Gut-liver axis and sensing microbes

'Detoxification' of gut-derived toxins and microbial products from gut-derived microbes is a major role of the liver. While the full repertoire of gut-derived microbial products that reach the liver in health and disease is yet to be explored, the levels of bacterial lipopolysaccharide (LPS), a component of Gram-negative bacteria, is increased in the portal and/or systemic circulation in several types of chronic liver diseases. Increased gut permeability and LPS play a role in alcoholic liver disease where alcohol impairs the gut epithelial integrity through alterations in tight junction proteins. In addition, non-alcoholic fatty liver disease is also associated with increased serum LPS levels and activation of the pro-inflammatory cascade plays a central role in disease progression. Microbial danger signals are recognized by pattern recognition receptors such as the Toll-like receptor 4 (TLR4). Increasing evidence suggests that TLR4-mediated signaling via the MyD88-dependent or MyD88-independent pathways may play different roles in liver diseases associated with increased LPS exposure of the liver as a result of gut permeability. For example, we showed that in alcoholic liver disease, the MyD88-independent, IRF3-dependent TLR4 cascade plays a role in steatosis and inflammation. Our recent data demonstrate that chronic alcohol exposure in the liver leads to sensitization of Kupffer cells to LPS via a mechanism involving upregulation of microRNA-155 in Kupffer cells. Thus, understanding the cell-specific recognition and intracellular signaling events in sensing gut-derived microbes will help to achieve an optimal balance in the gut-liver axis and ameliorate liver diseases.     

Acute ethanol treatment modulates Toll-like receptor-4 association with lipid rafts

BACKGROUND: Alcohol, a substance that is most frequently abused, suppresses innate immune responses to microbial pathogens. The host senses pathogens via Toll-like receptors (TLRs). Recent studies indicate that alcohol affects TLR signaling. METHODS: Here, we hypothesized that acute alcohol treatment may interfere with early steps of membrane-associated TLR2 and TLR4 signaling at the level of lipid rafts. Human monocytes and Chinese hamster ovary (CHO) cells, transfected with human TLR2, TLR4, or CD14, were stimulated with peptidoglycan (PGN, TLR2 ligand) or lipopolysaccharide (LPS, TLR4 ligand) with or without alcohol (50 mM) and analyzed for cytokine production (enzyme-linked immunosorbent assay), nuclear factor-kappaB (NF-kappaB) activation (electrophoretic mobility shift assay), membrane fluidity (fluorescent pyrene eximer formation), and partition of cellular membrane into cholesterol-rich, detergent-resistant domains (DRMs; Western blot). RESULTS: We determined that both TLR2 and TLR4 were located outside the rafts; flotillin, a DRM marker, was resident in the rafts, while CD14 was equally distributed in and outside the rafts in a steady-state condition. PGN forced TLR2 to migrate into DRMs. Engagement of TLR4 and CD14 with LPS induced their migration into the rafts. Alcohol prevented TLR4 partitioning; however, it did not affect TLR2 migration into the rafts. Furthermore, alcohol downregulated TLR4-induced, but not TLR2-induced, NF-kappaB activation and cytokine production in monocytes. We found that alcohol increased membrane fluidity and depleted cellular cholesterol in CHO cells without affecting cell viability. CONCLUSIONS: These data demonstrate for the first time that alcohol disturbs TLR4 and CD14 association with lipid rafts. We propose that alcohol-induced effects on lipid rafts may contribute to modulation of TLR4-CD14-triggered early cellular responses.     

Moderate alcohol intake in humans attenuates monocyte inflammatory responses: inhibition of nuclear regulatory factor kappa B and induction of interleukin 10

