Upregulation of antimicrobial peptide expression in slc26a3-/- mice with colonic dysbiosis and barrier defect

Abstract

Genetic defects in SLC26A3 (DRA), an intestinal Cl^-/HCO₃^- exchanger, result in congenital chloride diarrhea (CLD), marked by lifelong acidic diarrhea and a high risk of inflammatory bowel disease. Slc26a3^-/- mice serve as a model to understand the pathophysiology of CLD and search for treatment options. This study investigates the microbiota changes in slc26a3^-/- colon, the genotype-related causes for the observed microbiota alterations, its inflammatory potential, as well as the corresponding host responses. The luminal and the mucosa-adherent cecal and colonic microbiota of cohoused slc26a3^-/- and wt littermates were analyzed by 16S rRNA gene sequencing. Fecal microbiota transfer from cohoused slc26a3^-/- and wt littermates to germ-free wt mice was performed to analyze the stability and the inflammatory potential of the communities.The cecal and colonic luminal and mucosa-adherent microbiota of slc26a3^-/- mice was abnormal from an early age, with a loss of diversity, of short-chain fatty acid producers, and an increase of pathobionts. The transfer of slc26a3^-/- microbiota did not result in intestinal inflammation and the microbial diversity in the recipient mice normalized over time. A strong increase in the expression of Il22, Reg3β/γ, Relmβ, and other proteins with antimicrobial functions was observed in slc26a3^-/- colon from juvenile age, while the mucosal and systemic inflammatory signature was surprisingly mild. The dysbiotic microbiota, low mucosal pH, and mucus barrier defect in slc26a3^-/- colon are accompanied by a stark upregulation of the expression of a panel of antimicrobial proteins. This may explain the low inflammatory burden in the gut of these mice.

Keywords: Anion exchange; antimicrobial peptides; gut dysbiosis; inflammatory bowel disease; intestinal electrolyte transport.

Figure 1.

Altered fecal (colonic) and cecal luminal microbiota in the absence of slc26a3 – Relative abundance of the different phyla and bacterial families at different time points of the slc26a3^+/+ (wt) and slc26a3^−/− mice in the (a) colon and (c) cecum. Overall shifts (combination of all the time points) in the relative abundance of the taxonomic families in the (b) colon (n = 25/group) and in the (d) Cecum (n = 12 for slc26a3^+/+and 15 for slc26a3^−/−). Colon – n = 5 pairs for all timepoints; Cecum – Early: n = 2–3, Mid: n = 7–8 and Late: n = 3–4.

Figure 2.

Altered colonic mucosal adherent microbiota in the absence of slc26a3 – Relative abundance of the different phyla and bacterial families at different time points of the slc26a3^+/+ (wt) and slc26a3^−/− mice in the (a) colon and (d) cecum. Overall shifts (combination of all the time points) in the relative abundance of the taxonomic families and genus in the (b, c) colon (n= 12 for slc26a3^+/+and 17 for slc26a3^−/−) and in the (e,f) cecum (n = 11 for slc26a3^+/+and 14 for slc26a3^−/−). Colon Early: n = 5–7, Mid: n = 3–4 and Late: n = 4–6.; Cecum – Early: n = 4–5, Mid: n = 4–5 and Late: n = 3–4.

Figure 3.

Growth curves of different bacterial strains at different pH. Growth curve of (a) Bifidobacterium longum YL2 and (b) Muribaculum intestinale YL27. n = 3.

Figure 4.

Non transfer of mucosal inflammation into the germ-free mice (a) Lack of development of mucosal inflammation as seen by the gene expression analysis of Ly6g, Cd3e and Tnfα in the mid distal colon. (b) Immunohistochemistry of CD3 T cells in the mid distal colon. (n = 5). (c) No systemic inflammation observed in all groups as seen by gene expression analysis of Ly6g, Cd3e and Tnfα in the spleen.

Figure 5.

Improved and altered abundance of colonic taxonomic families post FMT (a) Relative abundance of the different phyla and families in the Donor wt, Recipient wt, Donor ko and Recipient ko. (b) Increase in the species richness post gavage transfer in the Recipient ko group. (c) Principal coordinate analysis depicting distinct microbial signatures between Donor ko and Recipient ko groups. (d,e.) Decrease in pro-inflammatory families Bacteroidaceae and Erysipelotrichaceae. (f) Increase in the relative abundance of Prevotellaceae and (g) protective Muribaculaceae . (h) Continued lowered abundance of Lachnospiraceae. n = 4 pairs for Donor wt, and Donor ko, n = 8 for Recipient wt and n = 7 for Recipient ko.

Figure 6.

Shifts in the gene expression of antimicrobial peptides in the absence of slc26a3^−/−. Unaltered (a-d) Cramp, Tff3, Defb4 and Defb14. Upregulated. (e-k) Reg3b, Reg3g, Il-22, Pla2g2a, Relmβ, Ang4, and iAlp.