Intestinal epithelial expression of TNFAIP3 results in microbial invasion of the inner mucus layer and induces colitis in IL-10-deficient mice

Abstract

Tumor necrosis factor-induced protein 3 (TNFAIP3; also known as A20) negatively regulates NF-κB and MAPK signals to control inflammatory responses. TNFAIP3 also protects against TNF-induced cell death. Intestinal epithelial cell (IEC) expression of TNFAIP3 improves barrier function and tight junction integrity and prevents dextran sulfate sodium (DSS)-induced IEC death and colitis. We therefore investigated the effects of TNFAIP3 expression in IEC on immune homeostasis in the intestines of immune-compromised mice. Villin-TNFAIP3 (v-TNFAIP3) transgenic mice were interbred with IL-10(-/-) mice (v-TNFAIP3 × IL-10(-/-)) and incidence, onset, and severity of colitis was assessed. v-TNFAIP3 × IL-10(-/-) mice displayed severe, early onset, and highly penetrant colitis that was not observed in IL-10(-/-) or v-TNFAIP3 mice. V-TNFAIP3 mice displayed altered expression of mucosal cytokines, increased numbers of mucosal regulatory T cells, and altered expression of mucosal antimicrobial peptides (AMPs). Microbial colonization of the inner mucus layer of v-TNFAIP3 mice was observed, along with alterations in the microbiome, but this was not sufficient to induce colitis in v-TNFAIP3 mice. The relative sterility of the inner mucus layer observed in wild-type and IL-10(-/-) mice was lost in v-TNFAIP3 × IL-10(-/-) mice. Thus IEC-derived factors, induced by signals that are inhibited by TNFAIP3, suppress the onset of inflammatory bowel disease in IL-10(-/-) mice. Our results indicate that IEC expression of TNFAIP3 alters AMP expression and allows microbial colonization of the inner mucus layer, which activates an IL-10-dependent anti-inflammatory process that is necessary to prevent colitis.

Keywords: A20; Ang4; NK-κB; Reg3; intestinal mucus.

Copyright © 2014 the American Physiological Society.

Figures

Fig. 1.

Epithelial expression of tumor necrosis factor-induced protein 3 (TNFAIP3) suppresses NF-κB activation and induces colitis in IL-10 knockout (KO) mice. Villin-TNFAIP3 transgenic mice (TNFAIP3tg) were interbred with IL-10−/− mice, and littermates were assessed for the activation of epithelial cell NF-κB and incidence and severity of colitis. TNFAIP3tg × IL-10KO, compared with IL-10KO mice, exhibited reduced body weight and failure to thrive apparent by 4 wk, significant by 5 wk of age (A); reduced length, increased weight, and visible turgidity of the colon (B); increased histological signs of inflammation (C); increased levels of activated CD4 T cells (D and E); and increased levels of FoxP3+ CD4 T cells in the colonic mucosa (F). G: reduced immunostaining for activated NF-κB (phospho-NF-κB) in the nuclei of epithelial cells. H: altered gene expression in the intestinal mucosa of noncolitic IL-10−/− mice. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

Fig. 2.

Epithelial expression of TNFAIP3 induces a Th1-type colitis in IL-10KO mice. ELISAs were performed on the colonic mucosa from IL-10KO vs. TNFAIP3tg × IL-10KO mice, and these revealed elevated levels of IFN-γ (A) and reduced levels of IL-4 (B) typical of a Th1-type of inflammation. There were also significantly reduced levels of IL-22 (C), thymic stromal lymphopoietin (TSLP) (F), and IL-25 (G) and a trend toward reduced IL-6 (H), in the mucosa of TNFAIP3tg × IL-10KO mice, compared with IL-10KO mice. No differences were observed in the mucosal levels of TNF (D), IL-12/23 (E), or IL-17 (I). *P < 0.05.

Fig. 3.

