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Although trichuriasis, a zoonotic disease, has recently become rare in Japan due to improved environmental hygiene, we herein report a 79-year-old man in whom a worm was incidentally found in the ascending colon during colonoscopy for positive fecal occult blood and was endoscopically removed. A genetic analysis identified the worm as Trichuris trichiura possessing mixed sequences from non-human primate and human origins. Despite controversy regarding Trichuris trichiura infection originating from Japanese macaques, according to some studies, it originates primarily from humans. This report suggests the efficacy of a genetic analysis for identifying infection sources.

Nickel, the most frequent contact allergy cause, is widely used for various metallic materials and medical devices. Autophagy is an intracellular protein degradation system and contributes to metal recycling. However, it is unclear the functions of nickel in autophagy. We here demonstrated that NiCl2 induced microtubule-associated protein 1 light chain 3 (LC3)-II and LC3 puncta, markers of autophagosomes. Bafilomycin A1 (BafA1) treatment did not enhance LC3 puncta under NiCl2 stimulation, suggesting that NiCl2 did not induce autophagic flux. In addition, NiCl2 promotes the accumulation of SQSTM1/p62 and increased SQSTM1/p62 colocalization with lysosomal-associated membrane protein 1 (LAMP1). These data indicated that NiCl2 attenuates autophagic flux. Interestingly, NiCl2 induced the expression of the high-molecular-weight (MW) form of SQSTM1/p62. Inhibition of NiCl2-induced reactive oxygen species (ROS) reduced the high-MW SQSTM1/p62. We also showed that NiCl2-induced ROS activate transglutaminase (TG) activity. We found that transglutaminase 2 (TG2) inhibition reduced high-MW SQSTM1/p62 and SQSTM1/p62 puncta under NiCl2 stimulation, indicating that TG2 regulates SQSTM1/p62 protein homeostasis under NiCl2 stimulation. Our study demonstrated that nickel ion regulates autophagy flux and TG2 restricted nickel-dependent proteostasis.

Ikarugamycin (IK) is an antibiotic which has been reported to have a variety of functions, such as inhibition of clathrin-mediated endocytosis (CME), anti-tumor effects and regulation of the immune system. Whether IK influences cytokine production is poorly understood. We have investigated the relationship between IK and production of tumor necrosis factor-α (TNF). TNF plays a pivotal role in pathogenesis of many diseases. Although the dynamics of soluble TNF (sTNF) has been widely explored so far, the functions of the membrane form of TNF (mTNF) have not been fully elucidated. We demonstrated that IK increases the amount of mTNF and prolongs the duration of TNF expression. This effect is unrelated to the shedding activity of disintegrin and metalloproteinase domain-containing protein 17 (ADAM 17). Our results revealed that there is a mechanism to terminate inflammation at the cellular level which IK dysregulates. Furthermore, IK can be a tool to study TNF signaling due to its effect of increasing mTNF expression.

Genome-wide association studies have identified autophagy-related susceptibility genes for inflammatory bowel disease (IBD); however, whether autophagy regulators can be utilized as therapeutic targets remains unclear. To identify novel microtubule-associated protein 1 light chain 3 (LC3)-interacting proteins in intestinal epithelial cells (IECs), we isolated primary IECs from green fluorescent protein (GFP)-LC3 mice. We performed immunoprecipitation with a GFP antibody and then analyzed co-immunoprecipitates by mass spectrometry. HADHA was identified as an LC3-interacting protein from primary IECs. The HADHA gene encodes the alpha subunit of the mitochondrial trifunctional protein. Given that HADHA catalyzes the last three steps of mitochondrial beta-oxidation of long-chain fatty acids, we investigated whether long-chain fatty acids induce autophagy in IECs. We found that palmitic acid induced autophagy in DLD-1, HT29, and HCT116 cells. HADHA was expressed in not only the mitochondria but also the cytosol. LC3 puncta co-localized with HADHA, which were enhanced by palmitic acid stimulation. However, LC3 puncta did not co-localize with Tom20, suggesting that HADHA was induced to associate with LC3 puncta at sites other than the mitochondria. Thus, HADHA may have extra-mitochondrial functions. Furthermore, we found that palmitic acid induced cell death in IECs, which was accelerated by bafilomycin A and chloroquine. These findings suggested that palmitic acid-induced autophagy supports the survival of IECs. Taken together, these results suggested that HADHA is involved in long-chain fatty acid-induced autophagy in IECs, thus providing new insights into the pathology of IBD and revealing novel therapeutic targets of IBD.

