Gender bias in autoimmunity is influenced by microbiota

Abstract

Gender bias and the role of sex hormones in autoimmune diseases are well established. In specific pathogen-free nonobese diabetic (NOD) mice, females have 1.3-4.4 times higher incidence of type 1 diabetes (T1D). Germ-free (GF) mice lost the gender bias (female-to-male ratio 1.1-1.2). Gut microbiota differed in males and females, a trend reversed by male castration, confirming that androgens influence gut microbiota. Colonization of GF NOD mice with defined microbiota revealed that some, but not all, lineages overrepresented in male mice supported a gender bias in T1D. Although protection of males did not correlate with blood androgen concentration, hormone-supported expansion of selected microbial lineages may work as a positive-feedback mechanism contributing to the sexual dimorphism of autoimmune diseases. Gene-expression analysis suggested pathways involved in protection of males from T1D by microbiota. Our results favor a two-signal model of gender bias, in which hormones and microbes together trigger protective pathways.

Copyright © 2013 Elsevier Inc. All rights reserved.

Figures

Figure 1. Gender-related differences in commensal microbiota composition

A. Alpha diversity of microbiota from 4 week-old and 10 week-old mice, grouped by gender, mean±SEM. Significance of changes in diversity between genders is reported using a non-parametric Mann-Whitney test. B. Principal component analysis of microbiota diversity at the family level from 4 week-old (8 males, 8 females), and 10 week-old mice (7 males, 7 females). C. Hierarchical clustering of 16S sequences of cecal samples from 4-week and 10-week old male and female mice were performed using the Euclidean distance metric and clustering by average linkage. The dendrogram shows the linkage points at increasing degree of dissimilarity. D. Principal component analysis of microbiota diversity at the family level from 13 wk old littermate mice, 3 females, 3 males, and 3 castrated males. E. Principal component analysis of microbiota diversity at the family level from 13 week old gnotobiotic littermate mice colonized with an SPF microbiota at birth. F. Relative abundances of bacterial families between male and female mice. Mean and standard error is marked for each family, significance (p value) is calculated using one tailed t-test. q values obtained by Benjamini-Hochberg approach to adjust for false discovery rate were above 0.05 in most cases (not shown) except experiment 4. See also Figure S1.

Figure 2. Influence of microbial lineages on the gender bias in T1D development in NOD mice

A. Histopathology (% of islets with infiltrates beyond peri-insulitis) in 13 wk old mice from male and female SPF NOD mice, from males castrated at 4 wks of age and from GF male and female mice. Data are represented as mean insulitis score±SEM. 30% infiltration was chosen as an arbitrary threshold B. Histopathology in 13 wk old gnotobiotic NOD mice reconstituted with indicated microbiota. Data are represented as mean insulitis score±SEM. Germ-free mice were reconstituted by natural acquisition of microbes from parents infected by gastric gavage during breeding. C. Diabetes incidence in NOD GF female mice and in gnotobiotic NOD female mice monocolonized with SFB. D. Diabetes incidence in NOD GF male mice and in gnotobiotic NOD male mice monocolonized with SFB. The differences between SFB-colonized and GF males were significant (p=0.005), as well as between SFB-colonized females and males (p=0.002). E. Diabetes incidence in MyD88-negative NOD gnotobiotic mice monocolonized with SFB. F. N=the number of mice per group. p values for incidence were determined using Kaplan-Meier statistics, for histopathology by Student’s t-test. See also Figure S2.

Figure 3. Microbiota and blood testosterone concentrations

A. The blood testosterone concentrations of the 13 wk old GF, SPF and gnotobiotic males and females of indicated ages. Mean testosterone concentration±SEM. Blood samples were collected between 10 am and 12 pm. C- castrated and MC – mock-castrated males. B. Islet histopathology in mice that demonstrated no gender bias (GF and VSL3 populated – open squares) and that demonstrated gender bias (SPF and SECS populated – black squares) plotted against blood testosterone concentrations. Data compiled from four experiments.

Figure 4. Analysis of the changes in gene expression driven by microbes and gender

A. SPF and GF NOD males and females were used as donors of the PLN. Of the four groups only one (SPF males) is protected from T1D. The logic of arrival at the gene set IV, specific to this group, is shown. B. Heat-map of expression of the genes from set IV. The intensity of the color corresponds to the strength of expression relative to the mean expression across all conditions. C. Gene set IV organized in a network using the STRING database. Genes encoding IFN-γ and IL-1β are highlighted in yellow.

Figure 5

A. Many genes belonging to set IV (Figure 4 A) have a signature characteristic of macrophages (highlighted in yellow) as determined by comparison to BioGPS murine RNA Gene Expression Atlas data. The intensity of the color corresponds to the strength of expression relative to the mean expression across all cell types. The complete expression map can be found in Figure S3. B. Enhanced presence of alternatively activated macrophages in age-matched adult male SFP mice compared to SPF female mice. CD11b+F4/80+ macrophages were stained with antibodies to transferrin receptor (TFRC, left panel) or to CD206. An example of CD206 staining is shown along with quantitation of both CD206+ cell number and the mean fluorescence intensity (MFI). 3 to 5 mice per group were used in these experiments. Data representative of 2–3 independent experiments. Data are represented as mean cell number or MFI±SEM.

Figure 6

A. Manipulation of sexual dimorphism of T1D in NOD mice by gene knock-outs (Chatenoud et al., 2001; Serreze et al., 2001; Serreze et al., 2000) as reported by T1DR or by an original paper for Casp1−/− mice(Schott et al., 2004). B. IFN-γ expression in the PLNs of males and females in GF, SPF and SFB-monocolonized NOD mice. Mean cytokine concentration±SEM. Tissues were collected from 13 week-old mice, homogenized and used for IFN-γ-specific ELISA. c - SPF males castrated at 4 weeks of age. C. IFN-γ-specific RNA expression in 12–13 week old SPF males and females was compared by RT- quantitative PCR. AU-arbitrary units. Mean±SEM. D. The percentage of IFN-γ-positive cells among CD4+ and CD8+ T cells from the PLNs of male, female and castrated male (c) NOD mice. Mean±SEM. E. Peritoneal macrophages from female, male and IFN-γ-negative male mice were pretreated or not with heat-inactivated SECS bacteria overnight before addition of G9C8 T cells and their cognate peptide. IFN-γ was measured by ELISA in the supernatants after overnight culture. Data from a representative experiment of three independent experiments. Mean±SEM. N= mice per group. p values were determined by Student’s t-test. See also Figure S4.