Constituting 60% of liver cells, hepatocytes are the principal site for PRR production[5,33]. They express mRNA for all TLRs and are responsive to multiple PAMPs, while respond fairly weakly to TLR2 and TLR4 ligands[5,9,33]. While TLR4 expression in hepatocytes is not upregulated by proinflammatory mediators, hepatocytes show increased responsiveness to TLR2 ligands under inflammatory conditions leading to up-regulation of TLR2 expression by LPS, TNF-alpha, bacterial lipoprotein, and IL-1β in an NF-κB-dependent manner[5,11,33,60,61].

Accounting for approximately 20% of non-parenchymal cells, KCs play a significant role in host defense by orchestrating the inflammatory response via functional properties, including phagocytosis, antigen processing and presentation, and secretion of proinflammatory mediators such as cytokines, prostanoids, nitric oxide, and reactive oxygen intermediates[5,9,11,33,62].

KCs express TLRs 2, 3, 4 and 9 and have a higher threshold for activation when compared with other immune cells given their milieu[5,9,33,63].

KCs are less responsive to “LPS tolerance” in the physiological environment, whereas upon activation, they produce several pro-inflammatory (IL-6, IL-12, IL-18 and TNFα) and anti-inflammatory (IL-10) mediators[33,64-66]. Additionally, KCs produce IFN-β, upregulate the expression of MHC-II/costimulatory molecules and promote T cell proliferation and IFN-γ production; when stimulated with TLR3/TLR4 ligands; TLR1/TLR8 ligands and TLR1/2/4/6 ligands, respectively[22,33].

Constituting < 1% of non-parenchymal cells, HSCs undergo an activation process after liver injury and become the main liver cell type that produce extracellular matrix, contributing onset of liver fibrosis[67-70].

HSCs express TLRs 4 and 9, whereas expression of TLR2 is induced by TLR4 stimulation in HSCs[68-70]. Activated HSCs express TLR4 and CD14 and respond to LPS upon the activation of IKK/NF-κB and c-Jun N-terminal kinase (JNK) as well as the secretion of proinflammatory cytokines such as transforming growth factor (TGF)-β, IL-6, IL-8 and several chemokines such as MCP-1, MIP-2, intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin[9,33,70]. TLR4 enhances TGF-β signaling, and stellate cell activation was shown to promote hepatic fibrosis[71]. In chimeric C3H/HeJ mice with TLR4 mutation in HSC or KCs, amelioration of hepatic fibrosis by LPS indicated a cardinal role for KCs and HSC in hepatic inflammation and fibrosis[9,72]. LPS was shown to downregulate the TGF-β pseudoreceptor BAMBI in quiescent HSCs to induce TGF-β signaling and stellate cell activation[71]. Additionally, TLR9 signaling activated via DNA from apoptotic hepatocytes was shown to modulate liver fibrosis via its effects on HSC differentiation through increased collagen production and inhibited HSC migration[73]. Hence, LPS and other TLR ligands are suggested to facilitate fibrogenic responses in the liver via their direct effects on HSCs[9,11,33].

Accounting for approximately 5% of non-parenchymal cell population in the liver, BECs are commonly exposed to several gut-derived microbes[74,75]. BECs mainly express TLRs 2, 3, 4 and 5, which are upregulated by IFN-γ stimulation[74,75]. TLR2 and TLR4 activation results in increased IRAK-M expression and provide negative feedback in human intrahepatic BECs[76].

Under normal conditions, increased IRAK-M expression is critical in preventing undesired induction of the TLR signaling cascade, while in case of inflammatory conditions, upregulation of BEC-associated TLRs leads to IFN-c and TNF-α exposure, participating in biliary pathogenic responses[9,75].

Making up 50% of the non-parenchymal cells, SECs function in hepatic perfusion and nutrient supply[66,77-79]. They express TLR3, 4 and 9 and show increased NF-κB activation and CD54 expression alongside a limited ability to trigger leukocyte adhesion after LPS stimulation[66,77-79]. Although these effects indicate a scavenging role and thus the likelihood of SECs acting as antigen presenting cells, the exact role of the TLR signaling in inflammatory process in SEC remains inconclusive[9,11,33,66,77-79].

