Intestinal epithelial cells as producers but not targets of chronic TNF suffice to cause murine Crohn-like pathology

Abstract

TNF plays a crucial role in the pathogenesis of Crohn disease. Dysregulated TNF production in mice that bear the genetic deletion of the TNF AU-rich regulatory elements (ARE) (Tnf(ΔARE/+) mice) results in TNF receptor I (TNFRI)-dependent spontaneous Crohn-like pathology. Current concepts consider intestinal epithelial cell (IEC) responses to TNF to be critical for intestinal pathology, but the potential contribution of IEC-derived TNF in disease pathogenesis has not been addressed. In this study we examined whether IEC are sufficient as cellular targets or sources of TNF in the development of intestinal pathology. Using IEC-specific reactivation of a hypomorphic Tnf(ΔAREneo) allele in mice, we show that selective chronic overproduction of TNF by IEC suffices to cause full development of Crohn-like pathology. Epithelial TNF overexpression leads to early activation of the underlying intestinal myofibroblast, a cell type previously identified as a sufficient target of TNF for disease development in the Tnf(ΔARE) model. By contrast, restricted TNFRI expression on IEC although sufficient to confer IEC apoptosis after acute exogenous TNF administration, fails to induce pathology following chronic specific targeting of IEC by endogenous TNF in Tnf(ΔARE/+) mice. Our results argue against IEC being early and sufficient responders to chronic TNF-mediated pathogenic signals and suggest that proinflammatory aberrations leading to chronic TNF production by IEC may initiate pathology in Crohn disease.

Fig. 1.

IEC are the major early source of TNF in the ileum of Tnf^ΔARE/+ mice. VillinCreTnf^ΔAREneo/+ mice overproduce TNF specifically by IEC. (A) Quantitative RT-PCR analysis comparing TNF expression in primary ileal IEC and the respective nonepithelial compartment (NEC) in individual mice. Littermate 4-wk-old WT (n = 5) and Tnf^ΔARE/+ (n = 7) mice were used. P values were calculated with two-tailed t test, unpaired for comparison of WT vs.Tnf^ΔARE/+ and paired for comparison of IEC vs. NEC. (B) PCR analysis for detection of the activated Tnf^ΔARE mutation in tissues and primary isolated IEC from VillinCreTnf^ΔAREneo/+ mice. MLN, mesenteric lymph node. (C) Quantitative RT-PCR analysis for TNF expression in primary IEC isolated from 2-mo-old WT (n = 3), VillinCre (n = 3), Tnf^ΔAREneo/+ (n = 3), and VillinCreTnf^ΔAREneo/+ (n = 4) littermates. (***P < 0.001; one-way ANOVA.) (D) TNF production determined by ELISA in culture supernatants from LPS-stimulated bone marrow-derived macrophages from 2-mo-old mice (n = 3 per genotype). (***P < 0.001; one-way ANOVA.) (E) TNF expression determined by flow cytometry following intracellular staining for TNF in TCRβ⁺-gated splenic T lymphocytes from 2-mo-old mice (n = 3–5 per genotype). (***P < 0.001; one-way ANOVA.) In B–E, data shown are representative of two independent experiments. In A–E, mice were analyzed individually. Error bars show SEM.

Fig. 2.

Intestinal epithelial TNF overexpression leads to Crohn-like IBD pathology. Histological examination of the ileum of VillinCreTnf^ΔAREneo/+ mice (A–C) and littermate controls (D–F). (A) Two-month-old VillinCreTnf^ΔAREneo/+ mice appear normal with no signs of inflammatory infiltration. (B) Severe pathology in a 4-mo-old VillinCreTnf^ΔAREneo/+ mouse characterized by massive inflammatory infiltration in the intestinal mucosa and submucosa. Intestinal villi are broadened and blunted. (C) Extensive submucosal and transmural inflammation in an 8-mo-old VillinCreTnf^ΔAREneo/+ mouse. (D) WT, (E) VillinCre, and (F) Tnf^ΔAREneo/+ littermate control mice are normal with no signs of intestinal inflammation at age 8 mo. Paraffin sections were stained with H&E. (Scale bars: 100 μm.) (G) Disease score distribution in the ileum of VillinCreTnf^ΔAREneo/+ and littermate mice at age 2 mo (*P < 0.05), 3 mo (***P < 0.001), 4 mo (***P < 0.001), and 8 mo (***P < 0.001).

