Upregulation of the ERRγ-VDAC1 axis underlies the molecular pathogenesis of pancreatitis.

Emerging evidence suggest that transcription factors play multiple roles in the development of pancreatitis, a necroinflammatory condition lacking specific therapy. Estrogen-related receptor γ (ERRγ), a pleiotropic transcription factor, has been reported to play a vital role in pancreatic acinar cell (PAC) homeostasis. However, the role of ERRγ in PAC dysfunction remains hitherto unknown. Here, we demonstrated in both mice models and human cohorts that pancreatitis is associated with an increase in ERRγ gene expression via activation of STAT3. Acinar-specific ERRγ haploinsufficiency or pharmacological inhibition of ERRγ significantly impaired the progression of pancreatitis both in vitro and in vivo. Using systematic transcriptomic analysis, we identified that voltage-dependent anion channel 1 (VDAC1) acts as a molecular mediator of ERRγ. Mechanistically, we showed that induction of ERRγ in cultured acinar cells and mouse pancreata enhanced VDAC1 expression by directly binding to specific site of the Vdac1 gene promoter and resulted in VDAC1 oligomerization. Notably, VDAC1, whose expression and oligomerization were dependent on ERRγ, modulates mitochondrial Ca2+ and ROS levels. Inhibition of the ERRγ-VDAC1 axis could alleviate mitochondrial Ca2+ accumulation, ROS formation and inhibit progression of pancreatitis. Using two different mouse models of pancreatitis, we showed that pharmacological blockade of ERRγ-VDAC1 pathway has therapeutic benefits in mitigating progression of pancreatitis. Likewise, using PRSS1R122H-Tg mice to mimic human hereditary pancreatitis, we demonstrated that ERRγ inhibitor also alleviated pancreatitis. Our findings highlight the importance of ERRγ in pancreatitis progression and suggests its therapeutic intervention for prevention and treatment of pancreatitis.

Antioxidant Analogue 6-Amino-2,4,5-Trimethylpyridin-3-ol Ameliorates Experimental Colitis in Mice.

BACKGROUND: Oxidative stress has been suggested to be a factor contributing to the disease severity of inflammatory bowel disease (IBD). BJ-1108, a derivative of 6-amino-2,4,5-trimethylpyridin-3-ol, is reported to significantly inhibit the generation of reactive oxygen species (ROS) in vitro. However, whether this molecule affects intestinal inflammation is largely unknown. We aimed to investigate the effect of BJ-1108 on dextran sulfate sodium (DSS)-induced experimental colitis in mice. METHODS: Colitis was induced in mice with DSS, and disease severity was estimated by evaluating body weight, colon length, histology, immune cell infiltration, and intestinal permeability. We examined the protective effects of BJ-1108 on barrier function using Caco-2 cells. Last, we estimated the impact of BJ-1108 on the phosphorylation of NF-kB, PI3K/AKT, and mitogen-activated protein kinases. RESULTS: Mice treated with BJ-1108 exhibited improved disease severity, as indicated by evaluations of body weight, histological scores, spleen weight, and infiltrates of T cells and macrophages. The administration of BJ-1108 inhibited the colonic mRNA expression of IL-6 and IL-1ß in vivo. Additionally, BJ-1108 limited intestinal permeability and enhanced the expression of tight junction (TJ) proteins such as claudin-1 and claudin-3 in the DSS-induced colitis model. In an in vitro model using Caco-2 cells, BJ-1108 ameliorated cytokine-induced ROS generation in a dose-dependent manner and remarkably recovered barrier dysfunction as estimated by evaluating transepithelial electrical resistance and TJ protein expression. BJ-1108 suppressed the NF-kB/ERK/PI3K pathway. CONCLUSIONS: This study demonstrated that BJ-1108 ameliorated intestinal inflammation in an experimental colitis mouse model, suggesting possible therapeutic implications for IBD.

Diabetes Primes Neutrophils for Neutrophil Extracellular Trap Formation through Trained Immunity.

