Regression of apoptosis-resistant colorectal tumors by induction of necroptosis in mice.

Cancer cells often acquire capabilities to evade cell death induced by current chemotherapeutic treatment approaches. Caspase-8, a central initiator of death receptor-mediated apoptosis, for example, is frequently inactivated in human cancers via multiple mechanisms such as mutation. Here, we show an approach to overcome cell death resistance in caspase-8-deficient colorectal cancer (CRC) by induction of necroptosis. In both a hereditary and a xenograft mouse model of caspase-8-deficient CRC, second mitochondria-derived activator of caspase (SMAC) mimetic treatment induced massive cell death and led to regression of tumors. We further demonstrate that receptor-interacting protein kinase 3 (RIP3), which is highly expressed in mouse models of CRC and in a subset of human CRC cell lines, is the deciding factor of cancer cell susceptibility to SMAC mimetic-induced necroptosis. Thus, our data implicate that it may be worthwhile to selectively evaluate the efficacy of SMAC mimetic treatment in CRC patients with caspase-8 deficiency in clinical trials for the development of more effective personalized therapy.

CD14 Plays a Protective Role in Experimental Inflammatory Bowel Disease by Enhancing Intestinal Barrier Function.

Intestinal homeostasis disturbance through intestinal barrier disruption presumably plays a key role in inflammatory bowel disease (IBD) development. Genetic and candidate gene analyses in an Il10-deficient IBD mouse model system identified Cd14 as a potentially protective candidate gene. The role of Cd14 in colitis development was determined using dextran sulfate sodium (DSS)-induced acute and an Il10-deficiency-induced chronic model of intestinal inflammation. Intestinal permeability was investigated by fluorescein isothiocyanate-dextran uptake assay, quantitative RT-PCR analysis of tight junction proteins, myosin light chain kinase, and proinflammatory cytokine expression. Immunohistological staining of occludin, Ki-67, NF-κB-p65, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was performed, and intestinal inflammation severity was evaluated histologically. Untreated B6-Cd14-/- mice and wild-type controls did not differ in intestinal barrier function. However, DSS-treated Cd14-deficient and B6-Il10-/-Cd14-/- mice exhibited more severe intestinal barrier disruption, with increased histological scores and proinflammatory cytokine expression, compared to controls. Therefore, Cd14 deficiency did not influence epithelial integrity under steady-state conditions but caused intestinal barrier dysfunction under inflammation. As expected, CD14 overexpression increased barrier integrity. No difference in intestinal epithelial NF-κB translocation was observed between the investigated groups. Intestinal myosin light chain kinase expression decreased in Cd14-deficient mice under steady-state conditions and in the chronic model, whereas no difference was detected in the DSS models. Thus, CD14 plays a protective role in IBD development by enhancing intestinal barrier function.

Activation of Epithelial Signal Transducer and Activator of Transcription 1 by Interleukin 28 Controls Mucosal Healing in Mice With Colitis and Is Increased in Mucosa of Patients With Inflammatory Bowel Disease.

BACKGROUND & AIMS: We investigated the roles of interleukin 28A (also called IL28A or interferon λ2) in intestinal epithelial cell (IEC) activation, studying its effects in mouse models of inflammatory bowel diseases (IBD) and intestinal mucosal healing. METHODS: Colitis was induced in C57BL/6JCrl mice (controls), mice with IEC-specific disruption of Stat1 (Stat1IEC-KO), mice with disruption of the interferon λ receptor 1 gene (Il28ra-/-), and mice with disruption of the interferon regulatory factor 3 gene (Irf3-/-), with or without disruption of Irf7 (Irf7-/-). We used high-resolution mini-endoscopy and in vivo imaging methods to assess colitis progression. We used 3-dimensional small intestine and colon organoids, along with RNA-Seq and gene ontology methods, to characterize the effects of IL28 on primary IECs. We studied the effects of IL28 on the human intestinal cancer cell line Caco-2 in a wound-healing assay, and in mice colon wounds. Colonic biopsies and resected tissue from patients with IBD (n = 62) and patients without colon inflammation (controls, n = 23) were analyzed by quantitative polymerase chain rection to measure expression of IL28A, IL28RA, and other related cytokines; biopsy samples were also analyzed by immunofluorescence to identify sources of IL28 production. IECs were isolated from patient tissues and incubated with IL28; signal transducer and activator of transcription 1 (STAT1) phosphorylation was measured by immunoblots and confocal imaging. RESULTS: Lamina propria cells in colon tissues of patients with IBD, and mice with colitis, had increased expression of IL28 compared with controls; levels of IL28R were increased in the colonic epithelium of patients with IBD and mice with colitis. Administration of IL28 induced phosphorylation of STAT1 in primary human and mouse IECs, increasing with dose. Il28ra-/-, Irf3-/-, Irf3-/-Irf7-/-, as well as Stat1IEC-KO mice, developed more severe colitis after administration of dextran sulfate sodium than control mice, with reduced epithelial restitution. Il28ra-/- and Stat1IEC-KO mice also developed more severe colitis in response to oxazolone than control mice. We found IL28 to induce phosphorylation (activation) of STAT1 in epithelial cells, leading to their proliferation in organoid culture. Administration of IL28 to mice with induced colonic wounds promoted mucosal healing. CONCLUSIONS: IL28 controls proliferation of IECs in mice with colitis and accelerates mucosal healing by activating STAT1. IL28 might be developed as a therapeutic agent for patients with IBD.

