supplemented with 10% fetal bovine serum (FBS; ExCellBio, Suzhou, China) and 1% penicillin‒streptomycin (P/S; Gibco, USA). CTLL-2 cells were maintained in RPMI-1640 medium (GIBCO, USA) supplemented with 10% FBS, 100 U/mL recombinant IL-2 (iCell Bioscience, Shanghai, China) and 1% P/S. Additional details regarding the cell culture conditions are provided in the supplementary information. Cell viability RAW264.7 viability in response to varying concentrations of Sema3A: RAW264.7 cells (2 × 10⁵ cells/mL) were treated with different concentrations of Sema3A (0.01, 0.1, 1, or 10 µg/mL) [ 12 ]. Each group had three replicate wells. The cells were incubated at 37℃ with 5% CO₂ for 24 or 48 hours. Subsequently, 10 µL of CCK-8 detection solution (Biosharp, Anhui, China) was added to each well, except for the untreated RAW264.7 wells. After 2 hours of incubation, the absorbance was measured at a wavelength of 450 nm using a microplate reader. Effect of Sema3A expressed by macrophages on T cells: The viability of CTLL-2 cells was evaluated after co-culture with RAW264.7 cells treated with Sema3A. The following three experimental groups were established: CTLL-2 cells, CTLL-2 cells cocultured with RAW264.7 cells (RCs), and CTLL-2 cells cocultured with Sema3A-treated RAW264.7 cells (STRCs). RAW264.7 cells or RAW264.7 cells treated with 1 µg/mL Sema3A in cell-free medium were inoculated in the upper chamber of a Transwell insert after 24 hours of culture, and the medium was replaced. Simultaneously, CTLL-2 cells (2 × 10 5 /mL) were seeded in the lower chamber of the Transwell system. After 24 hours of coculture, the cells from both chambers were transferred to 96-well plates, and 10 µL of CCK-8 solution was added to each well. Following a 2-hour incubation, the absorbance at 450 nm was measured using a microplate reader. RNA extraction and quantitative real-time PCR The mRNA expression of macrophage polarization-related factors, including TNF-α, iNOS, IL-10, Arg-1, IL-17RA, and IL-17A, was evaluated in vitro. RAW264.7 cells and Sema3A-treated RAW264.7 cells were cultured as described above. Total RNA was extracted using TRIzol reagent (Solarbio, Beijing, China) following the manufacturer’s instructions. The extracted RNA was reverse transcribed into complementary DNA (cDNA) using a reverse transcription kit (Vazyme, Nanjing, China) and a cDNA synthesis kit (Takara, Dalian, China). Real-time quantitative PCR (qRT‒PCR) was performed using the TB Green® Fast qPCR Mix Kit (Takara, Dalian, China). Relative mRNA expression levels were calculated using the 2-∆∆CT method. The primer sequences used in the present study are listed in Supplementary Table 1. Enzyme-linked immunosorbent assay (ELISA) The concentrations of IL-17A in RAW264.7 cells and Sema3A-treated RAW264.7 cells were measured using an ELISA kit (RX203066M; Ruixin, Fujian, China). The concentrations of TNF-α, IFN-γ, IL-2, IL-10 and TGF-β secreted by CTLL-2, RC or STRC in the culture supernatants were quantified using ELISA kits (SEA133Mu, HEA049Mu, SEA073Mu, SEA056Mu, and RX104768H Ruixin, Fujian, China) according to the manufacturer’s instructions. The absorbance was measured at a wavelength of 450 nm using a microplate reader. Flow cytometry Flow cytometry was used to analyze the expression of CD86 and CD206 in vitro to investigate the role of Sema3A in macrophage polarization. RAW264.7 cells (2×10 5 cells/mL) were seeded into 6-well plates with 2 mL of medium per well and incubated for 12 hours at 37°C in 5% CO 2 . Sema3A was added to the cells at a final concentration of 1 µg/mL, while PBS was administered to the other groups as a vehicle control. After 24 hours, the cells were harvested, washed twice with PBS, and incubated with antibodies against CD86 and CD206 for 15 minutes. After two washes with PBS, the cells were passed through a cell sorter and analyzed using a flow cytometer (Beckman Coulter, Inc., California, USA). To further investigate the effect of Sema3A on macrophage polarization in the presence of macrophage inducers, RAW264.7 cells were treated with M1 inducers, namely LPS (100 ng/mL, Beyotime Biotechnology, Shanghai) and IFN-γ (2.5 ng/mL, Beyotime Biotechnology), or M2 inducers, namely IL-4 (20 ng/mL, Procell, Wuhan) or Sema3A (1 µg/mL). Following a 24 and 48 hours culture period, the cells were harvested for analysis of CD86 and CD206 expression levels via flow cytometry. Additionally, an IL-17RA overexpression plasmid (ovIL-17RA) was used to verify the mechanism by which Sema3A influences macrophage polarization. The six experimental groups included untreated macrophages (control) and macrophages treated with Sema3A (1 µg/mL), ovIL-17RA (2 µg, Sangon, Shanghai, China), Sema3A (1 µg/mL) + ovIL-17RA (2 µg), IL-17A/F (100 ng/mL, MCE, New Jersey, USA), or Sema3A (1 µg/mL) + IL-17A/F (100 ng/mL). The proportions of M1 and M2 macrophages were analyzed using flow cytometry. Splenocytes were extracted from mice in all experimental groups to investigate the effect of Sema3A on macrophage polarization in vivo. Flow cytometry was used to analyze the proportions of splenocytes containing proinflammatory cytokines (IFN-γ + and IL-17A + ) and the Foxp3 + anti-inflammatory factor. Single-cell suspensions were prepared from spleen tissue, and 2 µL of BD GolgiPlug™ reagent was added per 1 mL of suspension. The mixture was incubated at 37°C in 5% CO₂ for 4–6 hours and then centrifuged at 1500 rpm for 5 minutes, after which the supernatant was discarded. Surface antibodies (CD3, CD4, CD8, and CD25) were added to the resuspended cells in 50 µL of dye dilution buffer, and the cells were incubated at 4 ℃ in the dark for 20‒30 minutes. After washing twice with staining buffer, 100 µL of BD cell fixation/membrane permeabilization solution was added, and the cells were incubated at 4℃ for 20 minutes. The cells were washed twice with 1× BD Perm/Wash buffer (1 mL per wash) and then incubated with 50 µL of 1× BD Perm/Wash buffer containing intracellular factor antibodies (IL-17A, IFN-γ, and Foxp3) for 30 minutes at 4 ℃ in the dark. After two additional washes, the cells were resuspended in staining buffer and analyzed using flow cytometry. To determine the M1/M2 macrophage ratio in mouse spleen tissue, single-cell suspensions were stained with antibodies (CD11b, F4/80, CD86, and CD206). After mixing and incubating at 4°C for 20 minutes, the cells were centrifuged to remove the supernatant, washed twice, resuspended in staining buffer, and analyzed using flow cytometry. RNA sequencing (RNA-seq) RAW264.7 cells in the logarithmic growth phase were seeded into a 6-well plate and cultured at 37℃ with 5% CO 2 for 12 hours. The treatment group was exposed to Sema3A (1 µg/mL), while the control group received an equivalent volume of PBS. After 24 hours of culture, the cells were collected, washed with PBS, and lysed with 1 mL of TRIzol. The lysate was mixed thoroughly with a pipette and then placed in liquid nitrogen for RNA sequencing. RNA extraction and sequencing were performed by Wuhan Life Origin Biotech Joint Stock Co., Ltd. using the Illumina HiSeq 2500 platform (Illumina, San Diego, CA). Three biological replicates were prepared for each group. The raw sequencing reads were preprocessed to filter out rRNA reads, sequencing adapters, short fragments, and other low-quality reads. Genome mapping was performed, and Cufflinks v2.1.1 was used with reference annotation to generate fragments per kilobase million (FPKM) values for known gene models. Differentially expressed genes were identified using Cuffdiff, with a significance threshold set by the false discovery rate (FDR) to control for multiple tests. Differential expression was determined using the following criterion: FDR ≤ 0.05. Fold changes in gene expression were calculated on the basis of the FPKM values for each sample. Western Blotting The protein expression levels of IL-17RA and IL-17A in RAW264.7 cells and Sema3A-treated RAW264.7 cells were evaluated using western blot analysis. RAW264.7 cells and Sema3A-treated RAW264.7 cells were collected and lysed via a protein extraction solution. The protein concentration in the lysate was quantified using a protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were separated by SDS‒PAGE and transferred onto PVDF membranes. After blocking, the membranes were incubated with primary antibodies at the recommended concentrations for 6 hours. The primary antibodies used included anti-IL-17RA, anti-IL-17A, and anti-β-actin antibodies (all from Sanying, Wuhan, China). β-actin was used as the internal reference gene. The membranes were subsequently incubated with secondary antibodies (anti-rabbit antibodies conjugated to horseradish peroxidase). The blots were washed four times with TBST (Solarbio, Beijing, China) and developed using a chemiluminescence kit (Millipore, Billerica, USA). Statistical analysis All the quantitative results are presented as the means ± standard deviations (SDs) (n ≥ 3). Statistical analysis was performed using a two-tailed unpaired Student’s t tests with GraphPad Prism 8.4 software. For comparisons involving more than two groups, one-way ANOVA was conducted. A value of * p < 0.05 was considered statistically significant. Results Sema3A treatment alleviates aGVHD in mice To investigate the effect of Sema3A on aGVHD, we constructed an aGVHD mouse model, as illustrated in Fig. 1 A, and analyzed the morphological and organ characteristics of each group. On Day 14 posttransplantation, the body weight of the aGVHD group was significantly lower than that of the S-aGVHD group ( p < 0.001, Fig. 1 B). Similarly, the clinical scores in the S-aGVHD group were significantly lower than those in the aGVHD group ( p = 0.0015, Fig. 1 C). As expected, none of the normal mice in the untreated group died, whereas the mice in the irradiated and aGVHD groups exhibited 100% mortality within 2 weeks. The survival rates of the mice in the BM group stabilized after 10 days. Importantly, the survival rate in the S-aGVHD group was significantly improved compared with that in the aGVHD group ( p = 0.0465, Fig. 1 D). These findings indicated that Sema3A treatment significantly reduces aGVHD symptoms, as evidenced by increased body weights, clinical scores, and survival rates (Fig. 1 B- 1 E). Additionally, we evaluated the organ weights (Figure S1 A-S1E) and examined the morphology of the spleen and colon. The spleen and colon morphologies of S-aGVHD mice were better than those of aGVHD mice (Fig. 1 F- 1 G). H&E staining of the colon, ileum, skin, liver and lung revealed severe tissue damage in the colon, ileum and skin of aGVHD mice, which was significantly ameliorated in the S-aGVHD group (Fig. 1 H- 1 K, S1 F- S1 H). These results demonstrated that Sema3A alleviates tissue damage in aGVHD mice. Sema3A alters the T cell ratio and macrophage polarization in aGVHD mice To verify the effects of Sema3A on