A transepithelial pathway delivers succinate to macrophages, thus perpetuating their pro-inflammatory metabolic state

The gut metabolite composition determined by the microbiota has paramount impact on gastrointestinal physiology. However, the role that bacterial metabolites play in communicating with host cells during inflammatory diseases is poorly understood. Here, we aim to identify the microbiota-determined output of the pro-inflammatory metabolite, succinate, and to elucidate the pathways that control transepithelial succinate absorption and subsequent succinate delivery to macrophages. We show a significant increase of succinate uptake into pro-inflammatory macrophages, which is controlled by Na+-dependent succinate transporters in macrophages and epithelial cells. Furthermore, we find that fecal and serum succinate concentrations were markedly augmented in inflammatory bowel diseases (IBDs) and corresponded to changes in succinate-metabolizing gut bacteria. Together, our results describe a succinate production and transport pathway that controls the absorption of succinate generated by distinct gut bacteria and its delivery into macrophages. In IBD, this mechanism fails to protect against the succinate surge, which may result in chronic inflammation.ope

Melatonin controls microbiota in colitis by goblet cell differentiation and antimicrobial peptide production through Toll-like receptor 4 signalling

Microbial dysbiosis has long been postulated to be associated with the pathogenesis of inflammatory bowel disease (IBD). Although evidence supporting the anti-colitic effects of melatonin have been accumulating, it is not clear how melatonin affects the microbiota. Herein, we investigated the effects of melatonin on the microbiome in colitis and identified involvement of Toll-like receptor (TLR) 4 signalling in the effects. Melatonin improved dextran sulfate sodium (DSS)-induced colitis and reverted microbial dysbiosis in wild-type (WT) mice but not in TLR4 knockout (KO) mice. Induction of goblet cells was observed with melatonin administration, which was accompanied by suppression of Il1b and Il17a and induction of melatonin receptor and Reg3β, an antimicrobial peptide (AMP) against Gram-negative bacteria. In vitro, melatonin treatment of HT-29 intestinal epithelial cells promotes mucin and wound healing and inhibits growth of Escherichia coli. Herein, we showed that melatonin significantly increases goblet cells, Reg3β, and the ratio of Firmicutes to Bacteriodetes by suppressing Gram-negative bacteria through TLR4 signalling. Our study suggests that sensing of bacteria through TLR4 and regulation of bacteria through altered goblet cells and AMPs is involved in the anti-colitic effects of melatonin. Melatonin may have use in therapeutics for IBD.ope

Genetic polymorphisms of the interleukin 23 receptor and interleukin 17A and their associations with inflammatory bowel diseases

Dept. of Medical Science/박사Inflammatory bowel disease (IBD), which includes mainly ulcerative colitis (UC) and Crohn’s disease (CD), is characterized by chronic relapsing intestinal inflammation. Currently, IBD is considered to be caused by a complex interaction of genetic, environmental, and other processes involving immunoregulatory factors of which play a principal role in the pathogenesis of IBD. Recently, a particular subset of T helper cells (Th17 cells) characterized by interleukin 17A (IL-17A or further referred to as IL-17) production was implicated as a critical mediators of autoimmune disease including IBD and the IL-23R gene is known to be a susceptibility gene related to IBD in Caucasian IBD patients, although it has not been detected in Asian populations. Moreover, while there are a few reports on the associations of IL-17A gene polymorphisms, it is still unknown whether IL-17A SNPs are associated with IBD susceptibility except rs2275913 and, if so, how these IL-17A SNPs exactly modulate IBD susceptibility.Thus, It was investigated the associations of genetic and epigenetic variations in IL-23R and IL-17A with the development of IBD in this study. The promoters and exon regions encompassing the intron junctions of IL-23R and IL-17A were analyzed in 728 subjects including 201 CD patients, 268 UC patients, and 259 healthy controls using DNA sequencing and denaturing high performance liquid chromatography. Associations of IL-23R and IL-17A polymorphisms with IBD susceptibility were analyzed and their gene-gene interactions including STAT4 SNPs were tested using logistic regression analysis. Jurkat cells and peripheral blood mononuclear cells (PBMC) were used for in vitro assay as follows. The transcription factor binding activity was determined using electrophoretic mobility shift assay, IL-17A mRNA expression levels by reverse-transcriptase polymerase chain reaction, and methylation status of IL-17A promoter by bisulfite sequencing and pyrosequencing. In CD, a case-control analysis showed that disease development was associated with the IL-23R variant G149R (odds ratios [OR] 0.32, 95% confidence intervals [CI] 0.15–0.68) and IL-17A variant IVS1+18G>C (OR 10.65, 95% CI 1.32–85.89). The analysis for UC showed an association with IL-23R variants G149R (OR 0.41, 95% CI 0.21–0.76), IVS4+17C>T (OR 2.89, 95% CI 1.20–6.96), and Q3H (OR 0.61, 95% CI 0.38-0.99), and IL-17A variants -737C>T (OR 1.50, 95% CI 1.06–2.13), -197G>A (OR 0.63, 95% CI 0.40–0.97), and IVS1+18 G>C (OR 8.93, 95% CI 1.12–70.99). As further evidence of the synergistic effect of the genes in this pathway in the development of IBD, a significant statistical gene-gene interaction among IL-23R, IL-17A and STAT4 were observed. The -877G, -737T, and -444A risk alleles of IL-17A displayed higher binding affinity of transcription factor complex and higher expression levels of IL-17A transcripts. DNA hypomethylation of the IL-17A promoter was observed in PBMCs from IBD patients with a methylation extent of IVS1+17 site and significant inverse correlation between IL-17A mRNA level. Finally, IL-17A mRNA expression was restored after exposure to demethylating agent in Jurkat cells. The results of this study provide insights into the genetic and epigenetic interactions in the IL-23R/IL-17 axis including STAT4 that are associated with elevated expression of IL-17 and IBD pathogenesis.ope

