Narelle Skinner

Regulation of Toll‐like receptor‐2 expression in chronic hepatitis B by the precore protein

Toll‐like receptors (TLRs) play a key role in the innate immune response. The aim of this study was to examine the expression of TLR2 and TLR4 in chronic hepatitis B (CHB). The TLR2 and TLR4 expression on hepatocytes and Kupffer cells from fresh liver biopsies was measured from 21 patients with untreated hepatitis B e antigen (HBeAg)‐positive and HBeAg‐negative CHB. Parallel studies were also undertaken on monocytes from their peripheral blood. Expression of TLR2 on hepatocytes, Kupffer cells, and peripheral monocytes was significantly reduced in patients with HBeAg‐positive CHB in comparison with HBeAg‐negative CHB and controls, whereas it was significantly increased in HBeAg‐negative CHB compared with controls. The level of TLR4 expression did not differ significantly between the groups. These results were confirmed in vitro using hepatic cell lines transduced with recombinant HBV baculovirus expressing wild‐type HBV (HBeAg‐positive), precore stop codon (G1896A) mutant HBV (HBeAg‐negative). The functional relevance of these findings was established by the demonstration of significantly reduced cytokine production (TNF‐α) and phospho‐p38 kinase expression in the presence of the HBeAg. In the absence of HBeAg, HBV replication was associated with up‐regulation of the TLR2 pathway leading to increased TNF‐α production. Conclusion: This study demonstrates a potentially important interaction between HBeAg, HBV, and the innate immune response. (HEPATOLOGY 2007;45:102–110.)

The Hepatitis B e antigen (HBeAg) targets and suppresses activation of the Toll-like receptor signaling pathway

BACKGROUND & AIMS Viruses target innate immune pathways to evade host antiviral responses. Recent studies demonstrate a relationship between hepatitis B disease states and the hosts innate immune response, although the mechanism of immunomodulation is unknown. In humans, the innate immune system recognizes pathogens via pattern recognition receptors such as the Toll-like receptors (TLR), initiating anti-inflammatory responses. TLR expression and pro-inflammatory cytokine production is reduced in hepatitis B e antigen (HBeAg)-positive patients following TLR stimulation. The aim of this study was to investigate interactions between TLR signaling pathways and the mature HBeAg protein localized in the cytosol. METHODS The ability of HBeAg to inhibit TLR signaling and association with TLR adapters was evaluated by immunoprecipitation, immunostaining, and reporter studies. RESULTS Our findings show that HBeAg co-localizes with Toll/IL-1 receptor (TIR)-containing proteins TRAM, Mal, and TLR2 at the sub-cellular level, which was not observed for Hepatitis B core antigen. Co-immunoprecipitation analysis demonstrated HBeAg interacted with TIR proteins Mal and TRAM, while a mutated HBeAg ablated interaction between Mal and MyD88. Importantly, HBeAg also disrupted homotypic TIR:TIR interaction critical for TLR-mediated signaling. Finally, HBeAg suppressed TIR-mediated activation of the inflammatory transcription factors, NF-κB and Interferon-β promoter activity. CONCLUSIONS Our study provides the first molecular mechanism describing HBeAg immunomodulation of innate immune signal transduction pathways via interaction and targeting of TLR-mediated signaling pathways. These finding suggest the mechanism as to how HBeAg evades innate immune responses contributing to the pathogenesis of chronic hepatitis B infection and the establishment of viral persistence.

Homologues of Insecticidal Toxin Complex Genes in Yersinia enterocolitica Biotype 1A and Their Contribution to Virulence

