Sex differences associate with late microbiome alterations after murine surgical sepsis

Abstract

Background: Sepsis-induced gut microbiome alterations contribute to sepsis-related morbidity and mortality. Given evidence for improved postsepsis outcomes in females compared with males, we hypothesized that female mice maintain microbiota resilience versus males.

Methods: Mixed-sex C57BL/6 mice underwent cecal ligation and puncture (CLP) with antibiotics, saline resuscitation, and daily chronic stress and were compared with naive (nonsepsis/no antibiotics) controls. For this work, the results of young (3-5 months) and old (18-22 months) adult mice were analyzed by sex, independent and dependent of age. Mice were sacrificed at days 7 and 14, and 16S rRNA gene sequencing was performed on fecal bacterial DNA. α and β diversity were determined by Shannon index and Bray-Curtis with principal coordinate analysis, respectively. False discovery rate (FDR) correction was implemented to account for potential housing effect.

Results: In control mice, there was no difference in α or β diversity between male and female mice (FDR, 0.76 and 0.99, respectively). However, male mice that underwent CLP with daily chronic stress had a decrease in microbiota α diversity at 7 days post-CLP (Shannon FDR, 0.005), which was sustained at 14 days post-CLP (Shannon FDR, 0.001), compared with baseline. In addition, male mice maintained differences in β diversity even at day 14 compared with controls (FDR, <0.0001). In contrast, female mice had a decreased microbiota α diversity (Shannon FDR, 0.03) and β diversity (FDR, 0.02) 7 days post-CLP but recovered their α and β diversity by post-CLP day 14 (Shannon FDR, 0.5, and FDR, 0.02, respectively). Further analysis of females revealed that only young female mice were not different (β diversity) post-CLP day 14 to controls.

Conclusion: Although sepsis-induced perturbations of the intestinal microbiota occur initially in both male and female C57BL/6 mice, females demonstrate different microbiota by day 14. This may be seen primarily in younger females. This difference in recovery may play a role in outcome differences between sexes after sepsis.

Graphical abstract

Figure 1

16S rRNA gene sequencing of the intestinal microbiota at baseline (“naive”) before CLP-DCS demonstrates no significant difference in α diversity by Chao1 (A) or β diversity by Bray-Curtis dissimilarity index (B). However, 7 and 14 days post–CLP-DCS demonstrated a significant difference in intestinal microbiota α diversity in male mice compared with baseline (C). While female mice also demonstrated a CLP-DCS–induced microbiota shift in diversity at 7 days, this change in diversity appears restored after 14 days (D).

Figure 2

Principal coordinate analysis of Bray-Curtis dissimilarity index is presented, which demonstrates no difference in β diversity in male B6 mice post–CLP-DCS at 7 days (A) but at 14 days is significantly different (B). In contrast, female B6 mice had a shift in the β diversity at 7 days (C), which appeared restored by 14 days (D). NS, not significant.

Figure 3

α Diversity by Chao1 of young and old mice that underwent CLP-DCS was analyzed for each sex and demonstrates that (A) young male mice do not have alterations in the α diversity of their intestinal microbiota after CLP-DCS but their old male counterparts do (B; FDR, 0.002). Similarly, young female mice that undergo CLP-DCS have stable α diversity after CLP-DCS (C), but old female mice were noted to have decreased α diversity over time after CLP-DCS (D; FDR, <0.001).

Figure 4

Principal coordinate analysis of Bray-Curtis dissimilarity index is presented for young and old male mice 14 days after CLP-DCS (A and B, respectively) as well as young and old female mice at 7 days post–CLP-DCS (C and D, respectively). This demonstrated a difference in the β diversity of the old male mice at 14 days post–CLP-DCS (FDR, <0.0001) and young female mice at 7 days post–CLP-DCS (FDR, 0.04).

Figure 5

Plasma measurement of estradiol (A) and testosterone (B) in male and female B6 mice at baseline, 7 days, and 14 days post–CLP-DCS demonstrated no significant difference in plasma levels within sexes at the indicated time points. NS, not significant.