Intestinal accumulation of microbiota-produced succinate caused by loss of microRNAs leads to diarrhea in weanling piglets

Abstract

Diarrheal disease is a common health problem with complex causality. Although diarrhea is accompanied by disturbances in microbial diversity, how gut microbes are involved in the occurrence of diarrhea remains largely unknown. Here, using a pig model of post-weaning stress-induced diarrhea, we aim to elucidate and enrich the mechanistic basis of diarrhea. We found significant alterations in fecal microbiome, their metabolites, and microRNAs levels in piglets with diarrhea. Specifically, loss of ssc-miRNA-425-5p and ssc-miRNA-423-3p, which inhibit the gene expression of fumarate reductase (frd) in Prevotella genus, caused succinate accumulation in piglets, which resulted in diarrhea. Single-cell RNA sequencing indicated impaired epithelial function and increased immune response in the colon of piglet with diarrhea. Notably, the accumulated succinate increased colonic fluid secretion by regulating transepithelial Cl-secretion in the epithelial cells. Meanwhile, succinate promoted colonic inflammatory responses by activating MyD88-dependent TLR4 signaling in the macrophages. Overall, our findings expand the mechanistic basis of diarrhea and suggest that colonic accumulation of microbiota-produced succinate caused by loss of miRNAs leads to diarrhea in weanling piglets.

Keywords: Diarrhea; microRNAs; microbiome; piglets; succinate.

Figure 1.

Piglets with diarrhea showed impaired intestinal morphology and inflammation. a Diarrhea score in weanling piglets. b Water content in the feces of weanling piglets. c Representative colonic morphology of the HE staining results. Arrow, loss of shape in colonic villi. d Representative colonic morphology under the transmission electron microscope. Arrow, unregularly aligned microvilli. Scale bar, 1 μm. e TNF-α level in colonic tissue. f Qualification of F4/80 protein expression. g Qualification of tight junction protein expression. HEA, healthy piglets without diarrhea (n = 11); DIA, diarrheal piglets (n = 13). Data are represented as mean ± SEM. *P < .05, determined by two-tailed Student’s t-test.

Figure 2.

Piglets with diarrhea exhibit alterations in the fecal microbiome. a PCoA based on weighted UniFrac distance. b Beta-diversity (Bray-Curtis distance) at the gene level. c Relative abundance of bacteria classified at a genus-level taxonomy. d Relative abundance of Prevotella via fecal microbe quantification using qPCR, Data are represented as mean ± SEM. *P < .05, determined by two-tailed Student’s t-test. e VIP scores of PLS-DA (The top 20 species were presented). HEA, healthy piglets without diarrhea (n = 11); DIA, diarrheal piglets (n = 13).

Figure 3.

Piglets with diarrhea exhibit alterations in the fecal metabolites. a PLS-DA score plot. b KEGG enrichment scatter plot. c-d Relative levels of succinate (c) and glutamate (d) in the feces based on the untargeted metabolomics analysis. e-f Levels of succinate (e) and glutamate (f) in the feces determined by Colorimetric Assay Kit. Data are represented as mean ± SEM. *P < .05, determined by two-tailed Student’s t-test. HEA, healthy piglets without diarrhea (n = 11); DIA, diarrheal piglets (n = 13).

Figure 4.

Effects of FMT and succinate on diarrhea in piglets. a Schematic design. b-d Diarrhea score (b), fecal water content (c) and succinate concentration (d) in diarrheal piglets transplanted with feces collected from healthy piglets or diarrheal piglets. n = 6, Data are represented as mean ± SEM. *P < .05, determined by one-way ANOVA. Saline to DG, diarrheal piglets were administrated with saline; HG to DG, feces were transplanted from healthy piglets to diarrheal piglets; DG to DG, feces were transplanted from diarrheal piglets to diarrheal piglets. e Schematic design. f Diarrhea score of healthy piglets treated with succinate through rectal administration. n = 6, Data are represented as mean ± SEM. *P < .05, determined by two-tailed Student’s t-test. g Schematic design. h, i Diarrhea score (h) and succinate concentration (i) in diarrheal piglets transplanted with heat-inactivated feces collected from healthy piglets or diarrheal piglets. n = 6, Data are represented as mean ± SEM. *P < .05, determined by one-way ANOVA. HG-HFMT, heat-inactivated feces were transplanted from healthy piglets to diarrheal piglets; DG-HFMT, heat-inactivated feces were transplanted from diarrheal piglets to diarrheal piglets.

