Obesity intensifies sex-specific interferon signaling to selectively worsen central nervous system autoimmunity in females

Abstract

Obesity has been implicated in the rise of autoimmunity in women. We report that obesity induces a serum protein signature that is associated with T helper 1 (Th1), interleukin (IL)-17, and multiple sclerosis (MS) signaling pathways selectively in human females. Females, but not male mice, subjected to diet-induced overweightness/obesity (DIO) exhibited upregulated Th1/IL-17 inflammation in the central nervous system during experimental autoimmune encephalomyelitis, a model of MS. This was associated with worsened disability and a heightened expansion of myelin-specific Th1 cells in the peripheral lymphoid organs. Moreover, at steady state, DIO increased serum levels of interferon (IFN)-α and potentiated STAT1 expression and IFN-γ production by naive CD4⁺ T cells uniquely in female mice. This T cell phenotype was driven by increased adiposity and was prevented by the removal of ovaries or knockdown of the type I IFN receptor in T cells. Our findings offer a mechanistic explanation of how obesity enhances autoimmunity.

Keywords: T cells; T helper 1 differentiation; autoimmunity; experimental autoimmune encephalomyelitis; interferon-α; multiple sclerosis; obesity; sex differences.

Figure 1:. Obesity uniquely elevates inflammatory pathways in female humans.

(A-D) Comparison of serum protein profiles of obese and non-obese male and female individuals with or without RRMS. Red and dark blue respectively denote proteins elevated or reduced by obesity at FDR of 0.1. Orange and light blue respectively denote proteins elevated or reduced by obesity at p value of ≤ 0.05. Analyses were adjusted for age and ethnicity. (E) Venn diagram showing the number of proteins increased with obesity between each comparison (p value ≤ 0.05). (F) Ingenuity Pathway Analysis (IPA) of signaling pathways altered in obesity. Shown are the Z-scores (X axis), 𢈒log₁₀ p-values (size indicated by symbol size), and the number of proteins in each pathway (heat map legend) as determined using IPA. (G) Heatmap depicting differentially abundant serum proteins that mapped to “IRF Activation”, “IL-17 signaling”, “Th1”, and “S100” signal pathways. See also Figure S1 and Table S2.

Figure 2:. Diet-induced obesity increases EAE severity, especially in female mice

EAE was induced in control (CTRL) and DIO C57BL/6J mice with MOG_35-55/CFA and PTX. (A-B) Clinical scores of (A) female and (B) male mice. (C) Day of symptom onset. (D) Peak clinical score. (E) Cumulative disease score. (F) Percent demyelinated white matter in thoracic spinal cord. (G-J) Representative images of H&E- and LFB-stained thoracic spinal cord sections. Scale bar=50 μm. (K-N) Total numbers of CD4⁺ T cells (K), CD8⁺T cells (L), CD45^hiCD11b⁺CD11c⁻ cells (M), and CD45^hiCD11b⁺CD11c⁺ cells (N) in the CNS at 2-4 days post-symptom onset. (O) Representative intracellular cytokine staining in the live CD4⁺ gate after PMA/Ionomycin restimulation. (P-S) Frequency (top) and number (bottom) of CD4⁺ T cells staining for the indicated cytokines. Data are presented as mean ± S.E.M. Data points in C-F, K-N, P-S are individual mice from one experiment that is representative of three performed. *p ≤ 0.05 between groups as determined by two-tailed Mann-Whitney U test (A,B) or two-way ANOVA and Bonferroni post-hoc test (C-S).

Figure 3:. DIO increases Th1 responses predominantly in female mice during EAE.

