Female mice are protected from MRSA gastrointestinal colonization in a microbiota dependent manner.

(A) MRSA colony forming units (CFU) per gram of stool following oral inoculation of B6 mice purchased from Jackson Laboratory (JAX). Males n=10, females n=13. (B) MRSA CFU in stool following oral inoculation of B6 mice generated from breeders in the NYU animal facility. Males n=13, females n=13. (C) Proportion of JAX and NYU female mice with detectable MRSA GI colonization over time. (D) MRSA CFU stool burden following oral inoculation of germ-free mice. Males n=8, females n=7. (E) MRSA CFU in stool following oral inoculation of mice with a defined minimal microbiota. Males n=12, females n=11. (F) MRSA CFU stool burden of female NYU or JAX mice housed separately or (G) co-housed with each other. NYU n=9, JAX n=11, cohoused NYU n=14, cohoused JAX n=14. Data points represent mean ± SEM from at least two independent experiments. Statistical analysis: area under the curve followed by a two-tailed t-test for (A), (B), (D)-(G) and Log-rank Mantel Cox test for (C). ns: not significant.

Microbiome is not sufficient to explain sex bias in MRSA GI colonization.

(A) Principal Coordinates Analysis (PCoA) based on Bray-Curtis distances of 16S sequences obtained from stool of JAX and NYU male and female mice prior to MRSA inoculation. Proportion of variance explained by each axes shown in parentheses. JAX M n=5, JAX F n=5, NYU M n=11, NYU F n=13. (B) Alpha diversity of the microbiomes of male and female JAX and NYU mice. Wilcoxon rank sum test used for pairwise statistical comparisons to NYU females; ns: not significant. (C) Relative abundance of bacterial families in female NYU and JAX mice prior to MRSA inoculation. (D) Relative abundance of bacterial families in male and female NYU mice prior to MRSA inoculation. (E) MRSA colony forming units (CFU) in stool following oral inoculation of JAX female (JAX F) and male (JAX M) recipients of fecal microbiome transplants (FMT) from female (NYU F) and male (NYU M) donor mice compared with JAX male controls (control JAX M) that did not receive an FMT. Mean MRSA burden ± SEM. Area under the curve analysis + 1 way ANOVA Sidak’s multiple comparisons test for (E). Control M n=6, M +NYU F stool n=7, M +NYU M stool n=6, F +NYU F stool n=6. ns: not significant.

MRSA GI colonization elicits sex-dependent gene expression changes in the gut.

(A) Volcano plot of differentially expressed genes identified by RNA-seq analyses of the intestinal lamina propria of NYU mice 2 dpi with MRSA compared with PBS mock inoculated NYU mice. (B) Ingenuity Pathway Analysis (IPA) of downregulated and upregulated genes in the intestinal lamina propria upon MRSA inoculation of male and female mice. (C) Volcano plot of differentially expressed genes in the intestinal lamina propria comparing mock treated males and females. (D) Volcano plot of differentially expressed genes in the intestinal lamina propria comparing males and females 2 dpi with MRSA. (E) Volcano plot of differentially expressed genes in the intestinal epithelial fraction of males and females prior to MRSA inoculation. (F) Volcano plot of differentially expressed genes in the intestinal epithelial fraction of males and females 2 dpi with MRSA. Four mice were used for each sequencing experimental condition. Genes shown in red have a false discovery rate (FDR) of <0.05 and an absolute log2 fold change (abslog2FC) of >1.2. Genes shown in blue have a FDR of <0.05. Genes linked to X and Y chromosomes were removed from volcano plots.

CD4+ T cells mediate colonization resistance in female mice.

(A) MRSA CFU in stool following oral inoculation of Rag2−/− and Rag2+/− mice bred at NYU. Male Rag2−/− n=12, male Rag2+/− n=12, female Rag2−/− n=12, female Rag2+/− n=11. (B) MRSA CFU in stool following oral inoculation of Igh-μ−/− and Igh-μ+/− mice bred at NYU. Male Igh-μ−/− n=6, male Igh-μ+/− n=6, female Igh-μ−/− n=8, female Igh-μ+/− n=5. (C) MRSA CFU in stool following oral inoculation of female NYU B6 mice injected I.P. with 250 μg of anti-CD4 depleting antibody or anti-IgG control. Anti-IgG n=8, anti-CD4 n=10. Data points represent mean ± SEM from at least two independent experiments. Statistical analysis: area under the curve analyzed by a 1 way ANOVA with Sidak’s multiple comparison test for (A) and (B) and a two-tailed t test for (C). ns: not significant.