ABSTRACT Yersinia enterocolitica is an enteric pathogen that consists of six biotypes: 1A, 1B, 2, 3, 4, and 5. Strains of the latter five biotypes can carry a virulence plasmid, known as pYV, and several well-characterized chromosomally encoded virulence determinants. Y. enterocolitica strains of biotype 1A lack the virulence-associated markers of pYV-bearing strains and were once considered to be avirulent. There is growing epidemiological, clinical, and experimental evidence, however, to suggest that some biotype 1A strains are virulent and can cause gastrointestinal disease. To identify potential virulence genes of pathogenic strains of Y. enterocolitica biotype 1A, we used genomic subtractive hybridization to determine genetic differences between two biotype 1A strains: an environmental isolate, Y. enterocolitica IP2222, and a clinical isolate, Y. enterocolitica T83. Among the Y. enterocolitica T83-specific genes we identified were three, tcbA, tcaC, and tccC, that showed homology to the insecticidal toxin complex (TC) genes first discovered in Photorhabdus luminescens. The Y. enterocolitica T83 TC gene homologues were expressed by Y. enterocolitica T83 and were significantly more prevalent among clinical biotype 1A strains than other Yersinia isolates. Inactivation of the TC genes in Y. enterocolitica T83 resulted in mutants which were attenuated in the ability to colonize the gastrointestinal tracts of perorally infected mice. These results indicate that products of the TC gene complex contribute to the virulence of some strains of Y. enterocolitica biotype 1A, possibly by facilitating their persistence in vivo.

Reduced Expression of Toll-Like Receptor 2 on Peripheral Monocytes in Patients with Chronic Hepatitis B

ABSTRACT Persistent infection with hepatitis B virus (HBV) likely depends on viral inhibition of host defenses. We report that chronic hepatitis B e antigen-positive HBV infection is associated with a significant reduction in peripheral blood monocyte expression of Toll-like receptor 2, a key component of innate immunity, thereby providing a mechanism by which wild-type HBV may establish persistent infection.

Acute allograft rejection in human liver transplant recipients is associated with signaling through toll-like receptor 4.

Background and Aims:  Toll‐like receptor (TLR) signaling is a crucial step in initiating adaptive immune responses. In addition to recognizing endotoxin, TLR4 also recognizes endogenous ligands (‘damage‐associated structures’), which are released into the circulation in the peri‐transplantation period. TLR2 to a lesser extent also recognizes these endogenous ligands. Multiple studies involving solid organ transplants demonstrate a clear association between TLR4 and allograft rejection. In the present study we assessed whether an association exists between TLR4 and TLR2‐dependent responses and acute liver allograft rejection.

Downregulation of Interleukin-18-Mediated Cell Signaling and Interferon Gamma Expression by the Hepatitis B Virus e Antigen

ABSTRACT The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection. IMPORTANCE It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.

Cyclosporine and tacrolimus have inhibitory effects on toll-like receptor signaling after liver transplantation.

Toll‐like receptors (TLRs) play a key role in transplantation biology. The effect of immunosuppression on TLR function after liver transplantation is unknown. Peripheral blood mononuclear cells (PBMCs) from 113 post–liver transplant patients and 13 healthy controls were stimulated with TLR‐specific ligands [lipopolysaccharide (TLR4), pan‐3‐cys (P3C) (TLR2), Poly (I:C) (PIC) (TLR3), R848 (TLR7/8), and CpG (TLR9)] for 24 hours. PBMCs from 5 healthy controls were also cultured with therapeutic concentrations of cyclosporine A (CYA) and tacrolimus (TAC). Cytokine production was measured with enzyme‐linked immunosorbent assays and flow cytometry. PBMCs from patients on calcineurin inhibitors after liver transplantation produced less interleukin‐6 (IL‐6) and tumor necrosis factor α (TNFα) in response to TLR2 stimulation (IL‐6: P=0.02; TNFα: P=0.01), TLR4 stimulation (IL‐6: P=0.02; TNFα: P=0.01), and TLR7/8 stimulation (IL‐6: P=0.02; TNFα: P=0.02), compared with healthy controls. Both CD56bright and CD56dim natural killer (NK) cells from patients on calcineurin inhibitors also produced less interferon‐γ (IFNγ) with TLR7/8 stimulation compared with healthy controls (CD56bright: P=0.002; CD56dim: P=0.004). Similar findings were demonstrated in healthy PBMCs cultured with CYA (PBMCs: TLR2, IL‐6: P=0.005; TLR4, IL‐6: P=0.03, TNFα: P=0.03; TLR7/8, IL‐6: P=0.02, TNFα: P=0.01; CD56dim NK cells: TLR7/8, IFNγ: P=0.03). TAC impaired TLR4‐mediated IL‐6 and TNFα production by PBMCs (IL‐6; P = 0.02; TNFα P = 0.009). In conclusion, patients on calcineurin inhibitors had impaired inflammatory cytokine production in response to TLR2, TLR4, and TLR7/8 stimulation compared comparison with healthy controls. Importantly, TAC and CYA appear to have different effects on TLR signaling. Impaired TLR function has important repercussions for risk of infection, graft rejection, and disease recurrence after transplantation, and the different immunosuppressive profiles of CYA and TAC may guide the choice of therapy to improve disease outcomes. Liver Transpl 19:1099‐1107, 2013.

