Enhanced NRP1 Expression in Dendritic Cells of Systemic Lupus Erythematosus and Its Impact on T Cell Proliferation

Enhanced NRP1 Expression in Dendritic Cells of Systemic Lupus Erythematosus and Its Impact on T Cell Proliferation | 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 Enhanced NRP1 Expression in Dendritic Cells of Systemic Lupus Erythematosus and Its Impact on T Cell Proliferation Yunjung Choi, Eun-Gyeong Lee, Kyoung Min Kim, Wan-Hee Yoo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3209000/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Neuropilin-1 (NRP1) is a transmembrane glycoprotein that acts as a receptor of class III/IV semaphorins known to play a role in the pathogenesis of autoimmune diseases. To date there has been only limited research into the role NRP1 plays in autoimmune inflammatory rheumatic diseases, including systemic lupus erythematosus (SLE). This study aimed to investigate the clinical and pathogenetic roles of NRP1 expression in lupus mouse models and patients with SLE. Methods NRP1 expression was measured by flow cytometry, polymerase chain reaction (PCR), and immunofluorescence assay using peripheral blood mononuclear cells (PBMCs) taken from both healthy controls and patients with SLE, as well as dendritic cells and renal tissues of both control mice and TLR-7 agonist-induced lupus mice. The correlation between NRP1 expression in PBMCs and disease activity markers were analyzed in patients with SLE (n = 57). To determine the effects of NRP1 on dendritic cells on T cells, as well as their mechanism, a proliferation assay was performed by flow cytometry, and the underlying signaling, including the MAPKs and NF-κB pathway, were examined with immunoblotting. Results The expression of NRP1 in dendritic cells and the kidneys was significantly higher in the lupus murine group than in the control group. The dendritic cells in the patients with SLE also showed a markedly higher expression of NRP1 than those of the healthy controls. The correlation analysis showed a significant positive relationship between NRP1 expression and disease activity markers, which included SLEDAI-2K score, as well as C3, C4 and anti-dsDNA antibody titers. The NRP1 antagonist (EG00229) decreased the capacity of dendritic cells on the proliferation of T cells under the condition of TLR7 agonist stimulation. It also downregulated the phosphorylation of ERK1/2 and NF-κB in dendritic cells. Conclusion Our results show that NRP1 is highly expressed in the dendritic cells of SLE patients, and its expression is significantly correlated with known disease activity markers. The inhibition of NRP1 in dendritic cells diminishes the proliferation of T cells, an effect that is mediated by the suppression of MAPKs and NF-kB signaling. These results indicate that dendritic cells with enhanced NRP1 expression alter immune functions by increasing T cell proliferation as part of the pathogenesis of SLE; accordingly, NRP1 may be a potential target in the search for a treatment for SLE. Biological sciences/Immunology Health sciences/Diseases Health sciences/Rheumatology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Systemic lupus erythematosus (SLE) is a potentially fatal autoimmune disease that may affect nearly every organ and tissue ( 1 ). Its development requires a complex interaction between genetic, environmental, viral, and hormonal factors. Through the occurrence of the complex interaction, immune dysregulation is initiated at the level of cytokines, T cells, B cells, and macrophages, after which immune tolerance to the self-antigen is lost and autoimmunity occurs. Along with these processes, complement activation, immune complex accumulation, and consequent tissue inflammation and damage result in a self-sustained autoimmune pathway ( 2 ). Dendritic cells have been focused on their pathogenic roles in the induction and progression of the broken immunological tolerance ( 3 ). High levels of self-RNA and self-DNA from apoptotic cells induce type I interferon (IFN) release from plasmacytoid dendritic cells, and promotes its own activation as well as the maturation of myeloid dendritic cells in an autocrine manner ( 4 ). Upon activation, dendritic cells exhibit an increased ability to present the self-antigen and induce the stronger proliferation of T cells ( 5 ). A consequent immune cascade that includes B cell activation, auto-antibody production, and immune complex formation occurs through at self-amplification loop. Dendritic cells are therefore regarded as a significant contributor to the pathogenesis of SLE by bridging the aberrant innate and adaptive immunity ( 6 ). Neuropilin-1 (NRP1) is a type I transmembrane protein with a molecular weight of 120 kDa that acts as a co-receptor for several extra-cellular ligands ( 7 ). The protein was initially identified as neuronal cell guidance and was first discovered as part of research to develop and regenerate Xenopus optic nerves ( 8 ). Recently, a wide range of physiological roles and pathologic involvements for NRP1 have been identified, including those in cardiovascular development, cell migration, angiogenesis, and cancer pathogenesis ( 9 ). Furthermore, NRP1 has attracted considerable attention for its immunoregulatory functions. NRP1 is also known as a receptor for class 3 semaphorins, which are intimately involved in the pathogenesis of autoimmune diseases ( 10 , 11 ). Furthermore, NRP1 expression in various kinds of immune cells, including myeloid cells such as monocytes, macrophages ( 12 , 13 ) and myeloid dendritic cells ( 14 ), plasmacytoid dendritic cells ( 15 , 16 ), and a small subset of regulatory T cells ( 17 ), has been confirmed. NRP1 expression in dendritic cells and resting T cells has been clearly documented in humans, and emerging evidence indicates that it plays a role in the initiation of primary immune responses by mediating the interaction between dendritic cells and T cells ( 18 ). For example, a co-culture of T lymphocytes with dendritic cells pretreated with blocking NRP1 antibody showed significantly decreased dendritic cell-induced T cell proliferation ( 14 ). These findings support the hypothesis that NRP1 is an essential component of dendritic cells that initiates their primary immune responses, and as such it may explain the aberrant immunity they show in response to autoimmune rheumatic diseases such as SLE. To date, however, studies regarding NRP1 expression in patients with SLE and its role in the pathogenesis of this disease are scarce. Building on previous findings suggesting the existence of NRP1 expression in the dendritic cells and their pathogenic role in SLE, this study was performed to investigate these aspects of NRP1 in lupus mouse models and patients with SLE. Materials and Methods Patients with SLE and healthy controls A total of 57 patients with SLE and 60 healthy controls were recruited from Jeonbuk National University Hospital in Jeonju, South Korea. All patients were between 18 to 75 years of age and fulfilled the 2012 Systemic Lupus International Collaborating Clinics (SLICC) Classification Criteria for SLE ( 19 ). The patients receiving over 1mg/kg/day prednisolone at the time of blood sampling were excluded from this study. SLE disease activity was evaluated using the SLE Disease Activity Index 2000 update (SLEDAI-2K) ( 20 ). Sixty age and gender-matched healthy control subjects were recruited. Written informed consent was obtained from all participants. This study was conducted consistent with the Declaration of Helsinki and was approved by the Ethics Committee of the Jeonbuk National University Hospital (CUH 2018-08-005-004). Lupus murine model induction BALB/C mice (7-9-week-old females) were purchased from Central Lab Animal Inc. All mice were maintained under a 12 h:12 h light/dark cycle, with the temperature at 24°C, and the humidity at 60% on the standard diet in a conventional cage. We adopted toll-like receptor (TLR) 7 agonist-induced mice as SLE murine models for our experiments. This model presents with the phenotypic findings of lupus including an elevated autoantibody levels, immune complex-deposited nephritis, and marked splenomegaly ( 21 ), and has been widely used for experiments in research into SLE pathogenesis ( 22 , 23 ). The lupus murine model was induced through the topical application of a TLR7 agonist. The skin on the back of the mice was treated topically 3 times weekly over 4 weeks with 100 µg of Resiquimod (R848, Enzo Life Sciences, NY, USA) mixed with 100 µL of acetone. The control group was treated with 100 µL of acetone topically in the same manner. At the end of the treatment, mice were euthanized via intraperitoneal administration of ketamine (75 mg/ kg) plus xylazine (10 mg/kg), followed by the collection of a blood sample through cardiac puncture. All experiments were conducted in accordance with National Institutes of Health guidelines and Animal Research: Reporting of In Vivo Experiments guidelines. The study protocol was approved by the Institutional Animal Care and Use Committee of Jeonbuk National University (JBNU 2020 − 0115). mRNA extraction and Real-Time PCR Total RNA was extracted using an RNA Extraction Kit (Bioneer, Daejeon, Korea) according to the manufacturer’s protocol, and cDNA was generated by using an Accupower RocketScript RT Premix, Rnase H Minus kit (Bioneer, Daejeon, Korea). Quantitative PCR was performed with SYBR Green Real time PCR Master mix (TOYOBO, Osaka, Japan) and a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Grand Island, NY, USA). Amplification conditions were as follows: 50℃ for 2 min, then 95℃ for 10 min for 1 cycle, followed by 40 cycles of 95℃ for 15 sec and 60℃ for 1 min. The following primers were used (forward and reverse, respectively): for humans, GAPDH, 5’-tggtatcgtggaaggactca-3’ and 5’-gcagggatgatgttctggag-3’; NRP1, 5’-gcctgactcaaatcctccag-3’ and 5’-acctggtgttttctgtccac-3’; ILT-4, 5’-tcagggcaaacactggacat-3’ and 5’-tcacggcagcatagaggttt-3’; for mice, GAPDH, 5’-acttgaagggtggagccaaa-3’ and 5’-gcaggatgcattgctgacaa-3’; NRP1, 5’-5’-gcctgactcaaatcctccag-3’ and 5’-acctggtgttttctgtccac-3’; ILT-4, 5’- tcagggcaaacactggacat-3’ and 5’-tcacggcagcatagaggttt-3’; for mice, GAPDH, 5’-acttgaagggtggagccaaa-3’ and 5’-gcaggatgcattgctgacaa-3’; NRP1, 5’- tgctctggaatgttgggcat-3’ and 5’-tggtcaccagacggatgttt-3’; Nephrin, 5’- aacatccagctcgtcagcat-3’ and 5’- aaagccaggtttccactcca-3’. The relative expression of the target genes was determined by normalizing the expression of each gene to GAPDH using the ΔΔCt method. Cell isolation and culture The peripheral blood of each participant was obtained via venipuncture. 10 ml of blood samples were diluted 1:1 with PBS (pH 7.4) and layered on Lymphoprep (Serumwerk Bernburg AG, Oslo, Norway) following centrifugation at 20°C for 30 min. PBMCs were isolated from the interphase between the Ficoll-Paque and serum layers, and washed twice with PBS. CD4 + T cells and monocytes were isolated from PBMCs using a MACS isolation kit (BD Biosciences, San Jose, CA, United States) by negative isolation through a CD8, CD11b, CD16, CD19, CD36, CD56, CD123, and CD235a γδ TCR biotinylated antibody cocktail (Human CD4 T Lymphocyte Enrichment Set; BD Biosciences, San Jose, CA, USA) as well as a CD3, CD45RA, CD19, CD56, and CD235a biotinylated antibody cocktail (Human Monocyte Enrichment Set; BD Biosciences, San Jose, CA, USA). CD4 + T cells or monocytes were purified using a magnetic board of over 90% purity, as assessed by the flow cytometry of CD4 + T cells with anti-Human CD4-FITC (Tonbo Biosciences, CA, USA) and monocytes with mouse anti-human CD14-FITC (BD pharmigen, USA). Cells were cultured in RPMI 1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Life Technologies Limited, UK), 100 U/mL penicillin (Gibco, Life Technologies Limited, UK), 100 mg/mL streptomycin (Gibco, Life Technologies Limited, UK), and 2 mM L-glutamine (Gibco, Life Technologies Limited, UK). Cells were grown at 37°C in a humidified 5% CO₂ atmosphere. Dendritic cell differentiation Isolated monocytes from human PBMCs were cultured in RPMI 1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Life Technologies Limited, UK), penicillin (100 U/mL) (Gibco, Life Technologies Limited, UK), streptomycin (100 mg/mL) (Gibco, Life Technologies Limited, UK), 2 mM L-glutamine, rh-granulocyte-macrophage colony-stimulating factor (GM-CSF) (50 ng/mL) (Peprotech, New Jersey Cranbury, USA), and rh-interleukin (IL)-4 (10 ng/mL) (Peprotech, New Jersey Cranbury, USA) over 6 days in a 96-well plate (Corning-falcon, USA) to obtain monocyte-derived dendritic cells (Mo-DC) as previously described ( 24 ). The generation of bone marrow-derived dendritic cells (BDMCs) was performed using a method previously described elsewhere ( 25 ). Briefly, bone marrow cells were isolated and prepared from the bone marrow of the femur and tibia in BALB/C mice as single-cell suspension. The aliquots of 1×10 6 bone marrow cells were placed in 24-well plates and cultured in RPMI 1640 containing 10% heat-inactivated FBS (Gibco, Life Technologies Limited, UK), 100 U/mL penicillin (Gibco, Life Technologies Limited, UK), 100 mg/mL streptomycin (Gibco, Life Technologies Limited, UK), 2 mM L-glutamine in the presence of rm-GM-CSF (50 ng/mL, Peprotech, New Jersey Cranbury, USA), and rm-IL-4 (10 ng/mL, Peprotech, New Jersey Cranbury, USA). On day 3, two-thirds of the medium was replaced. On day 5, the non-adherent cells were transferred into a six-well plate and cultured for two additional days. On day 7, the acquired dendritic cell morphology was confirmed by microscope and checked for CD11c fluorescence (BD Bioscience, San Jose, CA, USA) using flow cytometry. Then the harvested cells were used in the subsequent experiment. Glomeruli isolation The kidneys were harvested, and the medulla was removed carefully. The renal cortexes were