Isolated SECs from WT mice were shown to respond to TLR1, 2, 6 and 9 ligands via producing TNF-α; to TLR3 ligands by producing TNF-α, IL-6 and IFN-β; and to TLR4 ligands via production of TNF-α and IL-6[22,33]. Upon TLR8 ligand binding, SECs leads to TNF-α production alongside upregulation of major histocompatibility complex (MHC)-II and co-stimulatory molecules. Stimulation of SECs by TLR1, 2 or 6 ligands is suggested to be associated with activation of allogeneic T cells, as evaluated by the mixed lymphocyte reaction[22,33]. The SEC immune response is also modulated by LPS tolerance, which appears to be based on prostanoid expression rather than regulation at the level of TLR4 surface expression[78]. Although SECs have been suggested to be involved in the hepatic uptake of LPS in some studies, several studies have not confirmed such a role[33,80,81].

Comprising < 1% of non-parenchymal cells, hepatic DCs are recruited into the liver sinusoids during inflammation and then they may migrate to periportal and pericentral areas[5,33,82,83]. Plasmacytoid DCs (pDCs), myeloid DCs, lymphoid DCs, mixed lymphoid + myeloid DCs and natural killer DCs are amongst the DC subsets, whereas lymphoid and myeloid DCs are considered conventional DCs[33,82,83].

Each DC subset show distinct TLR expression pattern in humans with TLR1, 7 and 9 expression via pDCs, while expression of all TLRs excluding TLR9 by other DC subsets[20,33,84]. Cytokines TNF-α, IL-6 and IL-12 TLR7 are produced by hepatic pDCs upon TLR7 and TLR9 activation, whereas TNF-α and IL-6 in response to TLR2, TLR3 and TLR4 activation[50,85].

NAFLD and steatohepatitis is characterized by a pathologic spectrum that ranges from fatty liver (hepatic steatosis) to cirrhosis with intervening non-alcoholic steatohepatitis (NASH) and usually occurs in association with obesity and insulin resistance[13,72,88-90].

Increased serum PAMP levels were observed in both experimental models and in NAFLD patients[9,18,91-96]. A shift in microbial populations to adopt an “obese” phenotype in NAFLD is referred to as “metabolic endotoxaemia”, in which a high-fat diet is associated with elevated levels of LPS translocation[27,90,97].

While TLR2, TLR4 and TLR9 participate in the development of NASH and NAFLD, LPS-TLR4 is considered to be the main pathway for the progression of NAFLD[98-100]. The role of bacterial overgrowth has also been associated with development of NASH, emphasizing the interaction between bacterial overgrowth, gut permeability and liver injury[90,101,102].

While the role of adipose tissue macrophages in the development of NAFLD is not yet clear, KCs are known to play a pivotal role in the development of NAFLD alongside accompanying hepatic inflammation and related complications[18,98].

When inflammation occurs in NAFLD, NF-κB and transcriptional factor AP1 are activated, stimulating the production of TNF-α and IL-10, in particular, by KCs[23,103]. Studies in animal models indicated the likelihood of TLRs 2, 4 and 9 to participate in NAFLD onset or progression[9,18,91,104]. LPS/TLR4 and TLR9 signaling in KCs have been associated with both onset and progression of NAFLD by inducing reactive oxygen species (ROS)-dependent activation of X-box binding protein-1 and IL-1b, respectively, whereas induction of hepatic steatosis occurs independent of TLR2 signaling in KCs[18,104-106] (Figure 2).

While free fatty acids and denatured host DNA are considered to be potential candidates to activate TLR2, TLR4 and TLR9 signals, no clear-cut evidence exists to confirm their capacity to activate TLRs in NAFLD[18]. TLR4 signaling has been considered to play a major role in the pathogenesis of NAFLD that operates via KCs stimulation and increased ROS and TNF-α production[13].

ALD is described along a disease spectrum ranging from steatosis and steatohepatitis to fibrosis and cirrhosis and potential development of hepatocellular carcinoma (HCC)[90,107].

