Treatment with a Sphingosine-1-phosphate Receptor Five Agonist Enhances Recovery of Experimental Autoimmune Neuritis by Promoting Production of Schwann Cell Regeneration Factors

Treatment with a Sphingosine-1-phosphate Receptor Five Agonist Enhances Recovery of Experimental Autoimmune Neuritis by Promoting Production of Schwann Cell Regeneration Factors | 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 Treatment with a Sphingosine-1-phosphate Receptor Five Agonist Enhances Recovery of Experimental Autoimmune Neuritis by Promoting Production of Schwann Cell Regeneration Factors Takafumi Uchi, Shingo Konno, Hideo Kihara, Toshiki Fujioka This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4677686/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 Experimental autoimmune neuritis (EAN) serves as a model for studying autoimmune peripheral neuropathies. This study investigated the effects of a sphingosine-1-phosphate receptor 5 (S1Pr5) agonist on EAN recovery and nerve regeneration. Lewis rats with induced EAN were treated with the S1Pr5 agonist A-971432. Clinical scores, cytokine production, and expression of regeneration factors were analyzed. Histological examination of the cauda equina was also performed. Treatment with the S1Pr5 agonist promoted recovery of clinical symptoms in the later stages of EAN. While the agonist did not significantly modulate pathogenic cytokine expression, it enhanced the expression of Jun proto-oncogene and Sonic Hedgehog (Shh) mRNA, crucial molecules for peripheral nerve regeneration. Histological analysis revealed increased Shh-positive cells in the nerves of treated rats. These findings suggest that S1Pr5 agonists have the potential to enhance recovery from EAN by promoting nerve regeneration rather than by modulating the inflammatory response. This study provides insights into novel therapeutic approaches for autoimmune peripheral neuropathies, highlighting the role of S1Pr5 in nerve repair processes. Further research is needed to fully elucidate the mechanisms and validate these findings for potential clinical applications. Health sciences/Neurology Health sciences/Pathogenesis Guillain-Barré syndrome chronic inflammatory demyelinating polyneuropathy experimental autoimmune neuritis sphingosine-1-phosphate receptor Schwann cell regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are both autoimmune conditions that affect the peripheral nervous system which display unique clinical characteristics and progression patterns. GBS manifests acutely, with rapid symptom escalation over several weeks, while CIDP progresses chronically, with symptom development spanning months. Typically, GBS is preceded by infection, while CIDP lacks clear precipitation events. Symptoms of GBS generally plateau and improve, whereas CIDP symptoms persist and require ongoing treatment. Further, GBS usually occurs as a singular episode, while CIDP shows significant potential for relapse or continuous progression [ 1 – 3 ]. Despite these clinical variations, both disorders are linked by an underlying fundamental immune-mediated damage to peripheral nerves. Other similarities include their autoimmune nature, effects on the myelin sheath and nerve fibers, and symptoms, all of which stem from the erroneous attack of peripheral nerves by the immune system. This leads to inflammation and damage, primarily affecting the myelin sheath or axons, disrupting electrical signal transmission and causing symptoms such as weakness, numbness, and sometimes pain in the limbs. However, a high variability in symptom severity and progression is notable among individuals and diseases. Accurate identification of both commonalities and distinctions is essential for efficient diagnosis, therapeutic interventions, and patient care management. The presence of anti-ganglioside antibodies in 50–60% of GBS patients highlights the importance of humoral immune mechanisms in peripheral neuropathies [ 1 , 2 ]. The primary therapeutic approach for GBS is the administration of intravenous immunoglobulin [ 4 ], yet some individuals may suffer from persistent sequelae [ 5 ]. Ongoing clinical studies are assessing the effectiveness of combining complement C5 inhibitors like eculizumab with intravenous immunoglobulin to address complement-driven injury [ 6 ]. Treatment options for CIDP encompass corticosteroids, plasmapheresis, and intravenous immunoglobulin; these methods are effective, but corticosteroids have considerable adverse effects, and other treatments generally need regular application. After five years, only 26% of CIDP patients reach full remission while 39% continue to require treatment [ 7 ], underscoring the necessity for innovative therapeutic approaches. Experimental autoimmune neuritis (EAN) acts as a model to investigate autoimmunity-related peripheral neuritis and the development of therapies in humans [ 8 ]. IFN-γ-producing autoreactive T lymphocytes, which promote macrophage infiltration to peripheral nerves, induce EAN. EAN and CIDP exhibit similar pathological characteristics, particularly the predominance of cellular immune responses. The nerve pathology in CIDP often includes mild T-cell infiltration and pronounced macrophage presence in the endoneurium, critical for demyelination processes [ 9 ]. EAN characteristically shows multifocal infiltration of mononuclear cells around blood vessels, comprising lymphocytes, which lead to nerve demyelination through macrophage action [ 8 ]. S1P receptors, a group comprising five G protein-coupled receptors, play a significant role in peripheral nerve regeneration [ 10 – 12 ]. Our research indicates that the selective S1Pr1 and S1Pr5 agonist, siponimod, significantly reduces EAN severity by inhibiting lymphocyte migration, a process that depends on the S1P gradient, and involves the activation of the Jun proto-oncogene (C-Jun) and Sonic Hedgehog (Shh) pathways [ 13 ]. Peripheral nerves further demonstrate superior regenerative abilities compared to the central nervous system, a feature enhanced by S1Pr5, while their absence in rat models impedes nerve regeneration. Further research suggests a critical role of Schwann cell transformation in nerve repair, enhanced by S1Pr5 agonist application, which stimulates the Schwann cell repair mechanism and fosters effective nerve regeneration [ 10 ]. However, the reparative potential of S1Pr5 in the EAN-damaged nerves remains unclear. Therefore, this study aimed to evaluate the efficacy of the S1Pr5 agonist A-971432 in treating EAN and to explore the molecular mechanisms underlying the therapeutic effects of S1Pr5. Materials and Methods In vitro experiments Cell Culture and Sphingosine-1-phosphate receptor 5 agonist treatment Schwann cells of the IFRS1 lineage were obtained from COSMO BIO Co., Ltd. (catalog number: PMC-SWN-IFRS1C) and cultured in a specialized medium specifically designed for this line, purchased from COSMO BIO Co., Ltd. (catalog number: PMC-SWN-IFRS1C-COS). This medium was formulated to provide optimal support for the growth and maintenance of the IFRS1 lineage of Schwann cells, ensuring proper cultivation and suitability for experimental purposes. The cells were plated in 100 mm plastic culture dishes, and incubated at 37°C in a humidified environment with 5% CO2. The cultures were allowed to reach a subconfluent state over a 25-day period. On day 25, the cells were treated with various concentrations of the S1Pr5 agonist (0, 25, 50, 100, or 200 μM). The S1Pr5 agonist dissolved in dimethyl sulfoxide (DMSO), which also served as a vehicle control. The experimental setup included six conditions: four agonist concentrations and two control groups. One control group was administered a high concentration of DMSO, whereas the other remained untreated. The messenger RNA Analysis Twenty-four hours post-treatment, cells underwent RNA harvesting for the purpose of quantifying the mRNA expression of C-Jun and Shh through qRT-PCR. The RNA was extracted using Qiagen's RNeasy® Universal Mini kit, located in Tokyo, Japan, and cDNA was subsequently synthesized utilizing iScript RT SuperMix for qRT-PCR from Bio-Rad, also based in Tokyo, Japan. This cDNA was then employed as a template in real-time PCR, conducted using iTaq™ Universal SYBR ® Green Supermix from Bio-Rad, with gene-specific primers obtained from TAKARA BIO (Otsu, Japan) and Bio-Rad. Primers for sequencing are detailed in Supplementary data (Table 1). Semi-quantitative analyses of the mRNA levels of transcription factors associated with Schwann cell regeneration were conducted, referencing an endogenous control on the CFX96 Touch™ Real-Time PCR Detection System from BIO-RAD. These gene expression levels were expressed as relative copy numbers, calculated via the ΔΔCq method, offering a comparative assessment of mRNA levels. In vivo experiment Subjects The 30 female Lewis rats（Charles River Japan, Yokohama, Japan) used in this study were 7 weeks old at the start of the experiment. The rats were housed in the Toho University Ohashi Experimental Animal Laboratory. The gages were maintained at 25 °C with a 12-h light–dark cycle. The rats were provided with a comfortable and clean-living environment, which included proper bedding and enrichment to promote natural behaviors. The diet and hydration were carefully monitored to ensure adequate nutrition. Regular health checks were conducted to promptly identify