minced and digested with collagenase type V (Sigma, dissolved in HBSS) in a water bath at 37℃ for 20 minutes with pipetting at 5-min intervals. The digested mixture was transferred with cold Hanks Balanced Salt Solution (HBSS, Sigma-Aldrich) onto 100-µm, 70-µm, and 40-µm cell strainers to remove cell debris and small tubular fragments. New 40-µm cell strainers were then used to remove the remnant debris, and the retained glomeruli were transferred into another clean culture dish to remove the few residual tubular fragments. The floating glomeruli were collected and centrifuged at 290g at 4℃ for 5 minutes. The glomeruli were examined under a microscope and their purity confirmed by the expression of the glomerular marker using Real-time PCR and Western blot analysis. Renal histopathologic assessment and immunofluorescence assay Kidneys were harvested after perfusion with saline, and were fixed in 4% formaldehyde for 24 hr, embedded in paraffin, and separated into fixed sections of 10-µm thickness. After being stained with hematoxylin and eosin (H&E), and periodic acid–Schiff (PAS), renal pathology was evaluated according to a previously described scoring scale ( 26 ). Briefly, we assessed glomerular pathology in 20 glomeruli per kidney and interstitial/tubular pathology in randomly selected 10 high-power fields, semi-quantitatively on a scale of 0–3. For the immunofluorescence assay, the 10-µm-thick acetone fixed sections were stained with rabbit anti-mouse IgG-heavy and light chain antibody, FITC-conjugated (Bethyl Laboratories Inc., USA). Slides were then incubated at room temperature for 1 hr. Slides were stained with rat-anti mouse C3 (Abcam, Cambridge, United Kingdom) and then were incubated at 4℃ overnight. The slides were stained with goat anti-rat IgG-heavy and light chain antibody FITC-conjugated and then incubated for 1 hr at room temperature in a dark humid box. They were then mounted with mounting medium using DAPI (Abcam, Cambridge, United Kingdom). Staining for IgG and NRP1 was performed in the same manner, and the antibodies used are listed below. Slides were observed with a confocal microscope (Zeiss LSM 880, Oberkochen, Germany) and analyzed by ZEN 3.2 Zeiss Microscopy GmbH software (version 3.2.0.0000) (Zeiss LSM 880, Oberkochen, Germany). The fluorescence intensity (MFI) of the glomeruli in different groups was calculated using ImageJ software (National Institutes of Health, USA) for each section with 10 glomeruli. 1) For IgG staining: rabbit anti-mouse IgG-heavy and light chain antibody FITC-conjugated (Bethyl Laboratories Inc., USA), 2). For NRP1 staining: NRP1 antibody (eBioscience, Carlsbad, CA, USA) (1:200), with goat anti-rat IgG-heavy and light chain antibody FITC-conjugated (Bethyl Laboratories Inc., USA) Immunocytochemistry assay BMDCs were placed in a 35 mm confocal dish (SPL Life Sciences Co., Korea) at a number of 0.5×10 6 cells and incubated at 37°C for 2 hrs. The cells were fixed using 3.7% paraformaldehyde in PBS pH 7.4 for 10 minutes at room temperature and washed three times with cold PBS. The cells were incubated for 10 min with PBS containing 0.1% Triton X-100 at room temperature for permeabilization and washed in PBS three times. The washed cells were incubated with 1% BSA in PBST (PBS + 0.1% Tween 20) for 30 minutes to block the unspecific binding of the antibodies. The cells were stained in the diluted antibody in 1% BSA in PBST overnight at 4°C. After washing the cells in PBS, they were stained with the secondary antibody in 1% BSA in PBS for 1 hr at room temperature in the dark and washed in PBS. The cells were mounted with mounting medium with DAPI (Abcam). The following antibodies were used in the immunocytochemical analysis: Neuropilin-1 antibody (eBioscience, Carlsbad, CA, USA), Goat anti-rabbit IgG, highly Cross-Adsorbed Secondary Antibody, goat anti-rat IgG-heavy, and light chain antibody FITC-conjugated (Bethyl Laboratories Inc., USA). The slides were observed with a confocal microscope (Zeiss LSM 880, Oberkochen, Germany) and analyzed by ZEN 3.2 Zeiss Microscopy GmbH software (version 3.2.0.0000) (Zeiss, Oberkochen, Germany). Fluorescence intensity was calculated using ImageJ software (National Institutes of Health, USA) and the scores were evaluated through methods described previously ( 27 ). Cell cytotoxicity and proliferation assay BMDCs were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine at 37°C under 5% CO 2 . BMDCs were seeded at 5×10 4 cells/well in a 96-well plate and treated with various doses of R848 (0, 1, 2.5, 5, and 10 µg/mL) (Enzo Life Sciences, NY, USA) and EG00229 (Tocris Bioscience, United Kingdom) that had been dissolved in 0.2M NaOH (0, 10, 30, 100 and 300 µM) (Tocris Bioscience, United Kingdom) for 24 hrs. The 10 µL/well of the Lysis solution was added to the control group as a positive control. The cytotoxicity of the cells was evaluated using a LDH Cell Cytotoxicity Assay Kit (DoGenBio, Seoul, Korea). To assess the cell proliferation, BMDCs were seeded (5×10 4 cells/mL) in a 96-well plate. The BMDCs were pretreated with EG00229, an NRP1 antagonist (Tocris Bioscience, United Kingdom) (100 µM), for 2 hrs, followed by treatment with R848 for 24 hrs and 48 hrs. Cell proliferation was measured using a Cell Counting Kit-8 (Enzo Life Sciences, NY, USA). Absorbance was measured using a microplate reader (Bio-Rad) at 450nm. Serologic analysis and Urinalysis Anti-dsDNA antibodies (FUJIFILM Wako Shibayagi Corporation, Ishihara, Japan), urine albumin and creatinine (Exocell, Philadelphia, USA), and Human Neuropilin-1 Quantikine (CUSABIO, Houston, TX, USA) were quantified by ELISA consistent with the manufacturer’s instructions. NRP1 inhibition and co-culture condition CD4 + CD25 − T cells and CD11c + dendritic cells were isolated from the PBMCs of participants by BD FACSAria ІІІ flow cytometer sorting (> 95%). The CD4 + CD25 − T cells from the spleen of the BALB/C mice were sorted by BD FACSAria ІІІ (> 95%). Dendritic cells were pre-treated with EG00229 (100 µM) for 2 hrs and then stimulated by R848 (5 ug/mL) for 0.5 hr. CD4 + CD25 − T cells were incubated in the presence of anti-CD3 and anti-CD28 antibody coated beads (eBioscience, Carlsbad, CA, USA) as described previously ( 28 ). After 24 hrs of incubation, the T cells were labeled with 2.5 µM CFSE using a Cell Trace Cell Proliferation Kit (Molecular probes) and then co-cultured for 5 days with dendritic cells with target cells: the dendritic cell to target cell ratio was 1:1. The stained cells were detected using an FACSCalibur flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo software (TreeStar Inc., Ashland, OR, United States). Western blot analysis Proteins were extracted from isolated glomeruli and BMDCs using RIPA buffer containing protease inhibitors. (Thermo scientific, USA). Proteins were separated on SDS-PAGE gels (10%) and then transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon, Millipore). Membranes were blocked in 5% non-fat dry milk in TBST for 1 hr at room temperature with shaking and then probed overnight at 4℃ with primary antibodies (1:1,000) against Nephrin (Invitrogen, USA), Neuropilin-1 (Cell Signaling, Danvers, MA, USA), p-STAT1 (Cell Signaling, Danvers, MA, USA), STAT1 (Cell Signaling, Danvers, MA, USA), p-STAT3 (Cell Signaling, Danvers, MA, USA), STAT3 (Cell Signaling, Danvers, MA, USA), p-ERK 1/2 (Cell Signaling, Danvers, MA, USA), ERK 1/2 (Cell Signaling, Danvers, MA, USA), p-JNK (Cell Signaling, Danvers, MA, USA), JNK (Cell Signaling, Danvers, MA, USA), p-P38 (Cell Signaling, Danvers, MA, USA), P38 (Cell Signaling, Danvers, MA, USA), p-NF-κB, NF-κB (Cell Signaling, Danvers, MA, USA), p-IκB (Cell Signaling, Danvers, MA, USA), IκB (Cell Signaling, Danvers, MA, USA), and β-actin (Bioworld Technology Inc, USA). After three washes, the membranes were incubated with HRP-conjugated rabbit or mouse secondary antibody (1:3,000) at room temperature for 2 hrs. The reactive proteins were detected using an ECL (GE healthcare, USA) and the intensity of the bands was quantified using a Vilber Lourmat Fusion Fx7 system (Vilber Lourmat, Collégien, France). Statistical analysis The Mann-Whitney test with Bonferroni’s correction was used to compare two groups and the Kruskal-Wallis test was used to compare three or more groups for the analysis of pathology scores and IF scoring. All other parametrically distributed data, including flow cytometry, immunoblotting, and cell proliferation assay, were analyzed by a one-way ANOVA with a Turkey post hoc test. Spearmans’ rho test was applied to the analysis of correlation. SPSS 22.0 software (SPSS Inc., Chicago, Illinois, U.S.A.) was used for statistical analysis. P values of < 0.05 were considered statistically significant and results were expressed as mean ± SEM. Results Elevated expression of NRP1 in CD4 + T cells and dendritic cells in the SLE patients The expression of NRP1 was analyzed in CD4 + T cells and monocyte-derived dendritic cells (Mo-DCs) from the patients with SLE as well as their gender- and age-matched healthy controls using PCR. These cells were isolated from the PBMCs of lupus patients with a SLEDAI-2K score above 10 (indicating high disease activity). Cell purity was confirmed by flow cytometry, reaching about 80%. NRP1 expression was significantly higher in CD4 + T cells (9.45 ± 2.36 vs. 1.00 ± 0.16, p = 0.01) and Mo-DCs (8.10 ± 4.33 vs. 1.00 ± 0.37, p = 0.01) of lupus patients compared to the healthy controls [Fig. 1 A and 1 B]. The enhanced expression of NRP1 in CD4 + T cells and Mo-DCs in the lupus patients suggests a possible role for NRP1 in the pathogenesis of SLE. Thus, further experiments using the lupus murine and in vitro model were conducted to investigate the expression level of NRP1 and its roles in the pathogenesis of SLE. Induction of SLE murine model with TLR7 agonist To assess of NRP1 expression and its role in lupus pathogenesis, we induced a lupus model through the topical application of a TLR7 agonist ( 21 ). It is well documented that dendritic cells play a pivotal role in the pathogenesis of SLE through IFNα production upon TLR7 ligation. TLR7 activation leads to an increase in IFNα releases and, consequently, B cell expansion, the production of autoantibodies, and the activation of myeloid cells and autoreactive cells ( 29 , 30 ). It is also well demonstrated that this lupus murine model, upon TLR7 activation, has severe lupus-like systemic autoimmunity such as nephritis, and as a consequence, this animal model has become a well-established lupus murine model ( 23 , 31 , 32 ). First, we induced the animal model with topical application of a TLR7 agonist, resiquimod (R848), and confirmed the lupus-like systemic autoimmunity in this model. Phenotypically, the R848-treated mice showed marked splenomegaly and increased weight of the spleen [Fig. 2 A]. The immunofluorescence analysis of the glomeruli showed dominantly increased deposits of IgG and C3 in the R848-treated group compared to that of the control group [Fig. 2 B], suggesting glomerulonephritis with immune complex deposition, which is frequently associated with lupus renal involvement. Furthermore, histopathological assessment of the kidney in the R848-treated group revealed obvious mesangial hypercellularity and monocellular cell infiltration, as well as a significantly higher histologic score compared to the control group [Fig. 2 C]. The urine albumin to creatinine ratio (UACR) was significantly higher in the R848-treated group than in the control group (66.68 ± 16.81 vs. 11.73 ± 1.47, p = 0.02) [Fig. 2 D]. The anti-dsDNA antibody titer was also markedly higher in the R848-treated group than in the control group (526.53 ± 95.29 vs. 1.05 ± 0.16, p = 0.01) [Fig. 2 D]. These findings suggest that this animal model accurately reflects the manifestation of systemic autoimmunity observed in SLE. Consequently, we have verified the appropriateness of utilizing the TLR-7 agonist-induced lupus model to study the contribution of NRP1 in the progression of SLE. Increased expression of NRP1 in the glomeruli of the lupus murine model To investigate NRP1 expression in the lupus model and compare it to a control group, the immunofluorescence deposit of NRP1 was examined using an immunofluorescence assay. Compared to the kidney of the control group, the lupus mouse group showed significantly increased NRP1 deposits and enhanced fluorescence intensity (9.62 ± 2.34 vs. 3.32 ± 0.47, p < 0.01) [Fig. 3 A]. For quantification of NRP1 expression of the glomeruli in the protein level, glomeruli were isolated from the kidney and confirmed through the microscopic examination [Fig. 3 B], PCR, and western blotting. The western blot assay for NRP1 of the isolated glomeruli revealed greater expression in the lupus group (3.40 ± 0.41, p = 0.02) compared to the control mice [Fig. 3 C]. We next assessed the expression of NRP1 in dendritic cells in vitro . Dendritic cells differentiated from bone marrow cells were used for these experiments. The immunocytochemistry assay showed a significantly more intensity of the NRP1 in the BMDCs of the lupus mouse group (12.18 ± 0.16) than the BMDCs of the control mice (4.44 ± 0.06) (Fig. 4 ). These results confirm the enhanced expression of NRP1 in the lupus murine model in vivo and in vitro , and we accordingly assessed NRP1 expression in the dendritic cells of the patients with SLE and evaluated the correlation between the expression of NRP1 and SLE disease activity. Enhanced NRP1 expression in the dendritic cells from SLE patients and its correlation with disease activity CD11c + dendritic cells were isolated from the peripheral PBMCs of the patients with SLE (n = 10) and the healthy controls (n = 10) using flow cytometer sorting. As shown the Fig. 5 , we observed greater expression of NRP1 in the dendritic cells of the patients with SLE compared to those