Effects of sex and chronic cigarette smoke exposure on the mouse cecal microbiome

Abstract

Rationale: Chronic smoke exposure is associated with weight loss in patients with Chronic Obstructive Pulmonary Disease (COPD). However, the biological contribution of chronic smoking and sex on the cecal microbiome has not been previously investigated.

Methods: Adult male, female and ovariectomized mice were exposed to air (control group) or smoke for six months using a standard nose-only smoke exposure system. DNA was extracted from the cecal content using the QIAGEN QIAamp® DNA Mini Kit. Droplet digital PCR was used to generate total 16S bacterial counts, followed by Illumina MiSeq® analysis to determine microbial community composition. The sequencing data were resolved into Amplicon Sequence Variants and analyzed with the use of QIIME2®. Alpha diversity measures (Richness, Shannon Index, Evenness and Faith's Phylogenetic Diversity) and beta diversity (based on Bray-Curtis distances) were assessed and compared according to smoke exposure and sex.

Results: The microbial community was different between male and female mice, while ovariectomy made the cecal microbiome similar to that of male mice. Chronic smoke exposure led to significant changes in the cecal microbial community in both male and female mice. The organism, Alistipes, was the most consistent bacteria identified at the genus level in the cecal content that was reduced with chronic cigarette exposure and its expression was positively related to the whole-body weight of these mice.

Conclusion: Chronic smoke exposure is associated with changes in the cecal content microbiome; these changes may play a role in the weight changes that are observed in cigarette smokers.

Fig 1. Chronic smoke exposure reduced the ability to gain body weight in mice.

A) Whole-body weight over time is shown in mice stratified by smoke exposure (control vs. smoke-exposed). Whole-body weights at week 24 are shown in mice stratified by smoke exposure (B) and by both smoking and sex (C). In panel A, a two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test was applied, whereas in panels B and C a two-tailed unpaired t-test and one-way ANOVA with Bonferroni’s multiple comparisons test was used, respectively. C = control; S = smoke-exposed.

Fig 2. Chronic cigarette smoke exposure differentially impacted cecal microbial communities in mice.

A) PCoA plot based on Bray-Curtis distance matrix between control (n = 30) and smoke-exposed (n = 28) samples is shown. P-value obtained using the PERMANOVA built-in function from QIIME2^®’s diversity plugin. B and C) Relative abundance of most frequent taxa in control and smoke-exposed samples at the phylum and genus levels, respectively. Significant taxa differences (adj. p-value <0.05 based on the Benjamini-Hochberg method) were observed only at the genus level (*): Alistipes and Uncultured Bacteroidales bacterium (Family Muribaculaceae) showed a higher relative abundance in control samples, whereas Prevotellaceae NK3B31group and Bacteroides predominated in smoke-exposed samples. D) Differential taxa features identified by LEfSe (LDA score > 3.5) according to smoke exposure; red and blue bars represent taxa features with higher expression in the control and smoke-exposed groups, respectively. PCoA = Principal Component.

Fig 3. Comparison of mice cecal microbial communities between control and smoke-exposed ovariectomized females.

PCoA plot based on Bray-Curtis distance matrix between control ovariectomized (COF, n = 10) and smoke-exposed ovariectomized (SOF, n = 8) female mice is shown. P-value was obtained using the PERMANOVA built-in function from QIIME2^®’s diversity plugin. PCoA = Principal Component.

Fig 4. Effects of smoke exposure and sex on different alpha diversity metrics in mice cecal content.

Comparisons of different alpha diversity metrics between male (n = 20), female (n = 20) and ovariectomized females (n = 18) mice (A-D) and after stratification by smoking exposure and sex (E-H) are shown. P-values were obtained using the Kruskal-Wallis test (*), and the Benjamini-Hochberg method was applied to compute adjusted p-values for multiple pairwise comparisons. ASV = amplicon sequence variant, Faith’s PD = Faith’s Phylogenetic Diversity, CM = control male, SM = smoke-exposed male, CF = control female, SF = smoke-exposed female, COF = ovariectomized control female, and SOF = ovariectomized smoke-exposed female.

Fig 5. Sex hormones significantly affected the cecal microbial composition.

(A) PCoA plot based on Bray-Curtis distance matrix between male (n = 20), female (n = 20) and ovariectomized females (Ov Female, n = 18) mice is shown. Pairwise comparisons: female vs. ovariectomized female: adj. p = 0.002; female vs. male: adj. p = 0.002; ovariectomized female vs. male: adj. p = 0.10. (B) PCoA plot using the same samples after stratification by smoke exposure and sex: smoke-exposed female (SF, n = 10), smoke-exposed male (SM, n = 10), ovariectomized smoke-exposed female (SOF, n = 8), control female (CF, n = 10), control male (CM, n = 10), and ovariectomized control female (COF, n = 10) is shown. Main pairwise comparisons: control female and smoked-exposed female: adj. p-value = 0.004; control male and smoked-exposed male: adj. p-value = 0.002; ovariectomized control female and ovariectomized smoke-exposed female, adj. p-value = 0.002; control female and ovariectomized control female; adj. p-value = 0.002; smoke-exposed female vs. ovariectomized smoke-exposed female: adj. p = 0.003; control male and ovariectomized control female, adj. p-value = 0.10, and smoked-exposed male and ovariectomized smoke-exposed female, adj. p-value = 0.01. A list of all multiple pairwise comparisons is described in the S8 Table. All P-values were obtained using the PERMANOVA built-in function from QIIME2^®’s diversity plugin with adjustments for multiple pairwise comparisons according to the Benjamini-Hochberg method. PCoA = Principal Component, Ov Female = ovariectomized female.

Fig 6. Effects of smoke exposure and sex on the phylum and genus level in mice cecal content.

Average relative abundance of most frequent taxa at the phylum (A and B) and genus (C and D) levels in males, females and ovariectomized females (left) and after stratification by smoke exposure and sex (right). Ov Female = Ovariectomized Female, CM = control male, SM = smoke-exposed male, CF = control female, SF = smoke-exposed female, COF = ovariectomized control female, and SOF = ovariectomized smoke-exposed female. Taxa comparisons are described in detail in S9–S12 Tables.

Fig 7. Differential taxa features identified by LEfSe after stratification by smoke exposure and sex.

CF = control female, CM = control male, COF = ovariectomized control female, SF = smoke-exposed female, SM = smoke-exposed male, and SOF = ovariectomized smoke-exposed female.

Fig 8. Chronic smoke-induced weight loss is associated with reduced expression of the Alistipes genus in the cecal content.

A) Relative abundance of the bacterial genus Alistipes in mice stratified by smoke exposure and sex at 24 weeks post cigarette smoke exposure; p-value* was obtained using the Kruskal-Wallis test, and the Benjamini-Hochberg method was applied to compute adjusted p-values for multiple pairwise comparisons. Correlations between whole-body weight and the cecal relative abundance of Alistipes (%) are shown in male (B), female (C), and ovariectomized female (D) mice. Linear regression analyses were used in panels B-D. Red dot = control mice, blue dot = smoke-exposed mice. C = control, S = smoke-exposed, Ov Female = ovariectomized female.