and address any health issues. Additionally, all procedures and handling of the rats were performed by trained personnel to minimize stress and discomfort. Declarations All procedures regarding the use and handling of animals in research were reviewed and approved by the Toho University Animal Care and User Committee (approval numbers 20-442 and 24-567) and all the experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals in research. All the performed procedures and all the reported data were in accordance with the ARRIVE guidelines. Induction and evaluation of EAN Rats were subcutaneously injected with 150 µg of a synthetic peptide that corresponds to amino acid residues 53–78 of bovine P2 protein (TESPFKNTEISFKLGQEFEETTADNR, Operon, Tokyo, Japan), emulsified in an equal volume of Complete Freund's adjuvant (Sigma-Aldrich, MO, USA). The injections were administered to the right footpad with rats were under light anesthesia induced by sevoflurane (Mylan, Osaka, Japan) [14]. The motor function of the rats was observed daily, and a scoring system was used to assess paralysis of the tail, forelimbs, and left hind limb. The tail motility was scored from 0 to 3, with 0 indicating no clinical signs, 1 indicating paralysis of the tip, 2 indicating incomplete paralysis of the entire tail, and 3 indicating complete paralysis of the entire tail. Forelimbs were scored from 0 to 3, with 0 indicating no clinical signs, 1 indicating an inability to climb a fence using the forelimbs, 2 indicating an inability to walk, and 3 indicating complete paralysis. The left hind limb was scored from 0 to 3, with 0 indicating no clinical signs, 1 indicating paralysis of the toe only, 2 indicating incomplete dorsiflexion of the foot joint while walking, and 3 indicating complete paralysis, where the legs were dragged when walking. The rat with shallow breathing suspected respiratory muscle paralysis were euthanized with an overdose of sevoflurane before reaching that state. In this study, 30 rats were sacrificed across all phases, with 10 each on day 10 post-immunization (p.i.) for the subclinical to acute phase, day 14 p.i. for the early peak phase, and day 21 p.i. for the recovery phase. Sphingosine-1-phosphate receptor 5 agonist treatment The S1Pr5 agonist A-971432, (Cayman Chemical Co., Ltd., Michigan, USA) was in a solution of 0.5% carboxymethylcellulose (CMC) in phosphate-buffered saline (PBS) in preparation for oral administration. This solution was administered to the experimental rats, specifically those assigned to the S1Pr5 agonist group, at a daily dose of 1.0 mg/kg, starting from day 5 and continuing until day 20 p.i.. The purpose of this daily administration was to assess the effects of S1Pr5 agonist within the scope of the study. Tissue collection Rats were sedated using sevoflurane and underwent complete perfusion with ice-cold PBS on days 10, 14, and 21 p.i. Subsequently, the lumbar spinal cord was excised, and a portion of the cauda equina was preserved in 10% buffered formalin for histological examination. The other portion was kept in Allprotect® Tissue Reagent (Qiagen, KK, Tokyo, Japan) at -80°C in a deep freezer for subsequent analysis of mRNA and cytokines. Sequential analysis of cytokine production related to pathogenesis of EAN The cauda equina was subjected to enzyme-linked immunoassay (ELISA) using a 96-well plate (specifically the Rat IFN-γ and IL-10 DuoSet® ELISA Kit from biotech, USA). To achieve this, the cauda equina sample stored in Allprotect® tissue reagent was dissociated in 750 ml solution of 10 % radioimmunoprecipitation assay lysis buffer including protease inhibitor (Santa Cruz Biotechnology, CA, USA) using a bead tissue grinder (Shakeman™, Biomedical Science, Tokyo, Japan). The ELISA process was then initiated by diluting the capture antibody for each cytokine and coating each well. The plates were incubated overnight at room temperature, followed by washing to remove excess antibodies. Nonspecific sites were blocked using the reagent diluent (10% BSA dissolved in PBS), after which the plate was incubated again. Subsequently, 20 μg/ml of homogenized cauda equina solution or standards were added to the wells, covered, and incubated for two hours to allow antigen binding. After this incubation period, the plate was washed again before adding the detection antibody, followed by another incubation and washing step. Streptavidin-horseradish peroxidase was then introduced into the plate, which was incubated in the dark before being washed again. To develop color, a substrate solution was added to the plate and the reaction was stopped with a stop solution before mixing. Finally, the absorbance was measured at 450 nm with an iMark™ Microplate Absorbance Reader (BIO-RAD, Tokyo, Japan). Cytokine concentrations are expressed in pg/mL. Sequential analysis of messenger RNA expression of regeneration of peripheral nerv e Before RNA extraction, the cauda equina was crushed using a bead-type tissue grinder (Shakespeare Biomedical Science, Tokyo, Japan). The mRNA expression of the transcription factors C-Jun and Shh, which are related to Schwann cell regeneration, were subsequently analyzed. RNA extraction, cDNA synthesis, and mRNA expression analyses were performed in the same manner as described for Schwann cells. Histological analysis The cauda equina collected on day 14 p.i. was embedded in paraffin and cut into transverse sectional 5 μm thick slices. Luxol fast blue (LFB) staining and counterstaining with hematoxylin were applied to identify the areas of demyelination and infiltrating cells, respectively. Using the Hitsugi_planimetry software (https://hitsugi-edu-inst.main.jp/computer-software/Hitsugi-planimetry-manual), the ratio of demyelinated area and the number of infiltrating cells per square micrometer were quantified in 50 randomly sampling transverse sectional nerves of cauda equina from all groups. To validate the effect of the S1Pr5 agonist on cellular infiltration, we used specific antibodies to identify T lymphocytes and macrophages with the highest precision. T lymphocytes were detected using an anti-CD3 rabbit monoclonal antibody (SP7, Nichirei Biosciences Inc., Tokyo, Japan), whereas macrophages were identified using an anti-Iba-1 rabbit polyclonal antibody (GTX100042; GeneTex Inc., Irvine, CA, USA), following the standard antigen retrieval protocol recommended by the manufacturers. Shh was chosen as a marker for the regeneration of damaged Schwann cells because of its higher mRNA production compared to C-Jun. Anti-Shh rabbit polyclonal antibodies (St Johns Laboratory Ltd, London, UK) were applied to paraffin-embedded sections without an antigen retrieval protocol, according to the manufacturer's instructions. A peroxidase-labeled anti-rabbit IgG goat polyclonal antibody conjugated with an amino acid polymer (N-Histofine® Simple Stain MAX-PO (R)TM, Nichirei Biosciences Inc., Tokyo, Japan) was used to visualize these antibodies on tissue sections, followed by diaminobenzidine as a chromogen. Statistical analysis Analysis of the groups was performed with the Mann–Whitney U test, a non-parametric method. The R software (version 3.2.2; R Foundation for Statistical Computing, Vienna, Austria), utilizing the rms package [15], was employed to process the data, setting statistical significance at p < 0.05. Unless specified differently, results are expressed as mean ± SEM. Results In vitro experiment mRNA of expression regeneration factors from the Schwann cells. Our analysis identified a gradual increase in C-Jun expression with increasing concentrations of the S1Pr5 agonist, peaking at 50 μM (Fig.1A). A statistically significant increase in expression was further observed between the control and 50 μM S1Pr5 agonist-treated groups (3.0-fold increase, p<0.05). The expression dropped sharply at 200 μM, indicating a possible concentration-dependent effect, with a peak or optimal concentration for upregulation at 50 μM. Shh expression further showed a similar pattern, with increased expression at higher concentrations of the S1Pr5 agonist (Fig.1B). Statistically significant increases in expression compared to the control group were observed at both 50 and 100 μM NaCl (4.2 and 2.9-fold increases, respectively). The peak expression of Shh was observed at 50 μM, and, similar to that of C-Jun, we observed a decrease in expression at the highest concentration of 200 μM. In vivo experiment Clinical severity of rats Both groups had a clinical score of zero at the beginning of the observation period (Fig.2). The clinical score for the EAN group began to increase sharply from around day 8 p.i., reached a peak score around day 13 p.i., plateauing until around day 17 p.i., before declining slightly. The clinical score for the S1Pr5 agonist group similarly began to increase around day 8 p.i., but reached a slightly lower peak around day 13 p.i. compared to the EAN group. The score for the S1Pr5 agonist group also remained elevated until day 17 p.i., before declining again. Both groups showed a decline in the clinical score after day 17 p.i., with the EAN group decreasing to a score just below 8, and the S1Pr5 agonist group declining to a score of approximately 7 by day 21 p.i.. The treatment group had milder symptoms on day 18-21 p.i. than the EAN group (p < 0.05). Although some rats scored nine points, most sever score, none developed respiratory