Female mice have increased IL-17A+ CD4+ T cells and require neutrophils for MRSA clearance.

(A) Flow cytometry of cecal-colonic lamina propria CD4+ T cells as a percentage of CD45+ cells in male and female NYU mice treated with PBS or MRSA 2 dpi. (B) Flow cytometry of cecal-colonic lamina propria IL17A+ CD4+ T cells as a percentageof total CD4+ T cells in male and female NYU mice treated with PBS or MRSA 2 dpi. (C) Flow cytometry of cecal-colonic lamina propria γδ T cells as a percentage of CD45+ cells in male and female NYU mice treated with PBS or MRSA 2 dpi. (D) Flow cytometry of cecal-colonic lamina propria IL17A+ γδ T cells as a percentage of total CD4+ T cells in male and female NYU mice treated with PBS or MRSA 2 dpi. (E) MRSA CFU in stool following oral inoculation of Roryt−/− and Roryt+/−mice bred at NYU. Male Roryt+/− n=5, male Roryt−/−n=7, female Roryt+/− n=9, female Roryt−/− n=9. (F) MRSA CFU in stool following oral inoculation of female IL-17ra+/- and IL-17ra-/-mice bred at NYU. IL-17ra+/- n=6, IL-17ra-/-n=6. (G) MRSA CFU in stool following oral inoculation of Tcrd+/−and Tcrd−/− mice bred at NYU. Male Tcrd+/− n=6, male Tcrd−/− n=6, female Tcrd+/− n=8, and female Tcrd−/− n=12. (H) Flow cytometry of cecal-colonic lamina propria Ly6G+CD11b+ neutrophils as a percentage of CD45+ cells in male and female NYU mice treated with PBS or MRSA 2 dpi. (I) Quantification of mean fluorescence intensity (MFI) of surface CD11b on neutrophils by flow cytometry normalized to mock treated controls. (J) MRSA CFU in stool following oral inoculation of NYU B6 mice treated with anti-Ly6G neutrophil depleting antibody or anti-IgG control. Male anti-IgG n=6, male anti-Ly6G n=8, female anti-IgG n=12, and female anti-CD4+ n=15. Data points represent mean ± SEM from at least two independent experiments. Statistical analysis: 2 way ANOVA + Sidak’s multiple comparisons test for (A)-(D) and (H), area under the curve followed by a 1 way ANOVA + Sidak’s multiple comparisons test for (E), (G), (J) or a two-tailed t test for (F) and a two-tailed t test for (I). ns: not significant.

Sex hormones, not sex chromosomes, mediate MRSA colonization resistance in female mice.

(A) MRSA CFU in stool following oral inoculation of male or female mice that were irradiated and reconstituted with bone marrow (BM) from donor male or female mice. Female BM into female recipients (F --> F) n=8, male BM into male recipients (M--> M) n=7, female BM into male recipients (F--> M) n=8. (B) MRSA CFU in stool following oral inoculation of ovariectomized female mice or sham operated littermate controls. OVX n=10, sham n=10. (C) MRSA CFU in stool following oral inoculation of Esr1+/- and Esr1-/- female mice bred at NYU. Esr1+/- n=6, Esr1-/- n=6. (D) MRSA CFU in stool following oral inoculation of four core genotype mice. XX n=5, XY(-Sry) n=5. XY n=5, XX(+Sry) n=4. (E) CD4+ T cells from cecal-colonic lamina propria of XX females and XX(+Sry) males 2 dpi inoculation with MRSA or mock control. (F) Percentage of IL-17A+ CD4+ T cells in cecal-colonic lamina propria of XX females and XX(+Sry) males 2dpi inoculation with MRSA or mock control. Data points represent mean ± SEM from at least two independent experiments. Statistical analysis: area under the curve followed by a 1 way ANOVA with Sidak’s multiple comparisons for (A),(C) and two-tailed t-test for (B),(F) and 2 way ANOVA + Sidak’s multiple comparisons test for (D)-(E). ns: not significant.