(A-C) Representative staining of splenic IA^b-MOG-specific tetramer⁺CD44⁺ CD4⁺ T cells in male or female MOG_35-55/CFA-injected CTRL or DIO C57BL/6J mice. Frequencies (B) and total numbers (C) of IA^b-MOG-specific tetramer⁺ CD4⁺ T cells. Data points are individual mice. (D-F) Spleen and draining lymph node cells from mice were pooled and cultured in the presence of MOG_35-55. Supernatants were collected for ELISA measurement of IL-2 (D), IFN-γ (E) and IL-17A (F). Shown are mean ± S.E.M. of triplicate wells from one experiment of N=3 performed using pooled mice. (G-H) Spleen and draining lymph node cells were collected, pooled, and cultured with MOG_35-55 and IL-12p70 for 3 days and adoptively transferred into naive recipient male C57BL/6J mice. Mean ± S.E.M. daily clinical scores after transfer of cells from (G) female or (H) male mice. N=7-8 per group. *p ≤ 0.05 significance determined using a two-way ANOVA and Bonferroni post-hoc test (B-F) or two-tailed Mann-Whitney U test (G,H). In D-H, blue and red asterisks indicate differences in between CTRL and DIO males and between CTRL and DIO females, respectively.

Figure 4:. Increased Th1 responses in females with DIO are driven by T cell intrinsic differences

(A-M) Male and female C57BL/6J mice were treated to NCD (CTRL) or HFD (DIO) for 4 wk. (A) Number of splenic CD4⁺ T cells. (B-D) Frequency of splenic CD4⁺ T cells that were FoxP3⁺CD25⁺ (B), CD25⁺ (C) or CD44^hi (D). (E) Geometric mean fluorescent intensity (gMFI) of CD44 on CD4⁺ T cells. (F-G) CD4⁺CD25^hi (Treg) and CD4⁺CD44^low-intCD25⁻ (Tnaïve) cells from female (F) and male (G) mice were co-cultured at the indicated ratios with sex-matched Tnaive cells, irradiated splenocytes, and soluble anti-CD3. Proliferation of T cells was measured by [³H]-thymidine incorporation assay in counts per minute (CPM). (H-I) Splenic CD11c⁺ DCs from CTRL and DIO mice were co-cultured with sex-matched MOG_35-55-specific 2D2 CD4⁺ T cells with 0-40 μg/ml MOG_35-55. (H) Proliferation in CPM was measured by [³H]-thymidine incorporation assay. (I) IFN-γ in supernatants. (J-L) Naïve CD4⁺ T cells were sorted from NCD or DIO mice and were cultured with increasing amounts of anti-CD3/anti-CD28. (J) Proliferation was measured by [³H]-thymidine incorporation assay. IL-2 (K) and IFN-γ (L) in supernatants. (M) Relative mRNA expressions of Ifng, Tbx21, Stat1 and Stat4 in naïve CD4⁺ T cells stimulated with anti-CD3/anti-CD28 after normalization to beta-actin. (N-P) PBMCs were collected from human participants with healthy weight (20.0 to 24.9 kg/m²) or overweight (25.0-29.9 kg/m²) BMIs. Naïve CD45RA⁺ CD4⁺ T cells were stimulated in vitro with anti-CD3/anti-CD28-coated Dynabeads. IFN-γ (N) and IL-2 (O) were measured in supernatants by ELISA. Proliferation (CPM) (P) was measured by [H³]-thymidine incorporation assay. Data points are individual people. Data in mice are representative of 2-5 independent experiments. Significant differences were determined by two-way ANOVA with Bonferroni post-hoc test (A-E, H-P) or by Mann-Whitney two-tailed T test (F,G). *p ≤ 0.05.

Figure 5.. Ovarian hormones accentuate Th1 cytokine production by female T cells with DIO

Four-week-old female C57BL/6J mice underwent pre-pubertal ovariectomy (OVX) or sham surgery (sham). At 6 weeks of age, mice were placed on HFD or NCD for 4 weeks prior to data collection at 10 weeks of age. (A) % body weight change over 4 weeks of the diets. (B-C) Endpoint visceral adipose tissue (VAT) (B) and subcutaneous adipose tissue (SAT) (C) weight. (D-E) Naïve CD44^lo-int CD4⁺ cells were isolated from mice (N=5/group) and were cultured in presence of anti-CD3/anti-CD28. Cytokines in supernatants (D) and proliferation (CPM) (E) by [³H]-thymidine incorporation assay. Data points represent individual mice (A-C) or mean ± S.E.M of triplicate wells of pooled mice (D-E) in one experiment that was representative of 2 performed. Differences determined by two-way ANOVA and Bonferroni post-hoc test. *p ≤ 0.05.

