TRAV26-2 T-Cell Receptor Expression Is Associated With Mucosal Lymphocyte Response to Wheat Proteins in Patients With Functional Dyspepsia.

INTRODUCTION: An association between functional dyspepsia (FD) and wheat-containing foods has been reported in observational studies; however, an adaptive response has not been demonstrated. We examined whether antigens present in wheat could provoke a response from FD duodenal lymphocytes. METHODS: Lamina propria mononuclear cells (LPMCs) were isolated from duodenal biopsies from 50 patients with FD and 23 controls. LPMCs were exposed to gluten (0.2 mg/mL) or gliadin (0.2 mg/mL) for 24 hours. Flow cytometry was performed to phenotype lymphocytes. Quantitative PCR was used to measure the expression of gliadin-associated T-cell receptor alpha variant ( TRAV ) 26-2. RESULTS: In response to gliadin (but not gluten) stimulation, the effector Th2-like population was increased in FD LPMCs compared with that in controls and unstimulated FD LPMCs. Duodenal gene expression of TRAV26- 2 was decreased in patients with FD compared with that in controls. We identified a positive association between gene expression of this T-cell receptor variant and LPMC effector Th17-like cell populations in patients with FD, but not controls after exposure to gluten, but not gliadin. DISCUSSION: Our findings suggest that gliadin exposure provokes a duodenal effector Th2-like response in patients with FD, supporting the notion that food antigens drive responses in some patients. Furthermore, these findings suggest that altered lymphocyte responses to wheat proteins play a role in FD pathogenesis.

Defects in NLRP6, autophagy and goblet cell homeostasis are associated with reduced duodenal CRH receptor 2 expression in patients with functional dyspepsia.

Functional dyspepsia (FD) affects up to 15% of the population and is characterised by recurring upper gastrointestinal (GI) symptoms occurring in the absence of clinically identifiable pathology. Psychological stress is a key factor associated with the onset of FD and locally acting hypothalamic-pituitary-adrenal (HPA) axis hormones have been implicated in GI motility and barrier dysfunction. Recent pre-clinical work has identified mechanistic pathways linking corticotropin-releasing hormone (CRH) with the innate epithelial immune protein NLRP6, an inflammasome that has been shown to regulate GI mucus secretion. We recruited twelve FD patients and twelve healthy individuals to examine whether dysregulation of hypothalamic-pituitary adrenal (HPA) axis hormones and altered NLRP6 pathways were evident in the duodenal mucosa. Protein expression was assessed by immunoblot and immunohistochemistry in D2 duodenal biopsies. Plasma HPA axis hormones were assayed by ELISA and enteroid and colorectal cancer cell line cultures were used to verify function. FD patients exhibited reduced duodenal CRH-receptor 2, compared to non-GI disease controls, indicating a dysregulation of duodenal HPA signalling. The loss of CRH-receptor 2 correlated with reduced NLRP6 expression and autophagy function, processes critical for maintaining goblet cell homeostasis. In accordance, duodenal goblet cell numbers and mucin exocytosis was reduced in FD patients compared to controls. In vitro studies demonstrated that CRH could reduce NLRP6 in duodenal spheroids and promote mucus secretion in the HT29-MTX-E12 cell line. In conclusion, FD patients exhibit defects in the NLRP6-autophagy axis with decreased goblet cell function that may drive symptoms of disease. These features correlated with loss of CRH receptor 2 and may be driven by dysregulation of HPA signalling in the duodenum of FD patients.

Type 2 and type 17 effector cells are increased in the duodenal mucosa but not peripheral blood of patients with functional dyspepsia.

Background: Functional dyspepsia is characterised by chronic symptoms of post-prandial distress or epigastric pain not associated with defined structural pathology. Increased peripheral gut-homing T cells have been previously identified in patients. To date, it is unknown if these T cells were antigen-experienced, or if a specific phenotype was associated with FD. Objective: This study aimed to characterise T cell populations in the blood and duodenal mucosa of FD patients that may be implicated in disease pathophysiology. Methods: We identified duodenal T cell populations from 23 controls and 49 Rome III FD patients by flow cytometry using a surface marker antibody panel. We also analysed T cell populations in peripheral blood from 37 controls and 61 patients. Where available, we examined the number of duodenal eosinophils in patients and controls. Results: There was a shift in the duodenal T helper cell balance in FD patients compared to controls. For example, patients had increased duodenal mucosal Th2 populations in the effector (13.03 ± 16.11, 19.84 ± 15.51, p=0.038), central memory (23.75 ± 18.97, 37.52 ± 17.51, p=0.007) and effector memory (9.80±10.50 vs 20.53±14.15, p=0.001) populations. Th17 populations were also increased in the effector (31.74±24.73 vs 45.57±23.75, p=0.03) and effector memory (11.95±8.42 vs 18.44±15.63, p=0.027) subsets. Peripheral T cell populations were unchanged between FD and control. Conclusion: Our findings identify an association between lymphocyte populations and FD, specifically a Th2 and Th17 signature in the duodenal mucosa. The presence of effector and memory cells suggest that the microinflammation in FD is antigen driven.