Despite a strong association between alcohol and hepatotoxicity, the exact pathogenesis has not yet been elucidated[90]. Involvement of the gut microbiota via a “leaky” gut has been indicated in the development of ALD[18], whereas the role of alcohol has also been suggested in increasing gut permeability by disrupting tight junctions[108,109]. Increased plasma LPS levels and hepatic endotoxin levels, which leads to increased TLR4 signaling on KCs, HSC, LSECs and hepatocytes and thus the release of pro-inflammatory cytokines have been associated with inflammation and liver damage[9,107,108,110].

Recent studies indicate significant contribution of TLR4 signaling and thus the crucial role of both KCs and HSCs in development of gut-derived endotoxin related effects in ALD[18]. Chronic alcohol consumption is also associated with the increased expression of TLR1, TLR2, TLR4 and TLR6-TLR9, which further potentiates the secretion of the pro-inflammatory TNF-α in response to LPS[111].

KCs produce pro-inflammatory cytokines (TNF-α, IL-1, IL-6 and IL-8, chemokines) and profibrogenic factors (TGF-β) under post-LPS mediated TLR4-dependent stimulation, and consequent liver inflammation and stellate cell activation induce liver fibrosis[9,15,112,113]. The TLR4-dependent downstream signaling cascade in ALD was shown to proceed via the MyD88-independent pathway, possibly via adapter molecule TRIF[114]. Nonetheless, increased expression of not only TLR4 but also other TLRs such as TLR1, 2, 6, 7, 8 and 9 was shown in an experimental chronic alcohol model[115].

Although activation of KCs via TLR4 signaling is a key event in the pathogenesis of alcohol-induced liver injury[18], recent data emphasize the activation of TLR4 signaling in HSCs as well, indicating the their contribution to alcohol-induced hepatocyte injury, steatosis, inflammation, and fibrogenesis[18,116]. In HSCs, activated TLR4 signaling downregulates TGF-β pseudoreceptor BMP and activin membrane-bound inhibitor (BAMBI), resulting in enhancement of TGF-β signaling, whereas BAMBI downregulation is dependent on MyD88 but not TRIF[18,110]. The TLR4-TRIF-IRF3-dependent pathway associated with bone marrow-derived cells including KCs is considered to be more important than the TLR4-MyD88-dependent pathway in the development of alcoholic steatohepatitis[18,110,114].

Acting through upregulation of TLR4 and MD-2 and induction of a Th1-type immune response, bacterial DNA recognition by TLR9 was also shown to be associated with LPS induced liver injury[117], indicating the likelihood of TLR9 signaling to contribute to pathogenesis of ALD[18].

The development of hepatic fibrosis and consequent cirrhosis upon continued liver insults may occur in any type of chronic hepatic injury, including viral hepatitis, alcohol, autoimmune and metabolic disease[9,67].

Prolonged or repeated liver injury leads to a maladaptive interplay of hepatocytes, HSCs and KCs in association with TLR expression, eventually resulting in abnormal extracellular matrix protein deposition in the liver[35,67,118].

LPS-TLR4 activation is considered essential for hepatic fibrogenesis, whereas TLR4 is expressed on KCs and HSCs, the key mediators of hepatic fibrogenesis[27,75,80,81].

KCs express the highest levels of TLR4 and act as the principal target of LPS leading to release of several pro-inflammatory and pro-fibrogenic mediators[5,27,71,114,119]. However, HSCs are crucial in the pathogenesis of fibrosis and cirrhosis given their myofibroblastic phenotype and ability to produce collagen, the principal component of fibrotic tissue[9,120].

Activation of HSC occurs either via pro-inflammatory cytokines and growth factors secreted by LPS-TLR4-stimulated KCs, or directly via LPS-TLR4-dependent HSC stimulation[9,71]. LPS/TLR4 signaling in HSCs is essential for development of liver fibrosis and acts via stimulating production of chemokines that recruit KCs alongside enabling unrestricted activation of HSCs by KCs-derived profibrogenic cytokine TGF-β[11,13,103,121] (Figure 2).

TLR4 activation in HSCs is considered to be the main step for collagen production and the main mediator of fibrosis and cirrhosis[9,11,67,70,71].

