Toll‐like receptor signaling in liver regeneration,fibrosis and carcinogenesis

Toll‐like receptors (TLR) are the germline‐coded pattern recognition receptors that sense microbial products. This signaling orchestrates complex signaling pathways that induce expression of inflammatory genes for host defense against invading microorganisms. Recent studies illustrate the role of TLR on non‐infectious inflammatory diseases. The liver has a unique anatomy bridging with the intestine by portal vein and bile ducts. This allows delivery of products from intestinal microflora directly into the liver. Subsequently, microbial products cause acute and chronic inflammation through TLR signaling in the liver. Not only exogenous products, but endogenous denatured products released from dying cells also facilitate inflammation even in sterile conditions. Consequently, these responses elicit tissue repairing including liver regeneration and fibrogenesis. An aberrant regenerative response may lead to hepatic carcinogenesis. In this review, we highlight the recently accumulated knowledge about TLR signaling in liver regeneration, fibrosis and carcinogenesis.     

Significant association between toll‐like receptor gene polymorphisms and gallbladder cancer

Background: Toll‐like receptors (TLRs) are evolutionarily conserved cell surface receptors of innate immune system. Various polymorphisms in TLR genes have been identified and associated with susceptibility toward various malignancies such as prostate cancer, gastric cancer and colorectal cancer. The present study was undertaken to examine the potential association of two polymorphisms in TLR2 and TLR4 genes with gallbladder cancer (GBC) susceptibility. Methods: Genotypes and allelic frequencies of TLR2 and TLR4 gene polymorphisms were determined for 233 GBC patients and 257 cancer‐free controls randomly selected from the population, using polymerase chain reaction–restriction fragment length polymorphism. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated in a multivariate logistic regression analysis for the association of TLR polymorphisms with GBC. Results: ‘del’ allele carriers of TLR2 (Δ22) polymorphism were associated with a 1.54‐fold increased risk for GBC (95% CI=1.02–2.24; P_trend=0.091). The TLR4 Ex4+936C >T polymorphism (g.14143C>T; rs4986791) was also found to be significantly associated with the overall higher risk of GBC under a dominant mode of inheritance (OR=1.96; 95% CI=1.11–2.26; P_trend=0.021). The false‐positive report probability (FPRP) approach advocated that these results were noteworthy (FPRP<0.5). Subgroup analysis showed that TLR4 Ex4+936C>T polymorphism was associated with an increased risk of GBC in females and GBC cases with gallstones (OR=2.85 and 2.22 respectively). Conclusion: In summary, low‐penetrance variants in TLR genes may alter the susceptibility towards gallbladder cancer.     

Salvage effect of E5564, Toll‐like receptor 4 antagonist on d‐galactosamine and lipopolysaccharide‐induced acute liver failure in rats

Background and Aims: The transmembrane protein Toll‐like receptor 4 (TLR4), which exists mainly in macrophages such as Kupffer cells of the liver, plays an important role in recognizing and mediating macrophage activation and pro‐inflammatory cytokine release. Activation of the pro‐inflammatory cytokine cascade, including tumor necrosis factor‐alpha (TNF‐α), has a pivotal role in the progression of severe liver injury. D‐galactosamine (GalN) and lipopolysaccharide (LPS)‐induced liver injury in rats is an experimental model of fulminant hepatic failure, where TNF‐α plays a central role in the progression of liver injury. E5564, a synthetic analogue of the lipid A component of endotoxin, inhibits endotoxin‐stimulated inflammation and is under study for patients with sepsis. In the present study, we sought to explore the salvage effect of TLR4 antagonist E5564 on GalN+LPS‐induced acute liver failure (ALF) in rats. Methods: ALF was induced in male Wistar rats by the intraperitoneal injection of GalN (500 mg/kg) and LPS (50 µg/kg). Immediately after GalN+LPS injection, rats were treated with intravenous injection of E5564 (3 mg/kg). The cumulative survival rates of GalN+LPS‐induced ALF rats were compared between those with and without E5564 treatment. Results: The intravenous injection of E5564 reduced the elevation of serum total bilirubin, aspartate aminotransferase, alanine aminotransferase and TNF‐α levels in rats at 3 h after GalN+LPS injection, and improved the survival rate of GalN+LPS‐induced ALF rats at 24 h (8% vs 43%). Conclusions: TLR4 antagonist E5564 reduced GalN+LPS‐induced acute liver injury in rats and improved the overall survival rate of GalN+LPS‐induced ALF rats. It may contribute to the treatment of ALF through blocking endotoxin‐induced TNF‐α overproduction of macrophages.     