of healthy controls (healthy controls, 2.36 ± 0.76 vs. SLE, 4.50 ± 0.14, p = 0.001) in the immunocytochemistry analysis. Based on these results, we analyzed the correlation between the level of NRP1 expression and the disease activity of the patients with SLE. To determine the NRP1 expression, we conducted an analysis of isolated PBMCs obtained from a group of 57 patients with SLE and 57 age- and sex-matched healthy controls. PCR was utilized as the method for measuring NRP1 expression in these samples. As shown in Fig. 6 A, significantly more NRP1 was expressed in the patients with SLE than the healthy controls (healthy control, 0.99 ± 0.08 vs. SLE, 6.03 ± 0.96, p < 0.001). We then analyzed the correlation between NRP1 expression of PBMCs and disease activity markers of SLE, including SLEDAI score, C3, C4, and anti-dsDNA antibody titer. Table 1 described the baseline clinical characteristics of the patients with SLE. Seventeen patients were excluded due to the lack of some necessary clinical data. The mean age of patients was 37 years old and their mean SLEDAI-2K score was 7.4, which indicates moderate activity. Thirty percent of the patients have nephritis and their mean UACR was 1731 mg/g. Figure 6 B shows the significant correlations between NRP1 and disease activity markers; the positive correlation with the SLEDAI score (r = 0.56, p < 0.01) and the anti-dsDNA antibody titer (r = 0.31, p = 0.04), as well as the negative correlation with C3 (r= -0.36, p = 0.02) and C4 (r= -0.32, p = 0.04). These results suggest the possible involvement of NRP1 in the pathogenesis of SLE. Table 1 Clinical characteristics of patients with SLE Patients with SLE (N = 40) Sex, n (%) Male 5 (12.5%) Female 35 (87.5%) Age (yrs) 37.0 ± 15.1 WBC (x 10 3 /µL) 6.5 ± 3.8 Lymphocytes (x 10 3 ) 1.1 ± 0.5 Hemoglobin (g/dL) 10.8 ± 1.8 Platelet (x 10 3 /µL) 222.0 ± 120.4 C3 (mg/dL) 69.2 ± 27.5 C4 (mg/dL) 13.5 ± 10.3 Anti-dsDNA antibody (IU/mL) 141.4 ± 251.1 ESR (mm/hr) 36.5 ± 28.2 CRP (mg/L) 11.7 ± 23.3 SLEDAI-2K 7.4 ± 6.5 Nephritis, n (%) 12 (30%) III, n (%) 1 (8.3%) IV, n (%) 8 (66.7%) V, n (%) 3 (25.0%) UPCR (mg/g) 1731 ± 2487 Data are expressed as number (percentage) or mean ± S.D. CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; SLEDAI-2K, SLE activity index 2000; UPCR, urine protein to creatinine ratio Effect of NRP1 inhibition on viability and the function of dendritic cells Along with this finding, we used NRP1 antagonist (EG00029) to investigate the effect of NRP1 on the viability and function of dendritic cells. Referring to previous studies, including the experiments using EG00229 [33–35], allowed us to determine the range of the effective dose, and minimal cellular toxicity was confirmed through a serial single-dose toxicity study (Fig. 7 A). BMDCs were treated with a concentration of 100µM of EG00229 to assess the viability and function of these cells. The expression of NRP1 of BMDCs was significantly increased by the activation of TLR7 with R848, and this expression was decreased by EG00229 (Fig. 7 B). To assess the roles of NRP1 in determining the viability of BMDCs, a cell proliferation assay was performed for 24 hrs and 48 hrs using a CCK-8 assay. The treatment with R848 substantially increased the cell viability of BMDCs after both incubation periods. However, this increase was significantly inhibited when EG00229 was used (Fig. 7 C). A co-culture assay of CD4 + CD25 − T cells and dendritic cells treated with NRP1 antagonist was prepared to investigate the effects of NRP1 on dendritic cell-induced T cell proliferation. NRP1 antagonist-treated BMDCs from mice were co-cultured with CD4 + 25 − T cells labeled with CFSE at a ratio of 1:1 for 5 days. TLR7 agonist-treated dendritic cells significantly increased the proliferation of CD4 + CD25 − T cells after the co-culture of these two cells, and these effects were abrogated to a statistically significant degree when co-cultured NRP1 antagonist-treated dendritic cells and CD4 + CD25 − T cells (Control, 10.99 ± 0.55; R848-treated, 18.57 ± 2.08; EG00229-treated, 7.99 ± 0.56; EG00229 and R848-treated, 7.28 ± 0.72) (Fig. 8 A). To examine the impact of NRP1 on T cell proliferation mediated by human dendritic cells, we conducted a co-culture assay using CD4 + CD25- T cells and dendritic cells derived from healthy controls. In this assay, we treated the dendritic cells with an NRP1 antagonist, similar to the experiment performed with mice BMDCs. As shown in Fig. 8 B, the dendritic cells activated by the TLR7 agonist without pre-treatment with a NRP1 antagonist significantly increased CD4 + CD25 − T cell proliferation. However, this increased proliferation of CD4 + CD25 − T cells in TLR7 agonist treated dendritic cells was inhibited by the addition of NRP1 antagonist, EG00229. (Control, 35.49 ± 9.88; R848-treated, 53.68 ± 6.80; EG00229-treated, 44.31 ± 5.63; EG00229 and R848-treated, 41.80 ± 6.94). We also evaluated the effects of natural dendritic cells sorted from the PBMCs in the SLE patients on the proliferation of CD4 + CD25 − T cells (Fig. 8 C). We noted less proliferation of CD4 + CD25 − T cells co-cultured with the NRP1 antagonist-treated dendritic cells than CD4 + CD25 − T cells co-cultured with vehicle-treated dendritic cells (vehicle-treated, 59.57 ± 9.7; EG00229-treated, 41.03 ± 11.27, p = 0.05) (Fig. 8 C). Our co-culture assays suggest that the NRP1 component of dendritic cells plays a role in determining the viability and function of dendritic cells, potentially affecting activation and proliferation of T cells, which are the pivotal to the pathogenesis of SLE. Effect of NRP1 antagonist on the STAT, MAPK and NF-κB signaling in dendritic cells As NRP1 was hypothesized to affect the viability and function of dendritic cells, we examined the effects of NRP1 on the intracellular signaling pathways of dendritic cells. These pathways, including those associated with MAPK and NF-κB activation, have been shown to promote cell survival, proliferation [36]. NF-kB in particular is known to promote dendritic cell maturation into professional antigen-presenting cells [37]. The dendritic cells were stimulated with R848 with or without pretreatment of NRP1 antagonist and their signaling pathways were evaluated. Although TLR7 activation by R848 increased the ratio of p-STAT1/STAT1 and p-STAT3/STAT3, there were no significant differences after treatment with the NRP1 inhibitor, EG00229 (Fig. 9 A). In the MAPK pathways, the phosphorylation of ERK 1/2 and p38 of the dendritic cells was increased by R848 treatment, and this was attenuated by the NRP1 antagonist (Fig. 9 B). Furthermore, NRP1 inhibition also decreased the R848-induced phosphorylation of NF-κB of the dendritic cells, more so than in the R848-only treated group (Fig. 9 C). Our results suggest that upregulated expression of phosphorylated MAPK and NF-kB in dendritic cells by TLR7 activation is abolished by NRP1 antagonist treatment. In conclusion, these findings indicate that NRP1 enhances the survival and activation of dendritic cells through the activation of intracellular signaling pathways, specifically MAPK and NF-kB. Consequently, this leads to immune dysregulation by activating T cells, which are widely recognized for their crucial involvement in the pathogenesis of SLE. Discussion Recently, the involvement of NRP1 in the pathogenesis of various diseases has been discovered. As a regulator, NRP1 is now known to be involved in illnesses as diverse as eye diseases [38], angiogenesis [39], osteoblastogenesis [40], and tumorigenesis [41]. Research into NRP1 is particularly exciting in the area of tumor immunity, with evidence now showing that NRP1 is highly expressed in cancer cells and tissues, and is involved in cancer cell proliferation and metastasis ( 9 ). Based on these findings, NRP1 is now regarded as a predictor or prognostic marker for certain types of malignancies, and as a promising therapeutic target for antitumor immunity [9, 42–45]. Although malignancy and autoimmunity are distinct pathological conditions, they share similarities in terms of the underlying microenvironment that contributes to the immunopathological mechanisms, such as the aberrant activation of the immune or inflammatory system [46]. However, NRP1's involvement in the field of autoimmunity has not been thoroughly explored. To the best of our knowledge, this study is the first to investigate the role of NRP1 in the pathogenesis of SLE, which is considered a representative autoimmune disease. In the present study, we showed the significantly higher expression of NRP1 in those with SLE and the correlation of this expression with disease activity. NRP1 has modulatory effects on the function and viability of dendritic cells, and these effects are mediated by MAPKs and the NF-κB signaling pathway. We first confirmed higher levels of NRP1 in the Mo-DC and the PBMCs in patients with SLE than in healthy controls, and NRP1 expression in the PBMCs was significantly correlated with the disease activity of SLE. A limited number of studies regarding elevated NRP1 expression in SLE have been reported in the other specimens of the patients with SLE. A previous study has reported a significant increase in urinary NRP1 levels in individuals with SLE. This finding suggests that urinary NRP1 levels could serve as a potential discriminatory marker for indicating active lupus nephritis, distinguishing it from inactive lupus nephritis [47]. Similarly, Vadasz et al. showed a positive correlation between NRP1 deposits in the glomeruli and the clinicopathological parameters of nephritis [48]. Consistent findings were observed in our in vivo test that used a murine model, which showed higher fluorescence intensity in the kidneys of lupus models than in those of the control group. Taken together, the observed upregulation of NRP1 expression in the kidney, the primary organ affected in SLE, as well as in PBMCs, particularly in dendritic cells, suggests that NRP1 could play a role in the development and progression of immune abnormalities associated with the pathogenesis of SLE. The role of NRP1 in cell proliferation in other cell types, especially in cancer cell lines, has been elucidated. NRP1-positive lung cancer cells have shown higher clonogenic and self-renewal capacity, which is a tumor-initiating property, than NRP1 gene knockdown cells [49]. Additionally, studies have demonstrated that microRNA-145 and microRNA-205 can suppress the proliferation of uveal melanoma cells by targeting and inhibiting NRP1 expression [50]. Since the initial discovery of NRP1 expression in dendritic cells and its role in T cell proliferation a decade ago [14], extensive research has been conducted to explore the association between NRP1 and various pathologic conditions linked to immune dysregulation. In our study, we focused on the role of NRP1 in dendritic cells in the pathogenesis of SLE. Our results are consistent with the prior findings, inasmuch as they show the capacity for cell proliferation by NRP1. Accordingly, this study is significant as it is the first to identify NRP1's involvement in the pathogenesis of SLE, specifically as the causative mechanism behind the abnormal increase of pathological effector T cells. The mechanisms by which NRP1 increases cell viability and function may be multifactorial. In our study, we specifically examined the MAPKs and NF-κB signaling pathways as the underlying signaling pathways mediated by NRP1. These pathways are intricately involved in cell survival and the crucial function of antigen presentation to T cells. We showed that stimulation with a TLR7 agonist upregulated the phosphorylation of ERK 1/2 and p38, and NRP1 antagonists decreased NRP1 expression with significant attenuation of the activation of these MAPKs in dendritic cells. The activation of ERK 1/2 or p38 signaling in association with cell survival and proliferation is well documented in a broad variety of diseases including tumorigenesis [51, 52]. This study showed that inhibition of MAPK activation by an NRP1 antagonist decreases the proliferation of dendritic cells. Antigen-presentation is a key role of dendritic cells, and the activation and expansion of T cells is promoted following antigen recognition. NF-κB is already known as a major regulator of the antigen-presenting function as well as cell survival in dendritic cells [53, 54]. Consistent with these findings, we showed that NRP1 antagonists decrease the phosphorylation of NF-κB in dendritic cells and consequently reduce T cell expansion. These findings indicate that NRP1 affects the viability and function of dendritic cells on T cell proliferation via the phosphorylation of MAPKs and the NF-κB signaling pathways. In clinical practice, multiple disease activity markers are employed to effectively manage SLE. However, these markers have certain limitations, including the potential to yield complex, incorrect, or conflicting results. This study demonstrated a correlation between the expression of NRP1 of PBMCs and SLE disease activity markers, including C3, C4, anti-dsDNA antibody, and SLEDAI scores. Given the limited utility of available alternative SLE markers, we suggest that NRP1 may have a role to play as an additional disease activity marker. We further suggest that NRP1 expression in various immune cells and glomeruli could serve as a non-invasive and sensitive biomarker for predicting diverse clinical features, specific histopathologic characteristics of lupus nephritis, and responsiveness to medical treatment, although further studies are necessary to validate this suggestion. This study has some limitations inherent to how it sought determine the specific roles of NRP1 in the pathogenesis of SLE. First, the number of enrolled patients with SLE was small, so further studies with a large number of patients with SLE will be needed. Second, as we had