Intestinal inflammation alters mucosal carbohydrate foraging and monosaccharide incorporation into microbial glycans.

Endogenous carbohydrates released from the intestinal mucus represent a constant source of nutrients to the intestinal microbiota. Mucus-derived carbohydrates can also be used as building blocks in the biosynthesis of bacterial cell wall components, thereby influencing host mucosal immunity. To assess the uptake of endogenous carbohydrates by gut microbes in healthy mice and during intestinal inflammation, we applied azido-monosaccharides that can be tracked on bacterial cell walls after conjugation with fluorophores. In interleukin-10 deficient mice, changes in the gut microbiota were accompanied by decreased carbohydrate hydrolase activities and increased lumenal concentrations of host glycan-derived monosaccharides. Tracking of the monosaccharide N-azidoacetylglucosamine (GlcNAz) in caecum bacteria revealed a preferential incorporation of this carbohydrate by Xanthomonadaceae in healthy mice and by Bacteroidaceae in interleukin-10 deficient mice. These GlcNAz-positive Bacteroidaceae fractions mainly belonged to the species B. acidifaciens and B. vulgatus. Growth of Bacteroides species in the presence of specific monosaccharides changed their stimulatory activity toward CD11c+ dendritic cells. Expression of activation markers and cytokine production was highest after stimulation of dendritic cells with B. vulgatus. The variable incorporation of monosaccharides by related Bacteroides species underline the necessity to investigate intestinal bacteria down to the species level when addressing microbiota-host interactions.

Alleviation of Intestinal Inflammation by Oral Supplementation With 2-Fucosyllactose in Mice.

Milk oligosaccharides exert a prebiotic action that contributes to the development of the infant gut microbiota during lactation. Given that milk oligosaccharides remain intact after passage through stomach and small intestine, they can potentially influence the composition of the gut microbiota when ingested as dietary supplements after weaning. To address the regulatory effects of specific oligosaccharides in colitis linked to the microbiota composition, we have supplemented interleukin-10 null (Il10 -/-) mice with four fucosylated and sialylated oligosaccharides. We found that oral supplementation with 2-fucosyllactose significantly decreased the severity of colitis as displayed by reduced inflammatory marker expression, histological and diarrhea scores, an increased epithelial integrity and less pronounced colon shortening. Oral supplementation with 2-fucosyllactose led to a marked expansion of the commensal Ruminococcus gnavus, which was accompanied by an enhanced cecal concentration of propionate. Decreased activation of immune cells by R. gnavus was confirmed by reconstitution of antibiotic-treated Il10 -/- mice and by stimulation of dendritic cells in vitro. This study demonstrates that post-weaning administration of specific oligosaccharides can shift the composition of the gut microbiota to lessen chronic inflammation as observed in Il10 -/- mice. The expansion of R. gnavus sets a positive microbial environment at the cost of pro-inflammatory Gram-negative bacteria, thereby lowering intestinal inflammation.

TNFα sensitizes hepatocytes to FasL-induced apoptosis by NFκB-mediated Fas upregulation.

Although it is well established that TNFα contributes to hepatitis, liver failure and associated hepatocarcinogenesis via the regulation of inflammation, its pro-apoptotic role in the liver has remained enigmatic. On its own, TNFα is unable to trigger apoptosis. However, when combined with the transcriptional inhibitor GaLN, it can cause hepatocyte apoptosis and liver failure in mice. Moreover, along with others, we have shown that TNFα is capable of sensitizing cells to FasL- or drug-induced cell death via c-Jun N-terminal kinase (JNK) activation and phosphorylation/activation of the BH3-only protein Bim. In this context, TNFα could exacerbate hepatocyte cell death during simultaneous inflammatory and T-cell-mediated immune responses in the liver. Here we show that TNFα sensitizes primary hepatocytes, established hepatocyte cell lines and mouse embryo fibroblasts to FasL-induced apoptosis by the transcriptional induction and higher surface expression of Fas via the NFκB pathway. Genetic deletion, diminished expression or dominant-negative inhibition of the NFκB subunit p65 resulted in lower Fas expression and inhibited TNFα-induced Fas upregulation and sensitization to FasL-induced cell death. By hydrodynamic injection of p65 shRNA into the tail vein of mice, we confirm that Fas upregulation by TNFα is also NFκB-mediated in the liver. In conclusion, TNFα sensitization of FasL-induced apoptosis in the liver proceeds via two parallel signaling pathways, activation of JNK and Bim phosphorylation and NFκB-mediated Fas upregulation.

Inhibitor of Apoptosis Protein-1 Regulates Tumor Necrosis Factor-Mediated Destruction of Intestinal Epithelial Cells.