Neutrophils are primed for neutrophil extracellular trap (NET) formation during diabetes, and excessive NET formation from primed neutrophils compromises wound healing in patients with diabetes. Here, we demonstrate that trained immunity mediates diabetes-induced NET priming in neutrophils. Under diabetic conditions, neutrophils exhibit robust metabolic reprogramming comprising enhanced glycolysis via the pentose phosphate pathway and fatty acid oxidation, which result in the accumulation of acetyl-coenzyme A. Adenosine 5'-triphosphate-citrate lyase-mediated accumulation of acetyl-coenzyme A and histone acetyltransferases further induce the acetylation of lysine residues on histone 3 (AcH3K9, AcH3K14, and AcH3K27) and histone 4 (AcH4K8). The pharmacological inhibition of adenosine 5'-triphosphate-citrate lyase and histone acetyltransferases completely inhibited high-glucose-induced NET priming. The trained immunity of neutrophils was further confirmed in neutrophils isolated from patients with diabetes. Our findings suggest that trained immunity mediates functional changes in neutrophils in diabetic environments, and targeting neutrophil-trained immunity may be a potential therapeutic target for controlling inflammatory complications of diabetes.

Mitochondrial dysfunctions in T cells: focus on inflammatory bowel disease.

Mitochondria has emerged as a critical ruler of metabolic reprogramming in immune responses and inflammation. In the context of colitogenic T cells and IBD, there has been increasing research interest in the metabolic pathways of glycolysis, pyruvate oxidation, and glutaminolysis. These pathways have been shown to play a crucial role in the metabolic reprogramming of colitogenic T cells, leading to increased inflammatory cytokine production and tissue damage. In addition to metabolic reprogramming, mitochondrial dysfunction has also been implicated in the pathogenesis of IBD. Studies have shown that colitogenic T cells exhibit impaired mitochondrial respiration, elevated levels of mROS, alterations in calcium homeostasis, impaired mitochondrial biogenesis, and aberrant mitochondria-associated membrane formation. Here, we discuss our current knowledge of the metabolic reprogramming and mitochondrial dysfunctions in colitogenic T cells, as well as the potential therapeutic applications for treating IBD with evidence from animal experiments.

The Role of Pyruvate Metabolism in Mitochondrial Quality Control and Inflammation.

Pyruvate metabolism, a key pathway in glycolysis and oxidative phosphorylation, is crucial for energy homeostasis and mitochondrial quality control (MQC), including fusion/fission dynamics and mitophagy. Alterations in pyruvate flux and MQC are associated with reactive oxygen species accumulation and Ca2+ flux into the mitochondria, which can induce mitochondrial ultrastructural changes, mitochondrial dysfunction and metabolic dysregulation. Perturbations in MQC are emerging as a central mechanism for the pathogenesis of various metabolic diseases, such as neurodegenerative diseases, diabetes and insulin resistance-related diseases. Mitochondrial Ca2+ regulates the pyruvate dehydrogenase complex (PDC), which is central to pyruvate metabolism, by promoting its dephosphorylation. Increase of pyruvate dehydrogenase kinase (PDK) is associated with perturbation of mitochondria-associated membranes (MAMs) function and Ca2+ flux. Pyruvate metabolism also plays an important role in immune cell activation and function, dysregulation of which also leads to insulin resistance and inflammatory disease. Pyruvate metabolism affects macrophage polarization, mitochondrial dynamics and MAM formation, which are critical in determining macrophage function and immune response. MAMs and MQCs have also been intensively studied in macrophage and T cell immunity. Metabolic reprogramming connected with pyruvate metabolism, mitochondrial dynamics and MAM formation are important to macrophages polarization (M1/M2) and function. T cell differentiation is also directly linked to pyruvate metabolism, with inhibition of pyruvate oxidation by PDKs promoting proinflammatory T cell polarization. This article provides a brief review on the emerging role of pyruvate metabolism in MQC and MAM function, and how dysfunction in these processes leads to metabolic and inflammatory diseases.

Inhibition of Pyruvate Dehydrogenase Kinase 4 in CD4+ T Cells Ameliorates Intestinal Inflammation.

