Sex-Specific Gene-by-Vitamin D Interactions Regulate Susceptibility to Central Nervous System Autoimmunity

Vitamin D3 (VitD) insufficiency is postulated to represent a major modifiable risk factor for multiple sclerosis (MS). While low VitD levels strongly correlate with higher MS risk in white populations, this is not the case for other ethnic groups, suggesting the existence of a genetic component. Moreover, VitD supplementation studies in MS so far have not shown a consistent benefit. We sought to determine whether direct manipulation of VitD levels modulates central nervous system autoimmune disease in a sex-by-genotype-dependent manner. To this end, we used a dietary model of VitD modulation, together with the autoimmune animal model of MS, experimental autoimmune encephalomyelitis (EAE). To assess the impact of genotype-by-VitD interactions on EAE susceptibility, we utilized a chromosome substitution (consomic) mouse model that incorporates the genetic diversity of wild-derived PWD/PhJ mice. High VitD was protective in EAE in female, but not male C57BL/6J (B6) mice, and had no effect in EAE-resistant PWD/PhJ (PWD) mice. EAE protection was accompanied by sex- and genotype-specific suppression of proinflammatory transcriptional programs in CD4 T effector cells, but not CD4 regulatory T cells. Decreased expression of proinflammatory genes was observed with high VitD in female CD4 T effector cells, specifically implicating a key role of MHC class II genes, interferon gamma, and Th1 cell-mediated neuroinflammation. In consomic strains, effects of VitD on EAE were also sex- and genotype dependent, whereby high VitD: (1) was protective, (2) had no effect, and (3) unexpectedly had disease-exacerbating effects. Systemic levels of 25(OH)D differed across consomic strains, with higher levels associated with EAE protection only in females. Analysis of expression of key known VitD metabolism genes between B6 and PWD mice revealed that their expression is genetically determined and sex specific and implicated Cyp27b1 and Vdr as candidate genes responsible for differential EAE responses to VitD modulation. Taken together, our results support the observation that the association between VitD status and MS susceptibility is genotype dependent and suggest that the outcome of VitD status in MS is determined by gene-by-sex interactions

data_sheet_1_Sex-Specific Gene-by-Vitamin D Interactions Regulate Susceptibility to Central Nervous System Autoimmunity.docx

<p>Vitamin D3 (VitD) insufficiency is postulated to represent a major modifiable risk factor for multiple sclerosis (MS). While low VitD levels strongly correlate with higher MS risk in white populations, this is not the case for other ethnic groups, suggesting the existence of a genetic component. Moreover, VitD supplementation studies in MS so far have not shown a consistent benefit. We sought to determine whether direct manipulation of VitD levels modulates central nervous system autoimmune disease in a sex-by-genotype-dependent manner. To this end, we used a dietary model of VitD modulation, together with the autoimmune animal model of MS, experimental autoimmune encephalomyelitis (EAE). To assess the impact of genotype-by-VitD interactions on EAE susceptibility, we utilized a chromosome substitution (consomic) mouse model that incorporates the genetic diversity of wild-derived PWD/PhJ mice. High VitD was protective in EAE in female, but not male C57BL/6J (B6) mice, and had no effect in EAE-resistant PWD/PhJ (PWD) mice. EAE protection was accompanied by sex- and genotype-specific suppression of proinflammatory transcriptional programs in CD4 T effector cells, but not CD4 regulatory T cells. Decreased expression of proinflammatory genes was observed with high VitD in female CD4 T effector cells, specifically implicating a key role of MHC class II genes, interferon gamma, and Th1 cell-mediated neuroinflammation. In consomic strains, effects of VitD on EAE were also sex- and genotype dependent, whereby high VitD: (1) was protective, (2) had no effect, and (3) unexpectedly had disease-exacerbating effects. Systemic levels of 25(OH)D differed across consomic strains, with higher levels associated with EAE protection only in females. Analysis of expression of key known VitD metabolism genes between B6 and PWD mice revealed that their expression is genetically determined and sex specific and implicated Cyp27b1 and Vdr as candidate genes responsible for differential EAE responses to VitD modulation. Taken together, our results support the observation that the association between VitD status and MS susceptibility is genotype dependent and suggest that the outcome of VitD status in MS is determined by gene-by-sex interactions.</p

Sex-dependent DEGs mapping to the sex chromosomes.

Genes differentially expressed between males and females in: B6 mice only (B6; none in this category), ChrX.3 mice only (ChrX.3), or in both strains (both) which mapped to ChrX or ChrY, from the RNAseq data described in Fig 6. Direction of change: up indicates increased expression in males relative to females, down indicates increased expression in females relative to males. A cutoff filter of Padj = <0.05 was used to identify DEGs, without a fold change cutoff.</p

Genotype-dependent sex differences in IAV susceptibility of B6.Chr^PWD consomic mice.

