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Science 339 (6123): 1084-1088

Copyright © 2013 by the American Association for the Advancement of Science

Sex Differences in the Gut Microbiome Drive Hormone-Dependent Regulation of Autoimmunity

Janet G. M. Markle1,2, Daniel N. Frank3, Steven Mortin-Toth1, Charles E. Robertson4, Leah M. Feazel3, Ulrike Rolle-Kampczyk5, Martin von Bergen5,6,7, Kathy D. McCoy8, Andrew J. Macpherson8, and Jayne S. Danska1,2,9,*

1 Program in Genetics and Genome Biology, Hospital for Sick Children Research Institute, Toronto, Ontario M5G 1X8, Canada.
2 Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
3 Division of Infectious Diseases, University of Colorado School of Medicine, Aurora, CO 80045, USA.
4 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
5 Department of Metabolomics, Helmholtz Center for Environmental Research, 04318 Leipzig, Germany.
6 Department of Proteomics, Helmholtz Center for Environmental Research, 04318 Leipzig, Germany.
7 Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, 9000 Aalborg, Denmark.
8 Maurice Müller Laboratories, Universitätsklinik für Viszerale Chirurgie und Medizin (UVCM), University of Bern, 3008 Bern, Switzerland.
9 Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada.

Figure 1
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Fig. 1. Sex-biased autoimmunity in the NOD mouse depends on the microbiome. (A and B) The natural history of T1D in NOD/Jsd mice in SPF or GF conditions. (A) SPF females (n = 59) and SPF males (n = 69). ***P < 0.0001. (B) GF females (n = 73) and GF males (n = 54). P = 0.2115, log-rank (Mantel-Cox) comparisons of survival curves. (C) Serum testosterone concentrations were higher in GF versus SPF females (P = 0.0248) and lower in GF versus SPF males (asterisk represents significant differences between groups, P = 0.0049, Mann-Whitney test, n ≥ 10 per group). Testosterone in GF male sera was higher than in GF female sera (P = 0.0378). Box plots display the median, 25th percentile, and 75th percentile; whiskers display minimum and maximum values. (D) PCA plot, generated by analysis of more than 180 serum metabolites in SPF females, SPF males, GF females, and GF males (n ≥ 6 per group). Principal components PC1 and PC2, which explain 55.8% of the total variance observed, discriminate SPF female samples from male samples and discriminate both sexes in SPF housing from GF-housed females and males.


Figure 2
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Fig. 2. Sex-specific microbiome profiles emerge after puberty, and cecal microbiome transplantation can stably alter the microbiome of the host without inducing systemic immune priming. (A) 16S bacterial rRNA sequencing was used to define the microbiome profiles of NOD male and female SPF mice at various developmental time points. PCA was used to compare the top 30 differentially abundant bacterial families across six different groups (n = 5 per group): 3-week-old males (turquoise), 3-week-old females (red), 6-week-old males (pink), 6-week-old females (green), 14-week-old males (blue), and 14-week-old females (black). These groups were separated by principal components PC1 to PC3, collectively explaining 78.5% of the total variance. (B) Systemic immune priming against the commensal microbiome was evaluated in M->F, F->F, and unmanipulated female NOD mice (right histograms). No microbiome-dependent differences in commensal immunoglobulin G1 (IgG1) or IgG2b titers were detected among these groups (n > 10 per group; see fig. S2). Control mice were inoculated systemically with a bacterial isolate (black traces). (C to E) 16S sequencing comparisons of control males versus females at 14 weeks of age (C), female recipients of male microbiome (M->F) versus unmanipulated females (D), and female recipients of male microbiome (M->F) versus female recipients of female microbiome (F->F) (E) (n = 5 per control group, n = 10 per gavaged group). Bacterial genera shown represent those found to be significantly different in at least one of these pairwise comparisons [*P < 0.05, two-part statistic (35); see table S6].


Figure 3
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Fig. 3. Transplantation of the male microbiome results in hormonal and metabolic changes in the female recipient. (A) Serum testosterone was measured in unmanipulated NOD females and gavage recipients at indicated ages, from left to right: 14-week-old unmanipulated females, 7-week-old recipients of male bacteria, 7-week-old recipients of female bacteria, 14-week-old recipients of male bacteria, 14-week-old recipients of female bacteria, and 34-week-old recipients of male bacteria. Box plots display the median, 25th percentile, and 75th percentile; whiskers display minimum and maximum values. Testosterone levels were greater in male microbiome recipients at both 7 weeks and 14 weeks relative to unmanipulated females and to age-matched F->F recipients (groups marked A differ significantly from B, *P < 0.05, Mann-Whitney test, n ≥ 10 per group). (B to G) A panel of 183 metabolites was quantified in sera of unmanipulated females, M->F recipients, and F->F recipients. Volcano plots depict metabolites included in PC1 and PC2 of Fig. 1D. Metabolites that were also significantly different in the present experiment (exceeding –log P threshold of 2.08) are labeled. Comparisons of metabolite levels in F->F versus F [(B) and (E)] and M->F versus F [(C) and (F)] are shown (n ≥ 5 per group). The metabolomic assay was also applied to M->F recipients that had also been implanted with continuous-release pellets containing the AR antagonist flutamide [(D) and (G)].


Figure 4
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Fig. 4. Gavage of female NOD pups with male NOD-derived intestinal microbiome results in T1D protection, decreased insulitis severity, and decreased insulin Aab titer by an androgen-dependent mechanism. Female NOD weanlings were gavaged with cecal bacteria from either adult NOD males or adult NOD females. (A) T1D was assessed in unmanipulated NOD females (black, n = 59), F->F female recipients (red, n = 30), and M->F recipients (blue, n = 38). T1D survival curves did not differ between F and F->F groups. M->F recipients were protected from T1D relative to unmanipulated females (P < 0.0001, log-rank test). (B) Insulitis severity was assessed by established protocols (18) in (from left to right) unmanipulated females, recipients of the female microbiome (F->F), or recipients of the male microbiome (M->F), as well as a M->F group that simultaneously received a subcutaneous pellet secreting the AR antagonist flutamide. As a control for possible effects of male cells contaminating the gavage inoculum, females gavaged at weaning with male cells only were also included (middle histogram bar). Different letters indicate significantly different insulitis score distributions by {chi}2 test (Bonferroni-corrected α = 0.005, n ≥ 5 biological replicates per group). (C) Insulin Aab titer was assessed as another preclinical T1D-related phenotype. Different letters indicate significantly different group means by Mann-Whitney test (Bonferroni-corrected α = 0.005, n ≥ 5 per group). Error bars indicate SEM. (D) As a test for possible effects of microbiome manipulation and changes in host hormonal and metabolic status on T cell diabetogenicity, 107 purified splenic T cells were prepared from unmanipulated NOD females (black), M->F gavage recipients (blue), or M->F gavage recipients treated with flutamide to antagonize AR signaling (orange), then transferred to 4- to 5-week-old female NOD.SCID recipients by intravenous injection. Recipients were monitored for hyperglycemia (n > 7 per group). The latency of T1D in NOD.SCID recipients of T cells isolated from M->F gavage recipients was greater than in recipients of T cells isolated from either unmanipulated females or from M->F gavage recipients that had been treated with flutamide (P < 0.002, log-rank comparisons of survival curves).


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