Succinate-treated macrophages attenuate dextran sodium sulfate colitis in mice

The safety and effectiveness of adalimumab was demonstrated in a phase 3 trial in Japanese patients with intestinal Behçet's disease. The aim of this study was to evaluate the long-term safety and effectiveness of adalimumab in Japanese patients with intestinal Behçet's disease.ope

EW-7197, a novel ALK-5 kinase inhibitor, potently inhibits breast to lung metastasis.

Advanced tumors produce an excessive amount of transforming growth factor β (TGFβ), which promotes tumor progression at late stages of malignancy. The purpose of this study was to develop anti-TGFβ therapeutics for cancer. We synthesized a novel small-molecule TGFβ receptor I kinase (activin receptor-like kinase 5) inhibitor termed N-[[4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl]methyl]-2-fluoroaniline (EW-7197), and we investigated its potential antimetastatic efficacy in mouse mammary tumor virus (MMTV)/c-Neu mice and 4T1 orthotopic-grafted mice. EW-7197 inhibited Smad/TGFβ signaling, cell migration, invasion, and lung metastasis in MMTV/c-Neu mice and 4T1 orthotopic-grafted mice. EW-7197 also inhibited the epithelial-to-mesenchymal transition (EMT) in both TGFβ-treated breast cancer cells and 4T1 orthotopic-grafted mice. Furthermore, EW-7197 enhanced cytotoxic T lymphocyte activity in 4T1 orthotopic-grafted mice and increased the survival time of 4T1-Luc and 4T1 breast tumor-bearing mice. In summary, EW-7197 showed potent in vivo antimetastatic activity, indicating its potential for use as an anticancer therapy.ope

Nanocomposites-based targeted oral drug delivery systems with infliximab in a murine colitis model

Background: Infliximab (IFX), a TNF-α blocking chimeric monoclonal antibody, induces clinical response and mucosal healing in patients with inflammatory bowel disease (IBD). However, systemic administration of this agent causes unwanted side effects. Oral delivery of antibody therapeutics might be an effective treatment strategy for IBD compared to intravenous administration. Results: All three carriers had a high encapsulation efficiency, narrow size distribution, and minimal systemic exposure. There was a higher interaction between nanocomposite carriers and monocytes compared to lymphocytes in the PBMC of IBD patients. Orally administered nanocomposite carriers targeted to inflamed colitis minimized systemic exposure. All IFX delivery formulations with nanocomposite carriers had a significantly less colitis-induced body weight loss, colon shortening and histomorphological score, compared to the DSS-treated group. AC-IFX-L and EAC-IFX-L groups showed significantly higher improvement of the disease activity index, compared to the DSS-treated group. In addition, AC-IFX-L and EAC-IFX-L alleviated pro-inflammatory cytokine expressions (Tnfa, Il1b, and Il17). Conclusion: We present orally administered antibody delivery systems which improved efficacy in murine colitis while reducing systemic exposure. These oral delivery systems suggest a promising therapeutic approach for treating IBD.ope

TRIM31 promotes Atg5/Atg7-independent autophagy in intestinal cells.

Autophagyis responsible for the bulk degradation of cytosolic constituents and plays an essential role in theintestinalepithelium by controlling beneficial host-bacterial relationships.Atg5and Atg7 are thought to be critical forautophagy. However,Atg5- or Atg7-deficientcellsstill form autophagosomes and autolysosomes, and are capable of removing proteins or bacteria. Here, we report that humanTRIM31(tripartite motif), an intestine-specific protein localized in mitochondria, is essential for promoting lipopolysaccharide-inducedAtg5/Atg7-independentautophagy.TRIM31directly interacts with phosphatidylethanolamine in a palmitoylation-dependent manner, leading to induction of autolysosome formation. Depletion of endogenousTRIM31significantly increases the number ofintestinalepithelialcellscontaining invasive bacteria. Crohn's disease patients displayTRIM31downregulation. Human cytomegalovirus-infectedintestinalcellsshow a decrease inTRIM31expression as well as a significant increase in bacterial load, reversible by the introduction of wild-typeTRIM31. We provide insight into an alternativeautophagypathway that protects againstintestinalpathogenic bacterial infection.ope