KCs induce fibrogenesis by means of proinflammatory and profibrogenic cytokine secretion, whereas HSCs are the leading source of extracellular matrix production in the fibrotic liver[11,67].

TLR9 signaling-associated metabolic pathways are also considered important in the genesis of hepatic fibrosis in vivo, leading to activation of pathways such as IL-1 production and thus HSCs by upregulating profibrogenic genes, such as procollagen type I and tissue inhibitor metalloproteinase-1[16,69,103,104].

Moreover, a deficiency of TLR3-mediated NK cell-dependent apoptosis of HSCs has been linked to the progression of alcohol-induced liver fibrosis[122,123]. Upregulation of TLR2 was shown to promote liver inflammation and fibrogenesis in NASH[106] and HSCs activation and inflammation response during carbon tetrachloride-induced liver fibrosis mediated via MAPK and NF-jB signaling pathways[124], whereas TLR5 was also shown to be directly involved in the progression of fibrosis via activation of the NF-κB and MAPK signaling pathways[52].

Hepatitis B virus (HBV) is a DNA virus responsible for acute hepatitis, which is self-limiting in 80%-90% of adults and chronic in 10%-20% of cases[5,125]. Hepatitis B is associated with an increased risk of developing cirrhosis, hepatic decompensation and HCC, but prognosis shows interpersonal variation depending on the viral susceptibility and induction of antiviral immune response[126,127].

Indicating the role of TLRs in HBV infection, the activation of TLR3, TLR7 and TLR9 as well as TLR4 and TLR5, has been associated with blockage of viral replication via IFN-dependent inhibition of HBV[76,128,129]. Moreover, HBV leads to TLR downregulation alongside restriction of receptor activity, increasing the likelihood of persistent infection[27].

In vitro HBV studies on TLR expression in HepG2 cells revealed elevated expression of TLRs 2, 3, 4, 5, 6, 7 and 9 mRNA upon ligand binding along with an induced IFN response and abolished HBV DNA replication and RNA transcription, whereas no or very limited expression of TLRs 1, 8 and 10[9,130]. Furthermore, transfection of HBV-positive cell lines with TLR adaptor molecules was shown to be associated with elevated TLR activity and a consequent reduction in HBV DNA and mRNA levels[131], whereas HBV replication was completely abolished after injection of TLR3, TLR4, TLR5, TLR7 and TLR9 ligands into HBV transgenic mice[129].

TLR1, TLR2, TLR4 and TLR6 were shown to be downregulated in HBV-infected peripheral blood monocytes along with a decreased cytokine response to TLR2 and TLR4 ligands[132]. Downregulation of TLR2 on hepatocytes and hepatic KCs was demonstrated in HBeAg-positive CHB-infected patients, whereas upregulation of TLR2 and cytokine expression was observed in HBeAg-negative CHB patients[133]. Hence, HBeAg-induced downregulation of TLR2 via precore protein has been accused for the accelerated progression of disease in HBeAg-positive patients[9,133].

Although HBV is able to downregulate TLRs and thus avoid anti-viral pathways, prolonged infection and loss of HBeAg is considered likely to upregulate TLR signaling pathways such as TLR2 that are not primarily involved in anti-HBV responses while trigger hepatic inflammation and disease progression[11].

In vitro analysis of HBV-Met cells revealed that TLR-treated KCs and SECs to have a modulatory effect on HBV replication[134]. TLR3- and TLR4-stimulated KCs and TLR3-activated SECs were shown to affect HBV replication via MyD88-independent pathway[66]. HBV-suppressing effect was mediated by IFN-β in case of TLR3 ligand activation, whereas by cytokines of an undefined nature in case of TLR4-activated KCs[66].

HBV is a stealth virus and thus does not induce an IFN response during the early phase of infection, whereas its recognition by liver resident cells is considered likely to activate innate immune responses without IFN induction[107,135]. Notably, HBV was shown to be recognized by hepatic NPCs, mainly by KCs, leading to NF-κB-dependent induction of the release of the inflammatory cytokines IL-6, IL-8, TNF-α and IL-1β as well as reduced expression of transcription factors essential for HBV gene expression and replication including hepatocyte nuclear factor (HNF) 1α and HNF4α[136].

