Maria Jover-Cobos

Metabolic regulation of hepatitis B immunopathology by myeloid-derived suppressor cells

Infection with hepatitis B virus (HBV) results in disparate degrees of tissue injury: the virus can either replicate without pathological consequences or trigger immune-mediated necroinflammatory liver damage. We investigated the potential for myeloid-derived suppressor cells (MDSCs) to suppress T cell–mediated immunopathology in this setting. Granulocytic MDSCs (gMDSCs) expanded transiently in acute resolving HBV, decreasing in frequency prior to peak hepatic injury. In persistent infection, arginase-expressing gMDSCs (and circulating arginase) increased most in disease phases characterized by HBV replication without immunopathology, whilst L-arginine decreased. gMDSCs expressed liver-homing chemokine receptors and accumulated in the liver, their expansion supported by hepatic stellate cells. We provide in vitro and ex vivo evidence that gMDSCs potently inhibited T cells in a partially arginase-dependent manner. L-arginine–deprived T cells upregulated system L amino acid transporters to increase uptake of essential nutrients and attempt metabolic reprogramming. These data demonstrate the capacity of expanded arginase-expressing gMDSCs to regulate liver immunopathology in HBV infection.

Prevention of acute kidney injury in a rodent model of cirrhosis following selective gut decontamination is associated with reduced renal TLR4 expression

BACKGROUND & AIMS Superimposed infection and/or inflammation precipitate renal failure in cirrhosis. This study aimed at testing the hypothesis that increased gut bacterial translocation in cirrhosis primes the kidney to the effect of superimposed inflammation by upregulating expression of Toll-like receptor 4 (TLR4), NFκB, and cytokines. A well-characterized bile-duct ligated (BDL) model of cirrhosis, which develops renal failure following superimposed inflammatory insult with lipopolysaccharide (LPS), was used and selective gut decontamination was performed using norfloxacin. METHODS Sprague-Dawley rats were studied: Sham, Sham+LPS; BDL, BDL+LPS; an additional BDL and BDL+LPS groups were selectively decontaminated with norfloxacin. Plasma biochemistry, plasma renin activity (PRA) and cytokines and, protein expression of TLR4, NFκB, and cytokines were measured in the kidney homogenate. The kidneys were stained for TLR4, TLR2, and caspase-3. Endotoxemia was measured using neutrophil burst and Limulus amoebocyte lysate (LAL) assays. RESULTS The groups treated with norfloxacin showed significant attenuation of the increase in plasma creatinine, plasma and renal TNF-α and renal tubular injury on histology. The increased renal protein expression of TLR4, NFκB, and caspase-3 in the untreated animals was significantly attenuated in the norfloxacin treated animals. PRA was reduced in the treated animals and severity of endotoxemia was also reduced. CONCLUSIONS The results show for the first time that kidneys in cirrhosis show an increased expression of TLR4, NFκB, and the pro-inflammatory cytokine TNF-α, which makes them susceptible to a further inflammatory insult. This increased susceptibility to LPS can be prevented with selective decontamination, providing novel insights into the pathophysiology of renal failure in cirrhosis.

Increased renal expression and urinary excretion of TLR4 in acute kidney injury associated with cirrhosis

Patients with cirrhosis frequently develop renal dysfunction, a proportion of who do not fulfill criteria for hepatorenal syndrome (HRS). We hypothesized that the kidneys in these patients would exhibit histological and biomarker evidence of kidney injury. We looked specifically for TLR expression as they may mediate kidney injury.

Role of Toll‐Like Receptor 4 in Mediating Multiorgan Dysfunction in Mice With Acetaminophen Induced Acute Liver Failure

Strategies for the prevention of multiorgan dysfunction (MOD) in acetaminophen (APAP)‐induced acute liver failure (ALF) are an unmet need. Our study tested the hypothesis that sterile inflammation induced by APAP in a mouse model would activate toll‐like receptor 4 (TLR4) in the liver and extrahepatic organs and lead to the progression of ALF and MOD and that the administration of the novel TLR4 antagonist STM28 (a peptide formed of 17 amino‐acids) would prevent liver injury and associated MOD. ALF and, subsequently, MOD were induced in TLR4‐knockout (KO) mice (B6.B10ScN‐Tlr4 lpsdel /JthJ) and wild‐type (WT) mice (C57BL/6) with APAP (500 mg/kg). A second set of experiments was conducted to evaluate the effects of a pretreatment with a novel TLR4 antagonist, STM28, on APAP‐induced MOD in CD1 mice. Animals were sacrificed at the coma stage, and plasma, peripheral blood cells, liver, kidneys, and brain were collected. Biochemistry values and cytokines were measured. Liver and kidneys were studied histologically and were stained for TLR4 and activated Kupffer cells, and the expression of nuclear factor kappa B–p65 was quantified with western blotting. Brain water was measured in the frontal cortex. After APAP administration, TLR4‐KO (NFkBp65) mice were relatively protected from liver necrosis and end‐organ dysfunction and had significantly better survival than WT controls (P < 0.01). STM28 attenuated liver injury and necrosis, reduced creatinine levels, and delayed the time to a coma significantly. The increases in cytokines in the plasma and liver, including TLR4 expression and the activation of Kupffer cells, after APAP administration were reduced significantly in the STM28‐treated animals. The increased number of circulating myeloid cells was reduced significantly after STM28 treatment. In conclusion, these data provide evidence for an important role of the TLR4 antagonist in the prevention of the progression of APAP‐induced ALF and MOD. Liver Transpl 19:751–761, 2013..

