Identification and characterisation of a rare MTTP variant underlying hereditary non-alcoholic fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is a complex trait with an estimated prevalence of 25% globally. We aimed to identify the genetic variant underlying a four-generation family with progressive NAFLD leading to cirrhosis, decompensation, and development of hepatocellular carcinoma in the absence of common risk factors such as obesity and type 2 diabetes. Methods: Exome sequencing and genome comparisons were used to identify the likely causal variant. We extensively characterised the clinical phenotype and post-prandial metabolic responses of family members with the identified novel variant in comparison with healthy non-carriers and wild-type patients with NAFLD. Variant-expressing hepatocyte-like cells (HLCs) were derived from human-induced pluripotent stem cells generated from homozygous donor skin fibroblasts and restored to wild-type using CRISPR-Cas9. The phenotype was assessed using imaging, targeted RNA analysis, and molecular expression arrays. Results: We identified a rare causal variant c.1691T>C p.I564T (rs745447480) in MTTP, encoding microsomal triglyceride transfer protein (MTP), associated with progressive NAFLD, unrelated to metabolic syndrome and without characteristic features of abetalipoproteinaemia. HLCs derived from a homozygote donor had significantly lower MTP activity and lower lipoprotein ApoB secretion than wild-type cells, while having similar levels of MTP mRNA and protein. Cytoplasmic triglyceride accumulation in HLCs triggered endoplasmic reticulum stress, secretion of pro-inflammatory mediators, and production of reactive oxygen species. Conclusions: We have identified and characterised a rare causal variant in MTTP, and homozygosity for MTTP p.I564T is associated with progressive NAFLD without any other manifestations of abetalipoproteinaemia. Our findings provide insights into mechanisms driving progressive NAFLD. Impact and Implications: A rare genetic variant in the gene MTTP has been identified as responsible for the development of severe non-alcoholic fatty liver disease in a four-generation family with no typical disease risk factors. A cell line culture created harbouring this variant gene was characterised to understand how this genetic variation leads to a defect in liver cells, which results in accumulation of fat and processes that promote disease. This is now a useful model for studying the disease pathways and to discover new ways to treat common types of fatty liver disease.

Prednisolone or pentoxifylline for alcoholic hepatitis.

BACKGROUND: Alcoholic hepatitis is a clinical syndrome characterized by jaundice and liver impairment that occurs in patients with a history of heavy and prolonged alcohol use. The short-term mortality among patients with severe disease exceeds 30%. Prednisolone and pentoxifylline are both recommended for the treatment of severe alcoholic hepatitis, but uncertainty about their benefit persists. METHODS: We conducted a multicenter, double-blind, randomized trial with a 2-by-2 factorial design to evaluate the effect of treatment with prednisolone or pentoxifylline. The primary end point was mortality at 28 days. Secondary end points included death or liver transplantation at 90 days and at 1 year. Patients with a clinical diagnosis of alcoholic hepatitis and severe disease were randomly assigned to one of four groups: a group that received a pentoxifylline-matched placebo and a prednisolone-matched placebo, a group that received prednisolone and a pentoxifylline-matched placebo, a group that received pentoxifylline and a prednisolone-matched placebo, or a group that received both prednisolone and pentoxifylline. RESULTS: A total of 1103 patients underwent randomization, and data from 1053 were available for the primary end-point analysis. Mortality at 28 days was 17% (45 of 269 patients) in the placebo-placebo group, 14% (38 of 266 patients) in the prednisolone-placebo group, 19% (50 of 258 patients) in the pentoxifylline-placebo group, and 13% (35 of 260 patients) in the prednisolone-pentoxifylline group. The odds ratio for 28-day mortality with pentoxifylline was 1.07 (95% confidence interval [CI], 0.77 to 1.49; P=0.69), and that with prednisolone was 0.72 (95% CI, 0.52 to 1.01; P=0.06). At 90 days and at 1 year, there were no significant between-group differences. Serious infections occurred in 13% of the patients treated with prednisolone versus 7% of those who did not receive prednisolone (P=0.002). CONCLUSIONS: Pentoxifylline did not improve survival in patients with alcoholic hepatitis. Prednisolone was associated with a reduction in 28-day mortality that did not reach significance and with no improvement in outcomes at 90 days or 1 year. (Funded by the National Institute for Health Research Health Technology Assessment program; STOPAH EudraCT number, 2009-013897-42 , and Current Controlled Trials number, ISRCTN88782125 ).

