Mannose Receptor C type 2 influences autoimmune neuroinflammation and blood-brain barrier integrity

Mannose Receptor C type 2 influences autoimmune neuroinflammation and blood-brain barrier integrity | 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 Biological Sciences - Article Mannose Receptor C type 2 influences autoimmune neuroinflammation and blood-brain barrier integrity Bettina Zierfuss, Melanie Käch, Camille Sugère, Marion Morvan, and 28 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6917781/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Relapses in Multiple Sclerosis (MS) are driven by pathogenic immune cells breaching the disrupted blood–brain barrier (BBB), leading to tissue damage and eventual disease progression 1–3 . Among the most effective therapies are those that block immune cell infiltration across the BBB, a multistep process involving activation, adhesion, rolling, transendothelial migration, and extracellular matrix (ECM) degradation. Current treatments broadly suppress the immune system and can cause adverse side effects 4,5 , highlighting the need for targeted approaches. Here, we identified a previously unrecognized role of ECM remodeling pathways and Mannose Receptor C-type 2 (MRC2) in invasive T lymphocytes as a potential therapeutic target. Typically low under physiological conditions, MRC2 was upregulated on T lymphocytes upon inflammatory stimulation in vitro, enriched in the periphery of MS patients during relapse and progressive disease, and present in cerebrospinal fluid and active brain lesions co-localized with Col IV, the major ECM component of the BBB. Genetic deletion of MRC2 impaired T lymphocyte-mediated Col IV degradation, adhesion, and transendothelial migration, and leading to reduced neuroinflammation in a sex-dependent manner in an MS-like mouse model. These findings suggest MRC2 as a key regulator of T lymphocyte infiltration into the CNS and a promising target for MS therapy. Biological sciences/Neuroscience/Neuroimmunology Health sciences/Diseases/Neurological disorders/Multiple sclerosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Main Multiple Sclerosis (MS) is a chronic central nervous system (CNS) disease with a heterogeneous clinical presentation. Its’ pathophysiology involves overlapping inflammatory (e.g., relapses, new MRI lesions) and neurodegenerative (e.g., progression) processes, forming a continuum with inflammation dominating in the early, and neurodegeneration in the later stages 2,3,6 . Relapses result from immune cell infiltration, monocytes, T, and B lymphocytes, across a disrupted blood-brain barrier (BBB), a key regulator of molecule and cell trafficking between the periphery and the CNS 1 . This multistep process includes immune cell activation, adhesion, rolling along BBB-endothelial cells and transendothelial migration, followed by extracellular matrix (ECM) degradation to access the brain parenchyma. In progressive MS, immune cell infiltration into the CNS is less frequent, but in contrast, these cells accumulate behind a relatively intact BBB, sustaining compartmentalized inflammation 7 . Blocking interactions between encephalitogenic immune cells and BBB endothelial cells effectively reduces relapses in relapsing-remitting MS (RRMS). However, current therapies may impair regulatory immune function and CNS immunosurveillance 4,5 . We, and others, have previously identified the role of adhesion molecules (e.g., MCAM, DICAM) on highly invasive T lymphocytes in MS and its mouse model, experimental autoimmune encephalomyelitis (EAE) 8-10 . In this study, we investigated the mechanisms which may enable these cells to cross the BBB. Using exploratory proteomics, we highlighted an underappreciated role for pathways including ECM remodeling and collagen binding in inflammatory T lymphocytes. We identified Mannose Receptor C Type 2 (MRC2), also known as UPARAP, Endo180, or CD280, as a molecule expressed on these T lymphocytes with increased ability to transmigrate across the BBB and to degrade collagen IV (Col IV) 11,12 , a key structural component of the BBB and critical for its integrity 13,14 . Exploratory proteomics analysis To investigate mechanisms underlying CNS invasion by T lymphocytes, we performed proteomics on IL-23-polarized human MCAM⁺CD45RO⁺CD4⁺ memory T lymphocytes (Extended Fig.1A), previously shown to efficiently transmigrate across the BBB 8,9 . We identified 177 proteins with PSM >35 and PEP ≥1. Functional enrichment analysis via REACTOME 15 and GO terms revealed significant enrichment of immune pathways, ECM organization, cell-matrix adhesion, and hemostasis (Fig.1A-B). GO molecular function further highlighted ECM structural components and collagen binding in MCAM⁺ versus MCAM⁻ T lymphocytes (Fig.1C). Among the top five upregulated proteins was MRC2 (Fig.1D), a collagen-binding receptor involved in ECM degradation (including Col IV) and MMP activation 12,16 . In contrast, TIMP1, an MMP inhibitor, was downregulated in MCAM⁺ T lymphocytes, further highlighting the invasive phenotype of these cells. To identify co-regulatory mechanisms, we used NetworkAnalyst and ENCODE (BETA Minus algorithm) 17 to explore gene regulatory networks. MYC-Associated Zinc Finger Protein (MAZ) was identified as a shared transcription factor for MCAM and MRC2 (Extended Fig.1B) and has been implicated as an MS susceptibility gene 18 . MAZ was also predicted to regulate MMP9 and DICAM (MXRA8), suggesting a possible co-expression with MRC2 (Extended Fig.1C). These data indicate a not yet explored role for ECM remodeling in activated pro-inflammatory T lymphocytes. Supporting this, analysis of scRNA-seq data from freshly autopsied human brain tissue, along with re-analysis of recently published CD31-enriched murine BECs 19 , revealed that Col IV (α1 and α2 subunits), the substrate of MRC2, was mainly expressed by BECs. Additionally, immunofluorescence microscopy confirmed expression of Col IV on primary human BBB and meningeal ECs under resting and pro-inflammatory conditions (Extended Fig.2A-C). MRC2 is a marker for CNS-invasion MRC2 has been linked to MS, located in a risk-associated chromosomal region, with elevated mRNA levels in blood during MS relapse 20 . To assess T lymphocyte contributions, we performed flow cytometry on PBMCs from untreated, inactive RRMS (n=22), relapsing RRMS (n=5), progressive MS with sustained disability worsening (PPMS, n=6; SPMS, n=4), and matched healthy donors (Fig.2A-B, Extended Fig.3A, Extended Table 1). Peripheral MRC2⁺ T lymphocytes were low in healthy and inactive RRMS, but significantly increased during relapse, and in progressive MS (Fig.2A-B). Memory T lymphocytes, both central and effector memory subsets, showed higher abundance of MRC2, than naïve and effector cells in RRMS (Fig.2C, Extended Fig.3A). As MRC2 expression can be influenced by estrogen in vitro 21 , we tested for sex differences in PBMCs. Our results showed no significant differences in our RRMS patient cohort, nor in publicly available microarray datasets from healthy donors (Query data sets for GSE3365) 22 (Extended Fig.3B-C). Confocal microscopy of postmortem MS brain tissue revealed MRC2 colocalized with CD4⁺ and predominantly CD8⁺ T lymphocytes in lesions (Fig.2D, Extended Table 2), with higher frequencies in active versus pre-active lesions (Extended Fig.3D-E). Flow cytometry of autopsied human brain and blood from two MS and two ALS donors, who underwent medical aid in dying, confirmed higher MRC2⁺ T lymphocytes in periventricular lesions versus normal white matter (Fig.2E, Extended Table 3). CNS-infiltrating T lymphocytes showed a significantly higher frequency of MRC2 compared to peripheral T lymphocytes. Additional analysis of blood and CSF from untreated PwMS showed significantly increased MRC2 on CD4⁺ and CD8⁺ T lymphocytes in CSF versus blood (Fig.2F, Extended Table 4). Together, these findings indicate that MRC2 expression is elevated during relapse and disease progression and may present a marker for CNS-infiltrating T lymphocytes in MS. Phenotyping MRC2⁺ T lymphocytes To further characterize MRC2⁺ CD4⁺ and CD8⁺ T lymphocytes, we used a flow cytometry panel targeting ECM remodeling molecules and factors linked to CNS invasion and MS pathology 23-25 . Using PBMCs from untreated PwMS and healthy donors, we compared MRC2⁺ to MRC2⁻ cells per donor (Fig.3A, Extended Table 1). MRC2⁺ T lymphocytes from PwMS showed increased MMP2⁺ and MMP9⁺ frequencies and decreased TIMP-1 (CD8⁺ T lymphocytes). We also determined expression of MMP28, which was elevated (gMFI) in MRC2⁺ cells (Extended Fig.4A). UPAR expression showed no clear association with MRC2 in T lymphocytes (Extended Fig.4A). MRC2⁺ T lymphocytes also exhibited elevated TNF (Fig.3A), particularly in PwMS, but not GM-CSF or IFNγ (Extended Fig.4A). These findings suggest MRC2⁺ T lymphocytes possess a distinct invasive and pro-inflammatory phenotype, supporting a role in CNS tissue invasion. Inflammation