Hepatocyte free cholesterol lipotoxicity results from JNK1-mediated mitochondrial injury and is HMGB1 and TLR4-dependent.

BACKGROUND & AIMS: Free cholesterol (FC) accumulates in non-alcoholic steatohepatitis (NASH) but not in simple steatosis. We sought to establish how FC causes hepatocyte injury. METHODS: In NASH-affected livers from diabetic mice, subcellular FC distribution (filipin fluorescence) was established by subcellular marker co-localization. We loaded murine hepatocytes with FC by incubation with low-density lipoprotein (LDL) and studied the effects of FC on JNK1 activation, mitochondrial injury and cell death and on the amplifying roles of the high-mobility-group-box 1 (HMGB1) protein and the Toll-like receptor 4 (TLR4). RESULTS: In NASH, FC localized to hepatocyte plasma membrane, mitochondria and ER. This was reproduced in FC-loaded hepatocytes. At 40 µM LDL, hepatocyte FC increased to cause LDH leakage, apoptosis and necrosis associated with JNK1 activation (c-Jun phosphorylation), mitochondrial membrane pore transition, cytochrome c release, oxidative stress (GSSG:GSH ratio) and ATP depletion. Mitochondrial swelling and crystae disarray were evident by electron microscopy. Jnk1(-/-) and Tlr4(-/-) hepatocytes were refractory to FC lipotoxicity; JNK inhibitors (1-2 µM CC-401, CC-930) blocked apoptosis and necrosis. Cyclosporine A and caspase-3 inhibitors protected FC-loaded hepatocytes, confirming mitochondrial cell death pathways; in contrast, 4-phenylbutyric acid, which improves ER folding capacity did not protect FC-loaded hepatocytes. HMGB1 was released into the culture medium of FC-loaded wild type (WT) but not Jnk1(-/-) or Tlr4(-/-) hepatocytes, while anti-HMGB1 anti-serum prevented JNK activation and FC lipotoxicity in WT hepatocytes. CONCLUSIONS: These novel findings show that mitochondrial FC deposition causes hepatocyte apoptosis and necrosis by activating JNK1; inhibition of which could be a novel therapeutic approach in NASH. Further, there is a tight link between JNK1-dependent HMGB1 secretion from lipotoxic hepatocytes and a paracrine cytolytic effect on neighbouring cholesterol-loaded hepatocytes operating via TLR4.

Pharmacological cholesterol lowering reverses fibrotic NASH in obese, diabetic mice with metabolic syndrome.

BACKGROUND & AIMS: We have recently showed that hyperinsulinemia promotes hepatic free cholesterol (FC) accumulation in obese, insulin-resistant Alms1 mutant (foz/foz) mice with NASH. Here we tested whether cholesterol-lowering drugs reduce stress-activated c-Jun N-terminal kinase (JNK) activation, hepatocyte injury/apoptosis, inflammation, and fibrosis in this metabolic syndrome NASH model. METHODS: Female foz/foz and WT mice were fed HF (0.2% cholesterol) 16 weeks, before adding ezetimibe (5 mg/kg), atorvastatin (20 mg/kg), or both to diet, another 8 weeks. Hepatic lipidomic analysis, ALT, liver histology, Sirius Red morphometry, hepatic mRNA and protein expression and immunohistochemistry (IHC) for apoptosis (M30), macrophages (F4/80), and polymorphs (myeloperoxidase) were determined. RESULTS: In mice with NASH, ezetimibe/atorvastatin combination normalized hepatic FC but did not alter saturated free fatty acids (FFA) and had minimal effects on other lipids; ezetimibe and atorvastatin had similar but less profound effects. Pharmacological lowering of FC abolished JNK activation, improved serum ALT, apoptosis, liver inflammation/NAFLD activity score, designation as "NASH", macrophage chemotactic protein-1 expression, reduced macrophage and polymorph populations, and liver fibrosis. CONCLUSIONS: Cholesterol lowering with ezetimibe/atorvastatin combination reverses hepatic FC but not saturated FFA accumulation. This dampens JNK activation, ALT release, hepatocyte apoptosis, and inflammatory recruitment, with reversal of steatohepatitis pathology and liver fibrosis. Ezetimibe/statin combination is a potent, mechanism-based treatment that could reverse NASH and liver fibrosis.