Hypoxia regulates autophagy and nucleotide-binding oligomerization domain receptor, pyrin domain containing (NLRP)3, two innate immune mechanisms linked by mutual regulation and associated to IBD. Here we show that hypoxia ameliorates inflammation during the development of colitis by modulating autophagy and mammalian target of rapamycin (mTOR)/NLRP3 pathway. Hypoxia significantly reduces tumor necrosis factor α, interleukin (IL)-6 and NLRP3 expression, and increases the turnover of the autophagy protein p62 in colon biopsies of Crohn’s disease patients, and in samples from dextran sulfate sodium-treated mice and Il-10^−/− mice. In vitro, NF-κB signaling and NLRP3 expression are reduced through hypoxia-induced autophagy. We also identify NLRP3 as a novel binding partner of mTOR. Dimethyloxalylglycine-mediated hydroxylase inhibition ameliorates colitis in mice, downregulates NLRP3 and promotes autophagy. We suggest that hypoxia counteracts inflammation through the downregulation of the binding of mTOR and NLRP3 and activation of autophagy.

Objective: Western lifestyle and diet are major environmental factors playing a role in the development of IBD. Titanium dioxide (TiO₂) nanoparticles are widely used as food additives or in pharmaceutical formulations and are consumed by millions of people on a daily basis. We investigated the effects of TiO₂in the development of colitis and the role of the nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3 inflammasome.Design: Wild-type and NLRP3-deficient mice with dextran sodium sulfate-induced colitis were orally administered with TiO₂nanoparticles. The proinflammatory effects of TiO₂particles in cultured human intestinal epithelial cells (IECs) and macrophages were also studied, as well as the ability of TiO₂crystals to traverse IEC monolayers and accumulate in the blood of patients with IBD using inductively coupled plasma mass spectrometry.Results: Oral administration of TiO₂nanoparticles worsened acute colitis through a mechanism involving the NLRP3 inflammasome. Importantly, crystals were found to accumulate in spleen of TiO₂-administered mice. In vitro, TiO₂particles were taken up by IECs and macrophages and triggered NLRP3-ASC-caspase-1 assembly, caspase-1 cleavage and the release of NLRP3-associated interleukin (IL)-1β and IL-18. TiO₂also induced reactive oxygen species generation and increased epithelial permeability in IEC monolayers. Increased levels of titanium were found in blood of patients with UC having active disease.Conclusion: These findings indicate that individuals with a defective intestinal barrier function and pre-existing inflammatory condition, such as IBD, might be negatively impacted by the use of TiO₂nanoparticles.

Flavonoids may play an important role for adjunct nutritional therapy of chronic intestinal inflammation. In this study, we characterized the molecular mechanisms by which quercetin and its enteric bacterial metabolites, taxifolin, alphitonin, and 3, 4-dihydroxy-phenylacetic acid, inhibit tumor necrosis factor α (TNF)-induced proinflammatory gene expression in the murine small intestinal epithelial cell (IEC) line Mode-K as well as in heterozygous TNFΔARE/WT mice, a murine model of experimental ileitis. Quercetin inhibited TNF-induced interferon-γ-inducible protein 10 (IP-10) and macrophage inflammatory protein 2 (MIP-2) gene expression in Mode-K cells with effective inhibitory concentration of 40 and 44 μmol/L, respectively. Interestingly, taxifolin, alphitonin, and 3,4-dihydroxy-phenylacetic acid did not inhibit TNF responses in IEC, suggesting that microbial transformation of quercetin completely abolished its anti-inflammatory effect. At the molecular level, quercetin inhibited Akt phosphorylation but did not inhibit TNF-induced RelA/I-κB phosphorylation and IκB degradation or TNF-α-induced nuclear factor-κB transcriptional activity. Most important for understanding the mechanism involved, chromatin immunoprecipitation analysis revealed inhibitory effects of quercetin on phospho-RelA recruitment to the IP-10 and MIP-2 gene promoters. In addition, and consistent with the lack of cAMP response element binding protein (CBP)/p300 recruitment and phosphorylation/acetylation of histone 3 at the promoter binding site, quercetin inhibited histone acetyl transferase activity. The oral application of quercetin to heterozygous TNFΔARE/WT mice [10 mg/(d x kg body wt)] significantly inhibited IP-10 and MIP-2 gene expression in primary ileal epithelial cells but did not affect tissue pathology. These studies support an anti-inflammatory effect of quercetin in epithelial cells through mechanisms that inhibit cofactor recruitment at the chromatin of proinflammatory genes.