aGVHD in vivo, we extracted spleen cells from each group of mice for flow cytometry to analyze the ratios of CD4 + and CD8 + cells (Figure S2 A-S2C). The proportions of IFN-γ + and IL-17A + spleen cells were significantly greater in aGVHD mice compared with BM mice (IFN-γ + : 51.4 vs. 31.2, p < 0.001; IL-17A + : 16.0 vs. 5.64, p < 0.001; Fig. 2 A- 2 B). However, these proportions were significantly lower in S-aGVHD mice compared with aGVHD mice (IFN-γ + : 41.6 vs. 51.4, p = 0.0079; IL-17A + : 9.10 vs. 16.0, p = 0.0016; Fig. 2 A- 2 B). A lower proportion of Foxp3 + spleen cells was observed in aGVHD mice than in BM mice (8.66% vs. 5.58%, p = 0.0019; Fig. 2 C), and the proportion of Foxp3 + spleen cells was greater in the S-aGVHD group (13.2% vs. 8.66%, p = 0.0021; Fig. 2 C). Additionally, we analyzed the proportions of M1 and M2 macrophages in the spleens of the mice in each group. Compared with that in BM mice, the proportion of M1 macrophages significantly increased in aGVHD mice (17.2% vs. 7.22%, p < 0.001), whereas the proportion of M2 macrophages decreased in aGVHD mice (5.80% vs. 10.8%, p < 0.0001). Compared with the aGVHD group, the S-aGVHD group had a lower percentage of M1 macrophages (9.66% vs. 17.2%, p < 0.001) and a greater percentage of M2 macrophages (11.3% vs. 5.80%, p < 0.001) (Fig. 2 D- 2 E). Sema3A promotes M2 macrophage polarization in RAW264.7 cells To determine the optimal concentration and duration of Sema3A treatment, we performed cytotoxicity tests on cells treated with different concentrations of Sema3A for different durations; the highest nontoxic treatment was 1 µg/mL Sema3A for 24 hours, which was used in subsequent experiments (Figure S3A). To investigate the effect of Sema3A on macrophage polarization, flow cytometry was used to assess the proportions of M1 and M2 macrophages. Sema3A treatment significantly increased the proportion of M2 macrophages (17.5% vs. 0.15%, p < 0.0001; Fig. 3 A) but had no significant effect on the proportion of M1 macrophages in the absence of inducers (ns; Fig. 3 A). In the presence of the IL-4 M2 macrophage inducer, Sema3A further increased the proportion of M2 macrophages (38.3% vs. 25.1%, p < 0.001; Fig. 3 B). In the presence of the LPS and INF-γ M1 macrophage inducers, Sema3A treatment significantly reduced the proportion of M1 macrophages (48.7% vs. 63.2%, p < 0.001; Fig. 3 C). We also measured the mRNA expression levels of macrophage polarization-related factors. Consistent with the flow cytometry results, Sema3A treatment significantly reduced the mRNA expression levels of the TNF-α (0.548 vs. 1, p = 0.0248) and iNOS (0.535 vs. 1, p < 0.001) M1 macrophage-related factors, whereas the mRNA expression levels of the IL-10 (2.00 vs. 1, p = 0.0015) and Arg-1 (2.858 vs. 1, p < 0.001) M2 macrophage-related factors were significantly increased (Fig. 3 D- 3 G). Taken together, these results suggested that Sema3A promotes RAW264.7 M2 macrophage polarization and suppresses M1 macrophage polarization when an M1 macrophage inducer is present. Sema3A inhibits T cell function by promoting RAW264.7 M2 macrophages polarization The functions of M1 and M2 macrophages are almost oppositional [ 13 ]. M1 macrophages secrete classical inflammatory cytokines [ 14 ]. whereas M2 macrophages release immunosuppressive cytokines that impair T cells function and metabolism [ 15 ]. To investigate the effect of Sema3A on T cells through macrophages, we cocultured Sema3A-treated RAW264.7 cells with CTLL-2 cells. Cell viability assays revealed that coculture with RAW264.7 cells reduced CTLL-2 cell viability (24 h: 90.88% vs. 100%, p = 0.0127; 48 h: 40.60% vs. 100%, p < 0.0001; Fig. 4 A- 4 B). Compared with control RAW264.7 cells, Sema3A-treated RAW264.7 cells significantly inhibited T cell proliferation (24 h: 76.05% vs. 90.88%, p < 0.001; 48 h: 37.03% vs. 40.60%, p = 0.0064; Fig. 4 B). Subsequently, ELISA analysis of T cell cytokine secretion revealed that RAW264.7 cocultures (RCs) reduced the secretion of proinflammatory cytokines, including TNF-a, IFN-g and IL-2 (TNF-a: 255.725 vs. 269.775, p = 0.0031; IFN-γ: 703.875 vs. 747.925, p = 0.0042; IL-2: 190.175 vs. 211.825, p = 0.0183; Fig. 4 C- 4 E). Moreover, these proinflammatory factors were further suppressed in Sema3A-treated RAW264.7 cocultures (STRCs) compared with RCs alone (TNF-a: 234.525 vs. 255.725, p = 0.0022; IFN-g: 649.75 vs. 703.875, p = 0.0025; IL-2: 168.75 vs. 190.175, p = 0.0072, Fig. 4 C- 4 E). In contrast, the secretion of the IL-10 and TGF-b anti-inflammatory cytokines by T cells was increased in RAW264.7 co-cultures (RCs) (IL-10: 309.4 vs. 265.1, p < 0.001; TGF-b: 353.05 vs. 324.5, p = 0.0087, Fig. 4 F- 4 G) and further increased in the Sema3A-treated RAW264.7 cocultures (STRCs) (IL-10: 334.65 vs. 309.4, p = 0.0124; TGF-b: 413.6 vs. 353.05, p < 0.001, Fig. 4 F- 4 G). These findings suggested that Sema3A treatment promotes the secretion of anti-inflammatory cytokines and inhibits the secretion of proinflammatory cytokines. RNA-Seq reveals Sema3A inhibits the IL17/IL17RA signaling pathway in RAW264.7 cells To elucidate the molecular mechanism underlying Sema3A-mediated regulation of macrophage polarization, we conducted transcriptome sequencing on RAW264.7 cells treated with or without Sema3A. Differentially expressed genes were then identified through RNA-Seq by comparing the control and Sema3A-treated groups. Sema3A significantly decreased the mRNA expression of IL-17RA (Fig. 5 A). Gene Ontology (GO) and KEGG pathway analyses further elucidated the potential mechanisms (Fig. 5 C- 5 E) [ 16 ]. KEGG pathway analysis suggested that Sema3A may influence Th17 cell differentiation and the IL-17 signaling pathway. These findings indicated that Sema3A may participate in the IL-17 signaling pathway by transcriptionally regulating IL-17RA and IL-17 expression, thereby affecting macrophage polarization in RAW264.7 cells. Next, we assessed the mRNA expression levels of IL-17RA and IL-17A in Sema3A-treated and control RAW264.7 cells. As shown in Figs. 5 D and 5 E, compared with those in control RAW264.7 cells, the expression levels of IL-17RA (0.477 vs. 1.00, p = 0.0016) and IL-17A (0.378 vs. 1.00, p = 0.0027) in Sema3A-treated RAW264.7 cells were significantly lower. Consistent with these findings, western blot analysis revealed that the protein expression of IL-17RA and IL-17A was also significantly lower in Sema3A-treated RAW264.7 cells than in control cells (IL-17RA: 0.097 vs. 0.469, p < 0.001; Fig. 5 F; IL-17: 0.402 vs. 1.120, p < 0.001; Fig. 5 G). To further validate these results, we measured the secretion of IL-17A using ELISA. Compared with that in RAW264.7 control cells, the secretion of IL-17A in Sema3A-treated RAW264.7 cells was significantly lower (56.99 vs. 73.99, p = 0.0021; Fig. 5 H). Taken together, these results demonstrated that Sema3A downregulates the expression of IL-17RA and IL-17A. IL-17RA overexpression and IL-17A/F treatment suppress M2 macrophage polarization caused by Sema3A treatment To confirm whether Sema3A regulates macrophage polarization through the IL-17 signaling pathway, we analyzed the proportions of M1 and M2 macrophages using flow cytometry after separately overexpressing IL-17 receptor A (IL-17RA) or treating with IL-17A/F in the presence of Sema3A (Fig. 6 A). The proportion of M2 macrophages was significantly lower in the Sema3A + ovIL-17RA group and the Sema3A + IL-17A/F group compared with Sema3A group (Sema3A + ovIL-17RA, 9.55% vs. 17.6%, p < 0.001; Sema3A + IL-17A/F, 4.9% vs. 17.6%, p < 0.001; Fig. 6 A). Additionally, we assessed the mRNA expression of proinflammatory cytokines (TNF-α and iNOS) and anti-inflammatory cytokines (IL-10 and Arg-1). Compared with that in the Sema3A treatment alone group, the mRNA expressions of TNF-α (Sema3A + ovIL-17RA: 1.032 vs. 0.6407, p < 0.001; Sema3A + IL-17A/F; 1.181 vs. 0.6407, p < 0.001; Fig. 6 B) and iNOS (Sema3A + ovIL-17RA: 1.127 vs. 0.5727, p < 0.001; Sema3A + IL-17A/F: 1.522 vs. 0.5727, p < 0.001; Fig. 6 C) were significantly increased. Conversely, the expressions of IL-10 (Sema3A + ovIL-17RA: 1.338 vs. 1.803, p < 0.001; Sema3A + IL-17A/F: 1.288 vs. 1.803, p < 0.001; Fig. 6 D) and Arg-1 (Sema3A + ovIL-17RA: 1.188 vs. 1.717, p < 0.001; Sema3A + IL-17A/F: 1.298 vs. 1.717, p < 0.001; Fig. 6 E) were significantly suppressed. These findings indicated that Sema3A-induced M2 macrophage polarization is mediated through the IL-17 signaling pathway and that disruption of this pathway via IL-17RA overexpression or IL-17A/F treatment abrogates this effect. Discussion aGVHD is a prevalent and serious complication that arises following allo-HSCT [ 17 ]. Research has indicated that high macrophage infiltration in skin lesions is correlated with severe and refractory cases of aGVHD, highlighting the significant role of macrophages in aGVHD pathogenesis [ 18 ]. In aGVHD, most macrophages exhibit the M1 phenotype, which is characterized by the secretion of proinflammatory cytokines that contribute to tissue damage [ 19 ]. However, the influence of dominant cytokines in GVHD on macrophage polarization remains inadequately understood and is controversial. Sema3A has been shown to inhibit the proliferation of immune cells (such as T cells and B cells), promote the production of inflammatory cytokines [ 20 ], and play dual roles in inducing autoimmunity and maintaining immune tolerance. Our previous work demonstrated that Sema3A mediates the immunosuppressive effects of hDMSCs on T lymphocyte responses [ 7 ]. Previous studies have reported that Sema3A promotes the transition of M1 macrophages to the M2 phenotype and inhibits VEGF-induced endothelial cell proliferation, migration, and activation, and Sema3A suppresses tumor cell invasion while promoting apoptosis [ 21 ]. However, the role of Sema3A in aGVHD has not been explored. In the present study, the anti-inflammatory effects of Sema3A were confirmed in an aGVHD mouse model. The present findings demonstrated that Sema3A promotes M2 macrophage polarization and suppresses T cell proliferation and function, ultimately alleviating aGVHD symptoms. The present findings indicated that treatment with Sema3A significantly alleviates symptoms of aGVHD in model mice. Additionally, we observed an increase in the proportion of M2 macrophages in the spleens of aGVHD mice treated with Sema3A. Macrophages are components of innate and adaptive immunity, and they have both proinflammatory and anti-inflammatory effects [ 22 ]. The infiltration of macrophages plays a crucial role in the progression of GVHD, with variations in macrophage numbers and phenotypes occurring at different stages of the disease [ 23 ]. The present results suggested that primary macrophages in patients with aGVHD are preferentially polarized to the proinflammatory M1 phenotype. In addition, M1 macrophages exacerbate aGVHD, whereas M2 macrophages ameliorate aGVHD