IgG4-positive sclerosing cholangitis following autoimmune pancreatitis with deranged CA19.9.

Sclerosing cholangitis is an autoimmune condition characterized by lymphocytic infiltration within the biliary epithelium leading to multifocal stricturing of the biliary tree. Primary sclerosing cholangitis (PSC) is the most common type encountered clinically. However, a similar process may occur in conjunction with autoimmune pancreatitis (AIP), known as AIP-associated sclerosing cholangitis (AIP-SC). This subtype is associated with an elevated IgG(4) level and the presence of a number of autoantibodies. AIP-SC shows good response to steroid treatment, distinguishing it clinically from PSC. The authors report a case of AIP-SC in a patient who had previously undergone a biliary bypass for AIP-induced chronic pancreatitis. The presentation of jaundice and grossly elevated tumor marker, CA19.9, raised the concern of malignancy. The uncertainty of the diagnosis was resolved when AIP-SC was confirmed on liver biopsy, with a concomitantly elevated serum IgG(4) level. The disease went into remission with steroid treatment.

Epithelial inflammation is associated with CCL28 production and the recruitment of regulatory T cells expressing CCR10.

Mucosal tissues require constant immune surveillance to clear harmful pathogens while maintaining tolerance to self Ags. Regulatory T cells (Tregs) play a central role in this process and expression of alpha(E)beta(7) has been reported to define a subset of Tregs with tropism for inflamed tissues. However, the signals responsible for recruiting Tregs to epithelial surfaces are poorly understood. We have isolated a subset of CCR10-expressing CD25+CD4+Foxp3+ Tregs with potent anti-inflammatory properties from chronically inflamed human liver. The CCR10+ Tregs were detected around bile ducts that expressed increased levels of the CCR10 ligand CCL28. CCL28 was secreted by primary human cholangiocytes in vitro in response to LPS, IL-1beta, or bile acids. Exposure of CCR10+ Tregs to CCL28 in vitro stimulated migration and adhesion to mucosal addressin cell adhesion molecule-1 and VCAM-1. Liver-derived CCR10+ Tregs expressed low levels of CCR7 but high levels of CXCR3, a chemokine receptor associated with infiltration into inflamed tissue and contained a subset of alpha(E)beta7(+) cells. We propose that CXCR3 promotes the recruitment of Tregs to inflamed tissues and CCR10 allows them to respond to CCL28 secreted by epithelial cells resulting in the accumulation of CCR10+ Tregs at mucosal surfaces.

Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC), a chronic inflammatory liver disease characterized by progressive bile duct destruction, develops as an extra-intestinal complication of inflammatory bowel disease (IBD) (Chapman, R.W. 1991. Gut. 32:1433-1435). However, the liver and bowel inflammation are rarely concomitant, and PSC can develop in patients whose colons have been removed previously. We hypothesized that PSC is mediated by long-lived memory T cells originally activated in the gut, but able to mediate extra-intestinal inflammation in the absence of active IBD (Grant, A.J., P.F. Lalor, M. Salmi, S. Jalkanen, and D.H. Adams. 2002. Lancet. 359:150-157). In support of this, we show that liver-infiltrating lymphocytes in PSC include mucosal T cells recruited to the liver by aberrant expression of the gut-specific chemokine CCL25 that activates alpha4beta7 binding to mucosal addressin cell adhesion molecule 1 on the hepatic endothelium. This is the first demonstration in humans that T cells activated in the gut can be recruited to an extra-intestinal site of disease and provides a paradigm to explain the pathogenesis of extra-intestinal complications of IBD.