only a limited number of sorted dendritic cells from PBMCs in healthy controls and patients with SLE, we analyzed the expression of NRP1 in whole PBMCs, as opposed to isolated dendritic cells, to evaluate the correlation between NRP1 expression and disease activity markers. The PBMC count in patients with SLE was found to be significantly lower compared to healthy controls. Moreover, the population of dendritic cells accounted for only a small fraction (1–2%) of the total PBMCs [55]. To determine the precise roles of NRP1 in the pathogenesis of SLE and its potential as a disease activity marker, further studies involving isolated dendritic cells will be necessary. Thirdly, we did not assess the impact of NRP1 on the antigen-presenting function of dendritic cells in our study. Nevertheless, it is widely documented that T cell proliferation is contingent upon antigen presentation by dendritic cells [56]. While we did not specifically investigate the direct effects of NRP1 on the antigen-presenting function of dendritic cells, the significant increase in T cell expansion observed in our study provides crucial evidence of the enhanced function of dendritic cells mediated by NRP1. In conclusion, this study provided evidence of the increased expression of NRP1 in the PBMCs, as well as in the kidneys and dendritic cells, in both murine models and patients with SLE, as compared to the control group. NRP1 expression shows a significant correlation with disease activity in SLE patients. NRP1 modulates the viability and function of dendritic cells and enhance dendritic cell-induced T cell proliferation via the phosphorylation of intracellular MAPKs and NF-κB signaling pathways. Our study underscores the significant role of NRP1 in immune dysregulation during the progression of SLE, highlighting its potential as a promising therapeutic target for the effective management of this complex autoimmune disease. Declarations Acknowledgements The work was supported by a grant from the National Research Foundation of KOREA (NRF) funded by the Korea government (MSIT) (No. 2018R1D1A3B07041314 and No.2021R1G1A1094571), and by the Fund of the Biomedical Research Institute, Jeonbuk National University Hospital Author contributions Conceptualization, Y.C. and W.Y.; Data collection and investigation, K.K. and E.L.; draft manuscript preparation, Y.C. and E.L.; manuscript review and editing, W.Y. and Y.C.; supervision, W.Y. All authors have read and agreed to the final manuscript. Data availability statement The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. 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Verhoeckx et al. ) 161–167 (Springer Copyright 2015, The Author(s). 2015). Mayer, A., Zhang, Y., Perelson, A. S. & Wingreen, N. S. Regulation of T cell expansion by antigen presentation dynamics. Proc Natl Acad Sci U S A 116, 5914–5919, doi: 10.1073/pnas.1812800116 (2019). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3209000","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":223667103,"identity":"002283f2-3aea-40fe-b9c5-4eb135f2cebf","order_by":0,"name":"Yunjung Choi","email":"","orcid":"","institution":"Jeonbuk National University Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yunjung","middleName":"","lastName":"Choi","suffix":""},{"id":223667104,"identity":"bbd42b68-4ab1-493e-8778-4fa56b14d392","order_by":1,"name":"Eun-Gyeong Lee","email":"","orcid":"","institution":"Jeonbuk National University Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Eun-Gyeong","middleName":"","lastName":"Lee","suffix":""},{"id":223667105,"identity":"4f7ce83b-29a0-41e4-9c38-4be59bd7b5f2","order_by":2,"name":"Kyoung Min Kim","email":"","orcid":"","institution":"Jeonbuk National University Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kyoung","middleName":"Min","lastName":"Kim","suffix":""},{"id":223667106,"identity":"add1fb5b-49a1-4c69-9b2d-59d8fe58cfbd","order_by":3,"name":"Wan-Hee Yoo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYFACHoYDDBVsCVBeAh6VKFrOkKqFgbENrpQILQY3cg8eujmPL89cIoHxww+GtHwitOQlHM7dxlZsOSOBWbKHIceygaCW2zkGIC2JG24kMEgzMFQYELYFrGUOWAvzbxK0NIC1sAFtySGsRfL+G4PDOcfYig3OPGyz7DFII6yF78wZ4885NcfyDI4nH77xoyKZsBaFA2DqGBAzNgDdSVADA4N8A5iqIULpKBgFo2AUjFgAABmIP3MX8G+GAAAAAElFTkSuQmCC","orcid":"","institution":"Jeonbuk National University Medical School","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wan-Hee","middleName":"","lastName":"Yoo","suffix":""}],"badges":[],"createdAt":"2023-07-27 08:59:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3209000/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3209000/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":41248872,"identity":"4b8b941f-24a4-4cf9-a38b-9d0104618d7a","added_by":"auto","created_at":"2023-08-08 14:48:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":250911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of NRP1 and ILT4 in T and dendritic cells of the patients with SLE and healthy controls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of NRP1 was analyzed in CD4\u003csup\u003e+\u003c/sup\u003e T cells and Mo-DCs. Forty patients with SLE and forty age- and gender-matched healthy controls were enrolled to analyze the expression of NRP1 using PCR. The expression of NRP1 in CD4\u003csup\u003e+ \u003c/sup\u003eT cells (A), and the expression of NRP1 in Mo-DCs (B) is shown. Data are expressed as mean ± SEM. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. healthy controls.\u003c/p\u003e\n\u003cp\u003eILT4, immunoglobulin-like transcript 4; Mo-DCs, monocyte-derived dendritic cells; NRP1, neuropilin-1; SLE, systemic lupus erythematosus.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3209000/v1/f88c2336cba8651bf44f0c34.png"},{"id":41248877,"identity":"faa08f1b-0169-456d-8e13-a89ad758e0a2","added_by":"auto","created_at":"2023-08-08 14:48:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9679130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInduction and validation of TLR7 agonist-induced lupus murine model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBALB/C mice were treated topically with TLR7 agonist, 100µg of Resiquimod, 3 times per week for 4 weeks. (A) Comparisons of spleen size and spleen to weight ratio between control group (n=5) and R848-treated group (n=5). (B) Representative renal sections stained for IgG and C3 (original magnification, x 400) and MFI are shown, scale bar, 20µm. (C) Representative kidney sections stained with H\u0026amp;E and PAS stain (original magnification, x400) and histologic scores are shown. Black square indicates glomeruli, and magnified images of the glomeruli are shown next to original representatives. (D) Urine albumin to creatinine ratio and serum anti-dsDNA antibody in control group and R848-treated group were determined by ELISA. Data are expressed as mean ± SEM of three independent experiments per group. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs control group.\u003c/p\u003e\n\u003cp\u003eAnti-dsDNA antibody, anti-double stranded DNA antibody; H\u0026amp;E, hematoxylin and eosin; MFI, mean fluorescence intensity; PAS, Periodic acid–Schiff stain; TLR7, toll-like receptor 7\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3209000/v1/a46e6e1ccc07c7c884fbfc4e.png"},{"id":41248880,"identity":"c6a8452b-1886-4f36-9c23-15789d09cab0","added_by":"auto","created_at":"2023-08-08 14:48:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6491418,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of NRP1 in the kidney from lupus murine group and control group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative renal sections stained for NRP1 (original magnification, x 400) and MFI of lupus murine group (n=5) and control groups (n=5) are shown. Scale bar, 20µm. (B) Representatives of isolated glomeruli (arrows) (original magnification, x 400), scale bar, 20µm. (C) NRP1 of isolated glomeruli was semi-quantified by densitometric analysis and normalized to ACTIN expression. The samples derive from the same experiment and the gels/blots were processed in parallel. Data are expressed as mean ± SEM of three independent experiments per group. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs control group.\u003c/p\u003e\n\u003cp\u003eMFI, mean fluorescence intensity; NRP1, neuropilin-1\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3209000/v1/f74593d076d2277bd6b6ab65.png"},{"id":41248878,"identity":"e64a75cc-9c3f-48a0-a9ea-d10d39e3bb08","added_by":"auto","created_at":"2023-08-08 14:48:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2575897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of NRP1 in BMDCs from the lupus murine group and control group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunocytochemistry analysis of NRP1 in the BMDCs from the lupus murine group (n=3-5) and the control group (n=3-5). (Original magnification, x 630), scale bar, 10µm. The MFI from each group is shown and the data are expressed as mean ± SEM. Data were obtained from three paired experiments. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs control group\u003c/p\u003e\n\u003cp\u003eMFI, mean fluorescence intensity; NRP1, neurophilin-1\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3209000/v1/f44dfad83dd03fa5acead556.png"},{"id":41248874,"identity":"62393649-85c1-4598-b7c3-b1289b3673a6","added_by":"auto","created_at":"2023-08-08 14:48:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1210132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of NRP1 in dendritic cells from healthy controls and SLE patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCD11c\u003csup\u003e+\u003c/sup\u003e dendritic cells were isolated from the PBMCs of participants by BD FACSAria ІІІ flow cytometer sorting. Immunocytochemistry analysis of NRP1 was performed in these isolated dendritic cells from the healthy control (n=10) and the patients with SLE (n=10). Representative images and MFI of NRP1 in both groups are shown. Data are expressed as mean ± SEM. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs healthy control\u003c/p\u003e\n\u003cp\u003eMFI, mean fluorescence intensity; NRP1, neuropilin-1; SLE, systemic lupus erythematosus\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3209000/v1/b0466f6ce95d89f88283ec9f.png"},{"id":41249709,"identity":"dd545a99-7a52-41d1-8c22-3b56b4166c46","added_by":"auto","created_at":"2023-08-08 14:56:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":599104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between NRP1 expression and disease activity markers of SLE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) NRP1 expression of PBMCs compared between healthy controls (n=60) and patients with SLE (n=57). After excluding the patients for whom necessary clinical data was lacking, a total of 40 patients with SLE were analyzed for a correlation between NRP1 expression and disease activity markers such as (B) SLEDAI-2k scores, C3, C4, anti-dsDNA antibody. The NRP1 expression was assessed through PCR. The relationship between variables was evaluated using Speakmans’ rho correlation test.\u003c/p\u003e\n\u003cp\u003eAnti-dsDNA antibody, anti-double stranded antibody; SLEDAI-2k, systemic lupus erythematosus disease activity index 2000; NRP1, neuropilin-1\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3209000/v1/488f3af02a794b54bee331fd.png"},{"id":41248873,"identity":"09424161-fcc4-4a1a-8c7d-19fb0bc072b5","added_by":"auto","created_at":"2023-08-08 14:48:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":410453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NRP1 antagonist on the proliferation of dendritic cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The cytotoxicity of EG00229, an NRP1 antagonist, and R848, was evaluated according to various concentrations of BMDCs (n=3-5). The cell lysis buffer was used as a positive control. (B) Immunoblotting assay showing the inhibition of NRP1 by EG00229. The BMDCs were pretreated with EG00229 or vehicle for 2 hrs and stimulated by R848 or vehicle for 24 hrs. The samples derive from the same experiment and the gels/blots were processed in parallel. (C) The viability of BMDCs which were pre-treated with EG00229 or vehicle and activated by R848 or vehicle. Cell cytotoxicity and viability were measured with a CCK-8 kit. Data are expressed as mean ± SEM of three independent experiments per group. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. R848-treated, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. control\u003c/p\u003e\n\u003cp\u003eBMDCs, bone marrow-derived dendritic cells; NRP1, neuropilin-1\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3209000/v1/431622e6c89bb1b5471e08f8.png"},{"id":41248876,"identity":"11af6ef6-cae3-41f5-9f57-396decba7d3c","added_by":"auto","created_at":"2023-08-08 14:48:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1447214,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NRP1 inhibition of the dendritic cell on the proliferation of T cell\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDendritic cells treated with vehicle, R848, EG00029, or R848 with EG00029 were co-cultured with CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003e T cells labelled with CFSE at a ratio of 1:1 for 5 days. The dendritic cells were pretreated with EG00229 or vehicle for 2 hrs and stimulated with R848 or vehicle for 24 hrs, after which they were co-cultured with T cells for 5 days. Flow cytometric analysis of the proliferation of target cells in the co-culture assay of the treated-BMDCs and CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003e T cells, which were isolated from the spleens of the mice (A), in the co-culture assay of the treated-CD11c\u003csup\u003e+\u003c/sup\u003e dendritic cells and CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003e T cells isolated from PBMCs of healthy controls (B), and the patients with SLE (C). Results are presented as the mean ± SEM of three independent experiments per group (n=3-5). