Fig 2: CLRc regulates expression of Mcl and Mincle in monocytes and macrophages.(A) Expression of Mcl and Mincle assessed by microarray in spleens of (DA × PVG) × DA backcrossed rats subjected to EAE, affected (n = 95, score ≥1) and nonaffected (n = 51, score 0). (B) Gene expression in DA (n = 6) and CLRc (n = 6) spleens determined by qPCR (representative of 2 experiments). (C) Flow cytometry analysis of cells isolated from blood and spinal cord of naive DA (n = 5) and CLRc (n = 5), as well as DA (n = 7) and CLRc (n = 6) rats on day 13 p.i. assessing Mcl and Mincle protein expression by mean fluorescence intensity (MFI) (representative of 2 experiments). Ly, lymphocyte; Mi, microglia; Mo/Ma, monocyte/macrophage; Gr, granulocyte. (D) qPCR analysis of Mcl and Mincle expression in BMMas and MoDCs (BM-APCs) derived in vitro and meninges from naive and 7-day-p.i. DA (n = 4) and CLRc (n = 4) rats (representative of 2 experiments). (E) Immunofluorescent staining of rat spinal cord 11 days p.i. Representative images of staining for Mcl, Mincle, and nuclei (DAPI) (original magnification, ×40). Data are presented as the mean ± SEM or box plots with whiskers representing 5th to 95th percentile. All comparisons were analyzed with the Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: CNS-specific Mcl/Mincle silencing, as well as blockage of the endogenous ligand SAP130, protects from EAE.(A) MOG-immunized rats injected with Mcl (n = 8), Mincle (n = 8), Mcl/Mincle (n = 8), or scrambled control siRNA (n = 8) i.t. and i.c. at 7, 9, and 12 days p.i. were followed for clinical signs of EAE (representative of 3 experiments). (B) Characterization of proliferation and cytokine production of infiltrating cells isolated from spinal cord on day 12 p.i., stimulated in vitro with PMA/ionomycin/brefeldin A for 5 hours (proliferation and cytokine production) in rats injected with Mcl/Mincle (n = 5) or scrambled control siRNA (n = 6) and (C) quantification of IFN-γ– and IL-17–producing CD4+ T cells (representative of 2 experiments). (D) Clinical signs of EAE and disease parameters in DA rats treated with anti-SAP130 (n = 18) or rabbit IgG isotype control (n = 18) antibody i.t. and i.c. on days 2 and 7 p.i. (2 pooled experiments). Data are presented as the mean ± SEM. The following statistical tests were used: 1-way ANOVA with Dunnett’s multiple-comparisons test (A, for area under the curve [AUC] of clinical EAE and weight change), Mann-Whitney U test (A–D [for average, cumulative, and max EAE score], B, and C), unpaired 2-tailed t test (D, for AUC of clinical EAE and weight change), and χ2 test (A and D, for EAE incidence). *P < 0.05.

Fig 4: The MCL/ MINCLE signaling pathway is upregulated in MS patients and correlates with disease activity and progression.(A) Representative immunofluorescent staining of MS lesions: MCL or MINCLE (green), HLA-DR expression (LN3, red). Original magnification, ×40. (B and C) Gene expression analysis of PBMCs from MS patients and noninflammatory neurological disease controls (NINDCs) using RNA sequencing. (B) Expression of MCL, MINCLE, and CARD9 comparing MS patients in remission (n = 73), MS patients in relapse (n = 14), CIS patients (n = 28), and NINDCs (n = 36). (C) Expression of MCL, MINCLE, CARD9, and IL8 according to Expanded Disability Status Scale (EDSS) score. (D) PBMCs from RRMS patients in relapse (n = 9) and healthy controls (HC) (n = 11) were stimulated in vitro with TDB, LPS, or media. qPCR analysis of IL8 expression of adherent CD14+ fraction after 24-hour stimulation. (E) Flow cytometry analysis of IL-8 production gated on CD14+ monocytes after 48-hour stimulation. Data are presented as the mean ± SEM or box plots with whiskers representing 5th to 95th percentile. The following statistical tests were used: 1-way ANOVA with Dunnett’s multiple-comparisons test (B), Mann-Whitney U test (C and D), and unpaired 2-tailed t test (E). *P < 0.05; **P < 0.01.