K. Mehta

Mucosa-associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease

Background/aims Intestinal permeability with systemic distribution of bacterial products are central in the immunopathogenesis of alcoholic liver disease (ALD), yet links with intestinal immunity remain elusive. Mucosa-associated invariant T cells (MAIT) are found in liver, blood and intestinal mucosa and are a key component of antibacterial host defences. Their role in ALD is unknown. Methods/design We analysed frequency, phenotype, transcriptional regulation and function of blood MAIT cells in severe alcoholic hepatitis (SAH), alcohol-related cirrhosis (ARC) and healthy controls (HC). We also examined direct impact of ethanol, bacterial products from faecal extracts and antigenic hyperstimulation on MAIT cell functionality. Presence of MAIT cells in colon and liver was assessed by quantitative PCR and immunohistochemistry/gene expression respectively. Results In ARC and SAH, blood MAIT cells were dramatically depleted, hyperactivated and displayed defective antibacterial cytokine/cytotoxic responses. These correlated with suppression of lineage-specific transcription factors and hyperexpression of homing receptors in the liver with intrahepatic preservation of MAIT cells in ALD. These alterations were stronger in SAH, where surrogate markers of bacterial infection and microbial translocation were higher than ARC. Ethanol exposure in vitro, in vivo alcohol withdrawal and treatment with Escherichia coli had no effect on MAIT cell frequencies, whereas exposure to faecal bacteria/antigens induced functional impairments comparable with blood MAIT cells from ALD and significant MAIT cell depletion, which was not observed in other T cell compartments. Conclusions In ALD, the antibacterial potency of MAIT cells is compromised as a consequence of contact with microbial products and microbiota, suggesting that the ‘leaky’ gut observed in ALD drives MAIT cell dysfunction and susceptibility to infection in these patients.

CHAPTER 1:Betaine in Context

Betaine, an important nutritional component for humans, has been reviewed in the synopsis. Its chemical structure, synthesis, utilization and physiological significance have been discussed. Due to its ability to donate a methyl group, it plays a pivotal role in numerous pathways, including the methionine cycle. A betaine-deficient diet can disturb several cellular processes. Therefore, betaine supplementation to ameliorate certain pathological conditions has been envisaged.

Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells.

Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2, expressing iron-response-element-independent TFRC mRNA to promote cellular iron-overload and examined the effect of excess holotransferrin (5g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1, HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell-surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin-supplemented conditions for 24h and 48h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4h of treatment, while HFE expression altered later at 24h and 48h, suggesting that TFR2 may function prior to HFE in HAMP regulation.

Molecular Effects of Alcohol on Iron Metabolism

© 2016 Elsevier Inc. All rights reserved. This chapter explores the link between iron and alcohol metabolism. Chronic alcohol consumption alters the expression of numerous iron-related proteins, including the liver-secreted systemic iron-regulator hepcidin. Downregulation of hepcidin is the key reason for increased duodenal iron absorption, which causes high circulating and stored iron levels in alcoholics, similar to that attained in hereditary hemochromatosis. The resultant free-iron mediated cytotoxicity is a common determinant of the pathophysiology of cirrhosis, an advanced liver condition observed in both alcoholic liver disease (ALD), and congenital hemochromatosis. Often, alcohol consumption suppresses hematopoiesis and/or hinders normal erythrocyte maturation, which results in anemia. Due to these alcohol-induced changes in iron metabolism, several iron-related biomarkers, like serum levels of ferritin and the glycosylation status of transferrin, are used to detect ALD. Also, modulation of hepcidin expression has been explored as a therapy to limit duodenal iron absorption and thereby restrain the tissue injury mediated by excess iron.

Characterisation of hepcidin response to holotransferrin treatment in CHO TRVb-1 cells.