A20 (TNFAIP3) and ABIN-1 (TNIP1), two candidate inflammatory bowel disease (IBD) susceptibility genes, preserve intestinal homeostasis by cooperatively restricting intestinal epithelial cell death. A20 and ABIN-1 synergistically restrict both TNF-dependent and TNF-independent cell death., A20 (TNFAIP3) and ABIN-1 (TNIP1) are candidate susceptibility genes for inflammatory bowel disease and other autoimmune or inflammatory diseases, but it is unclear how these proteins interact in vivo to prevent disease. Here we show that intestinal epithelial cell (IEC)-specific deletion of either A20 or ABIN-1 alone leads to negligible IEC loss, whereas simultaneous deletion of both A20 and ABIN-1 leads to rapid IEC death and mouse lethality. Deletion of both A20 and ABIN-1 from enteroids causes spontaneous cell death in the absence of microbes or hematopoietic cells. Studies with enteroids reveal that A20 and ABIN-1 synergistically restrict death by inhibiting TNF-induced caspase 8 activation and RIPK1 kinase activity. Inhibition of RIPK1 kinase activity alone, or caspase inhibition combined with RIPK3 deletion, abrogates IEC death by blocking both apoptosis and necroptosis in A20 and ABIN-1 double-deficient cells. These data show that the disease susceptibility proteins A20 and ABIN-1 synergistically prevent intestinal inflammation by restricting IEC death and preserving tissue integrity.

We previously reported that IL-7−/−RAG−/− mice receiving naive T cells failed to induce colitis. Such abrogation of colitis may be associated with not only incomplete T cell maintenance due to the lack of IL-7, but also with the induction of colitogenic CD4+ T cell apoptosis at an early stage of colitis development. Moreover, NK cells may be associated with the suppression of pathogenic T cells in vivo, and they may induce apoptosis of CD4+ T cells. To further investigate these roles of NK cells, RAG−/− and IL-7−/−RAG−/− mice that had received naive T cells were depleted of NK cells using anti-asialo GM1 and anti-NK1.1 Abs. NK cell depletion at an early stage, but not at a later stage during colitogenic effector memory T cell (TEM) development, resulted in exacerbated colitis in recipient mice even in the absence of IL-7. Increased CD44+CD62L− TEM and unique CD44−CD62L− T cell subsets were observed in the T cell-reconstituted RAG−/− recipients when NK cells were depleted, although Fas, DR5, and IL-7R expressions in this subset differed from those in the CD44+CD62L− TEM subset. NK cell characteristics were the same in the presence or absence of IL-7 in vitro and in vivo. These results suggest that NK cells suppress colitis severity in T cell-reconstituted RAG−/− and IL-7−/−RAG−/− recipient mice through targeting of colitogenic CD4+CD44+CD62L− TEM and, possibly, of the newly observed CD4+CD44−CD62L− subset present at the early stage of T cell development.

SummaryInappropriate inflammasome activation contributes to multiple human diseases, but the mechanisms by which inflammasomes are suppressed are poorly understood. The NF-κB inhibitor A20 is a ubiquitin-modifying enzyme that might be critical in preventing human inflammatory diseases. Here, we report that A20-deficient macrophages, unlike normal cells, exhibit spontaneous NLRP3 inflammasome activity to LPS alone. The kinase RIPK3, but not the adaptor MyD88, is required for this response. In normal cells, A20 constitutively associates with caspase-1 and pro-IL-1β, and NLRP3 activation further promotes A20 recruitment to the inflammasome. Pro-IL-1β also co-immunoprecipitates with RIPK1, RIPK3, caspase-1, and caspase-8 in a complex that is modified with K63-linked and unanchored polyubiquitin. In A20-deficient macrophages, this pro-IL-1β-associated ubiquitination is markedly increased in a RIPK3-dependent manner. Mass spectrometric and mutational analyses reveal that K133 of pro-IL-1β is a physiological ubiquitination site that supports processing. Our study reveals a mechanism by which A20 prevents inflammatory diseases.