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. R848-treated, #\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. control,\u003c/p\u003e\n\u003cp\u003eBMDCs, bone marrow-derived dendritic cells; NRP1, neuropilin-1; PBMCs, peripheral blood mononuclear cell\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3209000/v1/f338a4526115b956eba9631b.png"},{"id":41248875,"identity":"66673455-1c37-430d-a842-f1cdc4e0e9be","added_by":"auto","created_at":"2023-08-08 14:48:05","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1331000,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of NRP1 inhibition on STATs, MAPKs, and NF-κB signaling in BMDCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot analysis of the expression of STAT 1/3 (A), MAPKs (B), and NF-kB signaling (C) (n=3-5). BMDCs were pretreated with EG00229 or vehicle for 2hrs and stimulated with R848 or vehicle for 0.5 hrs. The expression of reactive protein was semi-quantified by densitometric analysis and normalized to their unphosphorylated protein. The samples derive from the same experiment and the gels/blots were processed in parallel. Values are mean ± SEM. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. R848-treated, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. control\u003c/p\u003e\n\u003cp\u003eBMDCs, bone marrow-derived dendritic cells; MAPK, mitogen‑activated protein kinase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NRP1, neuropilin-1; STAT, signal transducer and activator of transcription\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3209000/v1/f54e235118b80f9dba931c93.png"},{"id":57686863,"identity":"d51b4de4-e773-44d8-88c7-e91f20b93544","added_by":"auto","created_at":"2024-06-04 10:18:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":38494696,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3209000/v1/05c41ede-6c40-47c6-bf1f-5c38068e56a0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced NRP1 Expression in Dendritic Cells of Systemic Lupus Erythematosus and Its Impact on T Cell Proliferation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSystemic lupus erythematosus (SLE) is a potentially fatal autoimmune disease that may affect nearly every organ and tissue (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Its development requires a complex interaction between genetic, environmental, viral, and hormonal factors. Through the occurrence of the complex interaction, immune dysregulation is initiated at the level of cytokines, T cells, B cells, and macrophages, after which immune tolerance to the self-antigen is lost and autoimmunity occurs. Along with these processes, complement activation, immune complex accumulation, and consequent tissue inflammation and damage result in a self-sustained autoimmune pathway (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Dendritic cells have been focused on their pathogenic roles in the induction and progression of the broken immunological tolerance (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). High levels of self-RNA and self-DNA from apoptotic cells induce type I interferon (IFN) release from plasmacytoid dendritic cells, and promotes its own activation as well as the maturation of myeloid dendritic cells in an autocrine manner (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Upon activation, dendritic cells exhibit an increased ability to present the self-antigen and induce the stronger proliferation of T cells (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). A consequent immune cascade that includes B cell activation, auto-antibody production, and immune complex formation occurs through at self-amplification loop. Dendritic cells are therefore regarded as a significant contributor to the pathogenesis of SLE by bridging the aberrant innate and adaptive immunity (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNeuropilin-1 (NRP1) is a type I transmembrane protein with a molecular weight of 120 kDa that acts as a co-receptor for several extra-cellular ligands (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The protein was initially identified as neuronal cell guidance and was first discovered as part of research to develop and regenerate \u003cem\u003eXenopus\u003c/em\u003e optic nerves (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Recently, a wide range of physiological roles and pathologic involvements for NRP1 have been identified, including those in cardiovascular development, cell migration, angiogenesis, and cancer pathogenesis (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Furthermore, NRP1 has attracted considerable attention for its immunoregulatory functions. NRP1 is also known as a receptor for class 3 semaphorins, which are intimately involved in the pathogenesis of autoimmune diseases (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Furthermore, NRP1 expression in various kinds of immune cells, including myeloid cells such as monocytes, macrophages (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) and myeloid dendritic cells (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), plasmacytoid dendritic cells (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), and a small subset of regulatory T cells (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), has been confirmed. NRP1 expression in dendritic cells and resting T cells has been clearly documented in humans, and emerging evidence indicates that it plays a role in the initiation of primary immune responses by mediating the interaction between dendritic cells and T cells (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). For example, a co-culture of T lymphocytes with dendritic cells pretreated with blocking NRP1 antibody showed significantly decreased dendritic cell-induced T cell proliferation (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). These findings support the hypothesis that NRP1 is an essential component of dendritic cells that initiates their primary immune responses, and as such it may explain the aberrant immunity they show in response to autoimmune rheumatic diseases such as SLE. To date, however, studies regarding NRP1 expression in patients with SLE and its role in the pathogenesis of this disease are scarce. Building on previous findings suggesting the existence of NRP1 expression in the dendritic cells and their pathogenic role in SLE, this study was performed to investigate these aspects of NRP1 in lupus mouse models and patients with SLE.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients with SLE and healthy controls\u003c/h2\u003e \u003cp\u003eA total of 57 patients with SLE and 60 healthy controls were recruited from Jeonbuk National University Hospital in Jeonju, South Korea. All patients were between 18 to 75 years of age and fulfilled the 2012 Systemic Lupus International Collaborating Clinics (SLICC) Classification Criteria for SLE (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The patients receiving over 1mg/kg/day prednisolone at the time of blood sampling were excluded from this study. SLE disease activity was evaluated using the SLE Disease Activity Index 2000 update (SLEDAI-2K) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Sixty age and gender-matched healthy control subjects were recruited. Written informed consent was obtained from all participants. This study was conducted consistent with the Declaration of Helsinki and was approved by the Ethics Committee of the Jeonbuk National University Hospital (CUH 2018-08-005-004).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLupus murine model induction\u003c/h2\u003e \u003cp\u003eBALB/C mice (7-9-week-old females) were purchased from Central Lab Animal Inc. All mice were maintained under a 12 h:12 h light/dark cycle, with the temperature at 24\u0026deg;C, and the humidity at 60% on the standard diet in a conventional cage. We adopted toll-like receptor (TLR) 7 agonist-induced mice as SLE murine models for our experiments. This model presents with the phenotypic findings of lupus including an elevated autoantibody levels, immune complex-deposited nephritis, and marked splenomegaly (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), and has been widely used for experiments in research into SLE pathogenesis (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The lupus murine model was induced through the topical application of a TLR7 agonist. The skin on the back of the mice was treated topically 3 times weekly over 4 weeks with 100 \u0026micro;g of Resiquimod (R848, Enzo Life Sciences, NY, USA) mixed with 100 \u0026micro;L of acetone. The control group was treated with 100 \u0026micro;L of acetone topically in the same manner. At the end of the treatment, mice were euthanized via intraperitoneal administration of ketamine (75 mg/ kg) plus xylazine (10 mg/kg), followed by the collection of a blood sample through cardiac puncture. All experiments were conducted in accordance with National Institutes of Health guidelines and Animal Research: Reporting of In Vivo Experiments guidelines. The study protocol was approved by the Institutional Animal Care and Use Committee of Jeonbuk National University (JBNU 2020\u0026thinsp;\u0026minus;\u0026thinsp;0115).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003emRNA extraction and Real-Time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using an RNA Extraction Kit (Bioneer, Daejeon, Korea) according to the manufacturer\u0026rsquo;s protocol, and cDNA was generated by using an Accupower RocketScript RT Premix, Rnase H Minus kit (Bioneer, Daejeon, Korea). Quantitative PCR was performed with SYBR Green Real time PCR Master mix (TOYOBO, Osaka, Japan) and a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Grand Island, NY, USA). Amplification conditions were as follows: 50℃ for 2 min, then 95℃ for 10 min for 1 cycle, followed by 40 cycles of 95℃ for 15 sec and 60℃ for 1 min. The following primers were used (forward and reverse, respectively): for humans, GAPDH, 5\u0026rsquo;-tggtatcgtggaaggactca-3\u0026rsquo; and 5\u0026rsquo;-gcagggatgatgttctggag-3\u0026rsquo;; NRP1, 5\u0026rsquo;-gcctgactcaaatcctccag-3\u0026rsquo; and 5\u0026rsquo;-acctggtgttttctgtccac-3\u0026rsquo;; ILT-4, 5\u0026rsquo;-tcagggcaaacactggacat-3\u0026rsquo; and 5\u0026rsquo;-tcacggcagcatagaggttt-3\u0026rsquo;; for mice, GAPDH, 5\u0026rsquo;-acttgaagggtggagccaaa-3\u0026rsquo; and 5\u0026rsquo;-gcaggatgcattgctgacaa-3\u0026rsquo;; NRP1, 5\u0026rsquo;-5\u0026rsquo;-gcctgactcaaatcctccag-3\u0026rsquo; and 5\u0026rsquo;-acctggtgttttctgtccac-3\u0026rsquo;; ILT-4, 5\u0026rsquo;- tcagggcaaacactggacat-3\u0026rsquo; and 5\u0026rsquo;-tcacggcagcatagaggttt-3\u0026rsquo;; for mice, GAPDH, 5\u0026rsquo;-acttgaagggtggagccaaa-3\u0026rsquo; and 5\u0026rsquo;-gcaggatgcattgctgacaa-3\u0026rsquo;; NRP1, 5\u0026rsquo;- tgctctggaatgttgggcat-3\u0026rsquo; and 5\u0026rsquo;-tggtcaccagacggatgttt-3\u0026rsquo;; Nephrin, 5\u0026rsquo;- aacatccagctcgtcagcat-3\u0026rsquo; and 5\u0026rsquo;- aaagccaggtttccactcca-3\u0026rsquo;. The relative expression of the target genes was determined by normalizing the expression of each gene to GAPDH using the ΔΔCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell isolation and culture\u003c/h2\u003e \u003cp\u003eThe peripheral blood of each participant was obtained via venipuncture. 10 ml of blood samples were diluted 1:1 with PBS (pH 7.4) and layered on Lymphoprep (Serumwerk Bernburg AG, Oslo, Norway) following centrifugation at 20\u0026deg;C for 30 min. PBMCs were isolated from the interphase between the Ficoll-Paque and serum layers, and washed twice with PBS.\u003c/p\u003e \u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells and monocytes were isolated from PBMCs using a MACS isolation kit (BD Biosciences, San Jose, CA, United States) by negative isolation through a CD8, CD11b, CD16, CD19, CD36, CD56, CD123, and CD235a γδ TCR biotinylated antibody cocktail (Human CD4 T Lymphocyte Enrichment Set; BD Biosciences, San Jose, CA, USA) as well as a CD3, CD45RA, CD19, CD56, and CD235a biotinylated antibody cocktail (Human Monocyte Enrichment Set; BD Biosciences, San Jose, CA, USA). CD4\u003csup\u003e+\u003c/sup\u003e T cells or monocytes were purified using a magnetic board of over 90% purity, as assessed by the flow cytometry of CD4\u003csup\u003e+\u003c/sup\u003e T cells with anti-Human CD4-FITC (Tonbo Biosciences, CA, USA) and monocytes with mouse anti-human CD14-FITC (BD pharmigen, USA).\u003c/p\u003e \u003cp\u003eCells were cultured in RPMI 1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Life Technologies Limited, UK), 100 U/mL penicillin (Gibco, Life Technologies Limited, UK), 100 mg/mL streptomycin (Gibco, Life Technologies Limited, UK), and 2 mM L-glutamine (Gibco, Life Technologies Limited, UK). Cells were grown at 37\u0026deg;C in a humidified 5% CO₂ atmosphere.