BACKGROUND: In contrast to the deleterious effects of chronic excessive alcohol consumption on the liver and cardiovascular system, modest alcohol intake, such as 1 to 2 drinks per day, has benefits on cardiovascular mortality. Little is known about the length of time or the amounts of alcohol consumed that may cause alterations in inflammatory cells such as monocytes that are crucial to atherosclerotic vascular disease. Here, we determine in vivo effects of acute alcohol consumption on inflammatory cytokine production and nuclear regulatory factor kappaB (NF-kappaB) binding in human monocytes. METHODS: Human blood monocytes were isolated by plastic adherence before and after acute alcohol consumption (2 ml vodka/kg body weight). Lipopolysaccharide (LPS)- and superantigen-induced tumor necrosis factor alpha (TNF alpha), interleukin (IL)-1beta, and IL-10 production were then determined in monocytes by ELISA. Nuclear regulatory factor-kappaB activity of monocytes before and after alcohol consumption was estimated by electromobility shift assay and promoter-driven reporter activity. IkappaBalpha was determined by Western blotting in the cytoplasmic extracts. RESULTS: Eighteen hours after moderate alcohol consumption, we found a significant reduction in monocyte production of inflammatory mediators, TNF-alpha and IL-1beta, in response to LPS or staphylococcal enterotoxin B stimulation. Acute alcohol consumption inhibited LPS-induced DNA binding of the p65/p50 NF-kappaB in monocytes that regulates the expression of both the TNF-alpha and the IL-1beta genes. Consistent with this, acute alcohol treatment (25 mM) significantly reduced LPS-induced activation of an NF-kappaB-driven reporter gene suggesting inhibition of this proinflammatory signaling pathway. Further, LPS-induced IkappaBalpha degradation was not affected by acute alcohol consumption indicating an IkappaBalpha-independent mechanism, as observed earlier in the in vitro acute alcohol studies. In contrast, monocyte production of the anti-inflammatory cytokine, IL-10, was augmented by acute alcohol intake. CONCLUSIONS: Our findings suggest that acute alcohol consumption has dual anti-inflammatory effects that involve augmentation of IL-10 and attenuation of monocyte inflammatory responses involving inhibition of NF-kappaB. These mechanisms may contribute to the beneficial effects of moderate alcohol use on atherosclerosis.     

Dose-independent antinociceptive interaction of endogenous ligands at the spinal level

Adenosine, agmatine and kynurenic acid are endogenous ligands acting on different (e.g. adenosine, NMDA, alpha(2)-adrenergic and imidazoline) receptors with a potential role in nociception at the spinal level. Their antinociceptive effects have already been investigated as monotherapy, but only a few studies have reported on their effects on the potency of other drugs. The purpose of the present study was carried out to analyse their interactions during continuous intrathecal co-administration in a carrageenan-induced thermal hyperalgesia model in rats. A paw withdrawal test was used for nociceptive testing. The intrathecal infusion (60 min) of these three drugs was administered alone or in combinations (kynurenic acid+adenosine or agmatine; adenosine+agmatine), which was followed by an additional 60-min observation period. Kynurenic acid alone was ineffective, while adenosine and agmatine alone caused a slight increase in pain threshold. However, independently of the applied doses all of the combinations significantly (p<0.05) increased the paw withdrawal latencies on the inflamed side during and after the infusion, but were almost ineffective on the normal side. The adenosine+kynurenic acid combination was the most effective: namely, that it relieved thermal hyperalgesia in all the applied dose combinations. Treatment with the kynurenic acid-containing combinations also caused dose-dependent side-effects (motor impairment and excitation), despite the fact that monotherapy with kynurenic acid in the applied dose (0.1 microg/min) did not result in adverse effects.     

Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins

Hepatitis C virus (HCV) is a leading cause of end-stage liver disease through sustained inflammation of the liver produced by the host's immune system. The mechanism for HCV evasion or activation of the immune system is not clear. TLRs are cellular activators of the innate immune system. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with TLR1 or TLR6. Here, we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing small interfering RNAs targeted to TLR2, TLR1, and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in human embryonic kidney-TLR2 cells and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-alpha and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1, and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1, or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. In conclusion, TLR2 may use TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins.     

Distinct Toll-like receptor expression in monocytes and T cells in chronic HCV infection

AIM: Hepatitis C virus often establishes chronic infections. Recent studies suggest that viral and bacterial infections are more common in HCV-infected patients compared to controls. Pathogens are recognized by Toll-like receptors (TLRs) to shape adaptive and innate immune responses. METHODS: In this study, to assess the ability of HCV-infected host to recognize invading pathogens, we investigated Toll-like receptor expression in innate (monocytes) and adaptive (T cells) immune cells by real-time PCR. RESULTS: We determined that RNA levels for TLRs 2, 6. 7, 8, 9 and 10 mRNA levels were upregulated in both monocytes and T cells in HCV-infected patients compared to controls. TLR4 was only upregulated in T lymphocytes, while TLR5 was selectively increased in monocytes of HCV-infected patients. MD-2, a TLR4 co-receptor, was increased in patients' monocytes and T cells while CD14 and MyD88 were increased only in monocytes. CONCLUSION: Our data reveal novel details on TLR expression that likely relates to innate recognition of pathogens and immune defense in HCV-infected individuals.     

