NHE8 plays an important role in mucosal protection via its effect on bacterial adhesion

Abstract

The Na⁺/H⁺ exchanger NHE8 is expressed on the apical membrane of intestinal epithelial cells and is particularly abundant in the colon. Our previous study showed that Muc2 expression was significantly reduced in NHE8-knockout (NHE8-/-) mice, suggesting that NHE8 plays a role in mucosal protection in the colon. The current study confirms and extends our studies on the role of NHE8 in mucosal protection. The number of bacteria attached on the distal colon was significantly increased in NHE8-/- mice compared with their wild-type littermates. As expected, IL-4 expression was markedly increased in NHE8-/- mice compared with wild-type mice. Immunohistochemistry showed disorganization in the mucin layer of NHE8-/- mice, suggesting a possible direct bacteria-epithelia interaction. Furthermore, NHE8-/- mice were susceptible to dextran sodium sulfate-induced mucosal injury. In wild-type mice, dextran sodium sulfate treatment inhibited colonic NHE8 expression. In Caco-2 cells, the absence of NHE8 expression resulted in higher adhesion rates of Salmonella typhimurium but not Lactobacillus plantarum. Similarly, in vivo, S. typhimurium adhesion rate was increased in NHE8-/- mice compared with wild-type mice. Our study suggests that NHE8 plays important roles in protecting intestinal epithelia from infectious bacterial adherence.

Keywords: NHE8; Salmonella; mucosal layer.

Fig. 1.

Quantitative analysis of bacterial groups in the distal colon in wild-type (WT) and Na⁺/H⁺ exchanger isoform 8 (NHE8)-knockout (NHE8^−/−) mice. Distal colon was collected, and luminal contents were removed immediately. Genomic DNA was extracted and used to quantify the relative abundance of major bacterial groups attached to the intestinal epithelial surface in the colon. Eubac, Eubacteria; Firm, Firmicutes; Bac, Bacteroidetes; Lac, Lactobacillus; gCocco, Clostridium coccoides; SFB, segmented filament bacteria. Values are means ± SE from 30 mice. *P ≤ 0.05.

Fig. 2.

Expression of inflammatory cytokine genes in the distal colon of NHE8^−/− mice. Colonic tissue was collected and used for total RNA isolation. Real-time PCR was used to determine cytokine gene expression. Values are means ± SE from 16 mice. *P ≤ 0.05.

Fig. 3.

Mucus and bacteria stain in the distal colon of NHE8^−/− mice. Distal colon was sectioned and used for Muc2 and 4′,6-diamidino-2-phenylindole staining. Muc2-positive goblet cells and overlaying mucus layers were detected by anti-MUC2C3 antiserum (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Bacteria were detected using the general bacterial probe EUB338-Alexa Fluor 555 (red). Top: low-power (×10) magnification; bottom: high-power (×40) magnification.

Fig. 4.

Expression of NHE8 and TNFα in dextran sodium sulfate (DSS)-treated WT mice. Distal colon was collected and used for RNA isolation and tissue lysate preparation. NHE8 and TNFα mRNAs were detected by real-time PCR. NHE8 and TNFα proteins were analyzed by Western blotting. A: NHE8 mRNA expression (top) and protein abundance (bottom). B: TNFα mRNA expression (top) and protein abundance (bottom). WT-N, control mice; WT-DSS, DSS-treated mice; MW, molecular weight; TBP, TATA-binding protein. Values are means ± SE from 20 mice. *P ≤ 0.05.

Fig. 5.

Susceptibility of NHE8^−/− mice to DSS treatment. NHE8^−/− mice were fed DSS water for 6 days, and body weight (A), death rate (B), and disease activity index (DAI; C) were recorded. Values are means ± SE from 20 mice. *P ≤ 0.05.

Fig. 6.

NHE8 knockdown and bacterial adhesion assay in Caco-2 cells. A: Caco-2 cells were transfected with 5 nM NHE8 small interfering RNA (siRNA). Total RNA was isolated 48 h after transfection, and real-time PCR was performed to determine NHE8 mRNA expression (top). Total protein was extracted 72 h after transfection, and Western blotting was used to evaluate NHE8 protein expression (bottom). siRNA-NC, negative control siRNA; siRNA-NHE8, NHE8 siRNA. Values are means ± SE from 3 separate experiments. *P ≤ 0.01. B: NHE8 siRNA-transfected Caco-2 cells were infected with Salmonella typhimurium and Lactobacillus plantarum. Number of bacteria attached to cells was calculated by quantitative PCR using species-specific primers. Values are means ± SE from 5 separate experiments. *P ≤ 0.05.

Fig. 7.

In vivo S. typhimurium adhesion assay. Wild-type and NHE8^−/− mice were gavaged with S. typhimurium. Distal colon was collected 7 days after infection. Number of S. typhimurium attached to the epithelial surface was calculated by semiquantitative PCR using S. typhimurium-specific primers (ST) and general bacterial 16S rRNA primers (16S). A: PCR amplification of S. typhimurium and 16S rRNA in control (−) and infected (+) wild-type mice. B: relative changes of S. typhimurium bound to colonic mucosal surface in wild-type and NHE8^−/− mice. Values are means ± SE from 16 mice. *P ≤ 0.05.