Hepatic Toll‐like receptor 3 expression in chronic hepatitis C genotype 1 correlates with treatment response to peginterferon plus ribavirin

Summary. Recent studies have shown that enhanced hepatic expression of several innate immune genes predicts non‐response to 48 weeks of peginterferon plus ribavirin in chronic hepatitis C genotype 1. This study aimed to further address how gene expression of TLR3/RIG‐I signalling correlates with the outcome of the 72‐week extended treatment regimen. Relative hepatic mRNA expression and copy numbers of positive‐ and negative‐strand hepatitis C virus (HCV) RNA were determined by real‐time PCR in 49 patients. Then, a 48‐week peginterferon‐α2b plus ribavirin treatment was commenced and extended to 72 weeks in cases of HCV RNA clearance after week 12. High rate of sustained virologic response was seen both in patients with early HCV clearance (85% [11/13]) and slow virologic responders (85% [11/13]) (per protocol analysis). The response was associated with low TLR3 expression (median, 0.9; range, 0–4.2 vs median, 1.9; range, 0.4–4.9; P = 0.004) but had no relation to the expression of TRIF (P = 0.315), RIG‐I (P = 0.953), IPS‐1 (P = 0.425), IRF3 (P = 0.329) and interferon‐β (P = 0.584). ROC curve analysis identified TLR3 expression of <1.5 as the best cut‐off for predicting response (positive and negative predictive values, 89% [16/18] and 70% [14/20], respectively). The expression was not affected by HCV replication but was higher in female patients (P = 0.043). Multivariate analysis showed TLR3 to be a single baseline predictor (odds ratio 18.5 [95% CI 3.2–111], P = 0.001). Low hepatic TLR3 expression is a novel predictor of response to peginterferon plus ribavirin in genotype 1 patients.     

Over‐expression of Toll‐like receptors and their ligands in small‐for‐size graft

Aim: Toll‐like receptors (TLRs) participate in several physiological and pathological processes of transplantation, including inflammation and allograft rejection, but the expression of TLRs and their ligands remains undetermined in small‐for‐size graft transplantation. Methods: A non‐arterialized partial liver transplantation model was used. The expression of TLR2 and TLR4 mRNA and protein, CD14 and Myeloid Differentiation‐2 (MD‐2) mRNA, as well as TLR2 and TLR4 exogenous ligands (endotoxin) and endogenous ligands [heat shock protein (HSP) 60 and 70] were assessed. The signaling pathways induced by TLR2 and TLR4 were also assessed. Results: In small‐for‐size liver graft, the expression of mRNA and protein of TLR2 and TLR4, CD14 and MD‐2 mRNA, as well as endogenous ligands of TLR2 and TLR4 such as HSP60 and HSP70 was quickly and significantly increased after reperfusion, and reached a peak at 3 h after reperfusion. The levels of exogenous ligands (endotoxin) were increased and reached a peak at 6 h after reperfusion. The appearance of TLR2 and TLR4 mRNA was accompanied by increased HSP 60 and 70 mRNA within 24 h after reperfusion. In the small‐for‐size group, the peak levels of TLRs and their endogenous ligands appeared earlier than those in the full size group; the peak levels of TLRs and their endogenous and exogenous ligands were higher than those in the full size group. Conclusion: TLR2 and TLR4, as well as their endogenous and exogenous ligands were activated in small‐for‐size liver graft transplantation.     