Peritubular capillary C4d staining in late acute renal allograft rejection – is it relevant?

Abstract: Background: In the early post-transplant period, renal allograft rejection with diffuse peritubular capillary (PTC) C4d deposition predicts poor graft survival. In the late post-transplant setting, that is, one or more yr after transplantation, the implication of diffuse PTC C4d deposition is still a topic of debate. The purpose of our study was to see if diffuse PTC C4d deposition, in late acute rejection (LAR), occurring more than one yr post-transplant, has any impact on graft survival and function. Methods: We selected cases, both cadaveric as well as living donor renal transplant recipients, in whom acute rejection with PTC C4d deposition was first detected after the first year post-transplant. Recipients with multiple acute rejection episodes during the first year post-transplant were excluded from the study. The first biopsy diagnosed with LAR was considered the index biopsy (n = 40). We formed two groups: group 1, C4d-positive LAR (n = 20), and group 2, C4d-negative LAR (n = 20). Groups were matched for maintenance and post-rejection immunosuppressive therapy, baseline serum creatinine levels before the time of the index biopsy, time from transplant to index biopsy, as well as chronic allograft damage index (CADI) score in the index biopsies. We compared the rate of graft loss, and the graft function of the surviving grafts at the end of the study period, as well as histologic parameters in the index biopsy specimens between the two groups. The mean follow-up period was 20 months. Results: No significant differences in the rate of graft loss or graft function were found between groups 1 and 2 at the end of the follow-up period. Histologically, PTC margination and transplant glomerulopathy were more common in the C4d-positive group, and this difference was statistically significant. There was no statistically significant difference in the degree of plasma cell infiltrates. Conclusions: Unlike in the acute setting, the presence or absence of PTC C4d staining in renal allografts with LAR may not have a predictive value regarding graft outcome.     

Pattern recognition receptors: a contemporary view on liver diseases

Pattern recognition receptors (PRRs) function as sensors of microbial danger signals enabling the vertebrate host to initiate an immune response. PRRs are present not only in immune cells but also in liver parenchymal cells and the complexity of the cell populations provide unique aspects to pathogen recognition and tissue damage in the liver. This review discusses the role of different PRRs in pathogen recognition in the liver, and focuses on the role of PRRs in hepatic inflammation, cholestasis, ischemia, repair and fibrosis. PRRs as novel therapeutic targets are evaluated.     

Critical role of toll-like receptors and the common TLR adaptor, MyD88, in induction of granulomas and liver injury

BACKGROUND/AIMS: Toll-like receptors (TLR) recognize pathogens and regulate innate immune activation. Here, we investigated the roles of TLR9 and the common TLR adaptor, MyD88, in liver injury. METHODS: C57BL6, TLR9(-/-), IFNgamma(-/-) or MyD88(-/-) mice were primed with Propionibacterium acnes, TLR9 (CpG) or TLR2 (lipoteichoic acid) ligands followed by LPS challenge. ALT, cytokines and liver histology were assessed. RESULTS: Selective priming through TLR9 but not TLR2 induced granulomas, elevated serum ALT, and sensitized C57BL6 mice to increased LPS-induced serum IL-6, IL-12 and IFNgamma levels. Further, TLR2 and TLR9 ligands synergized in induction of granulomas and sensitization to LPS-induced inflammation. IFNgamma induction by P. acnes, TLR2 and TLR9 ligands required MyD88. In MyD88(-/-) mice P. acnes failed to induce granulomas and both MyD88 and TLR9 deficiency prevented P. acnes-induced sensitization to LPS. Increased mRNA expression of genes of the TLR4 signaling complex (TLR4, CD14, MD-2, and MyD88) and the NADPH complexes (p47phox, p67phox, gp91phox, and p22phox) was induced by priming with P. acnes or TLR9 plus TLR2 suggesting mechanisms for LPS sensitization and liver injury. CONCLUSIONS: TLR9+/-TLR2 activation via MyD88-dependent pathways plays a pivotal role in liver sensitization and granuloma formation.