through the regulation of the immune microenvironment [ 24 ]. M2 macrophages are polarized by IL-4 and characterized by the expression of anti-inflammatory cytokines, such as IL-10 [ 25 ]. Mature macrophages not only synthesize Sema3A but also express its receptor, making macrophages both a source and a target of this factor [ 26 ]. In the present study, treatment of RAW264.7 cells with Sema3A in vitro significantly reduced the expression of M1 macrophage polarization markers, including TNF-a and iNOS, while simultaneously increasing the expression of M2 macrophage polarization markers, such as IL-10 and Arg-1. Moreover, Sema3A-treated RAW264.7 cells inhibited the proliferation of CTLL-2 cells, inhibited the secretion of proinflammatory factors by CTLL-2 cells, and promoted the secretion of anti-inflammatory factors by CTLL-2 cells. These findings provide strong evidence that Sema3A alleviates aGVHD by modulating macrophage polarization. IL-17 plays a crucial role in the regulation of aGVHD, and the IL-17 cytokine family comprises six members, namely, IL-17A, IL-17B, IL-17D, IL-17D, and IL-17E [ 27 ]. The role of Th17 cells and their associated cytokines, such as IL-17A, varies depending on the experimental conditions and GVHD model. For example, one study has reported that IL-17A deficiency ameliorates GVHD [ 28 ], whereas another study has reported that the absence of IL-17A-secreting cells exacerbates GVHD by promoting Th1 differentiation [ 29 ]. Another study has revealed that loss of IL-17 or blockade of the IL-17 signaling pathway reduces the severity of aGVHD in mouse models [ 30 ]. These findings underscore the complex and context-dependent roles of IL-17 in GVHD pathogenesis. The present study revealed that Sema3A-treated RAW264.7 cells present reduced mRNA expression of IL-17A and IL-17RA, accompanied by increased M2 macrophage polarization. Conversely, IL-17RA overexpression in Sema3A-treated RAW264.7 cells inhibits M2 macrophage polarization. Similarly, treatment of RAW264.7 cells with IL-17A/F significantly inhibits the increase of the M2 macrophages proportion caused by Sema3A. These results suggested that Sema3A regulates macrophage polarization through the IL-17 signaling pathway. However, the precise molecular mechanisms by which Sema3A modulates this pathway warrant further investigation. In summary, the present study reveals a novel function of Sema3A in the regulation of aGVHD in a mouse model. The present results indicate that Sema3A plays a pivotal role in promoting macrophage M2 polarization. Furthermore, the present findings provide insights into the potential molecular mechanism by which Sema3A modulates aGVHD, highlighting its interaction with the IL-17 signaling pathway. This study not only advances the understanding of the immunoregulatory role of Sema3A but also identifies it as a promising therapeutic target for modulating macrophage polarization to improve the outcomes of aGVHD treatment. Declarations Ethics approval and consent to participate All experimental procedures were approved by Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University and in compliance with the international regulations on care and protection of laboratory animals (AWUMEC2019050), and the study is reported in accordance with ARRIVE guidelines. Consent for publication Not applicable. Data availability statement The datasets generated and analysed during the current study are available in the Zenodo repository [10.5281/zenodo.17479653, https://zenodo.org/records/17479653]. Competing interests No potential conflict of interest was reported by the authors. Funding This work was supported by Chongqing Young and Middle aged Medical High end Talent Project (No. YXGD202467). Author’s contributions Li Gao designed the study. Hongyun Liu, Song Dong, Hanshan Yang, Bo Liu performed the experiments and acquired the data. Jiali Li, Li Gao, Xi Zhang analyzed the data. Jiali Li, Xixi Xiang, Bing Wang wrote the manuscript. Acknowledgments We thank all relevant partners from Xinqiao Hospital of Army Medical University for insightful comments and critical reading of this manuscript. References Ke Zhao,et al.Reduced morbidity and mortality of cGVHD in patients who received treatment with mesenchymal stromal cells for steroid-resistant aGVHD: long-term follow-up of a randomized phase 3 trial. Experimental Hematology & Oncology 2005, 95. Wang Y, et al .Consensus on the monitoring, treatment, and prevention of leukaemia relapse after allogeneic haematopoietic stem cell transplantation in China: 2024 update. Cancer Lett 2024, 605:217264. Xiao-Su Zhao, et al. Seeking biomakers for acute graft-versus-host disease:where we are and where we are heading？ Biomaker Research 2019, 7：17. Vadasz Z, Haj T, Toubi E. The role of B regulatory cells and Semaphorin3A in atopic diseases. Int Arch Allergy Immunol 2014, 163(4):245-251. Qamar T, Misra DP, Kar S. Semaphorins and its receptors: Emerging cellular biomarkers and therapeutic targets in autoimmune and inflammatory disorders. Life Sci 2025, 361:123281. Wu LF, et al. Nerve growth factor (Ngf) gene-driven semaphorin 3a (Sema3a) expression exacerbates thoracic aortic aneurysm dissection in mice. J Hypertens 2024, 42(5):816-827. Rui Wang BW et al. Sema3A Mediates the Negative Regulatory Effect of Dermal Mesenchymal Stromal Cells on T Lymphocyte Responses. Journal of Biological Regulators and Homeostatic Agents 2024, 38((4)):2933-2943. Hong C, et al. Neutrophils as regulators of macrophage-induced inflammation in a setting of allogeneic bone marrow transplantation. Stem Cell Reports 2022, 17(7):1561-1575. Kurokawa S, et al. Intravenous Semaphorin 3A Administration Maintains Cardiac Contractility and Improves Electrical Remodeling in a Mouse Model of Isoproterenol-Induced Heart Failure. Int Heart J 2023, 64(3):453-461. 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Kanehisa M, Furumichi M, Sato Y, Matsuura Y, Ishiguro-Watanabe M: KEGG: biological systems database as a model of the real world. Nucleic Acids Res 2025, 53(D1):D672-D677. Jamy O, Zeiser R, Chen YB. Novel developments in the prophylaxis and treatment of acute GVHD. Blood 2023, 142(12):1037-1046. Link-Rachner CS, Sockel K, Schuetz C. Established and Emerging Treatments of Skin GvHD. Front Immunol 2022, 13:838494. Wu K, et al . The gut microbial metabolite trimethylamine N-oxide aggravates GVHD by inducing M1 macrophage polarization in mice. Blood 2020, 136(4):501-515. Liu H, et al.Immunosuppressive Regulation of Dendritic Cells and T Cells in Allergic Rhinitis by Semaphorin 3A. Am J Rhinol Allergy 2021, 35(6):846-853. Teng Y, et al. Adenovirus-mediated delivery of Sema3A alleviates rheumatoid arthritis in a serum-transfer induced mouse model. Oncotarget 2017, 8(39):66270-66280. Hanaki R, et al .Donor-derived M2 macrophages attenuate GVHD after allogeneic hematopoietic stem cell transplantation. Immun Inflamm Dis 2021, 9(4):1489-1499. Cheng Q, et al : The S1P1 receptor-selective agonist CYM-5442 reduces the severity of acute GVHD by inhibiting macrophage recruitment. Cell Mol Immunol 2015, 12(6):681-691. Qi Wen, et al .M1 and M2 Macrophages Play Different Roles in the Pathogenesis of Acute Graft-Versus-Host Disease Post-Allotransplant By Modulating Immune Microenvironment. Blood 2020, 136:19-20. Wen Q, et al. G-CSF-induced macrophage polarization and mobilization may prevent acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 2019, 54(9):1419-1433. Kiseleva EP, Rutto KV.Semaphorin 3A in the Immune System: Twenty Years of Study. Biochemistry (Mosc) 2022, 87(7):640-657. Huangfu L, Li R, Huang Y, Wang S.The IL-17 family in diseases: from bench to bedside. Signal Transduct Target Ther 2023, 8(1):402. Y Hong, M Xu, Y Chen, N Li, XLM: Regulatory Effects of IL-17A Monoclonal Antibody and Fibronectin on Chronic Graft-Versus-Host Disease. Blood 2024, 144(3533). Dhital R, et al : Murine cytomegalovirus promotes renal allograft inflammation via Th1/17 cells and IL-17A. Am J Transplant 2022, 22(10):2306-2322. Normanton M, Marti LC.Current data on IL-17 and Th17 cells and implications for graft versus host disease. Einstein (Sao Paulo) 2013, 11(2):237-246. Additional Declarations No competing interests reported. Supplementary Files Fulllengthblots.pdf Bodyweightsofanimals.xlsx FigureS1.tif Figure S1. Histological features of organs in aGVHD mice. (A-E) The spleen (A), heart (B), liver (C), lung (D), kidney (E) index of mice in BM, aGVHD and S-aGVHD group. (F) Typical H&E staining images of liver and lung of mice in BM, aGVHD and S-aGVHD group. Scale bar, 20 μm. (G-H) Liver (G) and lung (H) histopathological score of mice in BM, aGVHD and S-aGVHD group. Quantification data are expressed as mean ± SD (n = 6). * p < 0.05, ** p < 0.01, **** p < 0.0001, NS: not significant. FigureS2.tif Figure S2. Effects of Sema3A on spleen CD4 + and CD8 + in aGVHD mice. The proportion of CD4 + and CD8 + cells in spleen of aGVHD mice, as assessed by CD4 and CD8 staining and flow cytometry. Representative images (A), quantification results of CD4 + cells (B) and CD8 + cells (C) are shown. Quantification data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, **** p < 0.0001, NS: not significant. FigureS3.tif Figure S3. Effects of Sema3A on cell viability of RAW264.7 cells. (A)Viability of RAW264.7 cells treated with indicated concentrations of Sema3A for 24 h and 48 h (n = 4), as detected with Cell Counting Kit-8 (CCK-8). Quantification data are expressed as mean ± SD (n = 4). * p < 0.05, NS: not significant. SupplementaryTable1.