Inflammatory bowel diseases encompass gastrointestinal illnesses typified by chronic inflammation, loss of epithelial integrity and gastrointestinal microbiota dysbiosis. In an effort to counteract these characteristic perturbations, we used stem cells and/or a probiotic therapy in a murine model of Dextran Sodium Sulfate induced colitis to examine both their efficacy in ameliorating disease and impact on niche-specific microbial communities of the lower GI tract. Colitis was induced in C57BL/6 mice by administering 3% DSS in drinking water for 10 days prior to administering one of three treatment plans: daily probiotic (VSL#3) supplementation for 3 days, a single tail vein injection of 1x10 (6) murine mesenchymal stem cells, or both. Ileal, cecal and colonic sections were collected for microbiota and histological analyses. Microbiota profiling revealed distinct bacterial community compositions in the ileum, cecum and colon of control untreated animals, all of which were predicted in silico to be enriched for a number of discrete KEGG pathways, indicating compositional and functional niche specificity in healthy animals. DSS-treatment perturbed community composition in all three niches with ileal communities exhibiting the greatest change relative to control animals. Each treatment group exhibited treatment-specific alterations in microbiota composition in the lower GI tract, though disease scores were only improved in VSL#3-treated animals. The ileal microbiota were most profoundly altered in composition in this group of animals and characterized by significant Enterobacteriaceae enrichment compared with colitic mice (P

It has been suggested that prolonged inflammatory bowel diseases (IBD) may lead to colitis-associated carcinogenesis (CAC). We previously observed that the NF-κB activation in colonic epithelial cells is associated with increased tumor necrosis factor receptor 2 (TNFR2) expression in CAC development. However, the mechanism by which epithelial NF-κB activation leading to CAC is still unclear. Myosin light chain kinase (MLCK) has been reported to be responsible for the epithelial permeability associated with TNF signaling. Therefore we focused on the role of MLCK expression via TNFR2 signaling on CAC development. Pro-tumorigenic cytokines such as IL-1β, IL-6 and MIP-2 production as well as INF-γ and TNF production at the lamina propria were increased in the setting of colitis, and further in tumor tissues in associations with up-regulated TNFR2 and MLCK expressions in the epithelial cells of a CAC model. The up-regulated MLCK expression was observed in TNF-stimulated colonic epithelial cells in a dose-dependent fashion in association with up-regulation of TNFR2. Silencing TNFR2, but not TNFR1, resulted in restoration of epithelial tight junction (TJ) associated with decreased MLCK expression. Antibody-mediated blockade of TNF signaling also resulted in restoration of TJ in association with suppressed MLCK expression, and interestingly, similar results were observed with suppressing TNFR2 and MLCK expressions by inhibiting MLCK in the epithelial cells. Silencing of MLCK also resulted in suppressed TNFR2, but not TNFR1, expression, suggesting that the restored TJ leads to reduced TNFR2 signaling. Such suppression of MLCK as well as blockade of TNFR2 signaling resulted in restored TJ, decreased pro-tumorigenic cytokines and reduced CAC development. These results suggest that MLCK may be a potential target for the prevention of IBD-associated tumor development.

The microRNA miR-210 is a signature of hypoxia. We found robust increase in the abundance of miR-210 (>100-fold) in activated T cells, especially in the TH17 lineage of helper T cells. Hypoxia acted in synergy with stimulation via the T cell antigen receptor (TCR) and coreceptor CD28 to accelerate and increase Mir210 expression. Mir210 was directly regulated by HIF-1α, a key transcriptional regulator of TH17 polarization. Unexpectedly, we identified Hif1a as a target of miR-210, which suggested negative feedback by miR-210 in inhibiting HIF-1α expression. Deletion of Mir210 promoted TH17 differentiation under conditions of limited oxygen. In experimental colitis, miR-210 reduced the abundance of Hif1a transcripts and the proportion of cells that produced inflammatory cytokines and controlled disease severity. Our study identifies miR-210 as an important regulator of T cell differentiation in hypoxia, which can limit immunopathology.

Nat. Immunol. 16, 618–627 (2015); published online 4 May 2015; corrected after print 21 May 2015 In the version of this article initially published, the filled circles in Figure 3b were incorrectly labeled 'A20+/flCD4-Cre'. The correct label is 'A20fl/flCD4-Cre'. The error has been corrected in the HTML and PDF versions of the article.