regulates MRC2 expression We investigated whether in vitro polarization affects MRC2 expression on T lymphocytes. Flow cytometry and confocal microscopy of CD4⁺ and CD8⁺ T lymphocytes showed highest MRC2 expression in IL-12-stimulated T lymphocytes (Fig.3B–C), which also exhibited the most IFNγ⁺ cells (Extended Fig.4B), suggesting IL-12–IFNγ signaling regulates MRC2 expression. We then analyzed transcription factor expression in IL-12, IL-23, and IL-4 conditions (Fig.3D). MRC2⁺ T lymphocyte frequency was significantly higher in T-bet⁺ (IL-12) and RORγt⁺ (IL-23) subsets, but not significantly different in GATA-3⁺ (IL-4) cells, when compared to their respective negative counterparts. These data suggest MRC2 is upregulated in activated pro-inflammatory human T lymphocytes. MRC2 promotes transendothelial migration To determine whether MRC2 plays a role in T lymphocyte migration, we performed in vitro assays using primary cultures of human BECs and IL-12-polarized CD4⁺ or CD8⁺ T lymphocytes. (Fig.4A, Extended Fig.5A) 27 . MRC2 blockade, using an anti-MRC2 blocking antibody, significantly reduced transendothelial migration of IL-12-stimulated T lymphocytes (Fig.4A). We also analyzed BECs post-migration of T lymphocytes. MRC2 blockade led to elevated surface expression of Col IV and Claudin-5 (Fig.4B-C), but not ICAM-1 (Extended Fig.5B-E), while IFNγ/TNF stimulation of BECs upregulated ICAM-1 mRNA levels but decreased both Col IV α chains COL4A1 and COL4A2 .Confocal microscopy showed strong co-localization of MRC2 and Col IV in perivascular CD4⁺ and CD8⁺ T lymphocytes within active MS lesions (Fig.4D-F). These findings suggest MRC2 facilitates T lymphocyte migration across the BBB possibly through interaction with its substrate Col IV. Genetic deletion of MRC2 affects T lymphocyte function To investigate the function of MRC2 on T lymphocytes in CNS invasion, we used MRC2 (UPARAP) KO mice and their wildtype (WT) littermates (Extended Fig.6A). CFSE-based proliferation assays showed that CD4⁺ and CD8⁺ T lymphocytes from KO and WT mice had similar proliferation rates upon IL-12 or IL-4 stimulation (Extended Fig.6B). Flow cytometry revealed no significant difference in IFNγ and IL-17a expression between genotypes (Extended Fig.6C-D). As in humans, IL-12 stimulation upregulated MRC2 on murine CD4⁺ and CD8⁺ T lymphocytes compared to IL-4 stimulation (Fig.5A), so we focused on IL-12-stimulated T lymphocytes. Using flow adhesion assays on WT murine BECs, MRC2 KO T lymphocytes showed reduced adhesion and rolling ability, indicated by fewer rolling cells and increased rolling velocity (Fig.5B, Extended Fig.6E), suggesting impaired early CNS invasion. To test native Col IV digestion and uptake, we cultured IL-12-stimulated T lymphocytes on DQ-Col IV–coated surfaces, allowing the measurement of green fluorescent intensity upon degradation of native Col IV (non-fluorescent) to its fluorescent degradation products (Extended Fig.6F, Fig. 5C). MRC2 KO T lymphocytes showed significantly reduced Col IV uptake compared to WT, indicated by lower fluorescence intensity. In MOG 35-55 -induced EAE (Extended Fig.7A), MRC2⁺ T lymphocytes were enriched in the CNS compared to their peripheral counterparts in the spleen (Fig.5D-E). Confocal imaging on postmortem tissue revealed that MRC2⁺ T lymphocytes were predominantly CD4⁺ T lymphocytes, and primarily localized in the spinal cord (Fig. 5F–G). These findings indicated that MRC2 supports T cell–mediated CNS invasion possibly by promoting Col IV degradation and BBB endothelial interaction. Genetic deletion of MRC2 dampens neuroinflammation To assess the role of MRC2 in vivo, we induced MOG 35-55 -dependent EAE in male and female MRC2 KO mice (n = 32) and their WT littermates (n = 24) (Extended Fig.7A). Female KO mice had significantly lower disease incidence (60%) when compared to female WT (87.5%), male WT (100%), and male KO (100%) mice (Extended Fig.7B, Fig.6A; χ2=17.68, 3 df, p < 0.0005). Affected MRC2 KO female animals also showed reduced weight loss and a milder disease course, with lower clinical and cumulative EAE scores, especially in the chronic phase (Extended Fig.7C-E, Fig.6B-D). Immunofluorescence of spinal cords from sick MRC2 KO female animals revealed fewer CD45⁺ immune cells compared to WT (Extended Fig.7F), and fewer infiltrating CD4⁺ and CD8⁺ T lymphocytes in the CNS (Fig.6E). Male MRC2 KO mice showed no clinical improvement, but reduced CD4⁺ T lymphocytes in spinal cord and CD8⁺ T lymphocytes in brain. Additionally, flow cytometry of CNS-infiltrating T lymphocytes showed elevated MMP2/TIMP2 ratios in affected WT mice, which were significantly reduced in MRC2 KO animals (Fig.6F-G) during the chronic disease. No differences were found in MMP9/TIMP1 ratios or IFNγ/IL-17a expression (Extended Fig.7G-H). In summary, MRC2 expression on T lymphocytes promotes CNS infiltration and disease severity in a sex-dependent manner, likely via ECM remodeling rather than cytokine production. DISCUSSION Current MS treatments often lack specificity, targeting broad immune populations and causing immunosuppression and increased infection risk 4,5 . This study aimed to uncover molecular mechanisms by which pathogenic T lymphocytes breach the BBB. We identified MRC2 as a key player in promoting T lymphocyte migration across the BBB in both humans, and mice during autoimmune neuroinflammation. Exploratory proteomics revealed enrichment of ECM pathways, including collagen binding and hemostasis, in highly invasive, pro-inflammatory (MCAM + ) T lymphocytes. MRC2 ranked among the top five upregulated proteins and is known for its role in ECM remodeling, particularly in degrading collagens including Col IV 12 . Col IV is a major BBB component and has been previously suggested in BBB integrity disturbance in MS 13,28 . While typically expressed at low levels on T lymphocytes, our data showed an upregulation of MRC2 upon activation 27 , making it a promising candidate for targeted therapy. Previously, MRC2 and its potential regulator (MAZ) have been assumed to be associated with increased risk to develop MS 18,20 . We confirmed increased frequencies of MRC2⁺ T lymphocytes in untreated PwMS during active relapse and observed associations in progressive MS with sustained disability worsening, particularly in SPMS. Interestingly, MRC2⁺ cells were enriched within the memory T lymphocyte subsets a population over-represented in myelin-reactive T lymphocytes in MS 29 . MRC2⁺ CD4⁺ and CD8⁺ T lymphocytes were significantly elevated in active lesions, especially co-localized with its substrate Col IV at the BBB in the perivascular space, suggesting a role in the CNS infiltration during the acute phase. To explore the functional role of MRC2 we used specific blocking antibodies and T lymphocytes from MRC2 KO mice for in vitro assays as well as the MOG 35 − 55 -induced EAE mouse model to assess in vivo CNS infiltration. All our data strongly pointed towards a role for MRC2 in promoting transendothelial migration of T lymphocytes across the BBB. We further explored downstream mechanisms by analyzing expression of MMPs and TIMPs, as well as related molecules from the hemostasis pathways like UPAR, and inflammatory cytokines. MMP2 and MMP9, critical for ECM degradation, were significantly elevated in MRC2⁺ T lymphocytes from untreated PwMS, while TIMPs were not, indicating a heightened invasive profile. In the EAE model, genetic MRC2 deletion significantly lowered the MMP2/TIMP2 ratio in CNS-infiltrating T lymphocytes. These findings suggest that MRC2 regulates MMP expression and activity in CNS-infiltrating T lymphocytes, potentially promoting tissue invasion. Nearly all T lymphocytes also expressed MMP28, but levels were significantly higher in MRC2⁺ T lymphocytes, suggesting a link between MRC2 expression and enhanced ECM remodeling capacity. While MRC2 is also known as UPAR-associated protein, we found UPAR expression was not consistently altered in peripheral MRC2⁺ T lymphocytes in our experiments, implying that MMP and cytokine activity in these cells may be UPAR-independent. However, UPAR and UPA were elevated in inflamed brain vasculature from PwMS 23 , pointing to potential interactions that require further investigation. We examined cytokine expression to understand whether MRC2 influences or opposingly, would be impacted by pro-inflammatory environments. MRC2⁺ T lymphocytes showed increased TNF expression (a downstream target of MMPs 26 ), but not GM-CSF or IFNγ, suggesting a specific inflammatory profile. In contrast, MRC2 deletion did not affect IFNγ or IL-17a expression, previously shown to play an important role in T lymphocyte pathology in EAE 30 , indicating that MRC2 primarily affects tissue invasion rather than cytokine production. A major mechanism by which MRC2 may promote T lymphocyte migration is via interaction with its substrate Col IV at the BBB. MRC2 KO T lymphocytes showed reduced capacity to degrade native Col