Hepatitis C virus (HCV) is a hepatotropic virus responsible for development of chronic hepatitis and related complications such as liver cirrhosis, liver failure or HCC[137,138].

Similarly to HBV, current evidence indicates that HCV selectively impairs activation of TLR signaling controlling HCV replication, while it concomitantly stimulates TLR pathways that generate a chronic inflammatory state leading to persistent liver injury[11,27,139,140].

HCV-induced inhibition of TLR signaling contributes to its chronicity related to virus dissemination, inflammation and eventual progression to fibrosis and cirrhosis[9,11].

Regulation of HCV replication by non-parenchymal liver cells occurs through the production of IFN-β upon their stimulation by TLR3 and TLR4[141]. The inhibitory effect of HCV proteins on TLR7 and TLR9, is also likely to prevent virus clearance[27]. Furthermore, activation of TLR2 along with TLR1 and TLR6 and possibly TLR4 by HCV core protein and NS3 promotes hepatic inflammation and injury[142-145].

In the presence of HCV, significantly decreased TLR7 expression along with TLR7-independent activation of IRF-7 pathway was demonstrated both in vitro and in vivo[146].

The NS3/4A serine protease of HCV, HCV NS3 protein and HCV NS5A act via three signaling pathways including the TLR3-TRIF-TBK1-IRF-3, TLRMyD88, and RIG-I/MDA5-IPS-1 pathways to enable HCV to evade innate immune signaling[33]. Moreover, LPS, the HCV core protein and IFN-γ have been suggested to amplify inflammatory monocyte/macrophage activation via formation of MyD88-IRAK complexes, increased NF-κB activation and increased production of TNF-α, leading to the loss of TLR tolerance[147].

Based on these findings, both host- and virus derived factors have been considered likely to act on macrophages to induce persistent inflammation during chronic HCV infection[53,107].

Diseases associated with uncontrolled innate immunity related to TLR ligand exposure in the liver (fibrosis, hepatitis B and C infection, ALD and NASH) are also among the etiologies for HCC. Therefore, it appears likely that TLRs play a role in the development of inflammation-associated liver cancer and are involved in the progression of HCC[18,107]. Hence, chronic hepatic inflammation and fibrosis, as regulated by TLR activation, promotes HCC formation in approximately 10% of cases of cirrhosis[9,54].

TLRs, TLR4 in particular, are considered to play a significant role in associating hepatic chronic inflammation and hepatocarcinoma[13]. A significant regression in liver tumors in TLR4 and MyD88 deficient mice indicates a prominent contribution of TLR signaling to hepatocarcinogenesis[23,148].

HCC has been indicated to be promoted via gut microbiota and TLR4 in association with increased production of proinflammatory cytokines (TNF-α, IL-6), hepatomitogen epiregulin expression and prevention of apoptosis, whereas a reduction in the development of HCC was shown via gut sterilization, germ-free status or TLR4 inactivation[18,149,150].

Activation of KCs via TLRs is considered to be involved in the process of tumorigenesis[18] by inducing proinflammatory cytokines and hepatomitogens responsible for enhanced development of HCC[150,151], whereas TLR4 expression on non-marrow-derived resident liver cells is considered to be required for the promotion of HCC[149].

TLR4 contributes significantly to hepatic inflammation and fibrosis, whereas upregulation of inflammatory factors such as COX-2 and NF-κB by TLR4 as well as the TLR adaptor protein Myd-88 is also important in hepatocarcinogenesis[148,152-155]. TLR3 expression is suggested to contribute to hepatocarcinoma via proapoptotic activity, while activation of TLR9 via CpG DNA of HBV has been associated with malignant transformation in liver cells[27,156,157].

Although, TLR2 binding with ligands such as HMGB1 and HSPA1A is associated with tumor enhancement, the effect of TLR2 activation is considered likely to differ according to the phase of HCC carcinogenesis, with anti-oncogenic potential slowing down the onset and development of HCC in earlier phases, whereas pro-oncogenic potential during later stages that promotes the progression of inflammation and fibrosis[158].

Activation of the NF-κB and JNK pathways and higher expression levels of IKKα and IKKβ are considered critical in the production of the cytokines related to TLR-induced liver damage and HCC progression[107].