BACKGROUND & AIMS: Despite recent evidence supporting the metabolic plasticity of CD4+ T cells, it is uncertain whether the metabolic checkpoint pyruvate dehydrogenase kinase (PDK) in T cells plays a role in the pathogenesis of colitis. METHODS: To investigate the role of PDK4 in colitis, we used dextran sulfate sodium (DSS)-induced colitis and T-cell transfer colitis models based on mice with constitutive knockout (KO) or CD4+ T-cell-specific KO of PDK4 (Pdk4fl/flCD4Cre). The effect of PDK4 deletion on T-cell activation was also studied in vitro. Furthermore, we examined the effects of a pharmacologic inhibitor of PDK4 on colitis. RESULTS: Expression of PDK4 increased during colitis development in a DSS-induced colitis model. Phosphorylated PDHE1α, a substrate of PDK4, accumulated in CD4+ T cells in the lamina propria of patients with inflammatory bowel disease. Both constitutive KO and CD4+ T-cell-specific deletion of PDK4 delayed DSS-induced colitis. Adoptive transfer of PDK4-deficient CD4+ T cells attenuated murine colitis, and PDK4 deficiency resulted in decreased activation of CD4+ T cells and attenuated aerobic glycolysis. Mechanistically, there were fewer endoplasmic reticulum-mitochondria contact sites, which are responsible for interorganelle calcium transfer, in PDK4-deficient CD4+ T cells. Consistent with this, GM-10395, a novel inhibitor of PDK4, suppressed T-cell activation by reducing endoplasmic reticulum-mitochondria calcium transfer, thereby ameliorating murine colitis. CONCLUSIONS: PDK4 deletion from CD4+ T cells mitigates colitis by metabolic and calcium signaling modulation, suggesting PDK4 as a potential therapeutic target for IBD.

Enhanced Ca2+-channeling complex formation at the ER-mitochondria interface underlies the pathogenesis of alcohol-associated liver disease.

Ca2+ overload-induced mitochondrial dysfunction is considered as a major contributing factor in the pathogenesis of alcohol-associated liver disease (ALD). However, the initiating factors that drive mitochondrial Ca2+ accumulation in ALD remain elusive. Here, we demonstrate that an aberrant increase in hepatic GRP75-mediated mitochondria-associated ER membrane (MAM) Ca2+-channeling (MCC) complex formation promotes mitochondrial dysfunction in vitro and in male mouse model of ALD. Unbiased transcriptomic analysis reveals PDK4 as a prominently inducible MAM kinase in ALD. Analysis of human ALD cohorts further corroborate these findings. Additional mass spectrometry analysis unveils GRP75 as a downstream phosphorylation target of PDK4. Conversely, non-phosphorylatable GRP75 mutation or genetic ablation of PDK4 prevents alcohol-induced MCC complex formation and subsequent mitochondrial Ca2+ accumulation and dysfunction. Finally, ectopic induction of MAM formation reverses the protective effect of PDK4 deficiency in alcohol-induced liver injury. Together, our study defines a mediatory role of PDK4 in promoting mitochondrial dysfunction in ALD.

Effect of Low-Dose Persistent Organic Pollutants on Mitochondrial Function: Human and in Vitro Evidence.

