High salt diet exacerbates colitis in mice by decreasing Lactobacillus levels and butyrate production - PubMed (original) (raw)

High salt diet exacerbates colitis in mice by decreasing Lactobacillus levels and butyrate production

Pedro M Miranda et al. Microbiome. 2018.

Abstract

Background: Changes in hygiene and dietary habits, including increased consumption of foods high in fat, simple sugars, and salt that are known to impact the composition and function of the intestinal microbiota, may explain the increase in prevalence of chronic inflammatory diseases. High salt consumption has been shown to worsen autoimmune encephalomyelitis and colitis in mouse models through p38/MAPK signaling pathway. However, the effect of high salt diet (HSD) on gut microbiota and on intestinal immune homeostasis, and their roles in determining vulnerability to intestinal inflammatory stimuli are unknown. Here, we investigate the role of gut microbiota alterations induced by HSD on the severity of murine experimental colitis.

Results: Compared to control diet, HSD altered fecal microbiota composition and function, reducing Lactobacillus sp. relative abundance and butyrate production. Moreover, HSD affected the colonic, and to a lesser extent small intestine mucosal immunity by enhancing the expression of pro-inflammatory genes such as Rac1, Map2k1, Map2k6, Atf2, while suppressing many cytokine and chemokine genes, such as Ccl3, Ccl4, Cxcl2, Cxcr4, Ccr7. Conventionally raised mice fed with HSD developed more severe DSS- (dextran sodium sulfate) and DNBS- (dinitrobenzene sulfonic acid) induced colitis compared to mice on control diet, and this effect was absent in germ-free mice. Transfer experiments into germ-free mice indicated that the HSD-associated microbiota profile is critically dependent on continued exposure to dietary salt.

Conclusions: Our results indicate that the exacerbation of colitis induced by HSD is associated with reduction in Lactobacillus sp. and protective short-chain fatty acid production, as well as changes in host immune status. We hypothesize that these changes alter gut immune homeostasis and lead to increased vulnerability to inflammatory insults.

Keywords: Butyrate; Colitis; Lactobacillus; MAPK-pathway; Microbiota; NaCl; Salt; Western diet.

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Conflict of interest statement

All experiments were approved by the McMaster University Animal Ethics Committee and were conducted under the Canadian guidelines for animal research.

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1

Fig. 1

High salt diet alters gut microbiota composition and function. a Principal coordinate analysis (PCoA) plots of unweighted UniFrac metric (left panel) and abundance Jaccard index (right panel) of fecal microbiota composition over time exposed to HSD. Gray arrows indicate the direction of the significant microbiota composition that shifts between groups. b Summary of the relative abundance of microbial genera present in 99.9% of the community overtime exposure to HSD. c Relative abundance of fecal bacterial genera (left panel) and OTUs (right panel) that significantly correlate with HSD (q < 0.05). The blue background represents the period exposed to HSD. d Heatmap of the 20 most significant KEGG pathways that correlate with HSD in fecal microbiota. e SCFA quantification at 0 and 4 weeks after HSD. Un. G (Unclassified Genus). AA (acetic acid), PrA (propionic acid), IbuA (isobutyric acid), BA (butyric acid), IvaA (isovaleric acid), PeA (pentanoic acid), LA (lactic acid). Values are presented as means ± SEM. n = 5–6 mice/group. *p < 0.05

Fig. 2

Fig. 2

High salt diet modulates immune expression in colonic lamina propria. a Volcano plot of gene expression in colonic lamina propria total cell extract of mice receiving HSD or control diet for 4 weeks, n = 9 mice/group. Pink dots: genes with p < 0.01. Blue dots: genes with 0.01 < p_ < 0.05, fold change > 2. b Heatmap of immune genes that changed with HSD (p < 0.05). Based on group average, Euclidean distance metric. c qRT-PCR analysis of differentially expressed genes in the two groups. Values are presented as means ± SEM, n = 5–6 colon/group, *p < 0.05, **p < 0.01, #p < 0.001, γ_p = 0.065. d Gene-cell function networks obtained from Ingenuity Pathway Analysis. On the left, network with the genes that contribute to the predicted inhibition of functions related to immune cell migration, and, on the right, network with the genes that contribute to the predicted upregulation of the immune cell viability and maturation

Fig. 3

Fig. 3

High salt diet exacerbates DSS colitis. a DSS colitis macroscopic scores at endpoint (day 7) in four groups of mice: control (control diet, no DSS), HSD (high salt diet, no DSS), control+DSS (control diet, DSS in water), HSD + DSS (high salt diet, DSS in water). b Body weight changes during DSS administration. c Colon length at endpoint. d Colonic MPO of the two groups of mice receiving DSS. e Survival curve of the two groups of mice with DSS colitis. f Heatmap of differentially expressed genes (p < 0.05) in the colon of mice with DSS colitis: C + D (control diet+DSS), HS + D (HSD + DSS). The heatmap was generated based on group average using Euclidean distance metric. Colitis scores, weight change, colon length, and MPO are presented as means ± SEM; n = 8–9 mice/group. *p < 0.05, **p < 0.01, #p < 0.001, ##p < 0.0001

Fig. 4

Fig. 4

High salt diet exacerbates DNBS colitis. a DNBS colitis macroscopic scores at endpoint (day 3) in four groups of mice: control (control diet, 50% EtOH vehicle), HSD (high salt diet, 50% EtOH vehicle), DNBS (control diet, DNBS in 50% EtOH vehicle), HSD + DNBS (high salt diet, DNBS in 50% EtOH vehicle). b Representative H&E-staining of colonic section of DNBS mice highlighting the ulcers in the mucosa. c Histological scores in colonic sections. d Survival curve of the two groups with DNBS colitis. Colitis and histology scores are presented as means ± SEM; n = 5–9 mice/group. *p < 0.05, **p < 0.01, #p < 0.001, ##p < 0.0001

Fig. 5

Fig. 5

The effect of high salt diet on exacerbation of colitis is dependent on gut microbiota. a DSS colitis macroscopic scores at endpoint (day 7) in three groups of mice: control (no DSS colitis), DSS (control diet, DSS in water), HSD + DSS (high salt diet, DSS in water). b Colon length of mice at endpoint. c Colonic MPO. d Histological scores of the DSS colitis. Values are presented as means ± SEM; n = 3–6 mice/group. *p < 0.05, **p < 0.01. e Heatmap of the gene expression of 68 genes related to immune, gut barrier, and neural function, in the colon tissue of germ-free mice after DSS administration, on control diet or HSD: C + D (control diet + DSS), HS + D (HSD + DSS). The heatmap was generated based on group average using Euclidean distance metric

Fig. 6

Fig. 6

HSD-signature microbiota requires constant exposure to high salt diet for its maintenance. a Experimental design. b Principal coordinate analysis (PCoA) of abundance Jaccard index from fecal microbiota composition data. c Summary of the relative microbial genera present in 99.9% of the community. Un. G (Unclassified Genus). d Relative abundance of Lactobacillus genus. e Total read count of each OTU present in the indicated donor fecal microbiota (in green triangles). Orange dots indicate the OTUs that were present in recipient mice at day 5 post-colonization. The horizontal dashed line separates the 15 most abundant OTUs from the others. f Body weight during DSS administration. g Macroscopic scores of DSS colitis at endpoint. h Colon length at endpoint

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