muscle paralysis. EAN pathogenesis-related cytokines in the cauda equina The production of IFN-γ and IL-10 production in the cauda equina were found to be markedly increased in the EAN and S1Pr5 groups compared to normal rats, which served as a negative control, but there was no significant difference between the groups between the different time points (Fig.3A and B). These results suggest that the inflammatory activity of infiltrating cells in the cauda equina was not significantly different between the groups. The IFN-γ levels further showed a pronounced response on day 10 p.i. (in the acute phase) that was moderated over time. However, the S1Pr5 agonist did not significantly mitigate this response. IL-10 levels were stable across different conditions and time points. Expression of regeneration factor mRNAs in the cauda equina during the peripheral nerve repair process C-Jun mRNA expression was significantly upregulated in the S1Pr5 agonist groups compared to normal rats at each time point, with the S1Pr5 group showing slightly higher expression than the EAN group at each time point (Fig. 4A). Shh expression was significantly higher in the S1Pr5 group than that in the normal and EAN groups. Significantly higher expression was further noted in the S1Pr5 group on days 10 and 12 p.i.. By day 21 p.i., the expression levels in the S1Pr5 group decreased, but remained higher than those in the EAN group, which showed no apparent increase (Fig. 4B). Histological examination of the cauda equina At the peak phase of clinical symptoms, on day 14 p.i., mononuclear infiltrating cells appeared patchy and multifocal in the cauda equina in all groups (Fig.5). The demyelination area that was LFB stain-negative area and the number of infiltrated mononuclear cells, which were hematoxylin stain-positive in the transverse sectional nerves, showed no difference between the groups (Fig.6). Immunohistochemical analysis demonstrated approximately the same degree of CD3-positive T lymphocytes and Iba-1-positive macrophages were counted in both groups (Fig.5). Moreover, we immunohistochemically stained for Shh to evaluate its role as a nerve trans-differential factor because Shh mRNA was significantly increased in the S1Pr5 agonist group compared to the EAN group. The number of Shh-positive cells significantly increased in the nerve of S1Pr5 positive group than the EAN group (Fig.7 and 8). Discussion Overall, this study identified the following results: 1) Treatment with an S1Pr5 agonist promoted the recovery of clinical symptoms in EAN. 2) The S1Pr5 agonist did not modulate the expression of cytokines related to the pathogenesis of EAN in the cauda equina; 3) The S1Pr5 agonist enhanced expression of C-Jun and Shh mRNA, which are crucial molecules for the regeneration of peripheral nerves, in the cauda equina. Pathogenic cytokines and molecules related to nerve repair in the cauda equina provide important insights into the effects of S1Pr5 agonists on the inflammatory and regenerative responses during neural injury in autoimmune neuritis. S1Pr5 agonists interact with S1Pr5, a receptor that responds to S1P, and plays a pivotal role in various physiological processes. S1P5 is predominantly expressed in the nervous and immune systems, influencing natural killer cell egress [16, 17]. Studies have further shown that selective agonists such as A-971432 stimulate S1Pr5, maintain the integrity of the blood-brain barrier, and exhibit therapeutic effects in conditions such as Huntington's disease [18]. Furthermore, S1Pr5 is present at synapses. and inhibits the spontaneous activity of neurons in culture, an action distinct from that of other S1PRs [19]. Furthermore, in the central nervous system, S1Pr5 is mainly present in oligodendrocyte progenitors and mature oligodendrocytes, and is thought to contribute to the regulation of the differentiation processes [20]. S1Pr5 significantly regulates tissue-resident memory (TRM) CD8 (+) cells [17], and is critical for controlling T cell migration and retention in peripheral tissues, such as the skin. S1Pr5 is further involved in T-lymphocyte trafficking and migration. It is typically downregulated in tissue-resident memory T lymphocytes, allowing them to remain in the peripheral tissue instead of circulating back into lymphoid organs. This downregulation is crucial for the retention and proper functioning of TRM cells, which provide localized and rapid immune responses upon re-exposure to pathogens. Furthermore, the expression of S1Pr5 is influenced by several factors, including the transcription factors T-bet and Zinc finger E-box-binding homeobox 2 (ZEB 2). T-bet and ZEB2 both upregulate S1Pr5 during differentiation of effector T cells from naïve T lymphocytes. However, in the tissue-resident memory formation process, local signals such as Transforming Growth Factor-β in the tissue microenvironment promote the downregulation of T-bet and ZEB2, subsequently leading to decreased S1Pr5 expression. The induction and control of murine EAN reportedly depends on CD4(+) and CD8(+) T lymphocytes, while B cells do not perpetuate the related inflammatory demyelination. EAN-induced CD8(-/-) mice have a milder clinical course, fewer histopathological changes, and a diminished T-cell response to the P0 peptide [21]. CD8(+) cells are related to the pathogenesis of EAN, the acute autoimmune neuritis model used in this study, are mainly circulating CD8(+) cells; however, the role of TRM cells in EAN has not yet been elucidated. Further experimental studies are required to fully elucidate these dynamics and understand the modifying potential of targeting S1Pr5 TRM CD8(+) cells. C-Jun acts primarily by promoting Schwann cell reprogramming and neuronal survival, and is essential for activating repair genes and the overall regenerative response. Shh supports regeneration through both direct and indirect pathways by promoting axonal survival, regulating myelin degradation, and aiding in neurite outgrowth. These molecules act cooperatively through distinct pathways and mechanisms during nerve regeneration to achieve effective nerve repair [22, 23]. This process is crucial, because defective C-Jun expression is associated with regenerative failure, notably in aging and chronic denervation scenarios. The enhancement of C-Jun levels has also been demonstrated to significantly improve nerve regeneration deficits, positioning the pathways that regulate C-Jun as potential therapeutic targets for nerve repair [24, 25]. Sonic Hedgehog signaling also plays a crucial role in the regeneration of peripheral nerves, although the exact underlying mechanisms are not yet fully understood. The key points from existing sources are as follows: Shh signaling regulates myelin degradation, which is essential for clearing debris and facilitating axonal regrowth; as such, inhibition of Shh signaling leads to increased myelin debris and abnormal axonal growth [26]. Shh is vital for maintaining neuroglial interactions and regulating the neural microenvironment, which is crucial for preventing demyelination and axonal degradation [23]. Shh facilitates the axonal outgrowth and branching of adult sensory neurons. Shh knockdown results in decreased outgrowth and branching, indicating its supportive role in neuron regeneration [27]. Shh expression is upregulated immediately after nerve injury, such as a sciatic nerve crush, and can be detected in Schwann cells proximal and distal to the site of the injury. This upregulation suggests that Shh signaling is involved in the early response to nerve injury. Shh produced in Schwann cells may regulate the homeostasis of adjacent vascular structures, indicating a role in neurovascular interactions during nerve regeneration [23]. Given its multifaceted role in nerve regeneration, targeting the Shh signaling pathway holds significant potential for therapeutic interventions. For example, enhancing Shh signaling could promote neuron survival, facilitate axonal outgrowth, and improve the overall regenerative process following nerve injury. Thus, the S1Pr5 agonist is a promising therapeutic agent for promoting peripheral nerve regeneration by leveraging the regenerative properties of Shh. This mechanism of action highlights the potential of targeted molecular therapies in nerve repair. This study has several limitations; first, while it highlights several critical considerations in translating findings from experimental models to human treatment protocols, particularly for autoimmune peripheral neuropathies such as GBS and CIDP, whether the doses used in this study are generally safe for humans requires careful adjustment and evaluation in clinical trials and other settings. Furthermore, our understanding of the complex mechanisms of target antigens and cellular immunity in GBS remains incomplete; further elucidation is required for these results to be applied to human therapy. Further, we found that the S1Pr5 agonist did not suppress the severity of EAN. AS such, it should be used with other medications that can decrease pro-inflammatory pathogenic cytokines or shift the cytokine balance to suppress cytokine dominance. These limitations not only emphasize the complexity of developing treatments based on animal models, but also underscore the necessity for further research to clarify these mechanisms and validate therapeutic approaches. Despite these challenges, the findings of this study present exciting prospects for innovative treatments of autoimmune peripheral neuropathies, suggesting that understanding and overcoming these barriers could significantly advance the field. Conclusion Overall, the results of this study show that S1Pr5 agonists have the potential to enhance recovery from peak severity in EAN. C-Jun and Shh play significant roles in the recovery of damaged nerves. Abbreviations EAN, experimental autoimmune neuritis; S1Pr, sphingosine-1-phosphate receptor; GBS, Guillain-Barré syndrome; EAE, experimental autoimmune encephalomyelitis; mRNA, messenger ribonucleic acid; CMC, carboxymethylcellulose; PBS, phosphate-buffered saline; LFB, Luxol fast blue; IFN-γ, interferon gamma; IL-10, interleukin-10; C-Jun, Jun proto-oncogene; Shh, sonic hedgehog signaling molecule. Declarations Data availability The datasets used in this study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding Statement This work was supported by JSPS KAKENHI Grant Number JP 24K10669. Author contributions SK designed and performed the experiments, analyzed the data, and wrote the manuscript. TU conceptualized and designed the study, analyzed the data, and wrote the manuscript. KH contributed to the methodology and analyzed the data. 