IV in vitro. Within active MS lesions, MRC2⁺ T lymphocytes co-localized with Col IV in perivascular areas, but not deeper parenchyma, supporting the idea that MRC2-Col IV interaction is critical for initial BBB crossing but not for further CNS navigation. Based on our collected data, we propose a hypothetical model in which MRC2⁺ T lymphocytes bind and intracellularly degrade Col IV at the BBB as summarized in Fig. 7. Together with MMP activation, this may contribute to the degradation of Claudin-5, further compromising BBB integrity and facilitating T lymphocyte entry into the CNS in MS. Although not further investigated in this study, adhesion molecules such as MCAM and DICAM on MRC2⁺ T lymphocytes may facilitate the initial steps of adhesion and rolling along BBB endothelial cells. Since Col IV is located on the abluminal site on post-capillary venules, the interaction of MRC2 and Col IV likely becomes critical during the final steps of CNS entry. Interestingly, we observed sex-dependent effects in the EAE mouse model. MRC2 deficiency led to differing disease outcomes in male and female mice, possibly due to ECM composition differences. Male brains reportedly have lower stiffness and altered collagen (including Col IV) and laminin expression 32 . Estrogen, protecting male and female mice against EAE development 33 , has been previously shown to regulate MRC2 21 . However, sex-dependent effects on expression were not recapitulated in human PBMC data. In our study, we did not investigate CNS-resident cells in this context, and which may also contribute to the sex-specific outcomes. Our use of a constitutive MRC2 KO mouse for MOG-induced EAE limits conclusions about T lymphocyte-specific effects, as other immune cells and CNS-resident cells may be involved. Future studies using conditional KO models and adoptive transfer EAE are needed to further investigate the role of MRC2 on autoimmune neuroinflammation outcomes. Additionally, our human data were drawn from a limited donor pool; larger cohorts are required to confirm the role of MRC2 as a disease marker at protein level, especially in progressive MS. An MRC2-targeting antibody and antibody-drug conjugate (ADC) have been previously developed 27 . The latter will be investigated in a clinical trial phase I/II study (ADCE-D01) in patients with metastatic and unresectable soft tissue sarcoma in the USA (currently enrolling) and is under regulatory review in the European Union [NCT06797999]. Preclinical data show good tolerability of these antibodies in mice 27 , suggesting an overall significant translational potential of MRC2 as a putative drug target in PwMS. However, these therapies have not yet been tested in the context of neuroinflammation. Future studies should therefore evaluate the safety and efficacy of MRC2-targeted interventions in models of autoimmune neuroinflammation like EAE. Such work may pave the way for new, more selective treatments for PwMS that minimize systemic immunosuppression while effectively preventing pathogenic T lymphocyte infiltration into the CNS. MATERIALS AND METHODS Statistics Statistical analyses were conducted using Graph Pad prism software (version 8). Normal distributed data was analyzed using unpaired or paired Student’s t test as indicated. For all data two-sided tests were performed. Additionally, for pairwise comparison, the ratio-paired Student’s t test was used. Otherwise for non-normally distributed data, Wilcoxon matched-paired signed rank test was performed. When more than 2 groups were compared, one-way ANOVA and Tukey’s (all comparisons), and Sidak’s (specific, independent comparison) multiple comparison tests were used. For non-normally distributed data Kruskal-Wallis test was carried out. To use a similar approach as repeated measures ANOVA but allowing for missing values (early harvest of animals for FACS / immunohistochemistry) and repeated measures, we used a mixed model implemented in Graph Pad prism (version 8) with a compound symmetry covariance structure, fit using Restricted Maximum Likelihood (REML) to analyze EAE curves and weight changes. Incidence of EAE disease was assessed by using Log-rank Mantel cox test. All performed tests are indicated in the figure legends. Declarations All non-human vertebrate experiments in this manuscript were approved by our animal ethic’s committee and by our veterinarian. Acknowledgments: We thank the Core facility of the Centre de Recherche du CHUM (CRCHUM) for their assistance; Jean Franҫois Schmouth, PhD from the platform for Genetic Engineering and Animal Modelling; Gaël Dulude, PhD and Philippe St-Onge, MSc from the platform for Flow Cytometry; Aurélie Cleret-Buhot, PhD from the core facility for Cellular Imaging; and Véronique Barrès, MSc and Liliane Meunier, MSc from the platform for Molecular Pathology; Alice Michallet Roy from the animal housing facility at the CRCHUM. We also would like to thank Dr. Thomas Bugge from the National Health Institutes (NIH) for providing the MRC2 KO mouse model. Language editing support was provided by ChatGPT. Schematics in Extended Figures 1A, 5A, 6E-F and 7A were created with BioRender.com. Funding: Canadian Institutes of Health Research (CIHR) grant and Canada Research Chair program (AP) Fonds de Recherche du Québec - Santé (FRQS) fellowship (BZ, FT, APF) Multiple Sclerosis Society Canada fellowship (BZ) MITACS Globalink Research Award (MKa, MIM) Université de Montréal Premier fellowship (CS) National MS Society postdoctoral fellowship (HD) CIHR fellowship (KT, FT, APF, MKr) Danish Cancer Society grant agreements No R231-A13820 (NB) Danish Cancer Society grant agreements R368-A21555 (NB) Novo Nordisk Foundation grant Nos. NNF19OC0058603 (NB) Novo Nordisk Foundation grant Nos NNF22OC0080995 (NB) NEYE Foundation (NB) Author contributions: Conceptualization: BZ, AP Methodology: BZ, MKa, CS, MIM, HLD, SL, SBA, WK, KT, CG, IA, FT, HM, CM, LB, APF, OT, OC, OS, MKr, HK, MD, ASH, PD, MG, GM, SO, NB, CL, NA, SEJZ Investigation: BZ, MKa, CS, MIM, SBA, OT, OC Visualization: BZ, MKa, CS, MIM, SBA, FT, OT, OC Funding acquisition: AP, BZ, NB Project administration: BZ, WK, LB Supervision: AP Writing – original draft: BZ Writing – review & editing: AP, NA, CL Competing interests: AP holds the Senior Canada Research Chair in MS Data and materials availability: All data are available in the main text or the supplementary materials. Raw data from scRNA-Seq will be deposited on Gene Expression Omnibus (GEO) upon publication. Code availability: Software code will be available upon publication. Correspondence and requests for materials should be addressed to [email protected] . 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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-6917781","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":476330304,"identity":"0e9a95cf-7ad7-4aec-ba13-07eb0e89e9f3","order_by":0,"name":"Bettina Zierfuss","email":"","orcid":"","institution":"Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM) / Université de Montréal","correspondingAuthor":false,"prefix":"","firstName":"Bettina","middleName":"","lastName":"Zierfuss","suffix":""},{"id":476330305,"identity":"0aa89532-2d67-4c13-abf8-d8b0a752f3d4","order_by":1,"name":"Melanie 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de Montréal (CRCHUM)","correspondingAuthor":false,"prefix":"","firstName":"Nathalie","middleName":"","lastName":"Arbour","suffix":""},{"id":476330334,"identity":"6d1c5082-279c-4132-b140-62d0dfebef77","order_by":30,"name":"Stephanie Zandee","email":"","orcid":"https://orcid.org/0000-0003-0812-676X","institution":"McGill University","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"Zandee","suffix":""},{"id":476330303,"identity":"74f5ecca-bd4a-4ebb-8af0-bd1dc2d87796","order_by":31,"name":"Alexandre Prat","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYPACGzhLhkgdCWkgkrEBSPAQq+UwCVr4G3iPffj543zi2vbe448rKux4GNgPP8CrReIAX/LMnoTbidvOnEtsPHMmmYeBJ80AvzUHeIwZeEBabuQYNja2MfMwSDDg1yIP1ML4J+EcVMu/eqAW9g94tRgAtTDzJByAamk4DNTCg98Ww8N8ycwyacnG286cMZzZcOw4DxtPTgFeLXLHew8zvrGxk912vMfgY0NNtRw/+/ENeLUwMKNHBBt+9SBAbHSPglEwCkbByAUA7UlDd6sPABUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6188-0580","institution":"Université de Montréal","correspondingAuthor":true,"prefix":"","firstName":"Alexandre","middleName":"","lastName":"Prat","suffix":""}],"badges":[],"createdAt":"2025-06-18 00:15:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6917781/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6917781/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86135572,"identity":"4be6fbfc-40bb-4b95-ae10-d3318552686c","added_by":"auto","created_at":"2025-07-07 07:44:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":306898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClassical functional gene enrichment analysis revealed next to Immune system-related pathways, ECM organization and Collagen binding as altered pathways on human activated MCAM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT lymphocytes compared to MCAM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT lymphocytes. (A) \u003c/strong\u003eOverrepresentation analysis using REACTOME webtool indicating significantly altered pathways highlighted in yellow. Next to “Immune system”, the pathways “Hemostasis” and “Extracellular matrix organization” were revealed. \u003cstrong\u003e(B)\u003c/strong\u003e GO Biological Processes and \u003cstrong\u003e(C)\u003c/strong\u003e GO Molecular Function pathways are shown. The size of the modules indicates hits per pathway; color scheme indicates adjusted p-values. Hemostasis, extracellular matrix and collagen-related pathways are highlighted in red. \u003cstrong\u003e(D) \u003c/strong\u003eHeat map depicts relative protein intensities from 2 donors per condition.