Recently, spontaneous HCC development was demonstrated in hepatocyte-specific TAK1 deleted (TAK1DHEP) mice along with a resistance for HCC development that occurs via deletion of MyD88, TLR4 or TLR9 signaling[159].

Alcohol and HCV are suggested to interact in causing progression of liver disease and malignancy, whereas TLR4, TLR4 downstream gene Nanog and activated LPS-TLR4 are also considered to contribute to this synergy via triggering proliferative and anti-apoptotic signals to non-marrow-derived resident liver cells and thus HCC progression[9,149,150,160].

Ischemia-reperfusion (I/R) injury in partial hepatectomy and liver transplantation is associated with the release of various endogenous ligands for hepatic tissue TLRs and thus the activation of complex signaling pathways that induce neutrophilic and T-lymphocytic tissue inflammation and injury[53,161,162].

Among the most studied TLRs in hepatic I/R, TLR4 was shown to participate in certain acute sterile injury models, including liver I/R, by mobilizing the immune system upon detection of endogenous ligands, whereas limited data are available on TLR2 and TLR9[163,164].

MyD88-independent activation of TLR4 by DAMPs is considered central to the inflammatory process observed in I/R lesions[165-167], whereas HSP, heparan sulfate, fibronectin, fibrinogen, hyaluronan and HMGB1 are known to act as endogenous ligands for TLR4 activation in hepatic I/R injury[5,163].

Release of HMGB1 activates the cell surface TLR4 on KCs and leads to a subsequent release of cytotoxic mediators (TNF-α, IL-6 and chemokine IP-10), alongside an inappropriate activation of the pro-apoptotic protein kinase JNK and stress-responsive NF-κB, all of which are mediators of cell injury[5,163,168,169]. Cellular expression of TLR4 is further upregulated via newly synthesized mediators such as TNF-α, leading to formation of a vicious cycle of proinflammatory cytokine production[61,163,170].

Downstream TLR4 signaling pathways in I/R injury seems to be independent of MyD88 signaling, whereas TRIF-dependent activation of the interferon response and IRF1 expression is considered critical for mediating I/R injury in hepatocytes in terms of releasing the danger signal HMGB1[164,171,172]. Hence, TLR4, IRF1 and HMGB1 are considered three important and interacting mediators of I/R injury[164].

Albeit not consistent, available data suggest that besides lack of TLR4, downregulation of TLR2 expression in the donor organ also suppress I/R injury[27,165,173]. Accordingly, given the amelioration of liver injury in I/R via non-selective inhibition of TLR2 and TLR4 activation by certain molecules such as bicyclol or N-acetylcysteine, role of TLRs in I/R lesion has been emphasized[27,174,175].

TLR9, which shows affinity toward both pathogen-derived and endogenous host DNA, is considered to play a crucial role in non-pathogen-induced hepatic I/R injury by causing neutrophil activation, liver necrosis, and inflammatory cytokine release[163,176,177].

Although TLR signaling dependent early activation of the innate immune system is consistently reported in the setting of I/R injury, additional studies are required to fully explore the roles of other TLRs and TLR signaling pathways in I/R injury[163,164].

Recognizing the mechanism of liver regeneration is important not only for managing acute liver failure and post-transplant hepatic dysfunction but also for disturbed liver regeneration in NASH or NAFLD and advanced liver fibrosis[178]. The deposition of excessive amounts of extracellular matrix, the presence of persistent inflammation, the transformation of SECs and HSCs, portal blood flow reduction and increased JNK activity are considered among the factors associated with the regenerative ability of fibrotic livers[178,179].

TLR/MyD88-mediated pathways are associated with onset of liver regeneration after partial hepatectomy (PH) via activation of NF-κB, release of TNF-α and IL-6 and the expression of the immediate early genes for cell replication in hepatocytes, whereas distinct TLR ligands responsible for the priming process have not yet been clarified[33,178]. No contribution of TLR2, TLR4 or TLR9 to MyD88-mediated pathways and no influence of TLR2 or TLR4 on proinflammatory cytokine production or gene replication have been reported for liver regeneration after PH[33,180,181].