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Hedging against neuropathic pain: role of hedgehog signaling in pathological nerve healing. Int. J. Mol. Sci. 21, 9115 (2020). Jessen, K. R. & Mirsky, R. The role of C-Jun and autocrine signaling loops in the control of repair Schwann cells and regeneration. Front. Cell. Neurosci. 15, 820216 (2021). Ramesh, R. et al. JUN regulation of injury-induced enhancers in Schwann cells. J. Neurosci. 42, 6506-6517 (2022). Yamada, Y., Ohazama, A., Maeda, T. & Seo, K. The Sonic Hedgehog signaling pathway regulates inferior alveolar nerve regeneration. Neurosci. Lett. 671, 114-119 (2018). Martinez, J. A. et al. Intrinsic facilitation of adult peripheral nerve regeneration by the Sonic hedgehog morphogen. Exp. Neurol. 271, 493-505 (2015). Additional Declarations No competing interests reported. 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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-4677686","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":330306062,"identity":"d6b9b258-90ef-49a0-beeb-64603fc8ada2","order_by":0,"name":"Takafumi Uchi","email":"","orcid":"","institution":"Toho University Ohashi Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Takafumi","middleName":"","lastName":"Uchi","suffix":""},{"id":330306063,"identity":"85ab2a59-09f1-48f9-baec-cb5bf5ac6e34","order_by":1,"name":"Shingo Konno","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYDCC+wcbDn78ZyPHIAHmHoCJs+HWcoP54GMJtjRjsJYDxGlhSzbgYTuc2ICmBTfgu91jJiHBw5zeL938gPlDxR2gCxMYP/xg4MvDpUXyzhkziQIJttyZc44ZMBw48wzowgRmyR4GtmJcWgwO5ABtMeDJ3XAjwYDhYNvhxP03EhikgX5JbMCnhSdBIt3gRvoHsJYGoC2/8Wq5kQb0/gGDBIMbOQYwLWx4bZE8c/jgY8mGBMOZM3IKDpw5c9iYgedhm2WPAW6/8B1vBEZlw395fon0jQ8qKg7LMbAnH77xo+IYzhBDAQcgFCPQSQbHEojSggxqSNcyCkbBKBgFwxUAAI7BXiI7PcOpAAAAAElFTkSuQmCC","orcid":"","institution":"Toho University Ohashi Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Shingo","middleName":"","lastName":"Konno","suffix":""},{"id":330306064,"identity":"9b4418b7-5673-4685-855e-403f2fe17058","order_by":2,"name":"Hideo Kihara","email":"","orcid":"","institution":"Toho University Ohashi Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Hideo","middleName":"","lastName":"Kihara","suffix":""},{"id":330306066,"identity":"5ae8409d-6573-4510-ac66-055dc9b78d89","order_by":3,"name":"Toshiki Fujioka","email":"","orcid":"","institution":"Toho University Ohashi Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Toshiki","middleName":"","lastName":"Fujioka","suffix":""}],"badges":[],"createdAt":"2024-07-03 05:12:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4677686/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4677686/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61313063,"identity":"957220af-ae44-4bd9-ae2a-89615fe3ebc9","added_by":"auto","created_at":"2024-07-29 11:28:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":35117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC-Jun and Shh messenger RNAs’ relative expression levels in response to S1Pr5 agonist’s varying concentrations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBar graph a represents the relative expression of C-Jun mRNA. The x-axis shows different conditions: control, dimethyl sulfoxide (DMSO, used as a solvent control), and increasing concentrations of the S1Pr5 agonist (25 μM, 50 μM, 100 μM, and 200 μM). The y-axis represents the relative expression level of C-Jun mRNA quantified using ΔΔCq, a method of quantifying gene expression in RT-PCR experiments. Bar graph b shows the relative expression of Shh. It has the same conditions on the x-axis as Chart A: control, DMSO, and increasing concentrations of the S1Pr5 agonist (25 μM, 50 μM, 100 μM, and 200 μM). The y-axis represents the relative expression level of Shh as ΔΔCq.\u003c/p\u003e\n\u003cp\u003eThe data are presented as the mean ± SEM. Statistical analysis was conducted using the Mann–Whitney U test, a robust non-parametric test, to compare the EAN and S1Pr5 groups. The significance level was set at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4677686/v1/212b19b09a1af8faa046e9fc.png"},{"id":61313065,"identity":"bd6f9df5-b3be-4559-b613-968b39c0ae8f","added_by":"auto","created_at":"2024-07-29 11:28:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":26215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical coarse of the EAN and S1Pr5 agonist groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe line graph represents the daily motor impairment profile of rats. The x-axis represents the time from day 0 to 21 p.i., while the y-axis indicates the clinical severity score, which ranges from 0 to 9. Both groups are represented in the graph; the EAN group is shown with black diamond markers, and the S1Pr5 agonist is depicted with gray square markers.\u003c/p\u003e\n\u003cp\u003eThe data are presented as mean ± SEM. Statistical analysis was conducted using the Mann–Whitney U test, a robust non-parametric test, to compare the EAN and S1Pr5 groups. The significance level was set at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4677686/v1/64937496904a6c5bfd397266.png"},{"id":61313728,"identity":"8d0191eb-271b-4f2b-8050-f9971465cc65","added_by":"auto","created_at":"2024-07-29 11:36:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytokines identified as EAN pathogenesis-related molecules in the cauda equina\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bar graph shows the relative expression levels of genes in the cauda equina, measured using quantitative PCR (assessed using the ΔΔCq method), at different time points post-immunization (p.i.). The x-axis shows three different time points: day 10 p.i., 14 p.i., and 21 p.i.. The y-axis represents the concentration of interferon-gamma (IFN-γ) and IL-10 (pg/ml) in the homogenate of cauda equina among the normal rats (white bar), the EAN (black bar), and the S1Pr5 agonist group (gray bar).\u003c/p\u003e\n\u003cp\u003eThe data are presented as mean ± SEM. Statistical analysis was conducted using the Mann–Whitney U test, a robust non-parametric test, to compare the EAN and S1Pr5 groups.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4677686/v1/a74336f60366d5ff328dbd96.png"},{"id":61315220,"identity":"059a3d13-fa0c-4ff7-b399-3a3c6a2af813","added_by":"auto","created_at":"2024-07-29 11:52:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":28526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequential changes in C-Jun and Shh mRNA expression in the cauda equina.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth bar graphs (a and b) present a significant comparison of the relative expression of C-Jun and Shh in the cauda equina among normal rats (white bar), the EAN group (black bar), and the S1Pr5 agonist group (gray bar). The data, crucial for understanding the molecular changes in the cauda equina, is normalized and presented using the ΔΔCq method, with measurements taken at various days post-immunization (p.i.).\u003c/p\u003e\n\u003cp\u003eBar graph a Relative expression of C-Jun in the cauda equina. Notably, both the EAN and S1Pr5 agonist groups exhibited increased C-Jun expression compared to normal rats, with the S1Pr5 group showing slightly higher levels than the EAN group. Bar graph b shows the relative expression of Shh in the cauda equina. Here, the EAN group demonstrated a stable expression pattern, whereas the S1Pr5 group showed significant elevations in Shh expression, particularly as compared with the EAN group on days 10 and 14 p.i.