Fig. 3.

Early activation of IMF in the ileum of VillinCreTnf^ΔAREneo/+ mice. (A) Gelatin zymogram showing MMP9 and MMP2 activity in whole-tissue extracts from the ileum of VillinCreTnf^ΔAREneo/+ and littermate control mice at age 2 mo. Lines represent individual mice. Data shown are representative of three independent experiments. (B) Gelatin zymogram showing MMP9 and MMP2 secretion from IMF isolated from VillinCreTnf^ΔAREneo/+ mice (n = 4) and littermates (n = 4 each) at age 2 mo. Lines represent duplicates of separate samples within a single experiment. WT IMF treated with TNF were used as positive control. Data are representative of two independent experiments. (C) Quantitative RT-PCR analysis for TIMP1 (**P < 0.01) and MMP13 (*P < 0.05) expression in IMF isolated from 2-mo-old VillinCreTnf^ΔAREneo/+ mice and the respective controls in three independent experiments. One sample t test was performed for statistical significance. Error bars show SD. (D) Discrimination of IMF (CD90.2⁺αSMA⁺CD31⁻ cells) among intestinal cell populations by FACS analysis. (E and F) ICAM1 expression measured on gated freshly isolated IMF. Analysis was performed in pooled tissues from WT (n = 3) and Tnf^ΔARE/+ (n = 3) 4-wk-old mice in three independent experiments (*P < 0.05) (E) and WT (n = 3), VillinCre (n = 3), Tnf^ΔAREneo/+ (n = 3), and VillinCreTnf^ΔAREneo/+ (n = 3) 2-mo-old mice in three independent experiments (**P < 0.01) (F). One sample t test was performed for statistical significance. Error bars show SD. MFI, mean fluorescence intensity.

Fig. 4.

Acute exogenous TNF administration leads to IEC apoptosis in a direct manner via epithelial TNFRI. (A–D) Ileal sections from TnfRI^−/− (n = 4), WT (n = 5), TnfRI^{flxneo/flxneo} (n = 5), and VillinCreTnfRI^{flxneo/flxneo} (n = 3) mice injected i.v. with 12 μg of murine recombinant TNF. Data represent two independent experiments. (A) Histological examination of H&E-stained sections. (B) TUNEL assay. (Scale bars: 50 μm.) (C) Representative E-cadherin immunostaining. (D) Colocalization of TUNEL and E-cadherin staining in serial paraffin sections. (E–H) Quantitation of TNF-induced IEC apoptosis. Data represent two independent experiments. (E) Number of cells collected from the small intestine of TNF-injected mice. P values (**P < 0.01, *P < 0.05) were calculated with two-tailed t test. Error bars show SEM. (F) Representative FACS analysis for the detection of the markers E-cadherin (epithelial) and CD45 (hemopoietic) on the cells collected. Mean ratio of E-cadherin⁺/CD45⁺ cells. Error bars show SEM. (G) DNA content analysis with propidium iodide (PI) staining in gated E-cadherin⁺/CD45⁻ cells: representative histograms. (H) Quantitation of PI staining data. Error bars show SD.

Fig. 5.

Chronic selective targeting of IEC by endogenous TNF in Tnf^ΔARE/+ mice is not sufficient for the development of intestinal pathology. (A–C) Histological examination of the ileum of 8-mo-old Tnf^ΔARE/+VillinCreTnfRI^{flxneo/flxneo} mice and littermate controls. (A) Tnf^ΔARE/+VillinCreTnfRI^{flxneo/flxneo} mice that express TNFRI selectively in IEC show normal tissue structure without signs of inflammation or tissue damage. (B) Severe intestinal pathology characterized by extensive mucosal and submucosal inflammation in positive-control Tnf^ΔARE/+TnfRI^flxneo/+ mice. (C) Negative-control Tnf^ΔARE/+TnfRI^{flxneo/flxneo} mice that do not express TNFRI appear normal. Paraffin sections were stained with H&E. (Scale bars: 100 μm.) (D) Incidence of IBD pathology in the ileum of Tnf^ΔARE/+VillinCreTnfRI^{flxneo/flxneo} mice and littermates at age 8 and 12 mo.