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 16 Feb, 2026 Reviewers agreed at journal 08 Feb, 2026 Reviewers invited by journal 06 Feb, 2026 Editor assigned by journal 27 Nov, 2025 Editor invited by journal 20 Nov, 2025 Submission checks completed at journal 19 Nov, 2025 First submitted to journal 19 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7904342","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":588130011,"identity":"053591df-eec9-4981-9491-e7c8e04743d2","order_by":0,"name":"Jiali Li","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiali","middleName":"","lastName":"Li","suffix":""},{"id":588130012,"identity":"88a31032-9018-4416-b9b1-8d422ea25dcf","order_by":1,"name":"Bing Wang","email":"","orcid":"","institution":"Children's Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Wang","suffix":""},{"id":588130013,"identity":"af1dde4f-143c-4424-9aa9-50ddd2da9198","order_by":2,"name":"Xixi Xiang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xixi","middleName":"","lastName":"Xiang","suffix":""},{"id":588130014,"identity":"16960f05-f898-4eab-ad98-c81eb1f3bc0a","order_by":3,"name":"Hongyun Liu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongyun","middleName":"","lastName":"Liu","suffix":""},{"id":588130015,"identity":"c72d9ca0-1d46-495d-9b94-62894f902dd9","order_by":4,"name":"Song Dong","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Song","middleName":"","lastName":"Dong","suffix":""},{"id":588130016,"identity":"ff390d51-886b-4966-8707-60853f65e823","order_by":5,"name":"Hanshan Yang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hanshan","middleName":"","lastName":"Yang","suffix":""},{"id":588130017,"identity":"877df75f-45ec-4135-a68b-bdd654354bf3","order_by":6,"name":"Bo Liu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Liu","suffix":""},{"id":588130018,"identity":"86c5e07e-0faa-42c3-af76-158eabf04bb6","order_by":7,"name":"Xi Zhang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Zhang","suffix":""},{"id":588130019,"identity":"ea629138-c947-45d8-90f1-26f1f3d9e624","order_by":8,"name":"Li Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYLCCBCBmgzH4mZkPPyCggbEBqFKCDcpgkGxnSzMgqAVISMAYDAbneRQk8KmXdz98/MGDmjt1fOxngYxfNvnGh3kYDBhqbKJxaTE8k5bYkHDsmQQbT15iQ2JfmuW2w7wHHjAcS8ttwKWlIcewIYHtMNAvQEZiz2EDs8N8CQaMDYdxa+l/A9TyD6iF/w1Iy38D42YeAwl8WuQlQIa3AbWAGAk/DhgYMBPQYiDxLHFGYt9hyTaJN4YzEhuSDSQOAwM5AY9f5PuTD3z88e0wv3x/jsHHH3/sDPj7Dx9+8KHGBrctB5B5jG1QRgIO5WBbUM36g0fpKBgFo2AUjFgAAPPjXwQThzr5AAAAAElFTkSuQmCC","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2025-10-20 09:38:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7904342/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7904342/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102421383,"identity":"21e17690-06fb-4d5c-9632-0d1d47e87e54","added_by":"auto","created_at":"2026-02-11 13:46:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":18865825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment of Sema3A alleviated aGVHD in a mouse model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eExperimental protocol of aGVHD mice. BM cells, splenic cells or Sema3A were intravenously injected into mice. Data were collected on day 14.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003eBody weight of aGVHD mice in each group every 2 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eClinical scores of aGVHD mice in BM, aGVHD and S-aGVHD group every 2 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eSurvival of aGVHD mice in each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003eTypical morphological images of mice in BM, aGVHD and S-aGVHD group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F-G) \u003c/strong\u003eSpleen (F) and colon (G) morphological images mice in BM, aGVHD and S-aGVHD group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003eTypical H\u0026amp;E staining images of the ileum, colon and skin of mice in BM, aGVHD and S-aGVHD group. Scale bar, 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I-K) \u003c/strong\u003eIleum (I), colon (J) and skin (K) histopathological score of mice in BM, aGVHD and S-aGVHD group. Quantification data are expressed as mean ± SD (n = 6). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/e77e3309d38a1701a26bf661.png"},{"id":102421381,"identity":"1d4b5762-1e99-4601-b069-cd3012368a73","added_by":"auto","created_at":"2026-02-11 13:46:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3942385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of Sema3A on spleen T cell subsets and macrophages in aGVHD mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe proportion of CD4\u003csup\u003e+\u003c/sup\u003e and IFN-g\u003csup\u003e+\u003c/sup\u003e cells in spleen of aGVHD mice, as assessed by CD4 and IFN-g staining and flow cytometry. Representative images (left) and quantification results (right) are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eThe proportion of CD4\u003csup\u003e+\u003c/sup\u003e and IL-17A\u003csup\u003e+\u003c/sup\u003e cells in spleen of aGVHD mice, as assessed by CD4 and IL-17A staining and flow cytometry. Representative images (left) and quantification results (right) are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e The proportion of CD25\u003csup\u003e+\u003c/sup\u003e and Foxp3-g\u003csup\u003e+\u003c/sup\u003e cells in spleen of aGVHD mice, as assessed by CD25 and Foxp3 staining and flow cytometry. Representative images (left) and quantification results (right) are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e The proportion of M1 macrophages in spleen of aGVHD mice, as assessed by CD86 staining and flow cytometry. Representative images (left) and quantification results (right) are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e The proportion of M2 macrophages in spleen of aGVHD mice, as assessed by CD206 staining and flow cytometry. Representative images (left) and quantification results (right) are shown. Quantification data are expressed as mean ± SD (n = 3). **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, NS: not significant.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/2bf767f3ab977342db3ccede.png"},{"id":102421382,"identity":"792a8fcc-cf94-4a55-9ef0-a67ba0159f59","added_by":"auto","created_at":"2026-02-11 13:46:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13410824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSema3A promotes polarization of M2 macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eThe M1 and M2 macrophages proportion of RAW264.7 cells treated with 1 μg/mL Sema3A for 24 h, as assessed by CD86 / CD206 staining and flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003eThe M1 and M2 macrophages proportion of RAW264.7 cells treated with 20 ng/mL IL-4 and 1 μg/mL Sema3A for 48 h, as assessed by CD86 / CD206 staining and flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003eThe M1 and M2 macrophages proportion of RAW264.7 cells treated with 2.5 ng/mL IFN-γ, 100 ng/mL LPS and 1 μg/mL Sema3A for 24 h, as assessed by CD86 / CD206 staining and flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D-G)\u003c/strong\u003e TNF-a (D), iNOS (E), IL-10 (F), Arg-1 (G) mRNA expression level in RAW264.7 cells treated with 1 μg/mL Sema3A for 24 h, as determined using qRT-PCR.\u003c/p\u003e\n\u003cp\u003eCells treated with PBS were used as controls. β-actin was used for qRT-PCR normalization. Quantification data are expressed as mean ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, NS: not significant.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/63b7e5876770f571a7a6239c.png"},{"id":102745732,"identity":"dc5df2c2-277d-46c7-b35b-5d1021e1f5db","added_by":"auto","created_at":"2026-02-16 08:53:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1675170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eM2 macrophages induced by Sema3A inhibit the proliferation and function of T cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eExperimental protocol of co-culture Sema3A-treated RAW264.7 cells and CTLL-2 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eViability of CTLL-2 cells in CTLL-2, RC and STRC groups for 24 h and 48 h, as detected with Cell Counting Kit-8 (CCK-8). Co-cultured cell groups were defined as RAW264.7+CTLL-2 (RC), Sema3A-treated RAW264.7 (treated with 1 μg/mL Sema3A) +CTLL-2 (STRC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C-G) \u003c/strong\u003eThe TNF-a (C), IFN-g(D), IL-2 (E), IL-10 (F), TGF-b(G) level of CTLL-2 cells in CTLL-2, RC and STRC groups, as detected by ELISA. Quantification data are expressed as mean ± SD (n = 4). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/63519be4cf1e0b7e94cb9824.png"},{"id":102421385,"identity":"31a65d36-e1f3-4d1e-b0e4-a22f34414633","added_by":"auto","created_at":"2026-02-11 13:46:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3570639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSema3A suppresses the IL17/IL17RA signaling axis in RAW264.7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRAW264.7 cells were treated with Sema3A for 24 h. Expression profiles of macrophages after Sema3A treatment revealed by high-throughput RNA-seq assay. Volcano map showing differential genes between Sema3A and Control groups. There are 87 up-regulated genes and 51 down-regulated genes in total. Notably, Cish, MgI2, H2-Aa, H2-Ea and H2-Eb1 were top 5 up-regulated genes while Il17ra, Ltb, Gm42793, Ndst1 and Mir6973a were top 5 down-regulated genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003eGene Ontology (GO) functional analysis reveals that after treated with Sema3A, Sema3A-regulated transcriptional events were mainly related with leukocyte mediated immunity, interaction between leukocyte, MHC class II protein complex assembly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003eKyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis reveals that exogenous administration of Sema3A will modulate macrophage Graft-versus-host disease, Cytokine-cytokine receptor interaction, cellular differentiation and function of helper T cells and Antigen processing and presentation processes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D-E)\u003c/strong\u003e IL-17RA (D) and IL-17A (E) mRNA expression level in RAW264.7 cells treated with 1 mg/mL Sema3A for 24 h, as determined using qRT-PCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F-G)\u003c/strong\u003e Western blot showed that the expression of IL-17RA (F) and IL-17A (G) was significantly down-regulated in Sema3A-treated macrophages. β-actin was used as western blotting loading control. Blot representative of three independent experiments. Representative images (left) and quantification results (right) are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003eThe IL-17A level in RAW264.7 cells treated with 1 mg/mL Sema3A for 24 h, as determined using ELISA. Cells treated with PBS were used as controls. β-actin was used for qRT-PCR normalization. Quantification data are expressed as mean ± SD (n = 3). **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/cc3079dab11b5a61c8f8731c.png"},{"id":102421384,"identity":"27963f80-d4ee-4e71-9a54-c9afc60e5801","added_by":"auto","created_at":"2026-02-11 13:46:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":20611306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of IL-17RA or supplementation of IL-17A/F inhibited M2 macrophage polarization induced by Sema3A treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe M1 and M2 macrophages proportion of RAW264.7 cells treated for 24 h, as assessed by CD86 / CD206 staining and flow cytometry. Sema3A, 1 μg/mL; ovIL-17RA, 2 μg/well; IL-17A/F, 100 ng/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B-E)\u003c/strong\u003e TNF-a (B), iNOS (C), IL-10 (D), Arg-1 (E)mRNA expression level in RAW264.7 cells treated for 24 h, as determined using qRT-PCR. Sema3A, 1 μg/mL; ovIL-17RA, 2 μg/well; IL-17A/F, 100 ng/mL. Quantification data are expressed as mean ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/d8c71f447bd71a6dcd91d7d3.png"},{"id":102750534,"identity":"16052635-5ea2-4ec8-a990-b62cb3bd3e2d","added_by":"auto","created_at":"2026-02-16 09:20:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":51181832,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/a8c62f09-e369-42cf-889c-bd07eef77b92.pdf"},{"id":102421378,"identity":"860f6ae7-fafa-4b69-8f8b-3d69fa5abad0","added_by":"auto","created_at":"2026-02-11 13:46:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":109050,"visible":true,"origin":"","legend":"","description":"","filename":"Fulllengthblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/4bf3156f8739f170fdb71bdb.pdf"},{"id":102421377,"identity":"74731395-a2b7-45c7-9eb7-4201f7b6ff6a","added_by":"auto","created_at":"2026-02-11 13:46:48","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11631,"visible":true,"origin":"","legend":"","description":"","filename":"Bodyweightsofanimals.