\u003c/p\u003e\n\u003cp\u003eThe data are presented as mean ± SEM. Statistical analysis was conducted using the Mann–Whitney U test, a robust non-parametric test, to compare the EAN and S1Pr5 groups. The significance level was set at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4677686/v1/f333d101174a4e68448dea78.png"},{"id":61313069,"identity":"525034fa-00cf-49c4-a692-b5e2006d729c","added_by":"auto","created_at":"2024-07-29 11:28:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2582879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological examination of CD3 cells and macrophages infiltrating into the cauda equina\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe top row of images shows a transverse serial section of a single cauda equina from the EAN group on day 14 after immunization (p.i.), stained from left to right with LFB stained with hematoxylin, anti-CD3 antibody, and anti-Iba-1 antibody. The lower section shows the S1Pr5 agonist group. Scale bars indicate 10 µm. Serial sections stained for CD3 revealed numerous T lymphocytes infiltrating the endoneurium, although fewer than macrophages. Serial sections stained for Iba-1 exhibited dense macrophage infiltration, primarily in the vicinity of the endoneurial vessels, which spread to the entire endoneurium.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4677686/v1/3b03a4d04f334af2949dc661.png"},{"id":61313729,"identity":"a3838eaf-b497-4c7d-b4c6-d4f2fae2efcc","added_by":"auto","created_at":"2024-07-29 11:36:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":38046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe demyelination area and number of infiltration cells in the cauda equina\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBar graph shows the demyelinated area, which indicates an LFB stain-negative area, while bar graph b shows the number of infiltrating cells, including lymphocytes and macrophages.\u003c/p\u003e\n\u003cp\u003eThere was no significant difference in the demyelination area (%), or mononuclear infiltration cells (number/μm2) analyzed randomly selected transverse section of 50 cauda equina on day 15 p.i.. Between the groups. The data are presented as mean ± SEM. Statistical analysis was conducted using the Mann–Whitney U test, a robust non-parametric test, to compare the EAN and S1Pr5 groups.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4677686/v1/fe7f945ca40d65cfa5fa4d86.png"},{"id":61314403,"identity":"41cec226-6185-47ff-88b5-9601867a37a8","added_by":"auto","created_at":"2024-07-29 11:44:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2171138,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical study of the cauda equina\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunostaining of Shh in the cauda equina from both groups on day 15 p.i.. Only a few Shh-positive cells were observed. Crescent-shaped Shh-positive cells were more frequently observed in the S1Pr5 agonist group than in the EAN group (black arrows).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4677686/v1/0cfd57196b29fe6781f3b1bb.png"},{"id":61313066,"identity":"328942e0-bcbe-4888-a860-6a223bbd165f","added_by":"auto","created_at":"2024-07-29 11:28:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":39547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of Shh-positive cells in the cauda equina of EAN and S1Pr5 groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bar graph shows the number of Shh-positive cells per square micrometer (μm²) in the cauda equina of rats from the EAN group (black bar) and the S1Pr5 group (gray bar). The y-axis represents the number of Shh-positive cells (×10⁻⁶ per μm²), while the x-axis shows the two experimental groups. The S1Pr5 group shows a higher number of Shh-positive cells compared to the EAN group, suggesting a differential expression of Shh in response to the experimental treatments. The data are presented as mean ± SEM. Statistical analysis was conducted using the Mann–Whitney U test, a robust non-parametric test, to compare the EAN and S1Pr5 groups.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4677686/v1/e1aa8fc6b21627d3e77da7e0.png"},{"id":63452933,"identity":"b9c5c2c1-cb6d-48ab-bc86-fa1ec6c06bb6","added_by":"auto","created_at":"2024-08-28 09:43:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6507205,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4677686/v1/e41e2a3c-fe3e-4923-bf88-43265f4160fa.pdf"},{"id":61313732,"identity":"0e1cef32-8db0-4ea4-8717-d5cef03642fb","added_by":"auto","created_at":"2024-07-29 11:36:46","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":14042,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4677686/v1/8d76240dd361a258f91f62f1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Treatment with a Sphingosine-1-phosphate Receptor Five Agonist Enhances Recovery of Experimental Autoimmune Neuritis by Promoting Production of Schwann Cell Regeneration Factors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGuillain-Barr\u0026eacute; syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are both autoimmune conditions that affect the peripheral nervous system which display unique clinical characteristics and progression patterns. GBS manifests acutely, with rapid symptom escalation over several weeks, while CIDP progresses chronically, with symptom development spanning months. Typically, GBS is preceded by infection, while CIDP lacks clear precipitation events. Symptoms of GBS generally plateau and improve, whereas CIDP symptoms persist and require ongoing treatment. Further, GBS usually occurs as a singular episode, while CIDP shows significant potential for relapse or continuous progression [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite these clinical variations, both disorders are linked by an underlying fundamental immune-mediated damage to peripheral nerves. Other similarities include their autoimmune nature, effects on the myelin sheath and nerve fibers, and symptoms, all of which stem from the erroneous attack of peripheral nerves by the immune system. This leads to inflammation and damage, primarily affecting the myelin sheath or axons, disrupting electrical signal transmission and causing symptoms such as weakness, numbness, and sometimes pain in the limbs. However, a high variability in symptom severity and progression is notable among individuals and diseases.\u003c/p\u003e \u003cp\u003eAccurate identification of both commonalities and distinctions is essential for efficient diagnosis, therapeutic interventions, and patient care management. The presence of anti-ganglioside antibodies in 50\u0026ndash;60% of GBS patients highlights the importance of humoral immune mechanisms in peripheral neuropathies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The primary therapeutic approach for GBS is the administration of intravenous immunoglobulin [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], yet some individuals may suffer from persistent sequelae [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Ongoing clinical studies are assessing the effectiveness of combining complement C5 inhibitors like eculizumab with intravenous immunoglobulin to address complement-driven injury [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Treatment options for CIDP encompass corticosteroids, plasmapheresis, and intravenous immunoglobulin; these methods are effective, but corticosteroids have considerable adverse effects, and other treatments generally need regular application. After five years, only 26% of CIDP patients reach full remission while 39% continue to require treatment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], underscoring the necessity for innovative therapeutic approaches.\u003c/p\u003e \u003cp\u003eExperimental autoimmune neuritis (EAN) acts as a model to investigate autoimmunity-related peripheral neuritis and the development of therapies in humans [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. IFN-γ-producing autoreactive T lymphocytes, which promote macrophage infiltration to peripheral nerves, induce EAN. EAN and CIDP exhibit similar pathological characteristics, particularly the predominance of cellular immune responses. The nerve pathology in CIDP often includes mild T-cell infiltration and pronounced macrophage presence in the endoneurium, critical for demyelination processes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. EAN characteristically shows multifocal infiltration of mononuclear cells around blood vessels, comprising lymphocytes, which lead to nerve demyelination through macrophage action [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eS1P receptors, a group comprising five G protein-coupled receptors, play a significant role in peripheral nerve regeneration [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our research indicates that the selective S1Pr1 and S1Pr5 agonist, siponimod, significantly reduces EAN severity by inhibiting lymphocyte migration, a process that depends on the S1P gradient, and involves the activation of the Jun proto-oncogene (C-Jun) and Sonic Hedgehog (Shh) pathways [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Peripheral nerves further demonstrate superior regenerative abilities compared to the central nervous system, a feature enhanced by S1Pr5, while their absence in rat models impedes nerve regeneration. Further research suggests a critical role of Schwann cell transformation in nerve repair, enhanced by S1Pr5 agonist application, which stimulates the Schwann cell repair mechanism and fosters effective nerve regeneration [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the reparative potential of S1Pr5 in the EAN-damaged nerves remains unclear. Therefore, this study aimed to evaluate the efficacy of the S1Pr5 agonist A-971432 in treating EAN and to explore the molecular mechanisms underlying the therapeutic effects of S1Pr5.