Iron overload coupled with low hepcidin levels are characteristics of hereditary haemochromatosis. To understand the role of transferrin receptor (TFR) and intracellular iron in hepcidin secretion, Chinese hamster ovary transferrin receptor variant (CHO TRVb-1) cells were used that express iron-response-element-depleted human TFRC mRNA (TFRC∆IRE). Results showed that CHO TRVb-1 cells expressed higher basal levels of cell-surface TFR1 than HepG2 cells (2.2-fold; p < 0.01) and following 5 g/L holotransferrin treatment maintained constitutive over-expression at 24h and 48 h, contrasting the HepG2 cells where the receptor levels significantly declined. Despite this, the intracellular iron content was neither higher than HepG2 cells nor increased over time under basal or holotransferrin-treated conditions. Interestingly, hepcidin secretion in CHO TRVb-1 cells exceeded basal levels at all time-points (p < 0.02) and matched levels in HepG2 cells following treatment. While TFRC mRNA expression showed expected elevation (2h, p < 0.03; 4h; p < 0.05), slc40a1 mRNA expression was also elevated (2 h, p < 0.05; 4 h, p < 0.03), unlike the HepG2 cells. In conclusion, the CHO TRVb-1 cells prevented cellular iron-overload by elevating slc40a1 expression, thereby highlighting its significance in the absence of iron-regulated TFRC mRNA. Furthermore, hepcidin response to holotransferrin treatment was similar to HepG2 cells and resembled the human physiological response.

Iron enhances hepatic fibrogenesis and activates TGF-β signaling in murine hepatic stellate cells

Background: Although excess iron induces oxidative stress in the liver, it is unclear whether it directly activates the hepatic stellate cells (HSC). Materials and Methods: We evaluated the effects of excess iron on fibrogenesis and transforming growth factor beta (TGF‐&bgr;) signaling in murine HSC. Cells were treated with holotransferrin (0.005‐5 g/L) for 24 hours, with or without the iron chelator deferoxamine (10 &mgr;M). Gene expressions (&agr;‐SMA, Col1‐&agr;1, Serpine‐1, TGF‐&bgr;, Hif1‐&agr;, Tfrc and Slc40a1) were analyzed by quantitative real time‐polymerase chain reaction, whereas TfR1, ferroportin, ferritin, vimentin, collagen, TGF‐&bgr; RII and phospho‐Smad2 proteins were evaluated by immunofluorescence, Western blot and enzyme‐linked immunosorbent assay. Results: HSC expressed the iron‐uptake protein transferrin receptor 1 (TfR1) and the iron‐export protein ferroportin. Holotransferrin upregulated TfR1 expression by 1.8‐fold (P < 0.03) and ferritin accumulation (iron storage) by 2‐fold (P < 0.01), and activated HSC with 2‐fold elevations (P < 0.03) in &agr;‐SMA messenger RNA and collagen secretion, and a 1.6‐fold increase (P < 0.01) in vimentin protein. Moreover, holotransferrin activated the TGF‐&bgr; pathway with TGF‐&bgr; messenger RNA elevated 1.6‐fold (P = 0.05), and protein levels of TGF‐&bgr; RII and phospho‐Smad2 increased by 1.8‐fold (P < 0.01) and 1.6‐fold (P < 0.01), respectively. In contrast, iron chelation decreased ferritin levels by 30% (P < 0.03), inhibited collagen secretion by 60% (P < 0.01), repressed fibrogenic genes &agr;‐SMA (0.2‐fold; P < 0.05) and TGF‐&bgr; (0.4‐fold; P < 0.01) and reduced levels of TGF‐&bgr; RII and phospho‐Smad2 proteins. Conclusions: HSC express iron‐transport proteins. Holotransferrin (iron) activates HSC fibrogenesis and the TGF‐&bgr; pathway, whereas iron depletion by chelation reverses this, suggesting that this could be a useful adjunct therapy for patients with fibrosis. Further studies in primary human HSC and animal models are necessary to confirm this.

Erratum to: HFE mRNA expression is responsive to intracellular and extracellular iron loading: short communication (Molecular Biology Reports, (2017), 44, 5, (399-403), 10.1007/s11033-017-4123-2)

The original article has been changed to reflect the correct co-author name: Sebastien Farnaud. The original article was corrected.