Male (M) and female (F) B6 control and B6.Chr5PWD (A-D) and B6.ChrX.3PWD (E-H) mice were infected with sex-adjusted doses of PR8 as in Fig 2B. The following comparisons were made: (A and E) male consomic vs. male B6 control, (B and F) female consomic vs. female B6 control, and (C, D, G and H) male vs. female of each consomic. Significance of difference in survival for each comparison assessed by Mantel-Cox test, while significance of difference in weight loss was assessed by two-way ANOVA (overall effect of sex). The P value for each comparison is indicated in each corresponding graph, in red font if below PTables 1 and 2.</p

Sex differences in genetically regulated host lung responses in B6 and ChrX.3^PWD mice.

Male and female B6 and B6.ChrX.3PWD (ChrX.3) mice were infected with PR8 IAV as in Fig 3. On day 6 post-infection, whole lung RNA was processed for RNA sequencing and analysis as described in Materials and Methods. (A) Principal component analysis was performed in DEseq2, demonstrating clustering of individual samples by sex and genotype, as annotated. (B, C, and D) DEseq2 was used to determine differentially expressed genes between X.3 and B6 mice within males or females, as annotated, using a cutoff of |Log2 (Fold Change)|>0.6 and Padjusted (Padj) B) A row-normalized gene expression heatmap showing DEGs in males, females, or both sexes, as indicated. Volcano plots (C) and Venn diagrams (D) illustrating differential gene expression between ChrX.3 and B6. (E) Biological pathway enrichment analysis on DEGs upregulated in ChrX.3 in males was performed using Gene Ontology as described in Materials and Methods. Top 20 enriched pathways are shown. (F) ImmGen MyGeneSet analysis was performed on DEGs upregulated in ChrX.3 in males as described in the Materials and Methods. Heatmap indicates relative expression of DEGs in cell populations of interest, with each column representing a cell type or cells state within the broader cell categories labeled across the top. (G) A Venn diagram indicating overlap between genes differentially expressed between males vs. females in ChrX.3 and B6 mice.</p

Survival analysis of IAV-challenged consomic strains using pooled sex data.

The indicated number (N) of each strain were challenged with IAV as described in Fig 3. Male and female data were pooled. Significance of differences in survival between B6 and each consomic strain were assessed using the Mantel-Cox test and the P value are reported, with PPWD represent pooled data from Chr17S and Chr17F strains. The low N for some strains (Chr13, Chr7, etc.) was a result of low availability of animals due to poor breeding performance.</p

Sex differences and normalization of IAV severity in male and female B6 mice.

(A) B6 male and female mice were challenged with equal doses of PR8 IAV, and survival was assessed. (B and C) B6 male and female mice were challenged with sex-adjusted estimated LD50 of PR8 IAV (33% lower dose for females). Significance of differences in survival between females and males was determined using the Mantel-Cox test, significance of differences in weight loss was determined by two-way ANOVA (overall effect of sex). P values are indicated below each panel title. The number of animals per condition is indicated in each panel.</p

DEGs from RNAseq analysis.

(A) DEGs between ChrX.3 females and B6 females. (B) DEGs between ChrX.3 males and B6 males. (C) DEGs between B6 males and B6 females. (D) DEGs between ChrX.3 males and ChrX.3 females. This is an Excel spreadsheet containing the full set of all genes in the analysis, in the with active DEG filters enabled, as indicated in each tab. (XLSX)</p

Survival analysis of IAV-challenged consomic strains using sex-stratified data.

Male and female consomic mice were challenged with IAV as described in Fig 3. Significance of differences in survival assessed using the Mantel-Cox test and the P value are reported, with P<0.05 shown in bold font. The following three comparisons were made: male vs. females of each consomic strain (Male vs. Female, column 1), consomic males vs. B6 males (Consomic M vs. B6 M, column 2), and consomic females vs. B6 females (Consomic F vs. B6 F, column 3).</p

PWD alleles on Chr16 provide complete protection from lethal IAV challenge.

(A–C) Male B6 and PWD mice were challenged with 2 LD50 or 5 LD50 doses of PR8 IAV (LD50 dose was pre-determined on B6 mice). Survival (A) and weight loss (B) are shown, with P values in each panel indicating overall significance of differences between the groups, as determined by Mantel-Cox test and 2-way ANOVA, respectively. (C) On day 22 post-infection, serum was collected from PWD mice and uninfected naïve PWD mice, and anti-IAV antibody titers were determined by ELISA (as described in the Materials and Methods). P values indicate the significance of difference between the indicated groups, as determined by student’s T test. (D, E) Male and female B6 and B6.Chr16PWD (Chr16) mice were challenged with ~LD50 of PR8 IAV. Survival (D) and weight loss (E) are shown, representing pooled data from males and females with P values in each panel indicating significance of differences between the strains, as determined by Mantel-Cox test and 2-way ANOVA, respectively. The number of animals per condition is indicated in each panel.</p