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6917781/v1/dc51ecfb33e0191f69b7453d.png"},{"id":86137684,"identity":"4ef893dd-78da-40a7-bac8-edb0d33b85f8","added_by":"auto","created_at":"2025-07-07 08:00:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":342408,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMRC2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT lymphocytes are present in blood and CNS tissue in PwMS. (A) \u003c/strong\u003eAssessment of MRC2\u003csup\u003e+\u003c/sup\u003eT lymphocytes using flow cytometry analysis of biobanked PBMCs from untreated PwMS in remission (RM, n = 22) and active relapse (RL, n = 5), \u003cstrong\u003e(B)\u003c/strong\u003e primary progressive (PP)MS (n = 6), and secondary progressive (SP)MS (n = 4) with sustained disability worsening, and compared to healthy controls (HC, n = 24). \u003cstrong\u003e(C)\u003c/strong\u003e Flow cytometry analysis of MRC2\u003csup\u003e+\u003c/sup\u003eT lymphocytes within different T lymphocyte subpopulations showing patients with RRMS (n = 7). \u003cstrong\u003e(D)\u003c/strong\u003e Confocal microscopy on postmortem CNS tissue from PwMS (10 specimen from biological n = 6, or biological n = 5) and non-neuroinflammatory disease (n = 4). WM = white matter; WML = white matter lesion; Pre = pre-active; Act = Active; scale bars, 50 µm. Insets provide a magnified view of the selected regions. \u003cstrong\u003e(E)\u003c/strong\u003e Flow cytometry analysis of PBMCs and freshly autopsied brain tissue showing periventricular WML (n = 4 specimen, from 2 donors with MS), and normal appearing white matter (NAWM, n = 4 specimen, from 1 donor with MS and 2 donors with ALS). T lymphocyte infiltrates were normalized to brain tissue weight. \u003cstrong\u003e(F)\u003c/strong\u003e Pairwise comparison showing flow cytometry analysis of blood and cerebrospinal fluid (CSF) from untreated PwMS (n = 6). * p \u0026lt; 0.05, **p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001, ns = not significant. For statistical analysis one-way ANOVA and Sidak’s test in \u003cstrong\u003e(A-B)\u003c/strong\u003e and Tukey’s test in \u003cstrong\u003e(C)\u003c/strong\u003e, unpaired Student’s t test in \u003cstrong\u003e(D)\u003c/strong\u003e, and ratio-paired Student’s t test in \u003cstrong\u003e(E-F)\u003c/strong\u003e were performed\u003cstrong\u003e. \u003c/strong\u003eData are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6917781/v1/2117fbcb606a2563ef7c4a63.png"},{"id":86136448,"identity":"0403dd9e-645c-4fe6-8b64-13ac4836c813","added_by":"auto","created_at":"2025-07-07 07:52:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":254325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMRC2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT lymphocytes show slightly increased MMP expression and are enriched in T lymphocytes after IL-12 stimulation. (A) \u003c/strong\u003eFlow cytometry analysis on biobanked PBMCs from untreated PwMS (n = 8 – 23, as indicated) and age-and sex-matched healthy donors (n = 4 – 9, as indicated), comparing MRC2\u003csup\u003e+\u003c/sup\u003eT lymphocytes to MRC2\u003csup\u003e-\u003c/sup\u003e counterparts. Exemplary flow cytometry plots and the frequency of the matrix metalloproteinase (MMP)2, MMP9, TIMP-1 and tumor necrosis factor (TNF), and geometric fluorescence intensities (gMFI) of MMP28 (assessed in a separate experiment), are shown. \u003cstrong\u003e(B)\u003c/strong\u003e Flow cytometry analysis of in vitro polarized CD4\u003csup\u003e+\u003c/sup\u003e (n = 6) or CD8\u003csup\u003e+\u003c/sup\u003eT lymphocytes (n = 10) stimulated with IL-12, IL-4 or IL-23 are shown and gMFI were normalized to unpolarized cells per experiment. \u003cstrong\u003e(C)\u003c/strong\u003e Microscopy on polarized CD8\u003csup\u003e+\u003c/sup\u003eT lymphocytes (n = 6) attached to objective slides by cytospin indicated enriched MRC2\u003csup\u003e+\u003c/sup\u003eT lymphocytes after IL-12-stimulation. Scale bars, 50 µm. \u003cstrong\u003e(D) \u003c/strong\u003eFlow cytometry analysis indicating frequency of the intracellular transcription factors T-bet\u003csup\u003e+\u003c/sup\u003e, GATA-3\u003csup\u003e+\u003c/sup\u003e, and RORγt\u003csup\u003e+\u003c/sup\u003e T lymphocytes (n = 4). * p \u0026lt; 0.05, **p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001, ns = not significant. For statistical analysis ratio-paired Student’s t test in \u003cstrong\u003e(A)\u003c/strong\u003e and \u003cstrong\u003e(D)\u003c/strong\u003e and one-way ANOVA and Tukey’s test in \u003cstrong\u003e(B)\u003c/strong\u003e were performed. Values are presented as mean ± SEM.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6917781/v1/e8cb6862a8b9c9ada08b32bb.png"},{"id":86137685,"identity":"eb1e0029-6582-494e-90e2-5946469c9bde","added_by":"auto","created_at":"2025-07-07 08:00:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":345548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMRC2 on T lymphocytes promotes transendothelial migration possibly linked to Col IV binding and / or degradation on human BECs. (A)\u003c/strong\u003e Average (from technical duplicates or triplicates) transendothelial migrated IL-12 stimulated T lymphocytes pre-treated with 50 µg / ml specific MRC2-blocking antibody or isotype control per donor (n = 4-6, as indicated). \u003cstrong\u003e(B-C)\u003c/strong\u003e Flow cytometry of BECs detached after transendothelial migration of pre-treated CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes. \u003cstrong\u003e(B)\u003c/strong\u003e Representative flow cytometry plots and \u003cstrong\u003e(C)\u003c/strong\u003e pairwise analysis for Col IV\u003csup\u003e+\u003c/sup\u003e, and Claudin-5\u003csup\u003e+\u003c/sup\u003eBECs (n = 4). \u003cstrong\u003e(D-F)\u003c/strong\u003e Confocal microscopy analysis of active MS WML on postmortem CNS tissue analyzing Col IV and MRC2 co-localization in CD4\u003csup\u003e+\u003c/sup\u003e (pooled data from 3 lesions in \u003cstrong\u003e(F)\u003c/strong\u003e) and CD8\u003csup\u003e+\u003c/sup\u003eT lymphocytes (pooled data from 5 lesions in \u003cstrong\u003e(F)\u003c/strong\u003e) in the perivascular space (PV) or the parenchyma (PC). Scale bars, 50 µm \u003cstrong\u003e(E)\u003c/strong\u003e Exemplary plot of Pearson’s correlation coefficient of fluorescence intensity line profiles of MRC2 and Col IV in perivascular CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003eT lymphocytes. * p \u0026lt; 0.05, **p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001, ns = not significant. For statistical analysis ratio-paired Student’s t test in \u003cstrong\u003e(B)\u003c/strong\u003e \u0026amp; \u003cstrong\u003e(D-E)\u003c/strong\u003e, and Kruskal-Wallis test in \u003cstrong\u003e(H)\u003c/strong\u003e were performed. Values show mean ± SEM. Boxplots show the median, with whiskers representing the 1.5x interquartile range.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6917781/v1/e434fd2d3b99572eb7034fe7.png"},{"id":86135575,"identity":"03f2338b-4830-4689-aafa-e5dd64d12ea0","added_by":"auto","created_at":"2025-07-07 07:44:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":419031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDysfunctional MRC2 on murine T lymphocytes results in reduced ability to adhere and roll along murine BECs, internalize and degrade native Col IV, and MRC2 is expressed on CNS-infiltrating T lymphocytes in autoimmune neuroinflammation.