Effects of antibiotics on expression and function of Toll-like receptors 2 and 4 on mononuclear cells in patients with advanced cirrhosis

BACKGROUND & AIMS Toll-like receptors (TLRs) are critical to innate immune responses. TLR4 recognises Gram-negative bacteria, whilst TLR2 recognises Gram-positive. We examined TLR expression and function in cirrhosis, and whether this is affected by antibiotic therapy. METHODS Sixty-four subjects were included (23 controls and 41 Child-Pugh C cirrhotic patients). Thirty patients were taking norfloxacin or trimethoprim-sulfamethoxazole as prophylaxis against bacterial peritonitis and 11 were not. In a second study, 8 patients were examined before and after commencement of antibiotics. Monocyte expression of TLR2 and 4 was determined by flow cytometry. Monocytes from the patients with paired samples were stimulated using TLR ligands and TNF-alpha production measured. RESULTS Patients not taking antibiotics had significantly decreased TLR4 expression compared with controls (0.74 vs. 1.0, p=0.009) and patients receiving antibiotics (0.74 vs. 0.98, p=0.02). There were no differences with regard to TLR2. In the patients with paired samples, TLR4 expression increased (0.74-1.49, p=0.002) following antibiotic use, whilst again, there was no change in TLR2 expression (0.99 vs. 0.92, p=0.20). TLR4-dependent TNF-alpha production increased following antibiotic use (1077 vs. 3620pg/mL, p<0.05), whilst TLR2-dependent production was unchanged. CONCLUSIONS TLR4 expression is decreased in patients with Child-Pugh C cirrhosis, but is restored by antibiotics targeting enteric Gram-negative bacteria. TLR4-dependent cytokine production also increases significantly following antibiotic therapy. This suggests that the high incidence of Gram-negative infection in cirrhotic patients is in part due to down-regulation of the TLR4-dependant immune response and that the efficacy of antibiotic prophylaxis is contributed to by modulation of innate immunity.

Toll-like receptor 3 and 7/8 function is impaired in hepatitis C rapid fibrosis progression post-liver transplantation.

Recurrence of hepatitis C (HCV) postliver transplant is universal, with a subgroup developing rapid hepatic fibrosis. Toll‐like receptors (TLRs) are critical to innate antiviral responses and HCV alters TLR function to evade immune clearance. Whether TLRs play a role in rapid HCV recurrence posttransplant is unknown. We stimulated peripheral blood mononuclear cells (PBMCs) from 70 patients with HCV postliver transplant with TLR subclass‐specific ligands and measured cytokine production, TLR expression and NK cell function. Rate of fibrosis progression was calculated using posttransplant liver biopsies graded by Metavir scoring (F0–4; R = fibrosis stage/year posttransplant; rapid fibrosis defined as >0.4 units/year). Thirty of 70 (43%) patients had rapid fibrosis progression. PBMCs from HCV rapid‐fibrosers produced less IFNα with TLR7/8 stimulation (p = 0.039), less IL‐6 at baseline (p = 0.027) and with TLR3 stimulation (p = 0.008) and had lower TLR3‐mediated monocyte IL‐6 production (p = 0.028) compared with HCV slow fibrosers. TLR7/8‐mediated NKCD56 dim cell secretion of IFNγ was impaired in HCV rapid fibrosis (p = 0.006) independently of IFNα secretion and TLR7/8 expression, while cytotoxicity remained preserved. Impaired TLR3 and TLR7/8‐mediated cytokine responses may contribute to aggressive HCV recurrence postliver transplantation through impaired immune control of HCV and subsequent activation of fibrogenesis.

Yersinia enterocolitica Biotype 1A: Not as Harmless as You Think

We have used subtractive hybridisation to identify genomic sequences in a clinical biotype 1A isolate that are absent from a non-clinical biotype 1A strain. We are currently investigating whether these differences contribute to the virulence of the biotype 1A strain.