SHP-2 Phosphatase Prevents Colonic Inflammation by Controlling Secretory Cell Differentiation and Maintaining Host-Microbiota Homeostasis

Abstract

Polymorphisms in the PTPN11 gene encoding for the tyrosine phosphatase SHP-2 were described in patients with ulcerative colitis. We have recently demonstrated that mice with an intestinal epithelial cell-specific deletion of SHP-2 (SHP-2(IEC-KO) ) develop severe colitis 1 month after birth. However, the mechanisms by which SHP-2 deletion induces colonic inflammation remain to be elucidated. We generated SHP-2(IEC-KO) mice lacking Myd88 exclusively in the intestinal epithelium. The colonic phenotype was histologically analyzed and cell differentiation was determined by electron microscopy and lysozyme or Alcian blue staining. Microbiota composition was analyzed by 16S sequencing. Results show that innate defense genes including those specific to Paneth cells were strongly up-regulated in SHP-2-deficient colons. Expansion of intermediate cells (common progenitors of the Goblet and Paneth cell lineages) was found in the colon of SHP-2(IEC-KO) mice whereas Goblet cell number was clearly diminished. These alterations in Goblet/intermediate cell ratio were noticed 2 weeks after birth, before the onset of inflammation and were associated with significant alterations in microbiota composition. Indeed, an increase in Enterobacteriaceae and a decrease in Firmicutes were observed in the colon of these mice, indicating that dysbiosis also occurred prior to inflammation. Importantly, loss of epithelial Myd88 expression inhibited colitis development in SHP-2(IEC-KO) mice, rescued Goblet/intermediate cell ratio, and prevented NFκB hyperactivation and inflammation. These data indicate that SHP-2 is functionally important for the maintenance of appropriate barrier function and host-microbiota homeostasis in the large intestine. J. Cell. Physiol. 231: 2529-2540, 2016. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.

Figure 1

Expression of antimicrobial peptides in the colon of SHP‐ 2^IEC‐KO mice. qPCR of Defα, Reg3β, Reg3γ, Lyz1, and Mmp7 mRNAs in total extracts from 3‐day‐old (A) and in enriched mucosal extracts from 2‐week‐old (B) SHP‐2^IEC‐KO mice versus controls. Relative expression was normalized with housekeeping genes (n ≥ 6 per group). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, NS: not significant. The error bars indicate SEM.

Figure 2

Lysozyme‐positive cells in the colon of young SHP‐2^{IEC‐ KO} mice. H&E staining (A), immunohistochemistry against lysozyme (B), and Alcian blue staining (C) were performed on 1‐day‐old, 1‐week‐old, 2‐week‐old, and 1‐month‐old control and SHP‐2^{IEC‐ KO} murine colons. Scale bars, 50 μm.

Figure 3

Expansion of intermediate cells in the colon of SHP‐ 2^IEC‐KO mice. (A) Electron microscopy of Paneth‐like cells from the small intestine and colon of 2‐week‐old control and SHP‐2^IEC‐KO mice. Scale bars, 2 μm (top panels) and 200 ηm (bottom panels). Co‐staining of lysozyme and Alcian blue was performed on small intestine (B) and colon (C) tissues of 2‐week‐old and 1‐month‐old control and mutant mice. Scale bars, 50 μm.

Figure 4

Altered microbiota composition in SHP‐2^IEC‐KO mice. Taxonomic microbiota composition at phylum level in SHP‐ 2^IEC‐KO and control littermates at the age of 4 days (A), 2 weeks (B), and 1 month (C) was determined by deep sequencing analysis. PCoA ordination plot constructed on the Bray Curtis distance matrix calculated on normalized data from SHP‐2^IEC‐KO and control littermates at the age of 2 weeks (D) and 1 month (E). Alpha‐diversity (within community) measures (richness, measured as observed species and diversity measured as Shannon's diversity index) in SHP‐ 2^IEC‐KO and control littermates at the age of 2 weeks (F) and 1 month (G). (H) Changes at genus level in SHP‐2^IEC‐KO and control littermates at the age of 2 weeks and 1 month (n ≥ 7 per group). *P ≤ 0.05. The error bars indicate SEM.

Figure 5

Loss of epithelial Myd88 signaling inhibits colitis in SHP‐2‐deficient mice. (A) Body weight of control, Myd88^IEC‐KO, SHP‐2^IEC‐KO, and SHP‐2;Myd88^IEC‐KO mice at 1 month (n ≥ 6 per group). **P ≤ 0.01, ***P ≤ 0.001; two‐tailed Student's t‐test. Error bars indicate SEM. (B) DAI of 1‐month‐old control, Myd88^IEC‐KO, SHP‐2^IEC‐KO, and SHP‐2; Myd88^IEC‐KO mice littermates were calculated by scoring stool softness, occult fecal blood, rectal bleeding, and colon hardness (n ≥ 7 per group). **P ≤ 0.01, ***P ≤ 0.001; Mann–Whitney U‐test. Error bars indicate interquartile range. (C) H&E staining from 1‐month‐old control, Myd88^IEC‐KO, SHP‐2^IEC‐KO, and SHP‐2; Myd88^IEC‐KO murine colons were performed. Scale bars: 100 μm. (D) Histological score from 1‐month‐old mice was calculated as described in the section Material and Methods (n ≥ 4 per group). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; Mann–Whitney U‐test. Error bars indicate interquartile range. (E) Mucosal enrichments from control, Myd88^IEC‐KO, SHP‐2^IEC‐KO, and Myd88^IEC‐KO; SHP‐2^{IEC‐ KO} colons aged of 1 month were analyzed by Western blot for the expression of phosphorylated RelA (S536). β‐actin served as loading control.

Figure 6

Loss of epithelial Myd88 increases the number of Goblet cells and decreases the number of intermediate cells in SHP‐2^{IEC‐ KO} mice. (A) Co‐staining for lysozyme and Alcian blue cells were performed on colon tissues of 2‐week‐old control, Myd88^{IEC‐ KO}, SHP‐2^IEC‐KO, and Myd88^IEC‐KO; SHP‐ 2^IEC‐KO mice. Scale bars, 100 μm. (B) Epithelial cells from 2‐week‐old murine proximal colon stained positively for Alcian blue were counted in 15 crypts (n ≥ 3 per group). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; two‐tailed Student's t‐test. The error bars indicate the SEM. (C) Epithelial cells from 2‐week‐old murine proximal colon stained positively for lysozyme were counted in 15 crypts (n ≥ 3 per group). **P ≤ 0.01, ***P ≤ 0.001; Mann–Whitney U‐test. Error bars indicate interquartile range.