Figures and data
Female mice are protected from MRSA gastrointestinal colonization in a microbiome 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 + Welch’s t test. 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 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 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 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+/− males bred at NYU. Rag2−/− n=12, Rag2+/− n=12. (B) MRSA CFU in stool following oral inoculation of Rag2−/− and Rag2+/− females bred at NYU. Rag2−/− n=12, Rag2+/− n=11. (C) MRSA CFU in stool following oral inoculation of Igh-μ−/− and Igh-μ+/−males bred at NYU. Igh-μ−/−n=6, Igh-μ+/− n=6. (D) MRSA CFU in stool following oral inoculation of Igh-μ−/− and Igh-μ+/− females bred at NYU. Igh-μ−/− n=8, Igh-μ+/− n=5. (E) 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 followed by a two-tailed t-test. ns: not significant.
Female mice have increased IL17+ 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 percentage of 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+/−males bred at NYU. Roryt+/−n=5, Roryt−/− n=7 (F) MRSA CFU in stool following oral inoculation of Roryt−/−and Roryt+/− females bred at NYU. Roryt+/− n=9, Roryt−/− n=9. (G) MRSA CFU in stool following oral inoculation of Tcrd+/− and Tcrd−/− males bred at NYU. Tcrd+/− n=6 and Tcrd−/− n=6. (H) MRSA CFU in stool following oral inoculation of Tcrd+/− and Tcrd−/− females bred at NYU. Tcrd+/− n=8 and Tcrd−/− n=12. (I) MRSA CFU in stool following oral inoculation of male NYU B6 mice treated with anti-Ly6G neutrophil depleting antibody or anti-IgG control. Male anti-IgG n=6, anti-Ly6G n=8. (J) MRSA CFU in stool following oral inoculation of female NYU B6 mice injected I.P. with 250 μg of of anti-Ly6G depleting antibody or anti-IgG control. Female anti-IgG n=12, anti-CD4+ n=15. Data points represent mean ± SEM from at least two independent experiments. Statistical analysis: 2WAY ANOVA + Sidak’s multiple comparisons test for (A)-(D) and area under the curve followed by a two-tailed t-test for (E)-(J). ns: not significant.
Female sex hormones, not sex chromosomes, mediate MRSA colonization resistance.
(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 four core genotype female mice (XX vs XY-Sry). XX n=5, XY(-Sry) n=5. (D) MRSA CFU in stool following oral inoculation of four core genotype male mice (XY vs XX+Sry). XY n=5, XX(+Sry) n=4. (E) CD4+ T cells from cecal-colonic lamina propria of XX females and XX(+Sry) males. (F) Percentage of IL-17A+ CD4+ T cells in cecal-colonic lamina propria of XX females and XX(+Sry) males. 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)-(C) and 2WAY ANOVA + Sidak’s multiple comparisons test for (D)-(E). ns: not significant.