Caspase-8 controls the gut response to microbial challenges by Tnf-α-dependent and independent pathways.

OBJECTIVES: Intestinal epithelial cells (IEC) express toll-like receptors (TLR) that facilitate microbial recognition. Stimulation of TLR ligands induces a transient increase in epithelial cell shedding, a mechanism that serves the antibacterial and antiviral host defence of the epithelium and promotes elimination of intracellular pathogens. Although activation of the extrinsic apoptosis pathway has been described during inflammatory shedding, its functional involvement is currently unclear. DESIGN: We investigated the functional involvement of caspase-8 signalling in microbial-induced intestinal cell shedding by injecting Lipopolysaccharide (LPS) to mimic bacterial pathogens and poly(I:C) as a probe for RNA viruses in vivo. RESULTS: TLR stimulation of IEC was associated with a rapid activation of caspase-8 and increased epithelial cell shedding. In mice with an epithelial cell-specific deletion of caspase-8 TLR stimulation caused Rip3-dependent epithelial necroptosis instead of apoptosis. Mortality and tissue damage were more severe in mice in which IECs died by necroptosis than apoptosis. Inhibition of receptor-interacting protein (Rip) kinases rescued the epithelium from TLR-induced gut damage. TLR3-induced necroptosis was directly mediated via TRIF-dependent pathways, independent of Tnf-α and type III interferons, whereas TLR4-induced tissue damage was critically dependent on Tnf-α. CONCLUSIONS: Together, our data demonstrate an essential role for caspase-8 in maintaining the gut barrier in response to mucosal pathogens by permitting inflammatory shedding and preventing necroptosis of infected cells. These data suggest that therapeutic strategies targeting the cell death machinery represent a promising new option for the treatment of inflammatory and infective enteropathies.

Regulation and pathophysiological role of epithelial turnover in the gut.

Cell death in the intestinal epithelium has to be tightly controlled. Excessive or misplaced epithelial cell death can result in barrier dysfunction and, as a consequence thereof, uncontrolled translocation of components of the microbial flora from the lumen into the bowel wall. Susceptibility to gastrointestinal infections or chronic inflammation of the gut, as observed in patients with inflammatory bowel disease, can be the result of such dysregulation. Conversely, defects in cell death initiation might lead to an irregular accumulation of epithelial cells and cause intestinal cancer development. Until recently, activation of caspases in the intestinal epithelium was considered as a potential contributor to barrier dysfunction and as a pathogenic factor in the development of intestinal inflammation. Thus blocking of caspases appeared to be a potential therapeutic option for patients with inflammatory bowel disease. Recent studies on necroptosis however demonstrated that also inhibition of caspases can cause barrier dysfunction and intestinal inflammation. Caspase-8 on top of its functions in the extrinsic apoptosis pathway also controls necroptosis and turns out to be an essential molecule in regulating tissue homeostasis in the gut. Epithelial caspase-8 therefore emerges as a checkpoint not only of cell survival and cell death, but also as a regulator of the mode of cell death. According to this model, both excessive activity as well as a lack of activity of caspase-8 results in epithelial cell death and intestinal inflammation and caspase-8 needs to be tightly controlled to warrant tissue homeostasis in the gut.