MIF and D-DT are potential disease severity modifiers in male MS subjects

Abstract

Little is known about mechanisms that drive the development of progressive multiple sclerosis (MS), although inflammatory factors, such as macrophage migration inhibitory factor (MIF), its homolog D-dopachrome tautomerase (D-DT), and their common receptor CD74 may contribute to disease worsening. Our findings demonstrate elevated MIF and D-DT levels in males with progressive disease compared with relapsing-remitting males (RRMS) and female MS subjects, with increased levels of CD74 in females vs. males with high MS disease severity. Furthermore, increased MIF and D-DT levels in males with progressive disease were significantly correlated with the presence of two high-expression promoter polymorphisms located in the MIF gene, a -794CATT_5-8 microsatellite repeat and a -173 G/C SNP. Conversely, mice lacking MIF or D-DT developed less-severe signs of experimental autoimmune encephalomyelitis, a murine model of MS, thus implicating both homologs as copathogenic contributors. These findings indicate that genetically controlled high MIF expression (and D-DT) promotes MS progression in males, suggesting that these two factors are sex-specific disease modifiers and raising the possibility that aggressive anti-MIF treatment of clinically isolated syndrome or RRMS males with a high-expresser genotype might slow or prevent the onset of progressive MS. Additionally, selective targeting of MIF:CD74 signaling might provide an effective, trackable therapeutic approach for MS subjects of both sexes.

Keywords: disease modifier; multiple sclerosis; sex differences.

Fig. 1.

Plasma levels of MIF in MS and HC subjects. MIF concentrations were evaluated in (A) all HC (n = 49) and MS (n = 287) subjects and stratified according to (B) gender and disease type or (C) gender and disease severity according to their EDSS scores. Data are presented as mean ± SD, *P < 0.05, **P < 0.01, one-way ANOVA with Tukey comparison (A and B) or t test of coefficient in ordinary linear regression as a test of linear trend (C). Scatterplot points were jittered slightly along the EDSS axis for better visualization.

Fig. 2.

Plasma levels of D-DT in MS and HC subjects. D-DT concentrations were evaluated in (A) all HC (n = 35) and MS (n = 123) subjects and stratified according to (B) gender and disease type or (C) gender and disease severity according to their EDSS scores. Data are presented as mean ± SD, analyzed for significant differences by one-way ANOVA with Tukey comparison (A and B) or t test of coefficient in ordinary linear regression as a test of linear trend (C). Scatterplot points were jittered slightly along the EDSS axis for better visualization.

Fig. 3.

MIF and D-DT relative expression in the brain and positive correlation between MIF and D-DT mRNA expression levels. (A) Relative mRNA expression levels of MIF, D-DT in three representative white matter areas of two males and three females SPMS subjects. Data are presented as mean ± SD, **P < 0.01, Student’s t test. (B) mRNA expression levels of MIF, D-DT and CD74 in PBMC of MS and HC subjects. Correlations were assessed by Spearman’s correlation test.

Fig. S1.

mRNA expression levels of MIF, D-DT, and CD74 in PBMC of MS and HC subjects. MIF, D-DT, and CD74 expression were evaluated in all HC and MS subjects and stratified according to (A) gender and disease type or (B) gender and disease severity according to their EDSS scores. Data are presented as mean ± SD, *P < 0.05, one-way ANOVA with Tukey comparison.

Fig. 4.

CD74 cell surface expression on CD11b⁺ cells of MS and HC subjects and inhibition of MIF and D-DT binding to recombinant human CD74 by the DRα1–hMOG-35-55 construct. CD74 cell surface mean fluorescent intensity on CD11b⁺ cells was evaluated in (A) all HC (n = 46) and MS (n = 123) subjects and stratified according to (B) gender and disease type or (C) gender and disease severity according to their EDSS scores. Data are presented as mean ± SD, analyzed for significant differences by one-way ANOVA with Tukey comparison (A and B) or t test of coefficient in ordinary linear regression as a test of linear trend (C). (D) HC PBMCs were incubated with 5 µg of DRα1–hMOG-35-55 or DR2β1 constructs for 1 h and analyzed for CD74 expression on CD11b⁺ monocytes. Data are presented as mean ± SD of reduction compared with untreated cells. *P < 0.05, Student’s t test. (E) Serial 1:2 dilutions of DRα1–hMOG-35-55 were prepared in 10 nM rhCD74 and the mixtures were applied to wells previously coated overnight with 30 nM of either hMIF or hD-DT. IC50s were computed by fitting the data to a one-site competition using the Prism program (DeltaGraph). IC50 DRα1–hMOG-35-55 vs. hMIF = 78.5, R² = 0.9720; IC50 for DRα1–hMOG-35-55 for hD-DT = 116.5, R² = 0.9821.

Fig. 5.

EAE disease course in WT, D-DT–KO, and MIF-KO mice. EAE was induced with mouse (m)MOG-35-55 peptide in C57BL/6 WT, D-DT–KO, and MIF-KO mice. Mean clinical EAE daily disease scores (Left) and CDI scores (Right) are shown. *P < 0.05, **P < 0.01, ***P < 0.001. Daily mean score curves were compared using Fan and Lin’s adaptive Neyman test (63) after discrete Fourier transformation of individual-level data, and CDIs were compared using Welch’s one-sided t test after augmenting the within-group variances to include the CDI estimation error (64).

Fig. 6.

Reduced CNS inflammation in D-DT–KO and MIF-KO mice. (A and B) Frequency of CD11b⁺CD45^high in spinal cords of WT, D-DT–KO, and MIF-KO male mice on day 18 postdisease induction. (C) CD74 cell surface expression (mean fluorescence intensity, MFI) on CD11b⁺ cells in spinal cords. (D) Absolute cell numbers in spleen of WT, D-DT–KO, and MIF-KO mice. (E) Frequencies of CD4⁺CD44^hiCD69⁻ and CD11b⁺CD74⁺ cells in spleen. Data are presented as mean ± SD *P < 0.05, **P < 0.01, ***P < 0.001 one-way ANOVA with Tukey comparison.