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDendritic cell differentiation\u003c/h2\u003e \u003cp\u003eIsolated monocytes from human PBMCs were cultured in RPMI 1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Life Technologies Limited, UK), penicillin (100 U/mL) (Gibco, Life Technologies Limited, UK), streptomycin (100 mg/mL) (Gibco, Life Technologies Limited, UK), 2 mM L-glutamine, rh-granulocyte-macrophage colony-stimulating factor (GM-CSF) (50 ng/mL) (Peprotech, New Jersey Cranbury, USA), and rh-interleukin (IL)-4 (10 ng/mL) (Peprotech, New Jersey Cranbury, USA) over 6 days in a 96-well plate (Corning-falcon, USA) to obtain monocyte-derived dendritic cells (Mo-DC) as previously described (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe generation of bone marrow-derived dendritic cells (BDMCs) was performed using a method previously described elsewhere (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Briefly, bone marrow cells were isolated and prepared from the bone marrow of the femur and tibia in BALB/C mice as single-cell suspension. The aliquots of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e bone marrow cells were placed in 24-well plates and cultured in RPMI 1640 containing 10% heat-inactivated FBS (Gibco, Life Technologies Limited, UK), 100 U/mL penicillin (Gibco, Life Technologies Limited, UK), 100 mg/mL streptomycin (Gibco, Life Technologies Limited, UK), 2 mM L-glutamine in the presence of rm-GM-CSF (50 ng/mL, Peprotech, New Jersey Cranbury, USA), and rm-IL-4 (10 ng/mL, Peprotech, New Jersey Cranbury, USA). On day 3, two-thirds of the medium was replaced. On day 5, the non-adherent cells were transferred into a six-well plate and cultured for two additional days. On day 7, the acquired dendritic cell morphology was confirmed by microscope and checked for CD11c fluorescence (BD Bioscience, San Jose, CA, USA) using flow cytometry. Then the harvested cells were used in the subsequent experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGlomeruli isolation\u003c/h2\u003e \u003cp\u003eThe kidneys were harvested, and the medulla was removed carefully. The renal cortexes were minced and digested with collagenase type V (Sigma, dissolved in HBSS) in a water bath at 37℃ for 20 minutes with pipetting at 5-min intervals. The digested mixture was transferred with cold Hanks Balanced Salt Solution (HBSS, Sigma-Aldrich) onto 100-\u0026micro;m, 70-\u0026micro;m, and 40-\u0026micro;m cell strainers to remove cell debris and small tubular fragments. New 40-\u0026micro;m cell strainers were then used to remove the remnant debris, and the retained glomeruli were transferred into another clean culture dish to remove the few residual tubular fragments. The floating glomeruli were collected and centrifuged at 290g at 4℃ for 5 minutes. The glomeruli were examined under a microscope and their purity confirmed by the expression of the glomerular marker using Real-time PCR and Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRenal histopathologic assessment and immunofluorescence assay\u003c/h2\u003e \u003cp\u003eKidneys were harvested after perfusion with saline, and were fixed in 4% formaldehyde for 24 hr, embedded in paraffin, and separated into fixed sections of 10-\u0026micro;m thickness. After being stained with hematoxylin and eosin (H\u0026amp;E), and periodic acid\u0026ndash;Schiff (PAS), renal pathology was evaluated according to a previously described scoring scale (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Briefly, we assessed glomerular pathology in 20 glomeruli per kidney and interstitial/tubular pathology in randomly selected 10 high-power fields, semi-quantitatively on a scale of 0\u0026ndash;3. For the immunofluorescence assay, the 10-\u0026micro;m-thick acetone fixed sections were stained with rabbit anti-mouse IgG-heavy and light chain antibody, FITC-conjugated (Bethyl Laboratories Inc., USA). Slides were then incubated at room temperature for 1 hr. Slides were stained with rat-anti mouse C3 (Abcam, Cambridge, United Kingdom) and then were incubated at 4℃ overnight. The slides were stained with goat anti-rat IgG-heavy and light chain antibody FITC-conjugated and then incubated for 1 hr at room temperature in a dark humid box. They were then mounted with mounting medium using DAPI (Abcam, Cambridge, United Kingdom). Staining for IgG and NRP1 was performed in the same manner, and the antibodies used are listed below. Slides were observed with a confocal microscope (Zeiss LSM 880, Oberkochen, Germany) and analyzed by ZEN 3.2 Zeiss Microscopy GmbH software (version 3.2.0.0000) (Zeiss LSM 880, Oberkochen, Germany). The fluorescence intensity (MFI) of the glomeruli in different groups was calculated using ImageJ software (National Institutes of Health, USA) for each section with 10 glomeruli. 1) For IgG staining: rabbit anti-mouse IgG-heavy and light chain antibody FITC-conjugated (Bethyl Laboratories Inc., USA), 2). For NRP1 staining: NRP1 antibody (eBioscience, Carlsbad, CA, USA) (1:200), with goat anti-rat IgG-heavy and light chain antibody FITC-conjugated (Bethyl Laboratories Inc., USA)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemistry assay\u003c/h2\u003e \u003cp\u003eBMDCs were placed in a 35 mm confocal dish (SPL Life Sciences Co., Korea) at a number of 0.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells and incubated at 37\u0026deg;C for 2 hrs. The cells were fixed using 3.7% paraformaldehyde in PBS pH 7.4 for 10 minutes at room temperature and washed three times with cold PBS. The cells were incubated for 10 min with PBS containing 0.1% Triton X-100 at room temperature for permeabilization and washed in PBS three times. The washed cells were incubated with 1% BSA in PBST (PBS\u0026thinsp;+\u0026thinsp;0.1% Tween 20) for 30 minutes to block the unspecific binding of the antibodies. The cells were stained in the diluted antibody in 1% BSA in PBST overnight at 4\u0026deg;C. After washing the cells in PBS, they were stained with the secondary antibody in 1% BSA in PBS for 1 hr at room temperature in the dark and washed in PBS. The cells were mounted with mounting medium with DAPI (Abcam). The following antibodies were used in the immunocytochemical analysis: Neuropilin-1 antibody (eBioscience, Carlsbad, CA, USA), Goat anti-rabbit IgG, highly Cross-Adsorbed Secondary Antibody, goat anti-rat IgG-heavy, and light chain antibody FITC-conjugated (Bethyl Laboratories Inc., USA). The slides were observed with a confocal microscope (Zeiss LSM 880, Oberkochen, Germany) and analyzed by ZEN 3.2 Zeiss Microscopy GmbH software (version 3.2.0.0000) (Zeiss, Oberkochen, Germany). Fluorescence intensity was calculated using ImageJ software (National Institutes of Health, USA) and the scores were evaluated through methods described previously (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell cytotoxicity and proliferation assay\u003c/h2\u003e \u003cp\u003eBMDCs were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. BMDCs were seeded at 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well in a 96-well plate and treated with various doses of R848 (0, 1, 2.5, 5, and 10 \u0026micro;g/mL) (Enzo Life Sciences, NY, USA) and EG00229 (Tocris Bioscience, United Kingdom) that had been dissolved in 0.2M NaOH (0, 10, 30, 100 and 300 \u0026micro;M) (Tocris Bioscience, United Kingdom) for 24 hrs. The 10 \u0026micro;L/well of the Lysis solution was added to the control group as a positive control. The cytotoxicity of the cells was evaluated using a LDH Cell Cytotoxicity Assay Kit (DoGenBio, Seoul, Korea). To assess the cell proliferation, BMDCs were seeded (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL) in a 96-well plate. The BMDCs were pretreated with EG00229, an NRP1 antagonist (Tocris Bioscience, United Kingdom) (100 \u0026micro;M), for 2 hrs, followed by treatment with R848 for 24 hrs and 48 hrs. Cell proliferation was measured using a Cell Counting Kit-8 (Enzo Life Sciences, NY, USA). Absorbance was measured using a microplate reader (Bio-Rad) at 450nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSerologic analysis and Urinalysis\u003c/h2\u003e \u003cp\u003eAnti-dsDNA antibodies (FUJIFILM Wako Shibayagi Corporation, Ishihara, Japan), urine albumin and creatinine (Exocell, Philadelphia, USA), and Human Neuropilin-1 Quantikine (CUSABIO, Houston, TX, USA) were quantified by ELISA consistent with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNRP1 inhibition and co-culture condition\u003c/h2\u003e \u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells and CD11c\u003csup\u003e+\u003c/sup\u003e dendritic cells were isolated from the PBMCs of participants by BD FACSAria ІІІ flow cytometer sorting (\u0026gt;\u0026thinsp;95%). The CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells from the spleen of the BALB/C mice were sorted by BD FACSAria ІІІ (\u0026gt;\u0026thinsp;95%). Dendritic cells were pre-treated with EG00229 (100 \u0026micro;M) for 2 hrs and then stimulated by R848 (5 ug/mL) for 0.5 hr. CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells were incubated in the presence of anti-CD3 and anti-CD28 antibody coated beads (eBioscience, Carlsbad, CA, USA) as described previously (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). After 24 hrs of incubation, the T cells were labeled with 2.5 \u0026micro;M CFSE using a Cell Trace Cell Proliferation Kit (Molecular probes) and then co-cultured for 5 days with dendritic cells with target cells: the dendritic cell to target cell ratio was 1:1. The stained cells were detected using an FACSCalibur flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo software (TreeStar Inc., Ashland, OR, United States).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eProteins were extracted from isolated glomeruli and BMDCs using RIPA buffer containing protease inhibitors. (Thermo scientific, USA). Proteins were separated on SDS-PAGE gels (10%) and then transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon, Millipore). Membranes were blocked in 5% non-fat dry milk in TBST for 1 hr at room temperature with shaking and then probed overnight at 4℃ with primary antibodies (1:1,000) against Nephrin (Invitrogen, USA), Neuropilin-1 (Cell Signaling, Danvers, MA, USA), p-STAT1 (Cell Signaling, Danvers, MA, USA), STAT1 (Cell Signaling, Danvers, MA, USA), p-STAT3 (Cell Signaling, Danvers, MA, USA), STAT3 (Cell Signaling, Danvers, MA, USA), p-ERK 1/2 (Cell Signaling, Danvers, MA, USA), ERK 1/2 (Cell Signaling, Danvers, MA, USA), p-JNK (Cell Signaling, Danvers, MA, USA), JNK (Cell Signaling, Danvers, MA, USA), p-P38 (Cell Signaling, Danvers, MA, USA), P38 (Cell Signaling, Danvers, MA, USA), p-NF-κB, NF-κB (Cell Signaling, Danvers, MA, USA), p-IκB (Cell Signaling, Danvers, MA, USA), IκB (Cell Signaling, Danvers, MA, USA), and β-actin (Bioworld Technology Inc, USA). After three washes, the membranes were incubated with HRP-conjugated rabbit or mouse secondary antibody (1:3,000) at room temperature for 2 hrs. The reactive proteins were detected using an ECL (GE healthcare, USA) and the intensity of the bands was quantified using a Vilber Lourmat Fusion Fx7 system (Vilber Lourmat, Coll\u0026eacute;gien, France).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe Mann-Whitney test with Bonferroni\u0026rsquo;s correction was used to compare two groups and the Kruskal-Wallis test was used to compare three or more groups for the analysis of pathology scores and IF scoring. All other parametrically distributed data, including flow cytometry, immunoblotting, and cell proliferation assay, were analyzed by a one-way ANOVA with a Turkey post hoc test. Spearmans\u0026rsquo; rho test was applied to the analysis of correlation. SPSS 22.0 software (SPSS Inc., Chicago, Illinois, U.S.A.) was used for statistical analysis. P values of \u0026lt;\u0026thinsp;0.05 were considered statistically significant and results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eElevated expression of NRP1 in CD4\u003csup\u003e+\u003c/sup\u003e T cells and dendritic cells in the SLE patients\u003c/h2\u003e \u003cp\u003eThe expression of NRP1 was analyzed in CD4\u003csup\u003e+\u003c/sup\u003e T cells and monocyte-derived dendritic cells (Mo-DCs) from the patients with SLE as well as their gender- and age-matched healthy controls using PCR. These cells were isolated from the PBMCs of lupus patients with a SLEDAI-2K score above 10 (indicating high disease activity). Cell purity was confirmed by flow cytometry, reaching about 80%. NRP1 expression was significantly higher in CD4\u003csup\u003e+\u003c/sup\u003e T cells (9.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36 vs. 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) and Mo-DCs (8.10\u0026thinsp;\u0026plusmn;\u0026thinsp;4.33 vs. 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) of lupus patients compared to the healthy controls [Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB]. The enhanced expression of NRP1 in CD4\u003csup\u003e+\u003c/sup\u003e T cells and Mo-DCs in the lupus patients suggests a possible role for NRP1 in the pathogenesis of SLE. Thus, further experiments using the lupus murine and \u003cem\u003ein vitro\u003c/em\u003e model were conducted to investigate the expression level of NRP1 and its roles in the pathogenesis of SLE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eInduction of SLE murine model with TLR7 agonist\u003c/h2\u003e \u003cp\u003eTo assess of NRP1 expression and its role in lupus pathogenesis, we induced a lupus model through the topical application of a TLR7 agonist (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). It is well documented that dendritic cells play a pivotal role in the pathogenesis of SLE through IFNα production upon TLR7 ligation. TLR7 activation leads to an increase in IFNα releases and, consequently, B cell expansion, the production of autoantibodies, and the activation of myeloid cells and autoreactive cells (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). It is also well demonstrated that this lupus murine model, upon TLR7 activation, has severe lupus-like systemic autoimmunity such as nephritis, and as a consequence, this animal model has become a well-established lupus murine model (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). First, we induced the animal model with topical application of a TLR7 agonist, resiquimod (R848), and confirmed the lupus-like systemic autoimmunity in this model. Phenotypically, the R848-treated mice showed marked splenomegaly and increased weight of the spleen [Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA]. The immunofluorescence analysis of the glomeruli showed dominantly increased deposits of IgG and C3 in the R848-treated group compared to that of the control group [Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB], suggesting glomerulonephritis with immune complex deposition, which is frequently associated with lupus renal involvement. Furthermore, histopathological assessment of the kidney in the R848-treated group revealed obvious mesangial hypercellularity and monocellular cell infiltration, as well as a significantly higher histologic score compared to the control group [Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC]. The urine albumin to creatinine ratio (UACR) was significantly higher in the R848-treated group than in the control group (66.68\u0026thinsp;\u0026plusmn;\u0026thinsp;16.81 vs. 11.