Reduced toll-like receptor 4 and substance P gene expression is associated with airway bacterial colonization in children

Neuro-immune interactions are increasingly relevant to human health and disease. The neuropeptide Substance P also has antibacterial activity and bears similarities to the innate immune antibacterial defensins. This suggests possible co-regulation of neuropeptide and innate immune mediators. In this study, non-bronchoscopic bronchoalveolar lavage (BAL) was performed on 69 children. BAL was examined for cellular profile, microbiology (bacteria, virus) and gene expression for TLRs 2, 3, 4; chemokine receptors (CCR3, CCR5, CXCR1); neurotrophins and neurokinin genes (TAC1, TAC3, CGRP, NGF). In children with bacterial colonization (n=10) there was an airway inflammatory response with increased BAL neutrophils, IL-8 protein, and CXCR1 expression. Substance P (TAC1) and TLR4 RNA expression were reduced in children with bacterial colonization. TLR3 mRNA was increased in 7.2% (n=5) children with rhinovirus, and there was a non-significant trend to increased TLR2. There is evidence for co-regulation of neurokinin (TAC1) and TLR4 gene expression in airway cells from children with airway bacterial colonization and their reduced expression may be associated with an impaired bacterial clearance.     

Monocytes from children with clinically stable cystic fibrosis show enhanced expression of Toll‐like receptor 4

Lung disease in patients with cystic fibrosis (CF) is characterized by recurrent bacterial respiratory infections and intense airway inflammation. Pattern recognition receptors such as Toll‐like receptor 2 (TLR2) and TLR4 identify bacterial pathogens and activate the innate immune response. We therefore hypothesized that increased expression of these receptors would be found on circulating immune cells from children with CF. A cohort of 66 young children (median age 3 years) with CF was studied and compared to both healthy controls (n = 14) and children without CF who were being investigated for recurrent respiratory infections (non‐CF disease controls; n = 17) of a similar age. Surface expression of TLR2 and TLR4 on peripheral blood monocytes was analyzed using flow cytometry. TLR4 expression was significantly higher in patients with CF compared to healthy controls (P = 0.017) and non‐CF disease controls (P = 0.025) but did not vary according to the presence or absence of pulmonary infection with Gram‐negative or Gram‐positive bacteria (P = 0.387) in the CF group. In contrast, TLR2 expression was similar across all three study groups (P = 0.930). The increased surface expression of TLR4 seen in young children with CF appears to be related to having CF per se and not related to current pulmonary infection. Pediatr. Pulmonol. © 2010 Wiley‐Liss, Inc.     

Polymorphism in 3′‐untranslated region of toll‐like receptor 4 gene is associated with protection from hepatitis B virus recurrence after liver transplantation

L. Zhou, B. Wei, C. Xing, H. Xie, X. Yu, L. Wu, S. Zheng. Polymorphism in 3′‐untranslated region of toll‐like receptor 4 gene is associated with protection from hepatitis B virus recurrence after liver transplantation
Transpl Infect Dis 2011: 13: 250–258. All rights reserved Background Hepatitis B virus (HBV) recurrence is one of the more severe complications following liver transplantation. Toll‐like receptors (TLRs) play a key role in human immunity by recognizing various bacteria, viruses, fungi, and parasites. Single nucleotide polymorphisms (SNPs) in the TLRs are thought to have an impact on the susceptibility to some pathogens. This study focused on the association between polymorphisms in the TLRs and HBV recurrence after liver transplantation in Han Chinese patients. Methods. A total of 41 tag SNPs in TLRs were detected by the snapshot technique in 125 patients with primary HBV‐related diseases receiving liver transplantation in our center from 2004 to 2008. Results. By comparing the genetic variations and clinical data between the HBV recurrence patients and nonrecurrence patients, we found that the variant genotype of rs11536889 (TLR4) was significantly associated with HBV recurrence after liver transplantation (P=0.040, odds ratio was 0.390, 95% confidence interval 0.159–0.957). Conclusion. Our findings indicate that polymorphism in 3′‐untranslated regions of the TLR4 gene may be related to protection from HBV recurrence after liver transplantation in Han Chinese patients.