Interplay between chronic inflammation and clonal haematopoiesis of indeterminate potential in Behçet's disease

BACKGROUND: Clonal haematopoiesis of indeterminate potential (CHIP) is a predisposition to haematological malignancy whose relationship with chronic inflammatory diseases, such as cardiovascular diseases, has been highlighted. Here, we aimed to investigate the CHIP emergence rate and its association with inflammatory markers in Behçet's disease (BD). METHODS: We performed targeted next-generation sequencing to detect the presence of CHIP using peripheral blood cells from 117 BD patients and 5004 healthy controls between March 2009 and September 2021 and analysed the association between CHIP and inflammatory markers. RESULTS: CHIP was detected in 13.9% of patients in the control group and 11.1% of patients in the BD group, indicating no significant intergroup difference. Among the BD patients of our cohort, five variants (DNMT3A, TET2, ASXL1, STAG2, and IDH2) were detected. DNMT3A mutations were the most common, followed by TET2 mutations. CHIP carriers with BD had a higher serum platelet count, erythrocyte sedimentation rate, and C-reactive protein level; older age; and lower serum albumin level at diagnosis than non-CHIP carriers with BD. However, the significant association between inflammatory markers and CHIP disappeared after the adjustment for various variables, including age. Moreover, CHIP was not an independent risk factor for poor clinical outcomes in patients with BD. CONCLUSIONS: Although BD patients did not have higher CHIP emergence rates than the general population, older age and degree of inflammation in BD were associated with CHIP emergence. © 2023. The Author(s).ope

Interleukin-33 regulates intestinal inflammation by modulating macrophages in inflammatory bowel disease

Interleukin 33 (IL-33) that signals through the ST2 receptor has emerged as a critical modulator in several inflammatory disorders, including inflammatory bowel disease (IBD). However, the precise mechanisms by which IL-33 modulates IBD are controversial. The aim of this study was thus to clarify the role of IL-33 in IBD. The plasma levels of IL-33 were significantly decreased, but soluble ST2 levels were increased in patients with IBD compared to healthy individuals. Moreover, IL-33 restored goblet cell numbers and induced macrophage switching from the M1 to the M2 phenotype. These effects were sufficient to ameliorate colitis in dextran sodium sulfate, trinitrobenzene sulfonic acid, and peritoneal cavity cell transfer models. IL-33 facilitated goblet cell restoration via modulating macrophages toward the M2 phenotype. In addition, wound healing was significantly faster in IL-33-treated human monocyte-derived macrophages than in control cells, which could be attributed to increased polarisation into M2 macrophages. We found that patients with IBD show decreased serum levels of IL-33 compared with healthy individuals and that IL-33 can attenuate colitis and aid tissue repair in mice. The mechanism by which IL-33 exerts these effects appears to involve the stimulation of differentiation of goblet cells and M2 macrophages.ope

The bifunctional autophagic flux by 2-deoxyglucose to control survival or growth of prostate cancer cells

BACKGROUND: Recent reports using metabolism regulating drugs showed that nutrient deprivation was an efficient tool to suppress cancer progression. In addition, autophagy control is emerging to prevent cancer cell survival. Autophagy breaks down the unnecessary cytoplasmic components into anabolic units and energy sources, which are the most important sources for making the ATP that maintains homeostasis in cancer cell growth and survival. Therefore, the glucose analog 2-deoxyglucose (2DG) has been used as an anticancer reagent due to its inhibition of glycolysis. METHODS: Prostate cancer cells (PC3) were treated with 2DG for 6 h or 48 h to analyze the changing of cell cycle and autophagic flux. Rapamycin and LC3B overexpressing vectors were administered to PC3 cells for autophagy induction and chloroquine and shBeclin1 plasmid were used to inhibit autophagy in PC3 cells to analyze PC3 cells growth and survival. The samples for western blotting were prepared in each culture condition to confirm the expression level of autophagy related and regulating proteins. RESULTS: We demonstrated that 2DG inhibits PC3 cells growth and had discriminating effects on autophagy regulation based on the different time period of 2DG treatment to control cell survival. Short-term treatment of 2DG induced autophagic flux, which increased microtubule associated protein 1 light chain 3B (LC3B) conversion rates and reduced p62 levels. However, 2DG induced autophagic flux is remarkably reduced over an extended time period of 2DG treatment for 48 h despite autophagy inducing internal signaling being maintained. The relationship between cell growth and autophagy was proved. Increased autophagic flux by rapamycin or LC3B overexpression powerfully reduced cell growth, while autophagy inhibition with shBeclin1 plasmid or chloroquine had no significant effect on regulating cell growth. CONCLUSION: Given these results, maintaining increased autophagic flux was more effective at inhibiting cancer cell progression than inhibition of autophagic flux, which is necessary for the survival of PC3 cells. Autophagic flux should be tightly regulated to maintain metabolic homeostasis for cancer cell growth and survival in PC3 cells and is a suitable target for cancer therapy.ope