BACKGROUND AND AIMS: Tumor necrosis factor (TNF) is a cytokine that promotes inflammation and contributes to pathogenesis of inflammatory bowel diseases. Unlike other cells and tissues, intestinal epithelial cells undergo rapid cell death upon exposure to TNF, by unclear mechanisms. We investigated the roles of inhibitor of apoptosis proteins (IAPs) in the regulation of TNF-induced cell death in the intestinal epithelium of mice and intestinal organoids. METHODS: RNA from cell lines and tissues was analyzed by quantitative polymerase chain reaction, protein levels were analyzed by immunoblot assays. BIRC2 (also called cIAP1) was expressed upon induction from lentiviral vectors in young adult mouse colon (YAMC) cells. YAMC cells, the mouse colon carcinoma cell line MC38, the mouse macrophage cell line RAW 264.7, or mouse and human organoids were incubated with second mitochondrial activator of caspases (Smac)-mimetic compound LCL161 or recombinant TNF-like weak inducer of apoptosis (TNFSF12) along with TNF, and cell death was quantified. C57BL/6 mice with disruption of Xiap, Birc2 (encodes cIAP1), Birc3 (encodes cIAP2), Tnfrsf1a, or Tnfrsf1b (Tnfrsf1a and b encode TNF receptors) were injected with TNF or saline (control); liver and intestinal tissues were collected and analyzed for apoptosis induction by cleaved caspase 3 immunohistochemistry. We also measured levels of TNF and alanine aminotransferase in serum from mice. RESULTS: YAMC cells, and mouse and human intestinal organoids, died rapidly in response to TNF. YAMC and intestinal crypts expressed lower levels of XIAP, cIAP1, cIAP2, and cFLIP than liver tissue. Smac-mimetics reduced levels of cIAP1 and XIAP in MC38 and YAMC cells, and Smac-mimetics and TNF-related weak inducer of apoptosis increased TNF-induced cell death in YAMC cells and organoids-most likely by sequestering and degrading cIAP1. Injection of TNF greatly increased levels of cell death in intestinal tissue of cIAP1-null mice, compared with wild-type C57BL/6 mice, cIAP2-null mice, or XIAP-null mice. Excessive TNF-induced cell death in the intestinal epithelium was mediated TNF receptor 1. CONCLUSIONS: In a study of mouse and human cell lines, organoids, and tissues, we found cIAP1 to be required for regulation of TNF-induced intestinal epithelial cell death and survival. These findings have important implications for the pathogenesis of TNF-mediated enteropathies and chronic inflammatory diseases of the intestine.

Analysis of Cell Death Induction in Intestinal Organoids In Vitro.

The intestinal epithelium has an important function in the absorption of nutrients contained in the food. Furthermore, it also has an important barrier function, preventing luminal pathogens from entering the bloodstream. This single cell layer epithelium is quite sensitive to various cell death-promoting triggers, including drugs, irradiation, and TNF family members, leading to loss of barrier integrity, epithelial erosion, inflammation, malabsorption, and diarrhea. In order to assess the intestinal epithelium-damaging potential of treatments and substances specific test systems are required. As intestinal tumor cell lines are a poor substitute for primary intestinal epithelial cells, and in vivo experiments in mice are costly and often unethical, the use of intestinal organoids cultured from intestinal crypts provide an ideal tool to study cell death induction and mechanisms in primary intestinal epithelial cells. This protocol describes the isolation and culture of intestinal organoids from murine small intestinal crypts, and the quantitative assessment of cell death induction in these organoids.

Cell death at the intestinal epithelial front line.

The intestinal epithelium represents the largest epithelial surface in our body. This single-cell-layer epithelium mediates important functions in the absorption of nutrients and in the maintenance of barrier function, preventing luminal microorganisms from invading the body. Due to its constant regeneration the intestinal epithelium is a tissue not only with very high proliferation rates but also with very prominent physiological and pathophysiological cell death induction. The normal physiological differentiation and maturation of intestinal epithelial cells leads to their shedding and apoptotic cell death within a few days, without disturbing the epithelial barrier integrity. In contrast excessive intestinal epithelial cell death induced by irradiation, drugs and inflammation severely impairs the vital functions of this tissue. In this review we discuss cell death processes in the intestinal epithelium in health and disease, with special emphasis on cell death triggered by the tumour necrosis factor receptor family.

Blocking TWEAK-Fn14 interaction inhibits hematopoietic stem cell transplantation-induced intestinal cell death and reduces GVHD.

Inhibition of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) system reduces intestinal cell death and disease development in several models of colitis. In view of the crucial role of TNF and intestinal cell death in graft-versus-host disease (GVHD) and the ability of TWEAK to enhance TNF-induced cell death, we tested here the therapeutic potential of Fn14 blockade on allogeneic hematopoietic cell transplantation (allo-HCT)-induced intestinal GVHD. An Fn14-specific blocking human immunoglobulin G1 antibody variant with compromised antibody-dependent cellular cytotoxicity (ADCC) activity strongly inhibited the severity of murine allo-HCT-induced GVHD. Treatment of the allo-HCT recipients with this monoclonal antibody reduced cell death of gastrointestinal cells but neither affected organ infiltration by donor T cells nor cytokine production. Fn14 blockade also inhibited intestinal cell death in mice challenged with TNF. This suggests that the protective effect of Fn14 blockade in allo-HCT is based on the protection of intestinal cells from TNF-induced apoptosis and not due to immune suppression. Importantly, Fn14 blockade showed no negative effect on graft-versus-leukemia/lymphoma (GVL) activity. Thus, ADCC-defective Fn14-blocking antibodies are not only possible novel GVL effect-sparing therapeutics for the treatment of GVHD but might also be useful for the treatment of other inflammatory bowel diseases where TNF-induced cell death is of relevance.