Chronic intestinal inflammation in mice expressing viral Flip in epithelial cells

Abstract

Viruses are present in the intestinal microflora and are currently discussed as a potential causative mechanism for the development of inflammatory bowel disease. A number of viruses, such as Human Herpesvirus-8, express homologs to cellular FLIPs, which are major contributors for the regulation of epithelial cell death. In this study we analyzed the consequences of constitutive expression of HHV8-viral FLIP in intestinal epithelial cells (IECs) in mice. Surprisingly, expression of vFlip disrupts tissue homeostasis and induces severe intestinal inflammation. Moreover vFlip^IEC-tg mice showed reduced Paneth cell numbers, associated with excessive necrotic cell death. On a molecular level vFlip expression altered classical and alternative NFκB activation. Blocking of alternative NFκB signaling by deletion of Ikka in vivo largely protected mice from inflammation and Paneth cell loss induced by vFLIP. Collectively, our data provide functional evidence that expression of a single viral protein in IECs can be sufficient to disrupt epithelial homeostasis and to initiate chronic intestinal inflammation.

Fig. 1

vFlip^IEC-tg mice spontaneously develop severe intestinal inflammation. a Strategy for generating vFlip^IEC-tg mice. Rosa26.vFLIP knockin mice were crossed to VillinCre mice to generate mice constitutively expressing vFlip in IECs. b Western Blot analysis of GFP using small intestinal IEC lysates from indicated mice. Actin was used as a loading control. c Representative pictures of GFP fluorescence staining of small intestinal cross sections from indicated mice. Scale bar 75 µm. d Representative endoscopic pictures from the small intestine of indicated mice. e Transcriptional analysis of Tnfa (n ≥ 10) and S100a9 (n ≥ 8) in the small intestine of control and vFlip^IEC-tg mice. Values are shown + SD and were calculated relative to control mice. Hprt was used as internal standard. Data were pooled from 4 individual experiments, **p < 0.01. f Representative pictures of H&E stained small intestinal cross sections from indicated mice. Scale bar upper panel 500 µm, lower panel 100 µm. g Representative pictures of immunohistochemical stainings of small intestinal cross sections from indicated mice using antibodies against F4/80, CD11c and CD4. Scale bar 75 µm

Fig. 2

vFlip^IEC-tg mice are characterized by reduced Paneth cell numbers and increased cell death. a Representative pictures of H&E and immunohistochemical Lysozyme staining of small intestinal cross sections of indicated mice. Scale bar 50 µm. b Transcriptional analysis of Ang4 (n = 11) and Lyz (n = 11) in the small intestine of indicated mice. Values are shown + SD and were calculated relative to control mice. Hprt was used as internal standard. Data were pooled from 4 individual experiments, **p < 0.01, ***p < 0.001. c Representative pictures of TUNEL/Cleaved Caspase-3 doublestaining of small intestinal cross sections from indicated mice. Scale bar upper panel: 75 µm, lower panel: 50 µm. d Quantification of cell death of IECs located at the villus or the crypt area. Villi: n(control) = 5, n(vFlip^IEC-tg) = 10, crypts: n(control) = 8, n(vFlip^IEC-tg) = 13, ***p < 0.001. e Transcriptional analysis of Rip3 (n ≥ 9) and Mlkl (n ≥ 9) in the small intestine of indicated mice. Values are shown + SD and were calculated relative to control mice. Hprt was used as internal standard. Data were pooled from 3 individual experiments, ***p < 0,001. f Western Blot analyses of small intestinal IEC lysates from indicated mice using antibodies against RIP3, Caspase-3, its cleaved forms and MLKL. Actin was used as a loading control. g Quantitative proteome analysis of known and putative STAT1 target genes in small intestinal epithelial lysates of control and vFlip^IEC-tg mice (n = 3). Values are shown + SD and were calculated relative to control mice, p < 0.05 for all target genes between both groups

Fig. 3

vFlip expression activates the NFκB pathway in IECs. a Representative pictures of an immunohistochemical P65 staining of small intestinal cross sections from indicated mice. Left panel: microscopic pictures, scale bar 50 µm. Right panel: confocal images of nuclear P65 translocation. Scale bar 7.5 µm. b Western Blot analyses of P65, phospho-P65, phospho-IκBα and iNOS using proteins isolated from small intestinal IECs of indicated mice. Actin was used as a loading control. c Western Blot analyses of P100 and P52 using proteins from small intestinal IECs of indicated mice. Actin was used as a loading control

Fig. 4

vFLIP-induced inflammation and Paneth cell loss depends on NFκB signaling. a Representative staining of H&E and immunohistochemical F4/80 and CD4 staining on small intestinal cross sections from indicated mice. Scale bar 100 µm. b Histology score of H&E stained small intestinal cross sections of control (n = 18), Ikka^ΔIEC (n = 10), vFlip^IEC-tg (n = 17) and Ikka^ΔIEC x vFlip^IEC-tg (n = 14) animals, *p < 0.05, **p < 0.01, ***p < 0.001. b Transcriptional analysis of Tnfa (n ≥ 9) in the small intestine of control and vFlip^IEC-tg mice. Values are shown + SD and were calculated relative to control mice. Hprt was used as internal standard. Data were pooled from 3 individual experiments, ***p < 0.001. c Representative pictures of an immunohistochemical lysozyme staining of small intestinal cross sections from indicated mice. Scale bar upper panel: 500 µm, lower panel: 100 µm. d Transcriptional analysis of Lyz (n ≥ 9) in the small intestine of control and vFlip^IEC-tg mice. Values are shown + SD and were calculated relative to control mice. Hprt was used as internal standard. Data were pooled from 3 individual experiments, **p < 0.01, ***p < 0.001