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/b4cc956f0e39e432ccc3539f.xlsx"},{"id":102421387,"identity":"49dc6aa4-b9b5-4d08-bb09-7b92c5d05f5f","added_by":"auto","created_at":"2026-02-11 13:46:49","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4159532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1. Histological features of organs in aGVHD mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-E)\u003c/strong\u003e The spleen (A), heart (B), liver (C), lung (D), kidney (E) index of mice in BM, aGVHD and S-aGVHD group.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) Typical H\u0026amp;E staining images of liver and lung of mice in BM, aGVHD and S-aGVHD group. Scale bar, 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G-H)\u003c/strong\u003e Liver (G) and lung (H) histopathological score of mice in BM, aGVHD and S-aGVHD group. Quantification data are expressed as mean ± SD (n = 6). *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, NS: not significant.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/7bb278e6a39cb931d7de8f2e.tif"},{"id":102421388,"identity":"d1029915-700d-4247-afd1-15521d761c5b","added_by":"auto","created_at":"2026-02-11 13:46:49","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1420122,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2. Effects of Sema3A on spleen CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e in aGVHD mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proportion of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e cells in spleen of aGVHD mice, as assessed by CD4 and CD8 staining and flow cytometry. Representative images (A), quantification results of CD4\u003csup\u003e+\u003c/sup\u003e cells (B) and CD8\u003csup\u003e+\u003c/sup\u003e cells (C) are shown. Quantification data are expressed as mean ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, NS: not significant.\u003c/p\u003e","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/aa73d604e81abc2dfbf5327b.tif"},{"id":102421386,"identity":"5f7f2934-b8ef-4660-9462-44e26b85b60b","added_by":"auto","created_at":"2026-02-11 13:46:49","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":418184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S3. Effects of Sema3A on cell viability of RAW264.7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eViability of RAW264.7 cells treated with indicated concentrations of Sema3A for 24 h and 48 h (n = 4), as detected with Cell Counting Kit-8 (CCK-8). Quantification data are expressed as mean ± SD (n = 4). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, NS: not significant.\u003c/p\u003e","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/576b8e708b9dd48b382351eb.tif"},{"id":102745612,"identity":"b76d9483-f2f0-48a3-ab16-aa38d3cfcfab","added_by":"auto","created_at":"2026-02-16 08:52:44","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":15051,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7904342/v1/173cd955807ce5c570683f67.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Semaphorin 3A reduces the severity of aGVHD by promoting M2 macrophage polarization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, aGVHD is a significant cause of morbidity and mortality among allogeneic HCT recipients [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Owing to the complex mechanisms underlying aGVHD, existing therapeutic approaches are inadequate for its complete prevention.\u003c/p\u003e \u003cp\u003eSemaphorin 3A (Sema3A) is a secreted protein that belongs to the class 3 semaphorin family. Over the past decade, Sema3A has emerged as one of the most active semaphorins (SEMAs) involved in regulating inflammatory responses. The increased expression levels of Sema3A in differentiated giant cells and activated T cells suggest that it plays a pivotal role in immune-mediated diseases [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In our previous study, we showed that Sema3A secreted by human dermal mesenchymal stromal cells decreases the expression of downstream factors, such as cyclin D1 and CDK4, thereby inhibiting T cell proliferation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the role of Sema3A in aGVHD and its specific regulatory mechanisms remain unexplored.\u003c/p\u003e \u003cp\u003eIn the present study, we established aGVHD model mice and treated them with Sema3A. Moreover, we integrated histopathological assessment, macrophage polarization profiling (in vivo and in vitro), and transcriptome pathway analyses and identified IL-17 signaling as a key component in the mechanism by which Sema3A mediates aGVHD pathogenesis.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMouse model of GVHD\u003c/h2\u003e \u003cp\u003ePreparation of mouse bone marrow cells and splenocytes: Male C57BL/6J donor mice (8\u0026ndash;12 weeks old) were purchased from Charles River Laboratory (Beijing, China). The femora and tibiae were washed 2\u0026ndash;3 times with PBS containing 2% FBS, and the ends of the bones were removed using ophthalmic scissors. A 10 mL syringe needle was then inserted into the bone marrow cavity, which was flushed with PBS containing 2% FBS. The resulting solution was collected in a 60 mm cell culture dish and passed through a 200-mesh cell strainer after gentle grinding. The cell suspension was transferred to a 50 mL centrifuge tube and centrifuged at 1500 rpm at 4℃ for 5 minutes. The supernatant was discarded, and the pellet was resuspended in PBS containing 2% FBS and filtered through a cell strainer. The bone marrow cell suspension was thus successfully prepared. For splenocyte preparation, the spleens were washed 2\u0026ndash;3 times with PBS containing 2% FBS, carefully ground using a syringe plunger, and suspended in PBS containing 2% FBS. The splenocyte suspension was prepared as described above.\u003c/p\u003e \u003cp\u003eEstablishment of the mouse model: A mouse model of MHC-mismatched HSCT was generated by transplanting bone marrow (BM) cells from C57BL/6J mice into male BALB/c mice previously irradiated with X-rays at a dose of 4.0 Gy on the day prior to transplantation and irradiated with X-rays at a dose of 3.5 Gy on the day of transplantation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The recipient male BALB/c mice were reconstituted with 5 \u0026times; 10⁶ BM cells from the donor C57BL/6 mice, either with or without 3 \u0026times; 10⁶ donor spleen cells. To investigate the effect of Sema3A (Sino Biological Inc., Beijing) on aGVHD, intravenous administration of Sema3A (0.5 mg/kg) was initiated concurrently with splenocyte and BM transplantation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], followed by repeated dosing every 3 days through 9 days posttransplantation. The experiment comprised the following 5 groups: the control group, which included 6 normal mice; the X-ray group, which included 10 irradiated mice; the BM group, in which 8 mice received 3 \u0026times; 10⁶ BM cells only; the aGVHD group, in which 19 mice received a mixture of 5 \u0026times; 10⁶ BM cells and 3 \u0026times; 10⁶ spleen cells from donor C57BL/6 mice; and the Sema3A-treated aGVHD (S-aGVHD) group, in which 18 mice received a mixture of 5 \u0026times; 10⁶ BM cells and 3 \u0026times; 10⁶ spleen cells from donor C57BL/6 mice plus Sema3A (0.5 mg/kg). Body weights and clinical scores were observed and recorded every two days up to 14 days posttransplantation, while survival rates were assessed up to 20 days after transplantation. At 14 days posttransplantation, the severity of clinical aGVHD was evaluated using a scoring system [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Histopathological changes in aGVHD target organs (liver, lung, skin, colon, and small intestine) were observed using HE staining, and histopathological scores for acute GVHD were determined following previously published protocols [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The euthanasia of mice is carried out by intraperitoneal injection of excessive phetobarbital sodium (St. Louis, MO, USA). According to the body weight of the mice, phenobarbital sodium solution was intraperitoneally injected at a dose of 150 mg/kg. The mice were observed at room temperature for 5\u0026ndash;8 minutes to determine death. Cervical dislocation was then performed as a secondary measure to ensure death. All animal experiments were approved by the Institutional Laboratory Animal Care and Use Committee of Xinqiao Hospital.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell lines and Cell culture:\u003c/h3\u003e\n\u003cp\u003eRAW264.7 and CTLL-2 cells were obtained from the Procell Cell Bank (Wuhan, China). RAW264.7 cells were cultured in DMEM (GIBCO, USA) supplemented with 10% fetal bovine serum (FBS; ExCellBio, Suzhou, China) and 1% penicillin‒streptomycin (P/S; Gibco, USA). CTLL-2 cells were maintained in RPMI-1640 medium (GIBCO, USA) supplemented with 10% FBS, 100 U/mL recombinant IL-2 (iCell Bioscience, Shanghai, China) and 1% P/S. Additional details regarding the cell culture conditions are provided in the supplementary information.\u003c/p\u003e\n\u003ch3\u003eCell viability\u003c/h3\u003e\n\u003cp\u003eRAW264.7 viability in response to varying concentrations of Sema3A: RAW264.7 cells (2 \u0026times; 10⁵ cells/mL) were treated with different concentrations of Sema3A (0.01, 0.1, 1, or 10 \u0026micro;g/mL) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Each group had three replicate wells. The cells were incubated at 37℃ with 5% CO₂ for 24 or 48 hours. Subsequently, 10 \u0026micro;L of CCK-8 detection solution (Biosharp, Anhui, China) was added to each well, except for the untreated RAW264.7 wells. After 2 hours of incubation, the absorbance was measured at a wavelength of 450 nm using a microplate reader.