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eIn vitro experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture and Sphingosine-1-phosphate receptor 5 agonist treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchwann cells of the IFRS1 lineage were obtained from COSMO BIO Co., Ltd. (catalog number: PMC-SWN-IFRS1C) and cultured in a specialized medium specifically designed for this line, purchased from COSMO BIO Co., Ltd. (catalog number: PMC-SWN-IFRS1C-COS). This medium was formulated to provide optimal support for the growth and maintenance of the IFRS1 lineage of Schwann cells, ensuring proper cultivation and suitability for experimental purposes. The cells were plated in 100 mm plastic culture dishes, and incubated at 37°C in a humidified environment with 5% CO2. The cultures were allowed to reach a subconfluent state over a 25-day period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn day 25, the cells were treated with various concentrations of the S1Pr5 agonist (0, 25, 50, 100, or 200 μM). The S1Pr5 agonist dissolved in dimethyl sulfoxide (DMSO), which also served as a vehicle control. The experimental setup included six conditions: four agonist concentrations and two control groups. One control group was administered a high concentration of DMSO, whereas the other remained untreated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe messenger RNA Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-four hours post-treatment, cells underwent RNA harvesting for the purpose of quantifying the mRNA expression of C-Jun and Shh through qRT-PCR. The RNA was extracted using Qiagen's RNeasy® Universal Mini kit, located in Tokyo, Japan, and cDNA was subsequently synthesized utilizing iScript RT SuperMix for qRT-PCR from Bio-Rad, also based in Tokyo, Japan. This cDNA was then employed as a template in real-time PCR, conducted using iTaq™ Universal SYBR\u003csup\u003e®\u003c/sup\u003e Green Supermix from Bio-Rad, with gene-specific primers obtained from TAKARA BIO (Otsu, Japan) and Bio-Rad. Primers for sequencing are detailed in Supplementary data (Table 1). Semi-quantitative analyses of the mRNA levels of transcription factors associated with Schwann cell regeneration were conducted, referencing an endogenous control on the CFX96 Touch™ Real-Time PCR Detection System from BIO-RAD. These gene expression levels were expressed as relative copy numbers, calculated via the ΔΔCq method, offering a comparative assessment of mRNA levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubjects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 30 female Lewis rats（Charles River Japan, Yokohama, Japan) used in this study were 7 weeks old at the start of the experiment. The rats were housed in the Toho University Ohashi Experimental Animal Laboratory. The gages were maintained at 25 °C with a 12-h light–dark cycle. The rats were provided with a comfortable and clean-living environment, which included proper bedding and enrichment to promote natural behaviors. The diet and hydration were carefully monitored to ensure adequate nutrition. Regular health checks were conducted to promptly identify and address any health issues. Additionally, all procedures and handling of the rats were performed by trained personnel to minimize stress and discomfort.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures regarding the use and handling of animals in research were reviewed and approved by the Toho University Animal Care and User Committee (approval numbers 20-442 and 24-567) and all the experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals in research. All the performed procedures and all the reported data were in accordance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInduction and evaluation of EAN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were subcutaneously injected with 150 µg of a synthetic peptide that corresponds to amino acid residues 53–78 of bovine P2 protein (TESPFKNTEISFKLGQEFEETTADNR, Operon, Tokyo, Japan), emulsified in an equal volume of Complete Freund's adjuvant (Sigma-Aldrich, MO, USA). The injections were administered to the right footpad with rats were under light anesthesia induced by sevoflurane (Mylan, Osaka, Japan) [14].\u003c/p\u003e\n\u003cp\u003eThe motor function of the rats was observed daily, and a scoring system was used to assess\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eparalysis of the tail, forelimbs, and left hind limb. The tail motility was scored from 0 to 3, with 0 indicating no clinical signs, 1 indicating paralysis of the tip, 2 indicating incomplete paralysis of the entire tail, and 3 indicating complete paralysis of the entire tail. Forelimbs were scored from 0 to 3, with 0 indicating no clinical signs, 1 indicating an inability to climb a fence using the forelimbs, 2 indicating an inability to walk, and 3 indicating complete paralysis. The left hind limb was scored from 0 to 3, with 0 indicating no clinical signs, 1 indicating paralysis of the toe only, 2 indicating incomplete dorsiflexion of the foot joint while walking, and 3 indicating complete paralysis, where the legs were dragged when walking. The rat with shallow breathing suspected respiratory muscle paralysis were euthanized with an overdose of sevoflurane before reaching that state.\u003c/p\u003e\n\u003cp\u003eIn this study, 30 rats were sacrificed across all phases, with 10 each on day 10 post-immunization (p.i.) for the subclinical to acute phase, day 14 p.i. for the early peak phase, and day 21 p.i. for the recovery phase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSphingosine-1-phosphate receptor 5 agonist treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe S1Pr5 agonist A-971432, (Cayman Chemical Co., Ltd., Michigan, USA) was in a solution of 0.5% carboxymethylcellulose (CMC) in phosphate-buffered saline (PBS) in preparation for oral administration. This solution was administered to the experimental rats, specifically those assigned to the S1Pr5 agonist group, at a daily dose of 1.0 mg/kg, starting from day 5 and continuing until day 20 p.i.. The purpose of this daily administration was to assess the effects of S1Pr5 agonist within the scope of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were sedated using sevoflurane and underwent complete perfusion with ice-cold PBS on days 10, 14, and 21 p.i. Subsequently, the lumbar spinal cord was excised, and a portion of the cauda equina was preserved in 10% buffered formalin for histological examination. The other portion was kept in Allprotect® Tissue Reagent (Qiagen, KK, Tokyo, Japan) at -80°C in a deep freezer for subsequent analysis of mRNA and cytokines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequential analysis of cytokine production related to pathogenesis of EAN\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cauda equina was subjected to enzyme-linked immunoassay (ELISA) using a 96-well plate (specifically the Rat IFN-γ and IL-10 DuoSet® ELISA Kit from biotech, USA). To achieve this, the cauda equina sample stored in Allprotect® tissue reagent was dissociated in 750 ml solution of 10 % radioimmunoprecipitation assay lysis buffer including protease inhibitor (Santa Cruz Biotechnology, CA, USA) using a bead tissue grinder (Shakeman™, Biomedical Science, Tokyo, Japan). The ELISA process was then initiated by diluting the capture antibody for each cytokine and coating each well. The plates were incubated overnight at room temperature, followed by washing to remove excess antibodies. Nonspecific sites were blocked using the reagent diluent (10% BSA dissolved in PBS), after which the plate was incubated again. Subsequently, 20 μg/ml of homogenized cauda equina solution or standards were added to the wells, covered, and incubated for two hours to allow antigen binding. After this incubation period, the plate was washed again before adding the detection antibody, followed by another incubation and washing step. Streptavidin-horseradish peroxidase was then introduced into the plate, which was incubated in the dark before being washed again. To develop color, a substrate solution was added to the plate and the reaction was stopped with a stop solution before mixing.\u003c/p\u003e\n\u003cp\u003eFinally, the absorbance was measured at 450 nm with an iMark™ Microplate Absorbance Reader (BIO-RAD, Tokyo, Japan). Cytokine concentrations are expressed in pg/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequential analysis of messenger RNA expression of regeneration of peripheral nerv\u003c/strong\u003ee\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBefore RNA extraction, the cauda equina was crushed using a bead-type tissue grinder (Shakespeare Biomedical Science, Tokyo, Japan). The mRNA expression of the transcription factors C-Jun and Shh, which are related to Schwann cell regeneration, were subsequently analyzed. RNA extraction, cDNA synthesis, and mRNA expression analyses were performed in the same manner as described for Schwann cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cauda equina collected on day 14 p.i. was embedded in paraffin and cut into transverse sectional 5 μm thick slices. Luxol fast blue (LFB) staining and counterstaining with hematoxylin were applied to identify the areas of demyelination and infiltrating cells, respectively. Using the Hitsugi_planimetry software (https://hitsugi-edu-inst.main.jp/computer-software/Hitsugi-planimetry-manual), the ratio of demyelinated area and the number of infiltrating cells per square micrometer were quantified in 50 randomly sampling transverse sectional nerves of cauda equina from all groups.