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) [Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD]. The anti-dsDNA antibody titer was also markedly higher in the R848-treated group than in the control group (526.53\u0026thinsp;\u0026plusmn;\u0026thinsp;95.29 vs. 1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) [Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD]. These findings suggest that this animal model accurately reflects the manifestation of systemic autoimmunity observed in SLE. Consequently, we have verified the appropriateness of utilizing the TLR-7 agonist-induced lupus model to study the contribution of NRP1 in the progression of SLE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIncreased expression of NRP1 in the glomeruli of the lupus murine model\u003c/h2\u003e \u003cp\u003eTo investigate NRP1 expression in the lupus model and compare it to a control group, the immunofluorescence deposit of NRP1 was examined using an immunofluorescence assay. Compared to the kidney of the control group, the lupus mouse group showed significantly increased NRP1 deposits and enhanced fluorescence intensity (9.62\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34 vs. 3.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) [Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA]. For quantification of NRP1 expression of the glomeruli in the protein level, glomeruli were isolated from the kidney and confirmed through the microscopic examination [Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB], PCR, and western blotting. The western blot assay for NRP1 of the isolated glomeruli revealed greater expression in the lupus group (3.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) compared to the control mice [Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC]. We next assessed the expression of NRP1 in dendritic cells \u003cem\u003ein vitro\u003c/em\u003e. Dendritic cells differentiated from bone marrow cells were used for these experiments. The immunocytochemistry assay showed a significantly more intensity of the NRP1 in the BMDCs of the lupus mouse group (12.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16) than the BMDCs of the control mice (4.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results confirm the enhanced expression of NRP1 in the lupus murine model \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, and we accordingly assessed NRP1 expression in the dendritic cells of the patients with SLE and evaluated the correlation between the expression of NRP1 and SLE disease activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEnhanced NRP1 expression in the dendritic cells from SLE patients and its correlation with disease activity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCD11c\u003csup\u003e+\u003c/sup\u003e dendritic cells were isolated from the peripheral PBMCs of the patients with SLE (n\u0026thinsp;=\u0026thinsp;10) and the healthy controls (n\u0026thinsp;=\u0026thinsp;10) using flow cytometer sorting. As shown the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, we observed greater expression of NRP1 in the dendritic cells of the patients with SLE compared to those of healthy controls (healthy controls, 2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 vs. SLE, 4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) in the immunocytochemistry analysis. Based on these results, we analyzed the correlation between the level of NRP1 expression and the disease activity of the patients with SLE. To determine the NRP1 expression, we conducted an analysis of isolated PBMCs obtained from a group of 57 patients with SLE and 57 age- and sex-matched healthy controls. PCR was utilized as the method for measuring NRP1 expression in these samples. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, significantly more NRP1 was expressed in the patients with SLE than the healthy controls (healthy control, 0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 vs. SLE, 6.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). We then analyzed the correlation between NRP1 expression of PBMCs and disease activity markers of SLE, including SLEDAI score, C3, C4, and anti-dsDNA antibody titer. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e described the baseline clinical characteristics of the patients with SLE. Seventeen patients were excluded due to the lack of some necessary clinical data. The mean age of patients was 37 years old and their mean SLEDAI-2K score was 7.4, which indicates moderate activity. Thirty percent of the patients have nephritis and their mean UACR was 1731 mg/g. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB shows the significant correlations between NRP1 and disease activity markers; the positive correlation with the SLEDAI score (r\u0026thinsp;=\u0026thinsp;0.56, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and the anti-dsDNA antibody titer (r\u0026thinsp;=\u0026thinsp;0.31, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04), as well as the negative correlation with C3 (r= -0.36, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) and C4 (r= -0.32, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04). These results suggest the possible involvement of NRP1 in the pathogenesis of SLE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical characteristics of patients with SLE\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePatients with SLE\u003c/p\u003e \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35 (87.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (yrs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;15.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC (x 10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocytes (x 10\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemoglobin (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlatelet (x 10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e222.0\u0026thinsp;\u0026plusmn;\u0026thinsp;120.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC3 (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.2\u0026thinsp;\u0026plusmn;\u0026thinsp;27.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC4 (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-dsDNA antibody (IU/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e141.4\u0026thinsp;\u0026plusmn;\u0026thinsp;251.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eESR (mm/hr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.5\u0026thinsp;\u0026plusmn;\u0026thinsp;28.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCRP (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;23.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSLEDAI-2K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNephritis, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (30%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (8.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 (66.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (25.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUPCR (mg/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1731\u0026thinsp;\u0026plusmn;\u0026thinsp;2487\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eData are expressed as number (percentage) or mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eCRP, C-reactive protein; ESR, erythrocyte sedimentation rate; SLEDAI-2K, SLE activity index 2000; UPCR, urine protein to creatinine ratio\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEffect of NRP1 inhibition on viability and the function of dendritic cells\u003c/h2\u003e \u003cp\u003eAlong with this finding, we used NRP1 antagonist (EG00029) to investigate the effect of NRP1 on the viability and function of dendritic cells. Referring to previous studies, including the experiments using EG00229 [33\u0026ndash;35], allowed us to determine the range of the effective dose, and minimal cellular toxicity was confirmed through a serial single-dose toxicity study (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). BMDCs were treated with a concentration of 100\u0026micro;M of EG00229 to assess the viability and function of these cells. The expression of NRP1 of BMDCs was significantly increased by the activation of TLR7 with R848, and this expression was decreased by EG00229 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). To assess the roles of NRP1 in determining the viability of BMDCs, a cell proliferation assay was performed for 24 hrs and 48 hrs using a CCK-8 assay. The treatment with R848 substantially increased the cell viability of BMDCs after both incubation periods. However, this increase was significantly inhibited when EG00229 was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA co-culture assay of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells and dendritic cells treated with NRP1 antagonist was prepared to investigate the effects of NRP1 on dendritic cell-induced T cell proliferation. NRP1 antagonist-treated BMDCs from mice were co-cultured with CD4\u003csup\u003e+\u003c/sup\u003e25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells labeled with CFSE at a ratio of 1:1 for 5 days. TLR7 agonist-treated dendritic cells significantly increased the proliferation of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells after the co-culture of these two cells, and these effects were abrogated to a statistically significant degree when co-cultured NRP1 antagonist-treated dendritic cells and CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells (Control, 10.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55; R848-treated, 18.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08; EG00229-treated, 7.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56; EG00229 and R848-treated, 7.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the impact of NRP1 on T cell proliferation mediated by human dendritic cells, we conducted a co-culture assay using CD4\u0026thinsp;+\u0026thinsp;CD25- T cells and dendritic cells derived from healthy controls. In this assay, we treated the dendritic cells with an NRP1 antagonist, similar to the experiment performed with mice BMDCs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, the dendritic cells activated by the TLR7 agonist without pre-treatment with a NRP1 antagonist significantly increased CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cell proliferation. However, this increased proliferation of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells in TLR7 agonist treated dendritic cells was inhibited by the addition of NRP1 antagonist, EG00229. (Control, 35.49\u0026thinsp;\u0026plusmn;\u0026thinsp;9.88; R848-treated, 53.68\u0026thinsp;\u0026plusmn;\u0026thinsp;6.80; EG00229-treated, 44.31\u0026thinsp;\u0026plusmn;\u0026thinsp;5.63; EG00229 and R848-treated, 41.80\u0026thinsp;\u0026plusmn;\u0026thinsp;6.94). We also evaluated the effects of natural dendritic cells sorted from the PBMCs in the SLE patients on the proliferation of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). We noted less proliferation of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells co-cultured with the NRP1 antagonist-treated dendritic cells than CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells co-cultured with vehicle-treated dendritic cells (vehicle-treated, 59.57\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7; EG00229-treated, 41.03\u0026thinsp;\u0026plusmn;\u0026thinsp;11.27, p\u0026thinsp;=\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Our co-culture assays suggest that the NRP1 component of dendritic cells plays a role in determining the viability and function of dendritic cells, potentially affecting activation and proliferation of T cells, which are the pivotal to the pathogenesis of SLE.