\u003c/p\u003e \u003cp\u003eEffect of Sema3A expressed by macrophages on T cells: The viability of CTLL-2 cells was evaluated after co-culture with RAW264.7 cells treated with Sema3A. The following three experimental groups were established: CTLL-2 cells, CTLL-2 cells cocultured with RAW264.7 cells (RCs), and CTLL-2 cells cocultured with Sema3A-treated RAW264.7 cells (STRCs). RAW264.7 cells or RAW264.7 cells treated with 1 \u0026micro;g/mL Sema3A in cell-free medium were inoculated in the upper chamber of a Transwell insert after 24 hours of culture, and the medium was replaced. Simultaneously, CTLL-2 cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/mL) were seeded in the lower chamber of the Transwell system. After 24 hours of coculture, the cells from both chambers were transferred to 96-well plates, and 10 \u0026micro;L of CCK-8 solution was added to each well. Following a 2-hour incubation, the absorbance at 450 nm was measured using a microplate reader.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and quantitative real-time PCR\u003c/h3\u003e\n\u003cp\u003eThe mRNA expression of macrophage polarization-related factors, including TNF-α, iNOS, IL-10, Arg-1, IL-17RA, and IL-17A, was evaluated in vitro. RAW264.7 cells and Sema3A-treated RAW264.7 cells were cultured as described above. Total RNA was extracted using TRIzol reagent (Solarbio, Beijing, China) following the manufacturer\u0026rsquo;s instructions. The extracted RNA was reverse transcribed into complementary DNA (cDNA) using a reverse transcription kit (Vazyme, Nanjing, China) and a cDNA synthesis kit (Takara, Dalian, China). Real-time quantitative PCR (qRT‒PCR) was performed using the TB Green\u0026reg; Fast qPCR Mix Kit (Takara, Dalian, China). Relative mRNA expression levels were calculated using the 2-∆∆CT method. The primer sequences used in the present study are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h3\u003e\n\u003cp\u003eThe concentrations of IL-17A in RAW264.7 cells and Sema3A-treated RAW264.7 cells were measured using an ELISA kit (RX203066M; Ruixin, Fujian, China). The concentrations of TNF-α, IFN-γ, IL-2, IL-10 and TGF-β secreted by CTLL-2, RC or STRC in the culture supernatants were quantified using ELISA kits (SEA133Mu, HEA049Mu, SEA073Mu, SEA056Mu, and RX104768H Ruixin, Fujian, China) according to the manufacturer\u0026rsquo;s instructions. The absorbance was measured at a wavelength of 450 nm using a microplate reader.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eFlow cytometry was used to analyze the expression of CD86 and CD206 in vitro to investigate the role of Sema3A in macrophage polarization. RAW264.7 cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL) were seeded into 6-well plates with 2 mL of medium per well and incubated for 12 hours at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Sema3A was added to the cells at a final concentration of 1 \u0026micro;g/mL, while PBS was administered to the other groups as a vehicle control. After 24 hours, the cells were harvested, washed twice with PBS, and incubated with antibodies against CD86 and CD206 for 15 minutes. After two washes with PBS, the cells were passed through a cell sorter and analyzed using a flow cytometer (Beckman Coulter, Inc., California, USA). To further investigate the effect of Sema3A on macrophage polarization in the presence of macrophage inducers, RAW264.7 cells were treated with M1 inducers, namely LPS (100 ng/mL, Beyotime Biotechnology, Shanghai) and IFN-γ (2.5 ng/mL, Beyotime Biotechnology), or M2 inducers, namely IL-4 (20 ng/mL, Procell, Wuhan) or Sema3A (1 \u0026micro;g/mL). Following a 24 and 48 hours culture period, the cells were harvested\u003c/p\u003e \u003cp\u003efor analysis of CD86 and CD206 expression levels via flow cytometry. Additionally, an IL-17RA overexpression plasmid (ovIL-17RA) was used to verify the mechanism by which Sema3A influences macrophage polarization. The six experimental groups included untreated macrophages (control) and macrophages treated with Sema3A (1 \u0026micro;g/mL), ovIL-17RA (2 \u0026micro;g, Sangon, Shanghai, China), Sema3A (1 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;ovIL-17RA (2 \u0026micro;g), IL-17A/F (100 ng/mL, MCE, New Jersey, USA), or Sema3A (1 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;IL-17A/F (100 ng/mL). The proportions of M1 and M2 macrophages were analyzed using flow cytometry.\u003c/p\u003e \u003cp\u003eSplenocytes were extracted from mice in all experimental groups to investigate the effect of Sema3A on macrophage polarization in vivo. Flow cytometry was used to analyze the proportions of splenocytes containing proinflammatory cytokines (IFN-γ\u003csup\u003e+\u003c/sup\u003e and IL-17A\u003csup\u003e+\u003c/sup\u003e) and the Foxp3\u003csup\u003e+\u003c/sup\u003e anti-inflammatory factor. Single-cell suspensions were prepared from spleen tissue, and 2 \u0026micro;L of BD GolgiPlug\u0026trade; reagent was added per 1 mL of suspension. The mixture was incubated at 37\u0026deg;C in 5% CO₂ for 4\u0026ndash;6 hours and then centrifuged at 1500 rpm for 5 minutes, after which the supernatant was discarded. Surface antibodies (CD3, CD4, CD8, and CD25) were added to the resuspended cells in 50 \u0026micro;L of dye dilution buffer, and the cells were incubated at 4 ℃ in the dark for 20‒30 minutes. After washing twice with staining buffer, 100 \u0026micro;L of BD cell fixation/membrane permeabilization solution was added, and the cells were incubated at 4℃ for 20 minutes. The cells were washed twice with 1\u0026times; BD Perm/Wash buffer (1 mL per wash) and then incubated with 50 \u0026micro;L of 1\u0026times; BD Perm/Wash buffer containing intracellular factor antibodies (IL-17A, IFN-γ, and Foxp3) for 30 minutes at 4 ℃ in the dark. After two additional washes, the cells were resuspended in staining buffer and analyzed using flow cytometry. To determine the M1/M2 macrophage ratio in mouse spleen tissue, single-cell suspensions were stained with antibodies (CD11b, F4/80, CD86, and CD206). After mixing and incubating at 4\u0026deg;C for 20 minutes, the cells were centrifuged to remove the supernatant, washed twice, resuspended in staining buffer, and analyzed using flow cytometry.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA sequencing (RNA-seq)\u003c/h3\u003e\n\u003cp\u003eRAW264.7 cells in the logarithmic growth phase were seeded into a 6-well plate and cultured at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e for 12 hours. The treatment group was exposed to Sema3A (1 \u0026micro;g/mL), while the control group received an equivalent volume of PBS. After 24 hours of culture, the cells were collected, washed with PBS, and lysed with 1 mL of TRIzol. The lysate was mixed thoroughly with a pipette and then placed in liquid nitrogen for RNA sequencing. RNA extraction and sequencing were performed by Wuhan Life Origin Biotech Joint Stock Co., Ltd. using the Illumina HiSeq 2500 platform (Illumina, San Diego, CA). Three biological replicates were prepared for each group. The raw sequencing reads were preprocessed to filter out rRNA reads, sequencing adapters, short fragments, and other low-quality reads. Genome mapping was performed, and Cufflinks v2.1.1 was used with reference annotation to generate fragments per kilobase million (FPKM) values for known gene models. Differentially expressed genes were identified using Cuffdiff, with a significance threshold set by the false discovery rate (FDR) to control for multiple tests. Differential expression was determined using the following criterion: FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05. Fold changes in gene expression were calculated on the basis of the FPKM values for each sample.\u003c/p\u003e\n\u003ch3\u003eWestern Blotting\u003c/h3\u003e\n\u003cp\u003eThe protein expression levels of IL-17RA and IL-17A in RAW264.7 cells and Sema3A-treated RAW264.7 cells were evaluated using western blot analysis. RAW264.7 cells and Sema3A-treated RAW264.7 cells were collected and lysed via a protein extraction solution. The protein concentration in the lysate was quantified using a protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were separated by SDS‒PAGE and transferred onto PVDF membranes. After blocking, the membranes were incubated with primary antibodies at the recommended concentrations for 6 hours. The primary antibodies used included anti-IL-17RA, anti-IL-17A, and anti-β-actin antibodies (all from Sanying, Wuhan, China). β-actin was used as the internal reference gene. The membranes were subsequently incubated with secondary antibodies (anti-rabbit antibodies conjugated to horseradish peroxidase). The blots were washed four times with TBST (Solarbio, Beijing, China) and developed using a chemiluminescence kit (Millipore, Billerica, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the quantitative results are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SDs) (n\u0026thinsp;\u0026ge;\u0026thinsp;3). Statistical analysis was performed using a two-tailed unpaired Student\u0026rsquo;s t tests with GraphPad Prism 8.4 software. For comparisons involving more than two groups, one-way ANOVA was conducted. A value of *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSema3A treatment alleviates aGVHD in mice\u003c/h2\u003e \u003cp\u003eTo investigate the effect of Sema3A on aGVHD, we constructed an aGVHD mouse model, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, and analyzed the morphological and organ characteristics of each group. On Day 14 posttransplantation, the body weight of the aGVHD group was significantly lower than that of the S-aGVHD group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Similarly, the clinical scores in the S-aGVHD group were significantly lower than those in the aGVHD group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0015, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). As expected, none of the normal mice in the untreated group died, whereas the mice in the irradiated and aGVHD groups exhibited 100% mortality within 2 weeks. The survival rates of the mice in the BM group stabilized after 10 days. Importantly, the survival rate in the S-aGVHD group was significantly improved compared with that in the aGVHD group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0465, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These findings indicated that Sema3A treatment significantly reduces aGVHD symptoms, as evidenced by increased body weights, clinical scores, and survival rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Additionally, we evaluated the organ weights (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-S1E) and examined the morphology of the spleen and colon. The spleen and colon morphologies of S-aGVHD mice were better than those of aGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). H\u0026amp;E staining of the colon, ileum, skin, liver and lung revealed severe tissue damage in the colon, ileum and skin of aGVHD mice, which was significantly ameliorated in the S-aGVHD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS1\u003c/span\u003eH). These results demonstrated that Sema3A alleviates tissue damage in aGVHD mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eSema3A alters the T cell ratio and macrophage