\u003c/p\u003e\n\u003cp\u003eTo validate the effect of the S1Pr5 agonist on cellular infiltration, we used specific antibodies to identify T lymphocytes and macrophages with the highest precision. T lymphocytes were detected using an anti-CD3 rabbit monoclonal antibody (SP7, Nichirei Biosciences Inc., Tokyo, Japan), whereas macrophages were identified using an anti-Iba-1 rabbit polyclonal antibody (GTX100042; GeneTex Inc., Irvine, CA, USA), following the standard antigen retrieval protocol recommended by the manufacturers.\u003c/p\u003e\n\u003cp\u003eShh was chosen as a marker for the regeneration of damaged Schwann cells because of its higher mRNA production compared to C-Jun. Anti-Shh rabbit polyclonal antibodies (St Johns Laboratory Ltd, London, UK) were applied to paraffin-embedded sections without an antigen retrieval protocol, according to the manufacturer's instructions. A peroxidase-labeled anti-rabbit IgG goat polyclonal antibody conjugated with an amino acid polymer (N-Histofine® Simple Stain MAX-PO (R)TM, Nichirei Biosciences Inc., Tokyo, Japan) was used to visualize these antibodies on tissue sections, followed by diaminobenzidine as a chromogen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of the groups was performed with the Mann–Whitney U test, a non-parametric method. The R software (version 3.2.2; R Foundation for Statistical Computing, Vienna, Austria), utilizing the rms package [15], was employed to process the data, setting statistical significance at p \u0026lt; 0.05. Unless specified differently, results are expressed as mean ± SEM.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIn vitro experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emRNA of expression regeneration factors from the Schwann cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur analysis identified a gradual increase in C-Jun expression with increasing concentrations of the S1Pr5 agonist, peaking at 50 μM (Fig.1A). A statistically significant increase in expression was further observed between the control and 50 μM S1Pr5 agonist-treated groups (3.0-fold increase, p\u0026lt;0.05). The expression dropped sharply at 200 μM, indicating a possible concentration-dependent effect, with a peak or optimal concentration for upregulation at 50 μM. Shh expression further showed a similar pattern, with increased expression at higher concentrations of the S1Pr5 agonist (Fig.1B). Statistically significant increases in expression compared to the control group were observed at both 50 and 100 μM NaCl (4.2 and 2.9-fold increases, respectively). The peak expression of Shh was observed at 50 μM, and, similar to that of C-Jun, we observed a decrease in expression at the highest concentration of 200 μM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical severity of rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth groups had a clinical score of zero at the beginning of the observation period (Fig.2). The clinical score for the EAN group began to increase sharply from around day 8 p.i., reached a peak score around day 13 p.i., plateauing until around day 17 p.i., before declining slightly. The clinical score for the S1Pr5 agonist group similarly began to increase around day 8 p.i., but reached a slightly lower peak around day 13 p.i. compared to the EAN group. The score for the S1Pr5 agonist group also remained elevated until day 17 p.i., before declining again. Both groups showed a decline in the clinical score after day 17 p.i., with the EAN group decreasing to a score just below 8, and the S1Pr5 agonist group declining to a score of approximately 7 by day 21 p.i.. The treatment group had milder symptoms on day 18-21 p.i. than the EAN group (p \u0026lt; 0.05). Although some rats scored nine points, most sever score, none developed respiratory muscle paralysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEAN pathogenesis-related cytokines in the cauda equina\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe production of IFN-γ and IL-10 production in the cauda equina were found to be markedly increased in the EAN and S1Pr5 groups compared to normal rats, which served as a negative control, but there was no significant difference between the groups between the different time points (Fig.3A and B). These results suggest that the inflammatory activity of infiltrating cells in the cauda equina was not significantly different between the groups. The IFN-γ levels further showed a pronounced response on day 10 p.i. (in the acute phase) that was moderated over time. However, the S1Pr5 agonist did not significantly mitigate this response. IL-10 levels were stable across different conditions and time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of regeneration factor mRNAs in the cauda equina during the peripheral nerve repair process\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC-Jun mRNA expression was significantly upregulated in the S1Pr5 agonist groups compared to normal rats at each time point, with the S1Pr5 group showing slightly higher expression than the EAN group at each time point (Fig. 4A). Shh expression was significantly higher in the S1Pr5 group than that in the normal and EAN groups. Significantly higher expression was further noted in the S1Pr5 group on days 10 and 12 p.i.. By day 21 p.i., the expression levels in the S1Pr5 group decreased, but remained higher than those in the EAN group, which showed no apparent increase (Fig. 4B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological examination of the cauda equina\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the peak phase of clinical symptoms, on day 14 p.i., mononuclear infiltrating cells appeared patchy and multifocal in the cauda equina in all groups (Fig.5). The demyelination area that was LFB stain-negative area and the number of infiltrated mononuclear cells, which were hematoxylin stain-positive in the transverse sectional nerves, showed no difference between the groups (Fig.6). Immunohistochemical analysis demonstrated approximately the same degree of CD3-positive T lymphocytes and Iba-1-positive macrophages were counted in both groups (Fig.5). Moreover, we immunohistochemically stained for Shh to evaluate its role as a nerve trans-differential factor because Shh mRNA was significantly increased in the S1Pr5 agonist group compared to the EAN group. The number of Shh-positive cells significantly increased in the nerve of S1Pr5 positive group than the EAN group (Fig.7 and 8).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOverall, this study identified the following results: 1) Treatment with an S1Pr5 agonist promoted the recovery of clinical symptoms in EAN. 2) The S1Pr5 agonist did not modulate the expression of cytokines related to the pathogenesis of EAN in the cauda equina; 3) The S1Pr5 agonist enhanced expression of C-Jun and Shh mRNA, which are crucial molecules for the regeneration of peripheral nerves, in the cauda equina. \u0026nbsp;Pathogenic cytokines and molecules related to nerve repair in the cauda equina provide important insights into the effects of S1Pr5 agonists on the inflammatory and regenerative responses during neural injury in autoimmune neuritis.\u003c/p\u003e\n\u003cp\u003eS1Pr5 agonists interact with S1Pr5, a receptor that responds to S1P, and plays a pivotal role in various physiological processes. S1P5 is predominantly expressed in the nervous and immune systems, influencing natural killer cell egress [16, 17]. Studies have further shown that selective agonists such as A-971432 stimulate S1Pr5, maintain the integrity of the blood-brain barrier, and exhibit therapeutic effects in conditions such as Huntington\u0026apos;s disease [18]. Furthermore, S1Pr5 is present at synapses. and inhibits the spontaneous activity of neurons in culture, an action distinct from that of other S1PRs [19]. Furthermore, in the central nervous system, S1Pr5 is mainly present in oligodendrocyte progenitors and mature oligodendrocytes, and is thought to contribute to the regulation of the differentiation processes [20].\u003c/p\u003e\n\u003cp\u003eS1Pr5 significantly regulates tissue-resident memory (TRM) CD8 (+) cells [17], and is critical for controlling T cell migration and retention in peripheral tissues, such as the skin. S1Pr5 is further involved in T-lymphocyte trafficking and migration. It is typically downregulated in tissue-resident memory T lymphocytes, allowing them to remain in the peripheral tissue instead of circulating back into lymphoid organs. This downregulation is crucial for the retention and proper functioning of TRM cells, which provide localized and rapid immune responses upon re-exposure to pathogens. Furthermore, the expression of S1Pr5 is influenced by several factors, including the transcription factors T-bet and Zinc finger E-box-binding homeobox 2 (ZEB 2). T-bet and ZEB2 both upregulate S1Pr5 during differentiation of effector T cells from na\u0026iuml;ve T lymphocytes. However, in the tissue-resident memory formation process, local signals such as Transforming Growth Factor-\u0026beta; in the tissue microenvironment promote the downregulation of T-bet and ZEB2, subsequently leading to decreased S1Pr5 expression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe induction and control of murine EAN reportedly depends on CD4(+) and CD8(+) T lymphocytes, while B cells do not perpetuate the related inflammatory demyelination. EAN-induced CD8(-/-) mice have a milder clinical course, fewer histopathological changes, and a diminished T-cell response to the P0 peptide [21]. CD8(+) cells are related to the pathogenesis of EAN, the acute autoimmune neuritis model used in this study, are mainly circulating CD8(+) cells; however, the role of TRM cells in EAN has not yet been elucidated. Further experimental studies are required to fully elucidate these dynamics and understand the modifying potential of targeting S1Pr5 TRM CD8(+) cells.