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEffect of NRP1 antagonist on the STAT, MAPK and NF-κB signaling in dendritic cells\u003c/h2\u003e \u003cp\u003eAs NRP1 was hypothesized to affect the viability and function of dendritic cells, we examined the effects of NRP1 on the intracellular signaling pathways of dendritic cells. These pathways, including those associated with MAPK and NF-κB activation, have been shown to promote cell survival, proliferation [36]. NF-kB in particular is known to promote dendritic cell maturation into professional antigen-presenting cells [37]. The dendritic cells were stimulated with R848 with or without pretreatment of NRP1 antagonist and their signaling pathways were evaluated. Although TLR7 activation by R848 increased the ratio of p-STAT1/STAT1 and p-STAT3/STAT3, there were no significant differences after treatment with the NRP1 inhibitor, EG00229 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). In the MAPK pathways, the phosphorylation of ERK 1/2 and p38 of the dendritic cells was increased by R848 treatment, and this was attenuated by the NRP1 antagonist (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Furthermore, NRP1 inhibition also decreased the R848-induced phosphorylation of NF-κB of the dendritic cells, more so than in the R848-only treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). Our results suggest that upregulated expression of phosphorylated MAPK and NF-kB in dendritic cells by TLR7 activation is abolished by NRP1 antagonist treatment. In conclusion, these findings indicate that NRP1 enhances the survival and activation of dendritic cells through the activation of intracellular signaling pathways, specifically MAPK and NF-kB. Consequently, this leads to immune dysregulation by activating T cells, which are widely recognized for their crucial involvement in the pathogenesis of SLE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRecently, the involvement of NRP1 in the pathogenesis of various diseases has been discovered. As a regulator, NRP1 is now known to be involved in illnesses as diverse as eye diseases [38], angiogenesis [39], osteoblastogenesis [40], and tumorigenesis [41]. Research into NRP1 is particularly exciting in the area of tumor immunity, with evidence now showing that NRP1 is highly expressed in cancer cells and tissues, and is involved in cancer cell proliferation and metastasis (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Based on these findings, NRP1 is now regarded as a predictor or prognostic marker for certain types of malignancies, and as a promising therapeutic target for antitumor immunity [9, 42\u0026ndash;45]. Although malignancy and autoimmunity are distinct pathological conditions, they share similarities in terms of the underlying microenvironment that contributes to the immunopathological mechanisms, such as the aberrant activation of the immune or inflammatory system [46]. However, NRP1's involvement in the field of autoimmunity has not been thoroughly explored. To the best of our knowledge, this study is the first to investigate the role of NRP1 in the pathogenesis of SLE, which is considered a representative autoimmune disease. In the present study, we showed the significantly higher expression of NRP1 in those with SLE and the correlation of this expression with disease activity. NRP1 has modulatory effects on the function and viability of dendritic cells, and these effects are mediated by MAPKs and the NF-κB signaling pathway. We first confirmed higher levels of NRP1 in the Mo-DC and the PBMCs in patients with SLE than in healthy controls, and NRP1 expression in the PBMCs was significantly correlated with the disease activity of SLE. A limited number of studies regarding elevated NRP1 expression in SLE have been reported in the other specimens of the patients with SLE. A previous study has reported a significant increase in urinary NRP1 levels in individuals with SLE. This finding suggests that urinary NRP1 levels could serve as a potential discriminatory marker for indicating active lupus nephritis, distinguishing it from inactive lupus nephritis [47]. Similarly, Vadasz et al. showed a positive correlation between NRP1 deposits in the glomeruli and the clinicopathological parameters of nephritis [48]. Consistent findings were observed in our \u003cem\u003ein vivo\u003c/em\u003e test that used a murine model, which showed higher fluorescence intensity in the kidneys of lupus models than in those of the control group. Taken together, the observed upregulation of NRP1 expression in the kidney, the primary organ affected in SLE, as well as in PBMCs, particularly in dendritic cells, suggests that NRP1 could play a role in the development and progression of immune abnormalities associated with the pathogenesis of SLE.\u003c/p\u003e \u003cp\u003eThe role of NRP1 in cell proliferation in other cell types, especially in cancer cell lines, has been elucidated. NRP1-positive lung cancer cells have shown higher clonogenic and self-renewal capacity, which is a tumor-initiating property, than NRP1 gene knockdown cells [49]. Additionally, studies have demonstrated that microRNA-145 and microRNA-205 can suppress the proliferation of uveal melanoma cells by targeting and inhibiting NRP1 expression [50]. Since the initial discovery of NRP1 expression in dendritic cells and its role in T cell proliferation a decade ago [14], extensive research has been conducted to explore the association between NRP1 and various pathologic conditions linked to immune dysregulation. In our study, we focused on the role of NRP1 in dendritic cells in the pathogenesis of SLE. Our results are consistent with the prior findings, inasmuch as they show the capacity for cell proliferation by NRP1. Accordingly, this study is significant as it is the first to identify NRP1's involvement in the pathogenesis of SLE, specifically as the causative mechanism behind the abnormal increase of pathological effector T cells.\u003c/p\u003e \u003cp\u003eThe mechanisms by which NRP1 increases cell viability and function may be multifactorial. In our study, we specifically examined the MAPKs and NF-κB signaling pathways as the underlying signaling pathways mediated by NRP1. These pathways are intricately involved in cell survival and the crucial function of antigen presentation to T cells. We showed that stimulation with a TLR7 agonist upregulated the phosphorylation of ERK 1/2 and p38, and NRP1 antagonists decreased NRP1 expression with significant attenuation of the activation of these MAPKs in dendritic cells. The activation of ERK 1/2 or p38 signaling in association with cell survival and proliferation is well documented in a broad variety of diseases including tumorigenesis [51, 52]. This study showed that inhibition of MAPK activation by an NRP1 antagonist decreases the proliferation of dendritic cells. Antigen-presentation is a key role of dendritic cells, and the activation and expansion of T cells is promoted following antigen recognition. NF-κB is already known as a major regulator of the antigen-presenting function as well as cell survival in dendritic cells [53, 54]. Consistent with these findings, we showed that NRP1 antagonists decrease the phosphorylation of NF-κB in dendritic cells and consequently reduce T cell expansion. These findings indicate that NRP1 affects the viability and function of dendritic cells on T cell proliferation via the phosphorylation of MAPKs and the NF-κB signaling pathways.\u003c/p\u003e \u003cp\u003eIn clinical practice, multiple disease activity markers are employed to effectively manage SLE. However, these markers have certain limitations, including the potential to yield complex, incorrect, or conflicting results. This study demonstrated a correlation between the expression of NRP1 of PBMCs and SLE disease activity markers, including C3, C4, anti-dsDNA antibody, and SLEDAI scores. Given the limited utility of available alternative SLE markers, we suggest that NRP1 may have a role to play as an additional disease activity marker. We further suggest that NRP1 expression in various immune cells and glomeruli could serve as a non-invasive and sensitive biomarker for predicting diverse clinical features, specific histopathologic characteristics of lupus nephritis, and responsiveness to medical treatment, although further studies are necessary to validate this suggestion.\u003c/p\u003e \u003cp\u003eThis study has some limitations inherent to how it sought determine the specific roles of NRP1 in the pathogenesis of SLE. First, the number of enrolled patients with SLE was small, so further studies with a large number of patients with SLE will be needed. Second, as we had only a limited number of sorted dendritic cells from PBMCs in healthy controls and patients with SLE, we analyzed the expression of NRP1 in whole PBMCs, as opposed to isolated dendritic cells, to evaluate the correlation between NRP1 expression and disease activity markers. The PBMC count in patients with SLE was found to be significantly lower compared to healthy controls. Moreover, the population of dendritic cells accounted for only a small fraction (1\u0026ndash;2%) of the total PBMCs [55]. To determine the precise roles of NRP1 in the pathogenesis of SLE and its potential as a disease activity marker, further studies involving isolated dendritic cells will be necessary. Thirdly, we did not assess the impact of NRP1 on the antigen-presenting function of dendritic cells in our study. Nevertheless, it is widely documented that T cell proliferation is contingent upon antigen presentation by dendritic cells [56]. While we did not specifically investigate the direct effects of NRP1 on the antigen-presenting function of dendritic cells, the significant increase in T cell expansion observed in our study provides crucial evidence of the enhanced function of dendritic cells mediated by NRP1.\u003c/p\u003e \u003cp\u003eIn conclusion, this study provided evidence of the increased expression of NRP1 in the PBMCs, as well as in the kidneys and dendritic cells, in both murine models and patients with SLE, as compared to the control group. NRP1 expression shows a significant correlation with disease activity in SLE patients. NRP1 modulates the viability and function of dendritic cells and enhance dendritic cell-induced T cell proliferation via the phosphorylation of intracellular MAPKs and NF-κB signaling pathways. Our study underscores the significant role of NRP1 in immune dysregulation during the progression of SLE, highlighting its potential as a promising therapeutic target for the effective management of this complex autoimmune disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by a grant from the National Research Foundation of KOREA (NRF) funded by the Korea government (MSIT) (No. 2018R1D1A3B07041314 and No.2021R1G1A1094571), and by the Fund of the Biomedical Research Institute, Jeonbuk National University Hospital\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Y.C. and W.Y.; Data collection and investigation, K.K. and E.L.; draft manuscript preparation, Y.C. and E.L.; manuscript review and editing, W.Y. and Y.C.; supervision, W.Y. All authors have read and agreed to the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLisnevskaia, L., Murphy, G. \u0026amp; Isenberg, D. Systemic lupus erythematosus. 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Proc Natl Acad Sci U S A 116, 5914\u0026ndash;5919, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1812800116\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1812800116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3209000/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3209000/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eNeuropilin-1 (NRP1) is a transmembrane glycoprotein that acts as a receptor of class III/IV semaphorins known to play a role in the pathogenesis of autoimmune diseases. To date there has been only limited research into the role NRP1 plays in autoimmune inflammatory rheumatic diseases, including systemic lupus erythematosus (SLE). This study aimed to investigate the clinical and pathogenetic roles of NRP1 expression in lupus mouse models and patients with SLE.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eNRP1 expression was measured by flow cytometry, polymerase chain reaction (PCR), and immunofluorescence assay using peripheral blood mononuclear cells (PBMCs) taken from both healthy controls and patients with SLE, as well as dendritic cells and renal tissues of both control mice and TLR-7 agonist-induced lupus mice. The correlation between NRP1 expression in PBMCs and disease activity markers were analyzed in patients with SLE (n\u0026thinsp;=\u0026thinsp;57). To determine the effects of NRP1 on dendritic cells on T cells, as well as their mechanism, a proliferation assay was performed by flow cytometry, and the underlying signaling, including the MAPKs and NF-κB pathway, were examined with immunoblotting.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe expression of NRP1 in dendritic cells and the kidneys was significantly higher in the lupus murine group than in the control group. The dendritic cells in the patients with SLE also showed a markedly higher expression of NRP1 than those of the healthy controls. The correlation analysis showed a significant positive relationship between NRP1 expression and disease activity markers, which included SLEDAI-2K score, as well as C3, C4 and anti-dsDNA antibody titers. The NRP1 antagonist (EG00229) decreased the capacity of dendritic cells on the proliferation of T cells under the condition of TLR7 agonist stimulation. It also downregulated the phosphorylation of ERK1/2 and NF-κB in dendritic cells.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur results show that NRP1 is highly expressed in the dendritic cells of SLE patients, and its expression is significantly correlated with known disease activity markers. The inhibition of NRP1 in dendritic cells diminishes the proliferation of T cells, an effect that is mediated by the suppression of MAPKs and NF-kB signaling. These results indicate that dendritic cells with enhanced NRP1 expression alter immune functions by increasing T cell proliferation as part of the pathogenesis of SLE; accordingly, NRP1 may be a potential target in the search for a treatment for SLE.\u003c/p\u003e","manuscriptTitle":"Enhanced NRP1 Expression in Dendritic Cells of Systemic Lupus Erythematosus and Its Impact on T Cell Proliferation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-08 14:48:00","doi":"10.21203/rs.3.rs-3209000/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8937a2ff-c1e9-4853-bd33-4f202307b4d0","owner":[],"postedDate":"August 8th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":23769274,"name":"Biological sciences/Immunology"},{"id":23769275,"name":"Health sciences/Diseases"},{"id":23769276,"name":"Health sciences/Rheumatology"}],"tags":[],"updatedAt":"2024-06-04T10:09:36+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-08 14:48:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3209000","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3209000","identity":"rs-3209000","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}