Figure 5.

Ssc-miRNA-425-5p and ssc-miRNA-423-3p inhibited the accumulation of succinate produced by Prevotella and prevented diarrhea. a Differentially expressed miRNAs in the feces of healthy and diarrheal piglets. b Schematic diagram of the putative binding sites of the seed sequence of ssc-miRNA-425-5p and ssc-miRNA-423-3p in the two genes (frdi and frdf) of Prevotella. N = A, G, C or T. c-e Succinate content (c), gene expression of frdi (d) and frdf (e) in the fermentation broth of feces from diarrheal piglets after treated with ssc-miRNA-425-5p and ssc-miRNA-423-3p. n = 4, Data are represented as mean ± SEM. *P < .05, determined by two-tailed Student’s t-test. f Schematic design. g, h Diarrhea score (g) and succinate concentration (h) in mice pretreated with miRNA antagomirs and then transplanted feces from diarrheal piglets. n = 6, Data are represented as mean ± SEM. *P < .05, **P < .01, determined by two-tailed Student’s t-test. i-k Diarrhea score (i), succinate concentration (j) and Prevotella abundance (k) in mice further treated with miRNA agomirs. n = 3, Data are represented as mean ± SEM. *P < .05, determined by one-way ANOVA. CONT-scramble, control mice; CONT-agomir, mice treated with miRNA agomir; anta-scramble, mice pretreated with miRNA antagomirs and transplanted feces from diarrheal piglets; anta-agomir, mice pretreated with miRNA antagomirs and transplanted feces from diarrheal piglets, and then treated with miRNA agomir.

Figure 6.

Single-cell RNA sequencing indicated impaired epithelial function and elevated immune response in piglets with diarrhea. a,b A t-SNE plot of epithelial cells of scRNA-seq data of colonic tissue from diarrheal piglet and healthy piglet showing clusters (a, merged results; b, separated results). c, d A t-SNE plot of immune cells of scRNA-seq data of colonic tissue from diarrheal piglet and healthy piglet showing clusters (c, merged results; d, separated results). e-i t-SNE plot showing expression of ANO9 (e), CLCA1 (f), IRF1 (g), IRF7 (h) and TNF (i) in colonic tissue from the indicated piglets at the single-cell level. HEA, healthy piglets without diarrhea; DIA, diarrheal piglets.

Figure 7.

Succinate accumulation in the colon increased epithelial fluid secretion and the immune response in piglets with diarrhea. a, b Gene expression of ANO9 (a) and CLCA1 (b) in colonic tissue determined by RT-qPCR. n = 6, Data are represented as mean ± SEM. **P < .01, determined by two-tailed Student’s t-test. c Protein qualification of ANO9 and CLCA1 in colonic tissue by the Wes Simple Western System. d Protein qualification of ANO9, CLCA1, NHE3 and DRA in IPEC-J2 cells. e Relative chloride level in the culture medium. f-h Gene expression of IRF1 (f), IRF7 (g) and TNF (h) in colonic tissue determined by RT-qPCR. n = 6, Data are represented as mean ± SEM. *P < .05, **P < .01, determined by two-tailed Student’s t-test. HEA, healthy piglets without diarrhea; DIA, diarrheal piglets. i Protein qualification of IRF7, IRF1, MyD88 and TLR4 in colonic tissue. j Protein qualification of SUCNR1 in RAW264.7 cells. k Protein qualification of IRF7, IRF1, MyD88 and TLR4 in RAW264.7 cells. l, m Levels of TNF-α (l) and IL-1β (m) in the culture medium. n = 3, Data are represented as mean ± SEM. *P < .05, determined by one-way ANOVA. CONT, control cells; SUC, cells treated with disodium succinate; TAK, cells treated with TAK-242; SUC+TAK, cells treated with disodium succinate and TAK-242.