\u003c/strong\u003e Flow cytometry of splenocyte-derived T lymphocytes to determine \u003cstrong\u003e(A)\u003c/strong\u003e %MRC2\u003csup\u003e+\u003c/sup\u003eT lymphocytes (n = 6 animal per condition). \u003cstrong\u003e(B)\u003c/strong\u003e Assessment of %rolling T lymphocytes (representative experiment, pooled data from two animals per genotype) and velocity of rolling cells (pooled data from three experiments and from six animals per genotype). \u003cstrong\u003e(C)\u003c/strong\u003e confocal microscopy (scale bars, 50 µm) to assess fluorescent Col IV maximum fluorescent intensity (MFI) in CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003eT lymphocytes. Results are pooled from four animals per genotype. Flow cytometry analysis of MRC2\u003csup\u003e+\u003c/sup\u003eT lymphocytes \u003cstrong\u003e(D)\u003c/strong\u003e in spleen and CNS in peak and chronic EAE disease, and \u003cstrong\u003e(E)\u003c/strong\u003e comparing MRC2\u003csup\u003e+\u003c/sup\u003eCNS-infiltrating T lymphocytes in peak and chronic disease in CNS (n = 4-7 animals per time point, as indicated). \u003cstrong\u003e(F-G)\u003c/strong\u003e Confocal microscopy (scale bars: 50 µm, 5 µm in magnified views of selected regions) on postmortem brain and spinal cord (Spc) tissue from mice in chronic EAE disease (pooled results from seven animals per tissue). * p \u0026lt; 0.05, **p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001, ns = not significant. For statistical analysis, unpaired Student’s t test in \u003cstrong\u003e(A, F-G)\u003c/strong\u003e, one-way ANOVA and Sidak’s test in \u003cstrong\u003e(B)\u003c/strong\u003e, Mann-Whitney test in \u003cstrong\u003e(C)\u003c/strong\u003e and paired Student’s test in \u003cstrong\u003e(D)\u003c/strong\u003e were performed. Values indicate mean ± SEM. Truncated violin plots are shown and indicate median.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6917781/v1/9c31cf7c26cbb6545a60366d.png"},{"id":86135579,"identity":"9d7349ee-b369-42f2-b09e-792376262223","added_by":"auto","created_at":"2025-07-07 07:44:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":313429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMRC2 dysfunction improves autoimmune neuroinflammation in vivo. (A) \u003c/strong\u003eSurvival curves indicate incidence of EAE disease in MRC2 KO (n = 27) mice and WT littermates (n = 23) (pooled data from three experiments). \u003cstrong\u003e(B)\u003c/strong\u003e Weight changes in gram (g) and in percentage (%) normalized to initial weight (pooled data from three experiments). \u003cstrong\u003e(C)\u003c/strong\u003e Clinical EAE scores of sick mice (pooled data from three experiments). \u003cstrong\u003e(D)\u003c/strong\u003e Cumulative scores of sick mice after 40 days post-immunization (n = 10, animals per genotype). \u003cstrong\u003e(E)\u003c/strong\u003e CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T lymphocyte infiltrates in brain and spinal cord in chronic EAE disease (n = 7-9 animals per group, as indicated). Scale bars: 50 µm, 10 µm in magnified views of selected regions. \u003cstrong\u003e(F-G)\u003c/strong\u003e Flow cytometry analysis of MMP\u003csup\u003e+\u003c/sup\u003e and TIMP\u003csup\u003e+\u003c/sup\u003e T lymphocytes in spleen, and CNS in chronic EAE disease (n = 3-7 animals per group as indicated). * p \u0026lt; 0.05, **p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001, ns = not significant. For statistical analysis Log-rank (Mantel-Cox test) in \u003cstrong\u003e(A)\u003c/strong\u003e, mixed effects model (REML) in \u003cstrong\u003e(B-C)\u003c/strong\u003e, unpaired Student’s t test in \u003cstrong\u003e(D, E, G)\u003c/strong\u003e, and two-way ANOVA and Sidak’s test in \u003cstrong\u003e(F)\u003c/strong\u003e were performed. Values indicate mean ± SEM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6917781/v1/cc69b7f04a69aebf4bf120ec.png"},{"id":86135577,"identity":"f0e3e8fa-cd69-4985-84e9-4ee8be6f1a95","added_by":"auto","created_at":"2025-07-07 07:44:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":192519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypothetical model illustrating the potential role of MRC2 in autoimmune neuroinflammation and in PwMS.\u003c/strong\u003e (Left panel) In healthy individuals and/or PwMS in remission, MRC2⁺ T lymphocytes are present at low frequencies in the periphery. The basement membrane (BM) at the blood-brain barrier (BBB) remains intact. (Right panel) During relapse or sustained disability worsening (SDW) in PwMS, the frequency of peripheral MRC2⁺ T lymphocytes increases. These cells may co-express cellular adhesion molecules (CAMs), including melanoma CAM (MCAM) and potentially other CAMs such as Dual Immunoglobulin Domain-Containing CAM (DICAM), facilitating initial adhesion to inflamed brain endothelial cells (BECs) (Step 1). Upon engagement with BECs, MRC2⁺ T lymphocytes may secrete matrix metalloproteinases (MMPs) and tumor necrosis factor (TNF) (a), leading to upregulation of Intercellular CAM (ICAM)-1 on BECs and contributing to the degradation of tight junction protein Claudin-5 and denaturation of collagen IV (Col IV) (b) (Step 2) and thus, may allow local firm adhesion (c). MRC2 may further support T cell transmigration across the BBB, either via paracellular or transendothelial routes, through its interaction with native or denatured Col IV (Step 3), ultimately resulting in perivascular accumulation of MRC2⁺ T lymphocytes (Step 4).The inset shows the proposed MRC2 recycling pathway: binding of MRC2 to native Col IV, followed by internalization, lysosomal degradation of ligand, and recycling of the receptor back to the cell surface. Inhibition of MRC2 by specific blocking antibody disrupts this recycling process, thereby interrupting the proposed cycle in T lymphocytes.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6917781/v1/f332021f7d80f61829933442.png"},{"id":86138136,"identity":"3c36e854-2f13-4237-b797-3bb56801bdd7","added_by":"auto","created_at":"2025-07-07 08:08:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3398730,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6917781/v1/bb93b022-e6ec-4e43-9810-58bece8d89c2.pdf"},{"id":86136446,"identity":"34aa55f6-0511-428c-9ccb-568b2bf75600","added_by":"auto","created_at":"2025-07-07 07:52:17","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26867,"visible":true,"origin":"","legend":"Supplementary Data Set - Proteomics Data","description":"","filename":"SupplementaryfileproteomicsdataZierfussetal.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6917781/v1/3b14e754c8907520fdc8e08f.xlsx"},{"id":86135570,"identity":"be89ce41-b13e-4fca-9c12-3d42a6745c06","added_by":"auto","created_at":"2025-07-07 07:44:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":56373,"visible":true,"origin":"","legend":"Supplementary material","description":"","filename":"SupplementarymaterialZierfussetal13.06.2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6917781/v1/d2bf6bbe195a17b3fc8f1e70.docx"},{"id":86135580,"identity":"1c37d321-7864-428d-9128-6a1efcc83be4","added_by":"auto","created_at":"2025-07-07 07:44:17","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2734675,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedTablesandFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6917781/v1/78578d079b635221247058ca.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Mannose Receptor C type 2 influences autoimmune neuroinflammation and blood-brain barrier integrity","fulltext":[{"header":"Main","content":"\u003cp\u003eMultiple Sclerosis (MS) is a chronic central nervous system (CNS) disease with a heterogeneous clinical presentation. Its\u0026rsquo; pathophysiology involves overlapping inflammatory (e.g., relapses, new MRI lesions) and neurodegenerative (e.g., progression) processes, forming a continuum with inflammation dominating in the early, and neurodegeneration in the later stages\u003csup\u003e2,3,6\u003c/sup\u003e. Relapses result from immune cell infiltration, monocytes, T, and B lymphocytes, across a disrupted blood-brain barrier (BBB), a key regulator of molecule and cell trafficking between the periphery and the CNS\u003csup\u003e1\u003c/sup\u003e. This multistep process includes immune cell activation, adhesion, rolling along BBB-endothelial cells and transendothelial migration, followed by extracellular matrix (ECM) degradation to access the brain parenchyma. In progressive MS, immune cell infiltration into the CNS is less frequent, but in contrast, these cells accumulate behind a relatively intact BBB, sustaining compartmentalized inflammation\u003csup\u003e7\u003c/sup\u003e. Blocking interactions between encephalitogenic immune cells and BBB endothelial cells effectively reduces relapses in relapsing-remitting MS (RRMS). However, current therapies may impair regulatory immune function and CNS immunosurveillance\u003csup\u003e4,5\u003c/sup\u003e. We, and others, have previously identified the role of adhesion molecules (e.g., MCAM, DICAM) on highly invasive T lymphocytes in MS and its mouse model, experimental autoimmune encephalomyelitis (EAE)\u003csup\u003e8-10\u003c/sup\u003e. In this study, we investigated the mechanisms which may enable these cells to cross the BBB.