In fact, given the inhibition of regenerative process via excessive TLR signaling produced by LPS injection after PH, the magnitude of TLR signaling is considered critical for intact liver regeneration[178,182].

TLR3 signaling, which utilizes a distinct adaptor protein, TRIF, is considered to attenuate the initiation of liver regeneration via TLR3-dependent NF-κB activation in hepatocytes and TLR3-induced IFN-γ through STAT1 and consequent induction of the IRF-1 and p21 pathways[178,183,184].

In addition, although a non-TLR MyD88-dependent pathway with IL-1 and IL-18 has been suggested to play a role in allograft rejection initially, findings on the existence of normal liver regeneration after PH in caspase 1-deficient mice indicate unremarkable participation of IL-1β and IL-18 in liver regeneration[178].

Although antibody formation against self-antigens is key to the development of autoimmune hepatic diseases, including autoimmune hepatitis, primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC)[185], recently the influence of gut microbiota on the propagation of these diseases has been indicated[90].

Given that the liver is considered a classical immunoprivileged site, TLR signals may act as an important promoter for overcoming this immunoprivilege and in–ducing hepatic autoimmune disease[11,13,186].

Previous studies have suggested regulator role of gut-derived products on T cell function within the liver[90], based on the connection between TLR4 signaling and the trapping of CD8+ T cells in the murine liver[187], as well as contribution of TLR9 to the homing and stimulation of hepatic NKT cells via a KC and IL-12 dependent process[188]. The role of LPS/TLR4 signaling has been indicated in the pathogenesis of PBC and PSC[13]. Monocytes from PBC patients have been suggested to show increased sensitivity to activation of selective TLRs (TLR2, TLR4, TLR3, TLR5 and TLR9), while the subsequent release of proinflammatory cytokines has been associated with development of self-tolerance and autoimmune progression[189] (Figure 2).

LPS was shown to accumulate in significant amounts in the biliary epithelia of PBC patients, whereas positivity for IgM antibodies against lipid A, an immunogenic and toxic component of LPS, is confirmed in 64% of PBC sera[190,191]. TLR4 expression is significantly elevated in BECs, periportal hepatocytes and blood monocytes of PBC patients[192,193], whereas LPS/TLR4 signaling has been associated with an increased release of proinflammatory cytokines such as IL-1b, IL-6, IL-8 and TNF-α[189]. TLR4 ligand-stimulated NK cells have been suggested to be associated with BEC damage in the presence of TLR3 ligand-activated monocytes among PBC patients[194]. Despite similar levels of TLRs in BECs isolated from livers from patients and controls, stimulation via TLR3 agonist poly I:C and co-culture with liver-infiltrating mononuclear cells resulted in elevated chemokine levels in livers from patients[195]. Moreover, when compared to patients with autoimmune hepatitis and Hepatitis C, patients with PBC showed higher levels of TLR3 and IFN-α/β in portal tracts and liver parenchyma[196]. Furthermore, TLR9 ligand (CpG) stimulation of peripheral blood monocytes from PBC patients was demonstrated to activate IgM-producing B cells and to increase TLR9 expression on these cells[197,198]. These findings emphasize the role of innate immunity not only in the pathogenesis and progression of PBC but also in the regulation of adaptive immune responses[9].

The role of TLRs in PSC has not been extensively studied[11]. Abnormal LPS accumulation was demonstrated in BECs in PSC[190]. Stimulating isolated BECs with anti-BEC antibodies from patients with PSC leads to increased expression of TLR4 along with higher levels of inflammatory cytokines in the presence of LPS[199].

Accordingly, increased LPS accumulation and TLR4 expression in BECs has been suggested to induce breakdown of self-tolerance and onset of bile duct damage in PBC and PSC thorough their stimulatory effects on selective pro-inflammatory cytokines with a critical role[13]. Given the signs of inflammatory bowel disease to exist in most patients with PSC and the likelihood of gut factors to induce response onset per se with no preceding immune cell dysfunction, future investigations are needed addressing the role of gut microbiota in conjunction with PSC and PBC to provide a better understanding of the mechanisms and treatment of these complex diseases[90].