polarization in aGVHD mice\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo verify the effects of Sema3A on aGVHD in vivo, we extracted spleen cells from each group of mice for flow cytometry to analyze the ratios of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e cells (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-S2C). The proportions of IFN-γ\u003csup\u003e+\u003c/sup\u003e and IL-17A\u003csup\u003e+\u003c/sup\u003e spleen cells were significantly greater in aGVHD mice compared with BM mice (IFN-γ\u003csup\u003e+\u003c/sup\u003e: 51.4 vs. 31.2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; IL-17A\u003csup\u003e+\u003c/sup\u003e: 16.0 vs. 5.64, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, these proportions were significantly lower in S-aGVHD mice compared with aGVHD mice (IFN-γ\u003csup\u003e+\u003c/sup\u003e: 41.6 vs. 51.4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0079; IL-17A\u003csup\u003e+\u003c/sup\u003e: 9.10 vs. 16.0, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0016; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). A lower proportion of Foxp3\u003csup\u003e+\u003c/sup\u003e spleen cells was observed in aGVHD mice than in BM mice (8.66% vs. 5.58%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0019; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and the proportion of Foxp3\u003csup\u003e+\u003c/sup\u003e spleen cells was greater in the S-aGVHD group (13.2% vs. 8.66%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0021; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Additionally, we analyzed the proportions of M1 and M2 macrophages in the spleens of the mice in each group. Compared with that in BM mice, the proportion of M1 macrophages significantly increased in aGVHD mice (17.2% vs. 7.22%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas the proportion of M2 macrophages decreased in aGVHD mice (5.80% vs. 10.8%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Compared with the aGVHD group, the S-aGVHD group had a lower percentage of M1 macrophages (9.66% vs. 17.2%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and a greater percentage of M2 macrophages (11.3% vs. 5.80%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSema3A promotes M2 macrophage polarization in RAW264.7 cells\u003c/h2\u003e \u003cp\u003eTo determine the optimal concentration and duration of Sema3A treatment, we performed cytotoxicity tests on cells treated with different concentrations of Sema3A for different durations; the highest nontoxic treatment was 1 \u0026micro;g/mL Sema3A for 24 hours, which was used in subsequent experiments (Figure S3A). To investigate the effect of Sema3A on macrophage polarization, flow cytometry was used to assess the proportions of M1 and M2 macrophages. Sema3A treatment significantly increased the proportion of M2 macrophages (17.5% vs. 0.15%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) but had no significant effect on the proportion of M1 macrophages in the absence of inducers (ns; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In the presence of the IL-4 M2 macrophage inducer, Sema3A further increased the proportion of M2 macrophages (38.3% vs. 25.1%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In the presence of the LPS and INF-γ M1 macrophage inducers, Sema3A treatment significantly reduced the proportion of M1 macrophages (48.7% vs. 63.2%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). We also measured the mRNA expression levels of macrophage polarization-related factors. Consistent with the flow cytometry results, Sema3A treatment significantly reduced the mRNA expression levels of the TNF-α (0.548 vs. 1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0248) and iNOS (0.535 vs. 1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) M1 macrophage-related factors, whereas the mRNA expression levels of the IL-10 (2.00 vs. 1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0015) and Arg-1 (2.858 vs. 1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) M2 macrophage-related factors were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Taken together, these results suggested that Sema3A promotes RAW264.7 M2 macrophage polarization and suppresses M1 macrophage polarization when an M1 macrophage inducer is present.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSema3A inhibits T cell function by promoting RAW264.7 M2 macrophages polarization\u003c/h2\u003e \u003cp\u003eThe functions of M1 and M2 macrophages are almost oppositional [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. M1 macrophages secrete classical inflammatory cytokines [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. whereas M2 macrophages release immunosuppressive cytokines that impair T cells function and metabolism [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To investigate the effect of Sema3A on T cells through macrophages, we cocultured Sema3A-treated RAW264.7 cells with CTLL-2 cells. Cell viability assays revealed that coculture with RAW264.7 cells reduced CTLL-2 cell viability (24 h: 90.88% vs. 100%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0127; 48 h: 40.60% vs. 100%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Compared with control RAW264.7 cells, Sema3A-treated RAW264.7 cells significantly inhibited T cell proliferation (24 h: 76.05% vs. 90.88%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 48 h: 37.03% vs. 40.60%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0064; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Subsequently, ELISA analysis of T cell cytokine secretion revealed that RAW264.7 cocultures (RCs) reduced the secretion of proinflammatory cytokines, including TNF-a, IFN-g and IL-2 (TNF-a: 255.725 vs. 269.775, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0031; IFN-γ: 703.875 vs. 747.925, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0042; IL-2: 190.175 vs. 211.825, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0183; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Moreover, these proinflammatory factors were further suppressed in Sema3A-treated RAW264.7 cocultures (STRCs) compared with RCs alone (TNF-a: 234.525 vs. 255.725, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0022; IFN-g: 649.75 vs. 703.875, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0025; IL-2: 168.75 vs. 190.175, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0072, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In contrast, the secretion of the IL-10 and TGF-b anti-inflammatory cytokines by T cells was increased in RAW264.7 co-cultures (RCs) (IL-10: 309.4 vs. 265.1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; TGF-b: 353.05 vs. 324.5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0087, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) and further increased in the Sema3A-treated RAW264.7 cocultures (STRCs) (IL-10: 334.65 vs. 309.4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0124; TGF-b: 413.6 vs. 353.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). These findings suggested that Sema3A treatment promotes the secretion of anti-inflammatory cytokines and inhibits the secretion of proinflammatory cytokines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA-Seq reveals Sema3A inhibits the IL17/IL17RA signaling pathway in RAW264.7 cells\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanism underlying Sema3A-mediated regulation of macrophage polarization, we conducted transcriptome sequencing on RAW264.7 cells treated with or without Sema3A. Differentially expressed genes were then identified through RNA-Seq by comparing the control and Sema3A-treated groups. Sema3A significantly decreased the mRNA expression of IL-17RA (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Gene Ontology (GO) and KEGG pathway analyses further elucidated the potential mechanisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. KEGG pathway analysis suggested that Sema3A may influence Th17 cell differentiation and the IL-17 signaling pathway. These findings indicated that Sema3A may participate in the IL-17 signaling pathway by transcriptionally regulating IL-17RA and IL-17 expression, thereby affecting macrophage polarization in RAW264.7 cells. Next, we assessed the mRNA expression levels of IL-17RA and IL-17A in Sema3A-treated and control RAW264.7 cells. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, compared with those in control RAW264.7 cells, the expression levels of IL-17RA (0.477 vs. 1.00, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0016) and IL-17A (0.378 vs. 1.00, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0027) in Sema3A-treated RAW264.7 cells were significantly lower. Consistent with these findings, western blot analysis revealed that the protein expression of IL-17RA and IL-17A was also significantly lower in Sema3A-treated RAW264.7 cells than in control cells (IL-17RA: 0.097 vs. 0.469, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eF; IL-17: 0.402 vs. 1.120, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). To further validate these results, we measured the secretion of IL-17A using ELISA. Compared with that in RAW264.7 control cells, the secretion of IL-17A in Sema3A-treated RAW264.7 cells was significantly lower (56.99 vs. 73.99, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0021; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Taken together, these results demonstrated that Sema3A downregulates the expression of IL-17RA and IL-17A.