\u003c/p\u003e\n\u003cp\u003eUsing exploratory proteomics, we highlighted an underappreciated role for pathways including ECM remodeling and collagen binding in inflammatory T lymphocytes. We identified Mannose Receptor C Type 2 (MRC2), also known as UPARAP, Endo180, or CD280, as a \u0026nbsp;molecule expressed on these T lymphocytes with increased ability to transmigrate across the BBB and to degrade collagen IV (Col IV)\u003csup\u003e11,12\u003c/sup\u003e, a key structural component of the BBB and critical for its integrity\u003csup\u003e13,14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExploratory proteomics analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate mechanisms underlying CNS invasion by T lymphocytes, we performed proteomics on IL-23-polarized human MCAM⁺CD45RO⁺CD4⁺ memory T lymphocytes (Extended Fig.1A), previously shown to efficiently transmigrate across the BBB\u003csup\u003e8,9\u003c/sup\u003e. We identified 177 proteins with PSM \u0026gt;35 and PEP \u0026ge;1. Functional enrichment analysis via REACTOME\u003csup\u003e15\u003c/sup\u003e and GO terms revealed significant enrichment of immune pathways, ECM organization, cell-matrix adhesion, and hemostasis (Fig.1A-B). GO molecular function further highlighted ECM structural components and collagen binding in MCAM⁺ versus MCAM⁻ T lymphocytes (Fig.1C). Among the top five upregulated proteins was MRC2 (Fig.1D), a collagen-binding receptor involved in ECM degradation (including Col IV) and MMP activation\u003csup\u003e12,16\u003c/sup\u003e. In contrast, TIMP1, an MMP inhibitor, was downregulated in MCAM⁺ T lymphocytes, further highlighting the invasive phenotype of these cells.\u003c/p\u003e\n\u003cp\u003eTo identify co-regulatory mechanisms, we used NetworkAnalyst and ENCODE (BETA Minus algorithm)\u003csup\u003e17\u003c/sup\u003e to explore gene regulatory networks. MYC-Associated Zinc Finger Protein (MAZ) was identified as a shared transcription factor for MCAM and MRC2 (Extended Fig.1B) and has been implicated as an MS susceptibility gene\u003csup\u003e18\u003c/sup\u003e. MAZ was also predicted to regulate MMP9 and DICAM (MXRA8), suggesting a possible co-expression with MRC2 (Extended Fig.1C). These data indicate a not yet explored role for ECM remodeling in activated pro-inflammatory T lymphocytes.\u003c/p\u003e\n\u003cp\u003eSupporting this, analysis of scRNA-seq data from freshly autopsied human brain tissue, along with re-analysis of recently published CD31-enriched murine BECs\u003csup\u003e19\u003c/sup\u003e, revealed that Col IV (\u0026alpha;1 and \u0026alpha;2 subunits), the substrate of MRC2, was mainly expressed by BECs. Additionally, immunofluorescence microscopy confirmed expression of Col IV on primary human BBB and meningeal ECs under resting and pro-inflammatory conditions (Extended Fig.2A-C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMRC2 is a marker for CNS-invasion\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMRC2 has been linked to MS, located in a risk-associated chromosomal region, with elevated mRNA levels in blood during MS relapse\u003csup\u003e20\u003c/sup\u003e. To assess T lymphocyte contributions, we performed flow cytometry on PBMCs from untreated, inactive RRMS (n=22), relapsing RRMS (n=5), progressive MS with sustained disability worsening (PPMS, n=6; SPMS, n=4), and matched healthy donors (Fig.2A-B, Extended Fig.3A, Extended Table 1). Peripheral MRC2⁺ T lymphocytes were low in healthy and inactive RRMS, but significantly increased during relapse, and in progressive MS (Fig.2A-B). Memory T lymphocytes, both central and effector memory subsets, showed higher abundance of MRC2, than na\u0026iuml;ve and effector cells in RRMS (Fig.2C, Extended Fig.3A). As MRC2 expression can be influenced by estrogen in vitro\u003csup\u003e21\u003c/sup\u003e, we tested for sex differences in PBMCs. Our results showed no significant differences in our RRMS patient cohort, nor in publicly available microarray datasets from healthy donors (Query data sets for GSE3365)\u003csup\u003e22\u003c/sup\u003e (Extended Fig.3B-C). Confocal microscopy of postmortem MS brain tissue revealed MRC2 colocalized with CD4⁺ and predominantly CD8⁺ T lymphocytes in lesions (Fig.2D, Extended Table 2), with higher frequencies in active versus pre-active lesions (Extended Fig.3D-E). Flow cytometry of autopsied human brain and blood from two MS and two ALS donors, who underwent medical aid in dying, confirmed higher MRC2⁺ T lymphocytes in periventricular lesions versus normal white matter (Fig.2E, Extended Table 3). CNS-infiltrating T lymphocytes showed a significantly higher frequency of MRC2 compared to peripheral T lymphocytes. Additional analysis of blood and CSF from untreated PwMS showed significantly increased MRC2 on CD4⁺ and CD8⁺ T lymphocytes in CSF versus blood (Fig.2F, Extended Table 4).\u003c/p\u003e\n\u003cp\u003eTogether, these findings indicate that MRC2 expression is elevated during relapse and disease progression and may present a marker for CNS-infiltrating T lymphocytes in MS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotyping MRC2⁺ T lymphocytes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further characterize MRC2⁺ CD4⁺ and CD8⁺ T lymphocytes, we used a flow cytometry panel targeting ECM remodeling molecules and factors linked to CNS invasion and MS pathology\u003csup\u003e23-25\u003c/sup\u003e. Using PBMCs from untreated PwMS and healthy donors, we compared MRC2⁺ to MRC2⁻ cells per donor (Fig.3A, Extended Table 1). MRC2⁺ T lymphocytes from PwMS showed increased MMP2⁺ and MMP9⁺ frequencies and decreased TIMP-1 (CD8⁺ T lymphocytes). We also determined expression of MMP28, which was elevated (gMFI) in MRC2⁺ cells (Extended Fig.4A). UPAR expression showed no clear association with MRC2 in T lymphocytes (Extended Fig.4A). MRC2⁺ T lymphocytes also exhibited elevated TNF (Fig.3A), particularly in PwMS, but not GM-CSF or IFN\u0026gamma; (Extended Fig.4A).\u003c/p\u003e\n\u003cp\u003eThese findings suggest MRC2⁺ T lymphocytes possess a distinct invasive and pro-inflammatory phenotype, supporting a role in CNS tissue invasion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInflammation regulates MRC2 expression\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated whether in vitro polarization affects MRC2 expression on T lymphocytes. Flow cytometry and confocal microscopy of CD4⁺ and CD8⁺ T lymphocytes showed highest MRC2 expression in IL-12-stimulated T lymphocytes (Fig.3B\u0026ndash;C), which also exhibited the most IFN\u0026gamma;⁺ cells (Extended Fig.4B), suggesting IL-12\u0026ndash;IFN\u0026gamma; signaling regulates MRC2 expression. We then analyzed transcription factor expression in IL-12, IL-23, and IL-4 conditions (Fig.3D). MRC2⁺ T lymphocyte frequency was significantly higher in T-bet⁺ (IL-12) and ROR\u0026gamma;t⁺ (IL-23) subsets, but not significantly different in GATA-3⁺ (IL-4) cells, when compared to their respective negative counterparts. These data suggest MRC2 is upregulated in activated pro-inflammatory human T lymphocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMRC2 promotes transendothelial migration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether MRC2 plays a role in T lymphocyte migration, we performed in vitro assays using primary cultures of human BECs and IL-12-polarized CD4⁺ or CD8⁺ T lymphocytes. (Fig.4A, Extended Fig.5A)\u003csup\u003e27\u003c/sup\u003e. MRC2 blockade, using an anti-MRC2 blocking antibody, significantly reduced transendothelial migration of IL-12-stimulated T lymphocytes (Fig.4A). We also analyzed BECs post-migration of T lymphocytes. MRC2 blockade led to elevated surface expression of Col IV and Claudin-5 (Fig.4B-C), but not ICAM-1 (Extended Fig.5B-E), while IFN\u0026gamma;/TNF stimulation of BECs upregulated \u003cem\u003eICAM-1\u003c/em\u003e mRNA levels but decreased both Col IV \u0026alpha; chains \u003cem\u003eCOL4A1\u003c/em\u003e and \u003cem\u003eCOL4A2\u003c/em\u003e .Confocal microscopy showed strong co-localization of MRC2 and Col IV in perivascular CD4⁺ and CD8⁺ T lymphocytes within active MS lesions (Fig.4D-F). These findings suggest MRC2 facilitates T lymphocyte migration across the BBB possibly through interaction with its substrate Col IV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic deletion of MRC2 affects T lymphocyte function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the function of MRC2 on T lymphocytes in CNS invasion, we used MRC2 (UPARAP) KO mice and their wildtype (WT) littermates (Extended Fig.6A). CFSE-based proliferation assays showed that CD4⁺ and CD8⁺ T lymphocytes from KO and WT mice had similar proliferation rates upon IL-12 or IL-4 stimulation (Extended Fig.6B). Flow cytometry revealed no significant difference in IFN\u0026gamma; and IL-17a expression between genotypes (Extended Fig.6C-D). As in humans, IL-12 stimulation upregulated MRC2 on murine CD4⁺ and CD8⁺ T lymphocytes compared to IL-4 stimulation (Fig.5A), so we focused on IL-12-stimulated T lymphocytes.