The molecular understanding of nutritional factors in the process of host factor–mediated activation of the intestinal epithelium may play an important role in the assessment of adjunct nutritional therapy for chronic intestinal inflammation. We characterized the molecular mechanisms of flavonoids including apigenin, luteolin, genistein, 3′-hydroxy-flavone, and flavone in inhibiting tumor necrosis factor-α (TNF)-induced interferon-induced protein (IP)-10 gene expression in the murine intestinal epithelial cell (IEC) line Mode-K. We demonstrated that 3′-hydroxy-flavone but not the chemical core structure flavone blocked TNF-α–induced nuclear factor (NF)-κB transcriptional activity and IP-10 expression at the level of NF-κB/IκBα phosphorylation/degradation by inhibiting IκB kinase activity. Although 3′-hydroxy-flavone effectively triggered p38 mitogen-activated protein kinase signaling and late caspase-3 cleavage, the induction of apoptotic cell death in TNF-activated IEC was not the primary mechanism inhibiting NF-κB transcriptional activity and IP-10 expression. In addition to the compound-specific inhibition of TNF-induced NF-κB DNA binding and NF-κB transcriptional activity, apigenin and luteolin selectively blocked Akt phosphorylation/activity. The ability of these polyphenolic compounds to target various signal transduction pathways was further supported by the observation that luteolin and 3′-hydroxy-flavone selectively induced interferon regulatory factor (IRF)-1 degradation. Finally, we showed that genistein blocked IP-10 but not IL-6 expression through NF-κB, IRF, and Akt independent mechanisms, demonstrating the functional diversity of flavonoids in inhibiting proinflammatory processes in IEC. In conclusion, we provide molecular evidence for the presence of characteristic inhibition patterns of these polyphenolic compounds to inhibit proinflammatory gene expression in IEC through the specific modulation of the NF-κB, IRF and Akt signaling pathways.

Summary: Bifidobacteria comprise a dominant microbial population group in the human intestinal tract with purported beneficial health effects on the host. In this study, we characterized the molecular mechanisms for the initial interaction of probiotic Bifidobacterium lactis strain BB12 with native and intestinal epithelial cell (IEC) lines. We showed that B. lactis‐monoassociated Fisher F344 rats transiently induce phosphorylation/activation of the NF‐κB transcriptionally active subunit RelA and the mitogen‐activated protein kinase (MAPK) p38 in native IEC at day 5 after initial bacterial colonization. In addition, Interleukin 6 (IL‐6) gene expression was significantly increased at day 5, demonstrating the physiological relevance of transient transcription factor activation in IEC. In contrast, Bacteroides vulgatus‐monoassociated Fisher rats revealed RelA but not p38 MAPK phosphorylation and failed to trigger significant IL‐6 gene expression in native IEC. Moreover, we demonstrated that B. lactis triggers NF‐κB RelA and p38 MAPK phosphorylation in IEC lines. Adenoviral delivery of mutant IKK‐β (Ad5dnIKKβ) and inhibition of the p38 MAPK pathway through the pharmacological inhibitor SB203580 significantly blocked B. lactis‐induced IL‐6 gene expression in IEC, suggesting that B. lactis triggers NF‐κB and MAPK signaling to induce gene expression in the intestinal epithelium. Regarding the mechanisms of bacteria epithelial cell cross‐talk, B. lactis‐induced IL‐6 gene expression was completely inhibited in TLR2 deficient mouse embryogenic fibroblasts (MEF TLR2^−/−) as well as TLR2ΔTIR transfected Mode‐K cells. In conclusion, we demonstrated that probiotic bacteria transiently trigger innate signal transduction and pro‐inflammatory gene expression in the intestinal epithelium at early stages of bacterial colonization.