Figure 6.. DIO increases type I IFN levels and T cell expression of ISGs and STAT1 in females

(A-C) Naïve (CD44^low-int) CD4⁺ T cells were isolated from female CTRL or DIO C57BL/6J mice (N=5 mice/group). Cells were snap frozen or stimulated with anti-CD3/anti-CD28 for 16 hr. cDNA was used to probe microarrays. (A) DAVID GO pathway analysis of top pathways upregulated with DIO (cut-off fold change > 1.5) in activated CD4⁺ T cells. (B) Genes upregulated in activated CD4⁺ T cells with DIO that overlapped with lists of genes enriched in iTreg, Th17, Th2, and Th1-lineage cells. (C) Venn diagram showing the overlap of DIO-upregulated genes in unstimulated and stimulated CD4⁺ T cells with those inducible in murine CD4⁺ T cells in response to type I or type II IFN. (D-G) Spleen and lymph node cells from C57BL/6J mice fed either a HFD or NCD were stained and gated on naïve (CD44^low-int) CD4⁺ T cells for total STAT1 (D), IL-18Rα (E), and IL-7Rα (F) or were stimulated in vitro with IFN-γ and gMFI and peak phosphorylated STAT1 (Y701) assessed in naïve (CD44^low-int) CD4⁺ T cells (G). (H) Correlation between STAT1 (Y701) gMFI in naïve T cells and % body weight change on diets. (I-K) Serum IFN-γ (I), pan-IFN-α (J), IFN-β (K) levels from female and male CTRL and DIO mice. (L) Serum IFN-α level in CTRL or DIO female mice, correlated with % weight change on the diets over 4 weeks. (M) Serum IFN-α levels in female sham or OVX mice after NCD or HFD. Data points in D-G, H-L are individual mice. Significance determined by one-way ANOVA with Tukey post-hoc test with FDR corrected (A,B), or 2-way ANOVA with Bonferroni post-hoc test (I, J, K, M), Mann-Whitney test (D-G) or Spearman’s test (H,L). *P ≤ 0.05.

Figure 7:. Loss of IFN-α signaling in female CD4⁺ T cells negates the effect of DIO on Th1 effector cell responses and EAE

(A) Schematic showing generation of T cell-specific IFNAR1-deficient mice. (B-D) % of body weight change over 4 weeks (B) and SAT (C) and VAT (D) weights at 10 weeks of age. (E-G) Frequencies of CD44⁺ (E), IL-18Rα⁺ (F), and expression of STAT1 (gMFI) (G) in splenic naive CD4⁺ T cells from CTRL and DIO flox and IFNAR-T-KO mice. (H) Levels of IFN-γ in culture supernatants at 48 hr after in vitro stimulation of naïve CD4⁺ T cells with anti-CD3/anti-CD28. Shown is mean ± S.E.M. of fold-change level (relative to flox CTRL) of 4 independent experiments. (I-L) Results of EAE studies in CTRL and DIO Flox and IFNAR-T-KO mice. (I) EAE clinical scores, (J) Peak score, (K) Cumulative disease score from day 0 to day 19, and (L) Day of disease onset. (M-O) Th cell responses in spleens of CTRL and DIO flox and IFNAR-T-KO mice at day 9 post-immunization (M) Representative staining of IFN-γ and CD44 in spleen CD4⁺ T cells. (N-O) Percentage of splenic CD4⁺ T cells expressing IFN-γ (N) or IL-17A (O) after PMA/Ionomycin stimulation. Values are mean ± S.E.M. Data are representative of at least 3 independent experiments. Significance was determined through two-way ANOVA and Bonferroni post-hoc test *p ≤ 0.05. Data in B-G, J-L, N-O are individual mice. See also Figure S6.