\u0026nbsp; \u003c/p\u003e\n\u003cp\u003eC-Jun acts primarily by promoting Schwann cell reprogramming and neuronal survival, and is essential for activating repair genes and the overall regenerative response. Shh supports regeneration through both direct and indirect pathways by promoting axonal survival, regulating myelin degradation, and aiding in neurite outgrowth. These molecules act cooperatively through distinct pathways and mechanisms during nerve regeneration to achieve effective nerve repair [22, 23].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis process is crucial, because defective C-Jun expression is associated with regenerative failure, notably in aging and chronic denervation scenarios. The enhancement of C-Jun levels has also been demonstrated to significantly improve nerve regeneration deficits, positioning the pathways that regulate C-Jun as potential therapeutic targets for nerve repair [24, 25]. Sonic Hedgehog signaling also plays a crucial role in the regeneration of peripheral nerves, although the exact underlying mechanisms are not yet fully understood. The key points from existing sources are as follows: Shh signaling regulates myelin degradation, which is essential for clearing debris and facilitating axonal regrowth; as such, inhibition of Shh signaling leads to increased myelin debris and abnormal axonal growth [26]. Shh is vital for maintaining neuroglial interactions and regulating the neural microenvironment, which is crucial for preventing demyelination and axonal degradation [23]. Shh facilitates the axonal outgrowth and branching of adult sensory neurons. Shh knockdown results in decreased outgrowth and branching, indicating its supportive role in neuron regeneration [27]. Shh expression is upregulated immediately after nerve injury, such as a sciatic nerve crush, and can be detected in Schwann cells proximal and distal to the site of the injury. This upregulation suggests that Shh signaling is involved in the early response to nerve injury. Shh produced in Schwann cells may regulate the homeostasis of adjacent vascular structures, indicating a role in neurovascular interactions during nerve regeneration [23]. Given its multifaceted role in nerve regeneration, targeting the Shh signaling pathway holds significant potential for therapeutic interventions. For example, enhancing Shh signaling could promote neuron survival, facilitate axonal outgrowth, and improve the overall regenerative process following nerve injury.\u003c/p\u003e\n\u003cp\u003eThus, the S1Pr5 agonist is a promising therapeutic agent for promoting peripheral nerve regeneration by leveraging the regenerative properties of Shh. This mechanism of action highlights the potential of targeted molecular therapies in nerve repair.\u003c/p\u003e\n\u003cp\u003eThis study has several limitations; first, while it highlights several critical considerations in translating findings from experimental models to human treatment protocols, particularly for autoimmune peripheral neuropathies such as GBS and CIDP, whether the doses used in this study are generally safe for humans requires careful adjustment and evaluation in clinical trials and other settings. Furthermore, our understanding of the complex mechanisms of target antigens and cellular immunity in GBS remains incomplete; further elucidation is required for these results to be applied to human therapy. \u0026nbsp;Further, we found that the S1Pr5 agonist did not suppress the severity of EAN. AS such, it should be used with other medications that can decrease pro-inflammatory pathogenic cytokines or shift the cytokine balance to suppress cytokine dominance. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese limitations not only emphasize the complexity of developing treatments based on animal models, but also underscore the necessity for further research to clarify these mechanisms and validate therapeutic approaches. Despite these challenges, the findings of this study present exciting prospects for innovative treatments of autoimmune peripheral neuropathies, suggesting that understanding and overcoming these barriers could significantly advance the field.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, the results of this study show that S1Pr5 agonists have the potential to enhance recovery from peak severity in EAN. C-Jun and Shh play significant roles in the recovery of damaged nerves.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eEAN, experimental autoimmune neuritis; S1Pr, sphingosine-1-phosphate receptor; GBS, Guillain-Barr\u0026eacute; syndrome; EAE, experimental autoimmune encephalomyelitis; mRNA, messenger ribonucleic acid; CMC, carboxymethylcellulose; PBS, phosphate-buffered saline; LFB, Luxol fast blue; IFN-\u0026gamma;, interferon gamma; IL-10, interleukin-10; C-Jun, Jun proto-oncogene; Shh, sonic hedgehog signaling molecule.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used in this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI Grant Number JP 24K10669.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSK designed and performed the experiments, analyzed the data, and wrote the manuscript. TU conceptualized and designed the study, analyzed the data, and wrote the manuscript. KH contributed to the methodology and analyzed the data. TF supervised the project, conceptualized and designed the study, analyzed the data, and wrote the manuscript. All the authors have read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKusunoki, S., Kaida, K. \u0026amp; Ueda, M. Antibodies against gangliosides and ganglioside complexes in Guillain-Barr\u0026eacute; syndrome: new aspects of research. Biochim. Biophys. Acta 1780, 441-444 (2008).\u003c/li\u003e\n\u003cli\u003eWillison, H. J. \u0026amp; Yuki, N. Peripheral neuropathies and anti-glycolipid antibodies. Brain 125, 2591-2625 (2002).\u003c/li\u003e\n\u003cli\u003eBunschoten, C. et al. Progress in diagnosis and treatment of chronic inflammatory demyelinating polyradiculoneuropathy. 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Intrinsic facilitation of adult peripheral nerve regeneration by the Sonic hedgehog morphogen. Exp. Neurol. 271, 493-505 (2015).\u003c/li\u003e\n\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":"Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, experimental autoimmune neuritis, sphingosine-1-phosphate receptor, Schwann cell regeneration","lastPublishedDoi":"10.21203/rs.3.rs-4677686/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4677686/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExperimental autoimmune neuritis (EAN) serves as a model for studying autoimmune peripheral neuropathies. This study investigated the effects of a sphingosine-1-phosphate receptor 5 (S1Pr5) agonist on EAN recovery and nerve regeneration. Lewis rats with induced EAN were treated with the S1Pr5 agonist A-971432. Clinical scores, cytokine production, and expression of regeneration factors were analyzed. Histological examination of the cauda equina was also performed. Treatment with the S1Pr5 agonist promoted recovery of clinical symptoms in the later stages of EAN. While the agonist did not significantly modulate pathogenic cytokine expression, it enhanced the expression of Jun proto-oncogene and Sonic Hedgehog (Shh) mRNA, crucial molecules for peripheral nerve regeneration. Histological analysis revealed increased Shh-positive cells in the nerves of treated rats. These findings suggest that S1Pr5 agonists have the potential to enhance recovery from EAN by promoting nerve regeneration rather than by modulating the inflammatory response. This study provides insights into novel therapeutic approaches for autoimmune peripheral neuropathies, highlighting the role of S1Pr5 in nerve repair processes. Further research is needed to fully elucidate the mechanisms and validate these findings for potential clinical applications.\u003c/p\u003e","manuscriptTitle":"Treatment with a Sphingosine-1-phosphate Receptor Five Agonist Enhances Recovery of Experimental Autoimmune Neuritis by Promoting Production of Schwann Cell Regeneration Factors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 11:28:41","doi":"10.21203/rs.3.rs-4677686/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":"61ef6aad-9db3-4ec6-a092-8678a87c6a80","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34964759,"name":"Health sciences/Neurology"},{"id":34964760,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2024-08-28T09:35:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-29 11:28:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4677686","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4677686","identity":"rs-4677686","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}