BACKGROUND: Chronic exposure to low-dose persistent organic pollutants (POPs) can induce mitochondrial dysfunction. This study evaluated the association between serum POP concentrations and oxygen consumption rate (OCR) as a marker of mitochondrial function in humans and in vitro cells. METHODS: Serum concentrations of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) were measured in 323 adults. The OCRs of platelets and peripheral blood mononuclear cells (PBMCs) were assessed in 20 mL of fresh blood using a Seahorse XF analyzer. Additionally, the in vitro effects of Arochlor-1254, ß-hexachlorocyclohexane, and p,p´-dichlorodiphenyltrichloroethane at concentrations of 0.1 pM to 100 nM were evaluated in human platelets, human PBMCs, and Jurkat T-cells. RESULTS: The association between serum POP concentrations and OCR differed depending on the cell type. As serum OCP concentrations increased, basal platelet OCR levels decreased significantly; according to the OCP quintiles of summary measure, they were 8.6, 9.6, 8.2, 8.0, and 7.1 pmol/min/µg (P trend=0.005). Notably, the basal PBMC OCR levels decreased remarkably as the serum PCB concentration increased. PBMC OCR levels were 46.5, 34.3, 29.1, 16.5, and 13.1 pmol/min/µg according to the PCB quintiles of summary measure (P trend <0.001), and this inverse association was consistently observed in all subgroups stratified by age, sex, obesity, type 2 diabetes mellitus, and hypertension, respectively. In vitro experimental studies have also demonstrated that chronic exposure to low-dose POPs could decrease OCR levels. CONCLUSION: The findings from human and in vitro studies suggest that chronic exposure to low-dose POPs can induce mitochondrial dysfunction by impairing oxidative phosphorylation.

DICAM Attenuates Experimental Colitis via Stabilizing Junctional Complex in Mucosal Barrier.

BACKGROUND: Adhesion molecules maintain the intestinal barrier function that is crucial to prevent intestinal inflammation. Dual immunoglobulin domain-containing adhesion molecule (DICAM) has been recently identified and known for the involvement in cell-cell adhesion through homophilic interaction and heterophilic interaction with integrin αVß3. We tested whether the change of DICAM expression affects the severity of colonic inflammation. METHODS: Colitis was induced with oral administration of 2.5% dextran sulfate sodium (DSS) in 8-week-old male mice for 5 days. The function of DICAM under inflammatory condition was investigated using loss-of-function and gain-of-function models such as DICAM-deficient mice and adenoviral transduction of DICAM into Caco-2 colonic epithelial cells. RESULTS: DICAM increased in parallel with the degree of inflammation after 5-day administration of DSS and decreased with the resolution of inflammation. DICAM was expressed in the epithelial junctional complex and colocalized with ZO-1. Treatment with TNF-α or IFN-γ in Caco-2 cells significantly increased DICAM in protein and RNA level. The DICAM knockout mice showed more severe DSS-induced colitis compared with WT littermates. Adenoviral transduction of DICAM into Caco-2 cells significantly attenuated the inflammation-mediated decrease of adhesion molecules, including ZO-1 and occludin. Furthermore, Caco-2 cells with DICAM overexpression maintained intestinal barrier function under IFN-γ treatment as estimated by transepithelial electrical resistance. CONCLUSION: Our study demonstrates that DICAM which is increased in an inflammatory condition has a protective role in experimental colitis by stabilizing the integrity of junctional complex in the intestinal mucosal barrier.

The Growth and Protein Expression of Inflammatory Bowel Disease-Associated Campylobacter concisus Is Affected by the Derivatives of the Food Additive Fumaric Acid.

Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal tract with multifactorial etiology. Both dietary factors and the microbe Campylobacter concisus have been found to be associated with the condition. The current study examined the effects of sodium fumarate, a neutralized product of the food additives fumaric acid and monosodium fumarate when in the intestinal environment, on the growth of C. concisus to determine the effects of these food additives on IBD-associated bacterial species. Through culture methods and quantification, it was found that neutralized fumaric acid, neutralized monosodium fumarate, and sodium fumarate increased the growth of C. concisus, with the greatest increase in growth at a concentration of 0.4%. Further examination of 50 C. concisus strains on media with added sodium fumarate showed that greatest growth was also achieved at a concentration of 0.4%. At a concentration of 2% sodium fumarate, all strains examined displayed less growth in comparison with those cultured on media without sodium fumarate. Using mass spectrometry, multiple C. concisus proteins showed significant differential expression when cultured on media with and without 0.4% sodium fumarate. The findings presented suggest that patients with IBD should consider avoiding excessive consumption of foods with fumaric acid or its sodium salts, and that the addition of 0.4% sodium fumarate alone to media may assist in the isolation of C. concisus from clinical samples.