P58 Evidence of dendritic cell dysfunction in cirrhosis and its restoration by Toll-like receptor 4 antagonism

Introduction Infection complicates the course of cirrhosis and is the main cause of admission of patients to hospital. Recent studies have shown evidence of immune paralysis during severe decompensation. Our studies have suggested that neutrophil dysfunction in alcoholic hepatitis patients may be associated with a defect of TLR4 signalling, which is a key pathogen response receptor. Aim The aims of this study were to characterise cellular immune function in a bile-duct ligated (BDL) model of cirrhosis and evaluate the role of TLR4 by using a selective antagonist, STM 28. (STM28 was kindly gifted by Professor Ken-ichi Tanamoto, Division of Microbiology, National Institute of Health Sciences, Tokyo 158-8501, Japan). Method 18 C57BL/6 mice were studied: Sham operated, BDL (2 weeks after ligation) and BDL treated with TLR4 antagonist STM-28 (20 ug IP, 5 days). Peripheral blood was stained with fluorochrome-labellel antibodies specific for peripheral blood myeloid cells, dendritic cells (DCs, plasmocytoid and myeloid, expression of CD86 in myeloid); monocytes (residents and inflammatory); total granulocytes and neutrophils; CD4/CD8 T-cells and the expression of CD25 in CD4 T cell population; B cells and NK cells. Flow cytometric analysis was performed on a FACS Canto II. Biochemistry was measured spectrophotometrically. Results A significant shift towards the myeloid subset of peripheral blood DCs was observed in BDL animals compared with sham (81% vs 54%, p=0.0002) accompanied by a significant decrease in the percentages of plasmocytoid DCs (2% vs 25%, p<0.0001), and an increased expression of CD86 in myeloid DCs (72% vs 55% p=0.05). There was an increase of TLR4 expression on monocytes and neutrophils (p=0.05). The percentage of CD3 T cell and CD8 T cell subpopulations were significantly lower only in BDL group (3% vs 14%, p=0.02; 1% vs 6%, p=0.02 respectively). No significant difference in NK cells was observed. TLR4 antagonist in BDL animals showed restoration towards plasmocytoid subset DCs, with a significant reduction in the percentage of myeloid DCs (67% vs 81%, p=0.003) but there was no significant difference in percentage of total myeloid cells, total granulocytes, neutrophils and monocyte populations. Liver function remained unaffected by the TLR4 antagonist. Conclusion In cirrhosis, myeloid subtype DCs are expanded and there is relative depletion of plasmacytoid DCs and lymphophenia. Treatment with a TLR4 antagonist redresses a shift of dendritic cells from the myeloid back to plasmacytoid type. As the DCs play a pivotal role in antigen presentation, TLR4 antagonism may play an important therapeutic role in decreasing susceptibility of cirrhotic patients to infection.

The Rise of Glutaminase in End-Stage Liver Diseases

Ammonia plays a major role in the pathogenesis of hepatic encephalopathy (HE). Systemic hyperammonemia has been largely found in patients with HE with underlying cirrhosis and acute liver failure. HE is associated with a poor prognosis of both acute and chronic liver disease (Bustamante, Rimola et al. 1999; Hui, Chan et al. 2002). Cirrhosis is the major cause of chronic liver dysfunction and affects 6.5 million people worldwide (Eurostat. 2010, as cited in Albrecht, 2010). HE is the hallmark for acute liver failure (ALF), a syndrome with very high mortality in which liver transplant is the often the only effective treatment.

PWE-267 Urinary TLR4: a novel biomarker to identify patients with acute kidney injury in patients with acute on chronic liver failure: Abstract PWE-267 Figure 1

Introduction Patients with stable cirrhosis often present with acute deterioration of cirrhosis secondary to precipitating illness which may progress to organ failure, a condition referred to as acute on chronic Liver failure (ACLF). A proportion of these patients develop renal dysfunction which do not fulfil criteria for the diagnosis of hepatorenal syndrome (HRS). We hypothesised that the kidneys in patients who develop renal dysfunction in ACLF would exhibit histological and biomarker evidence of acute kidney injury (AKI). Since ACLF is associated with systemic inflammatory response (SIRS) we aimed to look for Toll like receptor (TLR) 4 and 2 which recognise pathogens and when activated lead to apoptosis and production of cytokines. Methods Study 1: 74 subjects [healthy volunteers (6), compensated alcoholic cirrhosis (11), acute deterioration of alcoholic cirrhosis (57)] were included prospectively. Urinary biomarkers, kidney injury molecule-1 (KIM-1, a marker of renal injury), Glutathione S-Transferase (πGST, αGST; markers of proximal and distal tubular injury) (Commercial ELISA), and urinary TLR4 (Western Blotting) were measured. Study 2: Renal biopsies were available from 8 alcoholic cirrhosis patients (3 AKI; 5 HRS) which were stained for TLR4, TLR2 and, Caspase-3. Results Study 1: Nine patients developed AKI on the background of acute deterioration of cirrhosis and 3 had HRS. KIM-1, πGST and αGST were higher in patients with acute deterioration of cirrhosis compared with controls but did not differ in those with and without AKI. Urinary TLR4 values were significantly higher in patients with acute deterioration of cirrhosis with AKI (4.7±1.1) compared to controls (0.38±0.04) and stable cirrhosis (0.32±0.08) and patients with acute deterioration of cirrhosis without renal dysfunction (1.6±0.32) (p<0.01) respectively. Conclusion These data provide evidence for severe tubular injury and apoptosis in patients with AKI and identifies urinary TLR4, as a novel biomarker to identify AKI in Acute deterioration of cirrhosis.Abstract PWE-267 Figure 1 Competing interests None declared.