Altered gene expression in the intestinal mucosa of TNFAIP3tg mice. Total RNA was extracted from the intestinal mucosa of wild-type (WT) or villin-TNFAIP3 transgenic (TNFAIP3tg) littermates and assessed for gene expression by array. Selected mRNA levels from the complete set of data are shown graphically here. Genes with higher expression in TNFAIP3tg mucosa include indolamine 2,3 dioxygenase (INDO), solute carrier family 30, member 10 (SLC30A10), interferon-γ-inducible protein 47 (ifi47, also known as IRG-47), chemokine ligand 9 (CxCl9), and interferon-γ-induced GTPase (IgtP, also known as IRGM3). Genes with lower expression in TNFAIP3tg mice include pancreatic lipase-related protein precursor (Pnliprp), phospholipase A2 group VIC (Pla2g4c or cytosolic PLA2), intelectin b (itlnb), intelectin 1 (itln1), angiogenin-4 (Ang4), and 5′ nucleotides, ecto (Nt5e or CD73).

Fig. 4.

Increased Tregs in the colonic mucosa of TNFAIP3tg mice. Colonic lamina propria lymphocytes were isolated and analyzed for the presence of activated CD4 T-lymphocytes (A and B) and FoxP3+ CD4 T lymphocytes (C). Colonic mucosal tissue was assessed for the level of IL-25 (D), IL-23 (E), IL-17 (F), IL-12/23 (G), TSLP (H), IL-22 (I), TNF (J), and IL-10 (K). **P < 0.01.

Fig. 5.

Reduced expression of angiogenin-4 in the intestinal mucosa of TNFAIP3tg mice. Immunohistochemical localization (red) (A), immunoblots of Ang4 (B), and Reg3β protein in WT and TNFAIP3tg colon (C and D), showing decreased Ang4 expression in the colon of TNFAIP3tg mice.

Fig. 6.

Microbial invasion of the intestinal inner mucus layer in villin-TNFAIP3 transgenic mice. A: fluorescence in situ hybridization (FISH) with EUB338 probe for bacterial 16S rRNA (EUB) (red) and immunohistochemistry for Muc-2 (green) in Carnoy's-fixed paraffin-embedded colonic tissue. B: FISH and Hoescht (blue) staining to detect the spatial separation of bacteria from epithelial cells with quantification of the distance (μM) between the epithelial surface and the bacterial population. ****P < 0.001.

Fig. 7.

Altered microbiome of colonic mucosal samples in v-TNFAIP3 compared with littermate controls. Comparison between taxonomical population proportions of v-TNFAIP3 vs. littermate controls at both the phylum (A) and order (B) levels. Pie charts depict average proportions for N = 4 mice per group; significant differences were observed between Bacteroidetes and Firmicutes (A) and Bacteriodales and Lachnospiraceae (B), respectively. Tabular results of a 2-way ANOVA with multiple comparisons are also shown. α-Diversity metrics, phylogenetic diversity (PD) tree, Shannon and Simpson are also shown comparing between groups (C) as is β-diversity as assessed by principal coordinate analysis (PCoA) (D).

Fig. 8.

Altered production of antimicrobial peptides and invasion of the inner mucus layer in villin-TNFAIP3 × IL-10KO mice. Representative gene expression profile from an expression array showing mRNA that are increased (A) or decreased (B) in the colonic mucosa of TNFAIP3tg × IL-10KO mice, compared with IL-10KO mice. Immunohistochemistry and immunoblots showing decreased angiogenin-4 (C and D) and increased Reg3b protein (E and F) in the colonic mucosa of TNFAIP3tg × IL-10KO mice. G: FISH, Muc-2, and Hoescht staining showing microbial invasion of the colonic inner mucus layer and loss of spatial separation between the epithelial surface and bacteria in the intestines of TNFAIP3tg × IL-10KO mice, compared with IL-10KO mice. **P < 0.01, ***P < 0.005, ****P < 0.001.