Natural orifice transluminal endoscopic surgery with a snake-mechanism using a movable pulley.

BACKGROUND: Natural orifice transluminal endoscopic surgery is an emerging technique. We aimed to develop an advanced surgical robot mechanism for natural orifice surgery. METHODS: We propose the active-controlled overtube-type platform with multiple channels for an endoscopic camera and surgical tools. To make such a platform, we suggest an advanced snake mechanism comprising movable pulleys to make a snake mechanism with multiple degrees of freedom and high operating force. RESULTS: The stiffness and maneuverability of the active-controlled platform appeared satisfactory. Using prototypes and ex vivo experiments, we confirmed that the mechanism was suitable for a snake-like robotic platform for natural orifice surgery. CONCLUSIONS: The suggested snake mechanism using movable pulleys has the advantages of stiffness and maneuverability. This new mechanism can be an alternative platform for natural orifice surgery.

Zonula occludens toxins and their prophages in Campylobacter species.

BACKGROUND: We previously showed that zonula occludens toxin (Zot) encoded by Campylobacter concisus zot (808T) gene has the potential to initiate inflammatory bowel disease. This Zot protein caused prolonged intestinal epithelial barrier damage, induced intestinal epithelial and macrophage production of tumor necrosis factor-α and enhanced the responses of macrophages to other microbes. In order to understand the potential virulence of Zot proteins in other Campylobacter species, in this study we examined their presence, similarities, motifs and prophages. METHODS: The presence of Zot proteins in Campylobacter species was examined by searching for the Zot family domain in multiple protein databases. Walker A and Walker B motifs in Zot proteins were identified using protein sequence alignment. A phylogenetic tree based on Campylobacter zot genes was constructed using maximum-likelihood method. Campylobacter Zot proteins were compared using protein sequence alignment. The zot-containing prophages in Campylobacter species were identified and compared with known prophage proteins and other viral proteins using protein sequence alignment and protein BLAST. RESULTS: Twelve Zot proteins were found in nine Campylobacter species/subspecies. Among these Campylobacter species, three species had two Zot proteins and the remaining six species/subspecies had one Zot protein. Walker A and Walker B motifs and a transmembrane domain were found in all identified Campylobacter Zot proteins. The twelve Campylobacter zot genes from the nine Campylobacter species/subspecies formed two clusters. The ZotCampyType_1 proteins encoded by Cluster 1 Campylobacter zot genes showed high similarities to each other. However, ZotCampyType_2 proteins encoded by Cluster 2 Campylobacter zot genes were more diverse. Furthermore, the zot-containing Campylobacter prophages were identified. CONCLUSION: This study reports the identification of two types of Campylobacter Zot proteins. The high similarities of ZotCampyType_1 proteins suggest that they are likely to have similar virulence. ZotCampyType_2 proteins are less similar to each other and their virulent properties, if any, remain to be examined individually.

Investigation of the effects of pH and bile on the growth of oral Campylobacter concisus strains isolated from patients with inflammatory bowel disease and controls.

Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD). This study examined the impact of pH and bile on the growth of oral C. concisus strains isolated from patients with IBD and controls. The growth of 58 C. concisus strains on horse blood agar (HBA) plates following exposure to media with various pH values for different time points was examined. Furthermore, the growth of C. concisus strains on HBA plates containing different concentrations of ox bile was investigated. Following exposure to pH 2 for 30 min, none of the 58 oral C. concisus strains grew on HBA plates. Following exposure to pH 3.5 for 30 min, only four of 20 oral strains examined grew on HBA plates, with a log10 c.f.u. reduction of 0.7-2.5 compared to the same strains without low pH exposure. Exposure to pH 5 for 120 min had minimal effects on C. concisus growth. Approximately half of the oral strains (55.2%, 32/58) grew on HBA containing 2% bile. Bile inhibited the growth of C. concisus in a dose- and strain-dependent manner. These data suggest that both bacterial and intestinal environmental factors may play a role in the determination of C. concisus colonization in the different parts of the gastrointestinal tract and that increased gastric pH and reduced intestinal bile may be risk factors for increased gastric and intestinal C. concisus colonization.