\u003c/p\u003e\n\u003cp\u003eUsing flow adhesion assays on WT murine BECs, MRC2 KO T lymphocytes showed reduced adhesion and rolling ability, indicated by fewer rolling cells and increased rolling velocity (Fig.5B, Extended Fig.6E), suggesting impaired early CNS invasion. To test native Col IV digestion and uptake, we cultured IL-12-stimulated T lymphocytes on DQ-Col IV\u0026ndash;coated surfaces, allowing the measurement of green fluorescent intensity upon degradation of native Col IV (non-fluorescent) to its fluorescent degradation products (Extended Fig.6F, Fig. 5C). MRC2 KO T lymphocytes showed significantly reduced Col IV uptake compared to WT, indicated by lower fluorescence intensity. In MOG\u003csub\u003e35-55\u003c/sub\u003e-induced EAE (Extended Fig.7A), MRC2⁺ T lymphocytes were enriched in the CNS compared to their peripheral counterparts in the spleen (Fig.5D-E). Confocal imaging on postmortem tissue revealed that MRC2⁺ T lymphocytes were predominantly CD4⁺ T lymphocytes, and primarily localized in the spinal cord (Fig. 5F\u0026ndash;G).\u003c/p\u003e\n\u003cp\u003eThese findings indicated that MRC2 supports T cell\u0026ndash;mediated CNS invasion possibly by promoting Col IV degradation and BBB endothelial interaction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic deletion of MRC2 dampens neuroinflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the role of MRC2 in vivo, we induced MOG\u003csub\u003e35-55\u003c/sub\u003e-dependent EAE in male and female MRC2 KO mice (n = 32) and their WT littermates (n = 24) (Extended Fig.7A). Female KO mice had significantly lower disease incidence (60%) when compared to female WT (87.5%), male WT (100%), and male KO (100%) mice (Extended Fig.7B, Fig.6A; \u0026chi;2=17.68, 3 df, p \u0026lt; 0.0005). Affected MRC2 KO female animals also showed reduced weight loss and a milder disease course, with lower clinical and cumulative EAE scores, especially in the chronic phase (Extended Fig.7C-E, Fig.6B-D). Immunofluorescence of spinal cords from sick MRC2 KO female animals revealed fewer CD45⁺ immune cells compared to WT (Extended Fig.7F), and fewer infiltrating CD4⁺ and CD8⁺ T lymphocytes in the CNS (Fig.6E). Male MRC2 KO mice showed no clinical improvement, but reduced CD4⁺ T lymphocytes in spinal cord and CD8⁺ T lymphocytes in brain. Additionally, flow cytometry of CNS-infiltrating T lymphocytes showed elevated MMP2/TIMP2 ratios in affected WT mice, which were significantly reduced in MRC2 KO animals (Fig.6F-G) during the chronic disease. No differences were found in MMP9/TIMP1 ratios or IFN\u0026gamma;/IL-17a expression (Extended Fig.7G-H).\u003c/p\u003e\n\u003cp\u003eIn summary, MRC2 expression on T lymphocytes promotes CNS infiltration and disease severity in a sex-dependent manner, likely via ECM remodeling rather than cytokine production.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCurrent MS treatments often lack specificity, targeting broad immune populations and causing immunosuppression and increased infection risk\u003csup\u003e4,5\u003c/sup\u003e. This study aimed to uncover molecular mechanisms by which pathogenic T lymphocytes breach the BBB. We identified MRC2 as a key player in promoting T lymphocyte migration across the BBB in both humans, and mice during autoimmune neuroinflammation. Exploratory proteomics revealed enrichment of ECM pathways, including collagen binding and hemostasis, in highly invasive, pro-inflammatory (MCAM\u003csup\u003e+\u003c/sup\u003e) T lymphocytes. MRC2 ranked among the top five upregulated proteins and is known for its role in ECM remodeling, particularly in degrading collagens including Col IV\u003csup\u003e12\u003c/sup\u003e. Col IV is a major BBB component and has been previously suggested in BBB integrity disturbance in MS\u003csup\u003e13,28\u003c/sup\u003e. While typically expressed at low levels on T lymphocytes, our data showed an upregulation of MRC2 upon activation\u003csup\u003e27\u003c/sup\u003e, making it a promising candidate for targeted therapy. Previously, MRC2 and its potential regulator (MAZ) have been assumed to be associated with increased risk to develop MS\u003csup\u003e18,20\u003c/sup\u003e. We confirmed increased frequencies of MRC2⁺ T lymphocytes in untreated PwMS during active relapse and observed associations in progressive MS with sustained disability worsening, particularly in SPMS. Interestingly, MRC2⁺ cells were enriched within the memory T lymphocyte subsets a population over-represented in myelin-reactive T lymphocytes in MS\u003csup\u003e29\u003c/sup\u003e. MRC2⁺ CD4⁺ and CD8⁺ T lymphocytes were significantly elevated in active lesions, especially co-localized with its substrate Col IV at the BBB in the perivascular space, suggesting a role in the CNS infiltration during the acute phase.\u003c/p\u003e \u003cp\u003eTo explore the functional role of MRC2 we used specific blocking antibodies and T lymphocytes from MRC2 KO mice for in vitro assays as well as the MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e-induced EAE mouse model to assess in vivo CNS infiltration. All our data strongly pointed towards a role for MRC2 in promoting transendothelial migration of T lymphocytes across the BBB. We further explored downstream mechanisms by analyzing expression of MMPs and TIMPs, as well as related molecules from the hemostasis pathways like UPAR, and inflammatory cytokines. MMP2 and MMP9, critical for ECM degradation, were significantly elevated in MRC2⁺ T lymphocytes from untreated PwMS, while TIMPs were not, indicating a heightened invasive profile. In the EAE model, genetic MRC2 deletion significantly lowered the MMP2/TIMP2 ratio in CNS-infiltrating T lymphocytes. These findings suggest that MRC2 regulates MMP expression and activity in CNS-infiltrating T lymphocytes, potentially promoting tissue invasion. Nearly all T lymphocytes also expressed MMP28, but levels were significantly higher in MRC2⁺ T lymphocytes, suggesting a link between MRC2 expression and enhanced ECM remodeling capacity. While MRC2 is also known as UPAR-associated protein, we found UPAR expression was not consistently altered in peripheral MRC2⁺ T lymphocytes in our experiments, implying that MMP and cytokine activity in these cells may be UPAR-independent. However, UPAR and UPA were elevated in inflamed brain vasculature from PwMS\u003csup\u003e23\u003c/sup\u003e, pointing to potential interactions that require further investigation. We examined cytokine expression to understand whether MRC2 influences or opposingly, would be impacted by pro-inflammatory environments. MRC2⁺ T lymphocytes showed increased TNF expression (a downstream target of MMPs\u003csup\u003e26\u003c/sup\u003e), but not GM-CSF or IFNγ, suggesting a specific inflammatory profile. In contrast, MRC2 deletion did not affect IFNγ or IL-17a expression, previously shown to play an important role in T lymphocyte pathology in EAE\u003csup\u003e30\u003c/sup\u003e, indicating that MRC2 primarily affects tissue invasion rather than cytokine production. A major mechanism by which MRC2 may promote T lymphocyte migration is via interaction with its substrate Col IV at the BBB. MRC2 KO T lymphocytes showed reduced capacity to degrade native Col IV in vitro. Within active MS lesions, MRC2⁺ T lymphocytes co-localized with Col IV in perivascular areas, but not deeper parenchyma, supporting the idea that MRC2-Col IV interaction is critical for initial BBB crossing but not for further CNS navigation. Based on our collected data, we propose a hypothetical model in which MRC2⁺ T lymphocytes bind and intracellularly degrade Col IV at the BBB as summarized in Fig.\u0026nbsp;7. Together with MMP activation, this may contribute to the degradation of Claudin-5, further compromising BBB integrity and facilitating T lymphocyte entry into the CNS in MS. Although not further investigated in this study, adhesion molecules such as MCAM and DICAM on MRC2⁺ T lymphocytes may facilitate the initial steps of adhesion and rolling along BBB endothelial cells. Since Col IV is located on the abluminal site on post-capillary venules, the interaction of MRC2 and Col IV likely becomes critical during the final steps of CNS entry. Interestingly, we observed sex-dependent effects in the EAE mouse model. MRC2 deficiency led to differing disease outcomes in male and female mice, possibly due to ECM composition differences. Male brains reportedly have lower stiffness and altered collagen (including Col IV) and laminin expression\u003csup\u003e32\u003c/sup\u003e. Estrogen, protecting male and female mice against EAE development\u003csup\u003e33\u003c/sup\u003e, has been previously shown to regulate MRC2\u003csup\u003e21\u003c/sup\u003e. However, sex-dependent effects on expression were not recapitulated in human PBMC data. In our study, we did not investigate CNS-resident cells in this context, and which may also contribute to the sex-specific outcomes.