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIL-17RA overexpression and IL-17A/F treatment suppress M2 macrophage polarization caused by Sema3A treatment\u003c/h2\u003e \u003cp\u003eTo confirm whether Sema3A regulates macrophage polarization through the IL-17 signaling pathway, we analyzed the proportions of M1 and M2 macrophages using flow cytometry after separately overexpressing IL-17 receptor A (IL-17RA) or treating with IL-17A/F in the presence of Sema3A (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The proportion of M2 macrophages was significantly lower in the Sema3A\u0026thinsp;+\u0026thinsp;ovIL-17RA group and the Sema3A\u0026thinsp;+\u0026thinsp;IL-17A/F group compared with Sema3A group (Sema3A\u0026thinsp;+\u0026thinsp;ovIL-17RA, 9.55% vs. 17.6%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Sema3A\u0026thinsp;+\u0026thinsp;IL-17A/F, 4.9% vs. 17.6%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Additionally, we assessed the mRNA expression of proinflammatory cytokines (TNF-α and iNOS) and anti-inflammatory cytokines (IL-10 and Arg-1). Compared with that in the Sema3A treatment alone group, the mRNA expressions of TNF-α (Sema3A\u0026thinsp;+\u0026thinsp;ovIL-17RA: 1.032 vs. 0.6407, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Sema3A\u0026thinsp;+\u0026thinsp;IL-17A/F; 1.181 vs. 0.6407, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and iNOS (Sema3A\u0026thinsp;+\u0026thinsp;ovIL-17RA: 1.127 vs. 0.5727, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Sema3A\u0026thinsp;+\u0026thinsp;IL-17A/F: 1.522 vs. 0.5727, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) were significantly increased. Conversely, the expressions of IL-10 (Sema3A\u0026thinsp;+\u0026thinsp;ovIL-17RA: 1.338 vs. 1.803, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Sema3A\u0026thinsp;+\u0026thinsp;IL-17A/F: 1.288 vs. 1.803, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) and Arg-1 (Sema3A\u0026thinsp;+\u0026thinsp;ovIL-17RA: 1.188 vs. 1.717, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Sema3A\u0026thinsp;+\u0026thinsp;IL-17A/F: 1.298 vs. 1.717, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) were significantly suppressed. These findings indicated that Sema3A-induced M2 macrophage polarization is mediated through the IL-17 signaling pathway and that disruption of this pathway via IL-17RA overexpression or IL-17A/F treatment abrogates this effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eaGVHD is a prevalent and serious complication that arises following allo-HSCT [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Research has indicated that high macrophage infiltration in skin lesions is correlated with severe and refractory cases of aGVHD, highlighting the significant role of macrophages in aGVHD pathogenesis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In aGVHD, most macrophages exhibit the M1 phenotype, which is characterized by the secretion of proinflammatory cytokines that contribute to tissue damage [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the influence of dominant cytokines in GVHD on macrophage polarization remains inadequately understood and is controversial.\u003c/p\u003e \u003cp\u003eSema3A has been shown to inhibit the proliferation of immune cells (such as T cells and B cells), promote the production of inflammatory cytokines [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and play dual roles in inducing autoimmunity and maintaining immune tolerance. Our previous work demonstrated that Sema3A mediates the immunosuppressive effects of hDMSCs on T lymphocyte responses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previous studies have reported that Sema3A promotes the transition of M1 macrophages to the M2 phenotype and inhibits VEGF-induced endothelial cell proliferation, migration, and activation, and Sema3A suppresses tumor cell invasion while promoting apoptosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the role of Sema3A in aGVHD has not been explored. In the present study, the anti-inflammatory effects of Sema3A were confirmed in an aGVHD mouse model. The present findings demonstrated that Sema3A promotes M2 macrophage polarization and suppresses T cell proliferation and function, ultimately alleviating aGVHD symptoms.\u003c/p\u003e \u003cp\u003eThe present findings indicated that treatment with Sema3A significantly alleviates symptoms of aGVHD in model mice. Additionally, we observed an increase in the proportion of M2 macrophages in the spleens of aGVHD mice treated with Sema3A. Macrophages are components of innate and adaptive immunity, and they have both proinflammatory and anti-inflammatory effects [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The infiltration of macrophages plays a crucial role in the progression of GVHD, with variations in macrophage numbers and phenotypes occurring at different stages of the disease [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The present results suggested that primary macrophages in patients with aGVHD are preferentially polarized to the proinflammatory M1 phenotype. In addition, M1 macrophages exacerbate aGVHD, whereas M2 macrophages ameliorate aGVHD through the regulation of the immune microenvironment [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. M2 macrophages are polarized by IL-4 and characterized by the expression of anti-inflammatory cytokines, such as IL-10 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Mature macrophages not only synthesize Sema3A but also express its receptor, making macrophages both a source and a target of this factor [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the present study, treatment of RAW264.7 cells with Sema3A in vitro significantly reduced the expression of M1 macrophage polarization markers, including TNF-a and iNOS, while simultaneously increasing the expression of M2 macrophage polarization markers, such as IL-10 and Arg-1. Moreover, Sema3A-treated RAW264.7 cells inhibited the proliferation of CTLL-2 cells, inhibited the secretion of proinflammatory factors by CTLL-2 cells, and promoted the secretion of anti-inflammatory factors by CTLL-2 cells. These findings provide strong evidence that Sema3A alleviates aGVHD by modulating macrophage polarization.\u003c/p\u003e \u003cp\u003eIL-17 plays a crucial role in the regulation of aGVHD, and the IL-17 cytokine family comprises six members, namely, IL-17A, IL-17B, IL-17D, IL-17D, and IL-17E [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The role of Th17 cells and their associated cytokines, such as IL-17A, varies depending on the experimental conditions and GVHD model. For example, one study has reported that IL-17A deficiency ameliorates GVHD [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], whereas another study has reported that the absence of IL-17A-secreting cells exacerbates GVHD by promoting Th1 differentiation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Another study has revealed that loss of IL-17 or blockade of the IL-17 signaling pathway reduces the severity of aGVHD in mouse models [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These findings underscore the complex and context-dependent roles of IL-17 in GVHD pathogenesis. The present study revealed that Sema3A-treated RAW264.7 cells present reduced mRNA expression of IL-17A and IL-17RA, accompanied by increased M2 macrophage polarization. Conversely, IL-17RA overexpression in Sema3A-treated RAW264.7 cells inhibits M2 macrophage polarization. Similarly, treatment of RAW264.7 cells with IL-17A/F significantly inhibits the increase of the M2 macrophages proportion caused by Sema3A. These results suggested that Sema3A regulates macrophage polarization through the IL-17 signaling pathway. However, the precise molecular mechanisms by which Sema3A modulates this pathway warrant further investigation.\u003c/p\u003e \u003cp\u003eIn summary, the present study reveals a novel function of Sema3A in the regulation of aGVHD in a mouse model. The present results indicate that Sema3A plays a pivotal role in promoting macrophage M2 polarization. Furthermore, the present findings provide insights into the potential molecular mechanism by which Sema3A modulates aGVHD, highlighting its interaction with the IL-17 signaling pathway. This study not only advances the understanding of the immunoregulatory role of Sema3A but also identifies it as a promising therapeutic target for modulating macrophage polarization to improve the outcomes of aGVHD treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University and in compliance with the international regulations on care and protection of laboratory animals\u0026nbsp;(AWUMEC2019050), and the study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available in the Zenodo repository [10.5281/zenodo.17479653, https://zenodo.org/records/17479653].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Chongqing Young and Middle aged Medical High end Talent Project (No. YXGD202467).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLi Gao designed the study. Hongyun Liu, Song Dong, Hanshan Yang, Bo Liu\u0026nbsp;performed the experiments and acquired the data. Jiali Li, Li Gao,\u0026nbsp;Xi Zhang analyzed the data. Jiali Li, Xixi Xiang, Bing Wang wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all relevant partners from Xinqiao Hospital of Army Medical University for insightful comments and critical reading of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKe Zhao,et al.Reduced morbidity and mortality of cGVHD in patients who received treatment with mesenchymal stromal cells for steroid-resistant aGVHD: long-term follow-up of a randomized phase 3 trial. 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While Semaphorin 3A (Sema3A) is implicated in immune regulation during inflammatory conditions, its role in aGVHD pathogenesis was unclear. Using an aGVHD mouse model, intravenous recombinant Sema3A administration demonstrated therapeutic effects, significantly reducing tissue damage in target organs observed via histopathological analysis. Flow cytometry and immunofluorescence staining revealed an increased proportion of M2 macrophages (CD206⁺) in vivo. In vitro treatment of RAW264.7 murine macrophages with Sema3A promoted polarization towards the M2 phenotype, characterized by elevated expression of Arg1 and IL-10, and inhibited T cell function. Transcriptome sequencing of treated macrophages identified suppressed IL-17 signaling, which was validated by qPCR and western blot showing significant decreases in mRNA and protein levels of both IL-17A and its receptor IL-17RA. These findings indicate that Sema3A mitigates aGVHD by driving M2 macrophage polarization through inhibition of the IL-17 signaling pathway, thereby suppressing pathogenic T cell responses, highlighting its potential as a therapeutic target.\u003c/p\u003e","manuscriptTitle":"Semaphorin 3A reduces the severity of aGVHD by promoting M2 macrophage polarization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 13:46:38","doi":"10.21203/rs.3.rs-7904342/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-02-16T08:45:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24046571740235277992225509792202265727","date":"2026-02-08T23:42:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-06T14:23:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-27T14:27:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-20T13:37:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-19T12:53:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-19T12:49:12+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":"1afcdade-967f-4487-9185-e96031fb273b","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62563242,"name":"Biological sciences/Cell biology"},{"id":62563243,"name":"Health sciences/Diseases"},{"id":62563244,"name":"Biological sciences/Immunology"},{"id":62563245,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-02-11T13:46:39+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 13:46:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7904342","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7904342","identity":"rs-7904342","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}