Lymphocyte homing in the pathogenesis of extra-intestinal manifestations of inflammatory bowel disease.

Inflammatory bowel disease is associated with extra-intestinal manifestations which occur either at the same time as flares of bowel inflammation (skin and eye disease) or run a course that is independent to inflammation in the bowel (liver and some joint syndromes). It has been suggested that the skin and eye complications occur as a consequence of the recruitment of activated effector cells released from the gut into the circulation to extra-intestinal site where they cause acute damage. However, this does not explain how patients can develop primary sclerosing cholangitis many years after having their colon removed for colitis. We propose that long-lived populations of memory lymphocytes arise as a consequence of bowel inflammation and that these cells express homing receptors that direct their subsequent migration not only to the gut but also to the liver. These long-lived cells may recirculate to the liver for many years and, in the absence of a local activating stimulus, will not cause damage. However, if they are subsequently activated in the liver this will lead to the development of inflammation and tissue damage which promotes the recruitment of more mucosal lymphocytes resulting in persistent inflammation and disease. The recent findings that MAdCAM-1 and CCL25, previously thought to be restricted to the gut, are up-regulated in the liver during inflammatory liver diseases that complicate IBD support the concept that common mechanisms control lymphocyte recruitment to the inflamed liver and gut.

Hepatic expression of secondary lymphoid chemokine (CCL21) promotes the development of portal-associated lymphoid tissue in chronic inflammatory liver disease.

The chronic inflammatory liver disease primary sclerosing cholangitis (PSC) is associated with portal inflammation and the development of neolymphoid tissue in the liver. More than 70% of patients with PSC have a history of inflammatory bowel disease and we have previously reported that mucosal addressin cell adhesion molecule-1 is induced on dendritic cells and portal vascular endothelium in PSC. We now show that the lymph node-associated chemokine, CCL21 or secondary lymphoid chemokine, is also strongly up-regulated on CD34(+) vascular endothelium in portal associated lymphoid tissue in PSC. In contrast, CCL21 is absent from LYVE-1(+) lymphatic vessel endothelium. Intrahepatic lymphocytes in PSC include a population of CCR7(+) T cells only half of which express CD45RA and which respond to CCL21 in migration assays. The expression of CCL21 in association with mucosal addressin cell adhesion molecule-1 in portal tracts in PSC may promote the recruitment and retention of CCR7(+) mucosal lymphocytes leading to the establishment of chronic portal inflammation and the expanded portal-associated lymphoid tissue. This study provides further evidence for the existence of portal-associated lymphoid tissue and is the first evidence that ectopic CCL21 is associated with lymphoid neogenesis in human inflammatory disease.

Homing of mucosal lymphocytes to the liver in the pathogenesis of hepatic complications of inflammatory bowel disease.

Primary sclerosing cholangitis is strongly linked to inflammatory bowel disease, but any model to explain the development of primary sclerosing cholangitis must take into account the fact that it usually runs a course independent from inflammation in the bowel, illustrated by the fact that this disease can develop many years after proctocolectomy. Thus, liver disease can develop in the absence of a diseased colon and cannot be explained solely by release of toxic factors from the inflamed gut. We propose the existence of an enterohepatic circulation of lymphocytes, whereby some mucosal lymphocytes generated in the gut during active inflammatory disease subsequently persist as longlived memory cells capable of recirculation through the liver. Under the right conditions, these dual-homing lymphocytes might become activated in the liver resulting in hepatic inflammation that is independent from inflammation in the gut. Recent reports that some lymphocyte homing-receptors are shared by the liver and gut provide a molecular basis for this hypothesis and explain the distribution of extraintestinal disease in inflammatory bowel disease.