\u003c/p\u003e \u003cp\u003eOur use of a constitutive MRC2 KO mouse for MOG-induced EAE limits conclusions about T lymphocyte-specific effects, as other immune cells and CNS-resident cells may be involved. Future studies using conditional KO models and adoptive transfer EAE are needed to further investigate the role of MRC2 on autoimmune neuroinflammation outcomes. Additionally, our human data were drawn from a limited donor pool; larger cohorts are required to confirm the role of MRC2 as a disease marker at protein level, especially in progressive MS. An MRC2-targeting antibody and antibody-drug conjugate (ADC) have been previously developed\u003csup\u003e27\u003c/sup\u003e. The latter will be investigated in a clinical trial phase I/II study (ADCE-D01) in patients with metastatic and unresectable soft tissue sarcoma in the USA (currently enrolling) and is under regulatory review in the European Union [NCT06797999]. Preclinical data show good tolerability of these antibodies in mice\u003csup\u003e27\u003c/sup\u003e, suggesting an overall significant translational potential of MRC2 as a putative drug target in PwMS. However, these therapies have not yet been tested in the context of neuroinflammation. Future studies should therefore evaluate the safety and efficacy of MRC2-targeted interventions in models of autoimmune neuroinflammation like EAE. Such work may pave the way for new, more selective treatments for PwMS that minimize systemic immunosuppression while effectively preventing pathogenic T lymphocyte infiltration into the CNS.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using Graph Pad prism software (version 8). Normal distributed data was analyzed using unpaired or paired Student\u0026rsquo;s t test as indicated. For all data two-sided tests were performed. Additionally, for pairwise comparison, the ratio-paired Student\u0026rsquo;s t test was used. Otherwise for non-normally distributed data, Wilcoxon matched-paired signed rank test was performed. When more than 2 groups were compared, one-way ANOVA and Tukey\u0026rsquo;s (all comparisons), and Sidak\u0026rsquo;s (specific, independent comparison) multiple comparison tests were used. For non-normally distributed data Kruskal-Wallis test was carried out. To use a similar approach as repeated measures ANOVA but allowing for missing values (early harvest of animals for FACS / immunohistochemistry) and repeated measures, we used a mixed model implemented in Graph Pad prism (version 8) with a compound symmetry covariance structure, fit using Restricted Maximum Likelihood (REML) to analyze EAE curves and weight changes. Incidence of EAE disease was assessed by using Log-rank Mantel cox test. All performed tests are indicated in the figure legends.\u003c/p\u003e \n"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eAll non-human vertebrate experiments in this manuscript were approved by our animal ethic\u0026rsquo;s committee and by our veterinarian.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe thank the Core facility of the Centre de Recherche du CHUM (CRCHUM) for their assistance; Jean Franҫois Schmouth, PhD from the platform for Genetic Engineering and Animal Modelling; Ga\u0026euml;l Dulude, PhD and Philippe St-Onge, MSc from the platform for Flow Cytometry; Aur\u0026eacute;lie Cleret-Buhot, PhD from the core facility for Cellular Imaging; and V\u0026eacute;ronique Barr\u0026egrave;s, MSc and Liliane Meunier, MSc from the platform for Molecular Pathology; Alice Michallet Roy from the animal housing facility at the CRCHUM. We also would like to thank Dr. Thomas Bugge from the National Health Institutes (NIH) for providing the MRC2 KO mouse model. Language editing support was provided by ChatGPT. Schematics in Extended Figures 1A, 5A, 6E-F and 7A were created with BioRender.com.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCanadian Institutes of Health Research (CIHR) grant and Canada Research Chair program (AP)\u003c/p\u003e\n\u003cp\u003eFonds de Recherche du Qu\u0026eacute;bec - Sant\u0026eacute; (FRQS) fellowship (BZ, FT, APF)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMultiple Sclerosis Society Canada fellowship (BZ)\u003c/p\u003e\n\u003cp\u003eMITACS Globalink Research Award (MKa, MIM)\u003c/p\u003e\n\u003cp\u003eUniversit\u0026eacute; de Montr\u0026eacute;al Premier fellowship (CS)\u003c/p\u003e\n\u003cp\u003eNational MS Society postdoctoral fellowship (HD)\u003c/p\u003e\n\u003cp\u003eCIHR fellowship (KT, FT, APF, MKr)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDanish Cancer Society grant agreements No R231-A13820 (NB)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDanish Cancer Society grant agreements R368-A21555 (NB)\u003c/p\u003e\n\u003cp\u003eNovo Nordisk Foundation grant Nos. NNF19OC0058603 (NB)\u003c/p\u003e\n\u003cp\u003eNovo Nordisk Foundation grant Nos NNF22OC0080995 (NB)\u003c/p\u003e\n\u003cp\u003eNEYE Foundation (NB)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: BZ, AP\u003c/p\u003e\n\u003cp\u003eMethodology: BZ, MKa, CS, MIM, HLD, SL, SBA, WK, KT, CG, IA, FT, HM, CM, LB, APF, OT, OC, OS, MKr, HK, MD, ASH, PD, MG, GM, SO, NB, CL, NA, SEJZ\u003c/p\u003e\n\u003cp\u003eInvestigation: BZ, MKa, CS, MIM, SBA, OT, OC\u003c/p\u003e\n\u003cp\u003eVisualization: BZ, MKa, CS, MIM, SBA, FT, OT, OC\u003c/p\u003e\n\u003cp\u003eFunding acquisition: AP, BZ, NB\u003c/p\u003e\n\u003cp\u003eProject administration: BZ, WK, LB\u003c/p\u003e\n\u003cp\u003eSupervision: AP\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: BZ\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: AP, NA, CL\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAP holds the Senior Canada Research Chair in MS\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the supplementary materials. Raw data from scRNA-Seq will be deposited on Gene Expression Omnibus (GEO) upon publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSoftware code will be available upon publication.\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to [email protected].\u003c/p\u003e\n\u003cp\u003eReprints and permissions information is available at www.nature.com/reprints.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZierfuss, B., Larochelle, C. \u0026amp; Prat, A. Blood-brain barrier dysfunction in multiple sclerosis: causes, consequences, and potential effects of therapies. \u003cem\u003eLancet Neurol\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 95-109, doi:10.1016/s1474-4422(23)00377-0 (2024).\u003c/li\u003e\n\u003cli\u003eLublin, F. 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F., Jr.\u003cem\u003e et al.\u003c/em\u003e Low-Dose Estrogen Therapy Ameliorates Experimental Autoimmune Encephalomyelitis in Two Different Inbred Mouse Strains1. \u003cem\u003eThe Journal of Immunology\u003c/em\u003e \u003cstrong\u003e166\u003c/strong\u003e, 2080-2089, doi:10.4049/jimmunol.166.3.2080 (2001).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6917781/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6917781/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRelapses in Multiple Sclerosis (MS) are driven by pathogenic immune cells breaching the disrupted blood\u0026ndash;brain barrier (BBB), leading to tissue damage and eventual disease progression\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. Among the most effective therapies are those that block immune cell infiltration across the BBB, a multistep process involving activation, adhesion, rolling, transendothelial migration, and extracellular matrix (ECM) degradation. Current treatments broadly suppress the immune system and can cause adverse side effects\u003csup\u003e4,5\u003c/sup\u003e, highlighting the need for targeted approaches. Here, we identified a previously unrecognized role of ECM remodeling pathways and Mannose Receptor C-type 2 (MRC2) in invasive T lymphocytes as a potential therapeutic target. Typically low under physiological conditions, MRC2 was upregulated on T lymphocytes upon inflammatory stimulation in vitro, enriched in the periphery of MS patients during relapse and progressive disease, and present in cerebrospinal fluid and active brain lesions co-localized with Col IV, the major ECM component of the BBB. Genetic deletion of MRC2 impaired T lymphocyte-mediated Col IV degradation, adhesion, and transendothelial migration, and leading to reduced neuroinflammation in a sex-dependent manner in an MS-like mouse model. These findings suggest MRC2 as a key regulator of T lymphocyte infiltration into the CNS and a promising target for MS therapy.\u003c/p\u003e","manuscriptTitle":"Mannose Receptor C type 2 influences autoimmune neuroinflammation and blood-brain barrier integrity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-07 07:44:13","doi":"10.21203/rs.3.rs-6917781/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8b9df069-710d-473d-8d13-e332dc4266c5","owner":[],"postedDate":"July 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":50581026,"name":"Biological sciences/Neuroscience/Neuroimmunology"},{"id":50581027,"name":"Health sciences/Diseases/Neurological disorders/Multiple sclerosis"}],"tags":[],"updatedAt":"2025-07-07T07:44:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-07 07:44:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6917781","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6917781","identity":"rs-6917781","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}