Campylobacter concisus and inflammatory bowel disease.

Investigation of the possible role of Campylobacter concisus (C. concisus) in inflammatory bowel disease (IBD) is an emerging research area. Despite the association found between C. concisus and IBD, it has been difficult to explain how C. concisus, a bacterium that is commonly present in the human oral cavity, may contribute to the development of enteric diseases. The evidence presented in this review shows that some C. concisus strains in the oral cavity acquired zonula occludens toxin (zot) gene from a virus (prophage) and that C. concisus Zot shares conserved motifs with both Vibrio cholerae Zot receptor binding domain and human zonulin receptor binding domain. Both Vibrio cholerae Zot and human zonulin are known to increase intestinal permeability by affecting the tight junctions. Increased intestinal permeability is a feature of IBD. Based on these data, we propose that a primary barrier function defect caused by C. concisus Zot is a mechanism by which zot-positive C. concisus strains may trigger the onset and relapse of IBD.

Examination of the Anaerobic Growth of Campylobacter concisus Strains.

Campylobacter concisus is an oral bacterium that is associated with intestinal diseases. C. concisus was previously described as a bacterium that requires H2-enriched microaerobic conditions for growth. The level of H2 in the oral cavity is extremely low, suggesting that C. concisus is unlikely to have a microaerobic growth there. In this study, the anaerobic growth of C. concisus was investigated. The growth of fifty-seven oral C. concisus strains and six enteric C. concisus strains under various atmospheric conditions including anaerobic conditions with and without H2 was examined. The atmospheric conditions were generated using commercially available gas-generation systems. C. concisus putative virulence proteins were identified using mass spectrometry analysis. Under anaerobic conditions, 92% of the oral C. concisus strains (52/57) and all six enteric strains grew without the presence of H2 and the presence of H2 greatly increased C. concisus growth. An oral C. concisus strain was found to express a number of putative virulence proteins and the expression levels of these proteins were not affected by H2. The levels of H2 appeared to affect the optimal growth of C. concisus. This study provides useful information in understanding the natural colonization site and pathogenicity of C. concisus.

The effects of oral and enteric Campylobacter concisus strains on expression of TLR4, MD-2, TLR2, TLR5 and COX-2 in HT-29 cells.

Campylobacter concisus, a Gram-negative bacterium that colonizes the human oral cavity, has been shown to be associated with inflammatory bowel diseases (IBD). The effects of different C. concisus strains on intestinal epithelial expression of Toll like receptors (TLR) have not been investigated. This study examined the effects of C. concisus strains isolated from patients with IBD and controls on expression of TLR4, its co-receptor myeloid differentiation factor (MD)-2; TLR2, TLR5, cyclooxygenase-2 (COX-2) and interleukin (IL)-8 in HT-29 cells.Fourteen oral and enteric C. concisus strains isolated from patients with IBD and healthy controls were co-incubated with HT-29 cells. Expression of TLR4, MD-2, TLR2, TLR5 and COX-2 in HT-29 cells in response to C. concisus infection was examined by Western blot, flow cytometry analysis and immunofluorescent staining visualized by confocal microscope. Production of IL-8 was evaluated by enzyme-linked immunosorbent assay.Both oral and enteric C. concisus strains upregulated expression of TLR4 in HT-29 cells. The levels of glycosylated TLR4 (Gly-TLR4) and surface TLR4 induced by C. concisus strains isolated from patients with IBD were significantly higher than those induced by C. concisus strains isolated from the healthy controls. Four C. concisus strains isolated from patients with IBD induced more than two-fold increase of surface expression of MD-2. C. concisus did not affect expression of TLR2 and TLR5. All C. concisus strains induced production of IL-8 and COX-2 in HT-29 cells.This study shows that some C. concisus strains, most from patients with IBD, upregulate surface expression of TLR4 and MD-2 in HT-29 cells. These data suggest that a potential role of specific C. concisus strains in modulating the intestinal epithelial responses to bacterial LPS needs to be investigated.