Toll-Like Receptor Response To Hepatitis B Virus Infection And Potential Of TLR Agonists As Immunomodulators For Treating Chronic Hepatitis B: An Overview Part 1

Abstract:

Chronic hepatitis B virus (HBV) infection remains a major global health problem. The immunopathology of the disease, especially the interplay between HBV and host innate immunity, is poorly understood. Moreover, inconsistent literature on HBV and host innate immunity has led to controversies. However, recently, there has been an increase in the number of studies that have highlighted the link between innate immune responses, including Toll-like receptors (TLRs), and chronic HBV infection. 

TLRs are the key sensing molecules that detect pathogen-associated molecular patterns and regulate the induction of pro- and anti-inflammatory cytokines, thereby shaping adaptive immunity. The suppression of TLR response has been reported in patients with chronic hepatitis B (CHB), as well as in other models, including tree shrews, suggesting an association of TLR response in HBV chronicity. Additionally, TLR agonists have been reported to improve the host's innate immune response against HBV infection, highlighting the potential of these agonists as immunomodulators for enhancing CHB treatment. In this study, we discuss the current understanding of host innate immune responses during HBV infection, particularly focusing on the TLR response and TLR agonists as immunomodulators.

Chronic hepatitis B virus (HBV) infection can affect the immune system. HBV infection suppresses the immune system's response to the virus, resulting in chronic infection.

In patients with chronic hepatitis B, immunity is suppressed. This is due to the virus' attack on immune cells and the inflammatory response caused by the infection. The patient's immune system may not be able to respond effectively to the virus, causing the virus to persist in the body.

In terms of treatment, immune modifiers can boost patients' immunity and help them clear the virus. However, these drugs can also cause an overactive immune system, which can lead to autoimmune diseases.

In conclusion, there is a complex interplay between chronic HBV infection and immunity. Therapeutic strategies to slow viral replication and enhance immunity should be combined with individualized treatment to achieve the best therapeutic effect. From this point of view, we need to improve our immunity. Cistanche has the effect of significantly improving immunity. Meat ash contains various biologically active components, such as polysaccharides, two mushrooms, Huang Li, etc. These ingredients can stimulate meat. Various types of cells of the immune system, increase their immune activity.

Click cistanche tubulosa benefits

Keywords:

hepatitis B virus; chronic; infection; toll-like receptor; TLR agonists.

1. Introduction

Hepatitis B virus (HBV) infection, which causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC), remains a major global health problem despite the availability of an effective preventive vaccine for HBV [1,2]. According to a recent estimate, 296 million people were affected by chronic HBV infection in 2019, with an annual 1.5 million new infections [3]. The current treatment involves the administration of pegylated interferon alpha (Peg-IFN-α) and nucleus (t)ide analogs (NAs), including three nucleoside analogs (lamivudine, entecavir, and telbivudine) and three nucleotide analogs (adefovir, tenofovir disoproxil, and tenofovir alafenamide). However, these drugs can only suppress HBV replication; they cannot eliminate HBV infection, mostly because of the persistent nature of the covalently closed circular DNA (cccDNA) and/or integrated HBV DNA in hepatocytes [4]. 

HBV is an enveloped, circular, and partially double-stranded relaxed circular DNA (cDNA) virus [5]. HBV belongs to the family Hepadnaviridae, which contains a genome of approximately 3.2 kb with four overlapping open reading frames, encoding seven proteins, including polymerase, core, pre-core, three envelope/surface proteins (large, middle, and small), and X protein [6–8]. The envelope and core proteins are structural proteins, whereas X and polymerase are non-structural proteins [8]. Sodium taurocholate co-transporting polypeptide (NTCP) has been demonstrated as an entry receptor for HBV [9], and the interaction is host-specific [10]. A complex series of events occurs between a virus and the host immune system, which determines the outcome of infection [11]. 

Similarly, the outcome of HBV infection largely depends on the virus–host interactions, where, in 95% of the immunocompetent adults the infection is cleared, but in over 90% of the infected neonates this fails and they develop chronic infection [12,13]. Chronic HBV infection is detected by the continuous expression of hepatitis B surface antigen (HBsAg) for at least 6 months after the initial infection. There are inconsistent data on HBV–host interactions, particularly innate immune responses. For example, the lack of innate immune response, including induction of interferons (IFNs) and IFN-stimulated genes (ISGs), in acute HBV-infected chimpanzees [14] the woodchuck model of HBV infection [15] and patients with HBV infection [16] highlights HBV as a stealth virus. A recent study demonstrated that HBV remains invisible to pattern recognition receptors (PRRs) [17], which could be due to the ability of HBV proteins to inhibit or evade the host's innate immune system [18–22]. Notably, in our previous study, we observed a significant suppression of interferon regulatory factor 7 (IRF-7) and ISG15, as well as no increase in IFN-β production at 1- or 3-days post-infection in the tree shrew model [23], which was suggestive of the inhibition of innate immune response at an early stage of infection in this model. 

However, it is usually difficult to identify the exact time-point of initial infection of the host, which makes it difficult to characterize the early stages of HBV infection. Moreover, the differences in innate immune response, which may be due to the differences in HBV genotype, infection state, or host genetics, remain largely undefined. Nevertheless, numerous recent studies have indicated the sensing of HBV by PRRs, including Toll-like receptors (TLRs) [24,25]. The innate immune response plays an important role as the first line of immune defense against many viral infections [26]. TLRs, a retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)- like receptors (NLRs) and C-type lectin receptors play an essential role in sensing the invading pathogens, including viruses, and initiating an innate immune response. This leads to the synthesis of IFNs and cytokines through several distinct signaling pathways, thereby limiting infection and promoting adaptive immune responses [27,28]. 

Although the role of adaptive immunity in controlling HBV infection is well documented, the role of innate immunity in this regard is yet to be largely explored [18]. Moreover, the mechanisms behind HBV-specific immune responses [29] remain to be investigated, which may pave the way for the development of new strategies for the treatment of chronic HBV infection. TLRs are evolutionarily conserved key molecules of innate immunity, which are involved in the early detection of invading microorganisms by sensing pathogen-associated molecular patterns (PAMPs) [30–32]. TLRs are type I transmembrane proteins containing three domains: an extracellular leucine-rich repeat domain that recognizes specific PAMPs, a single transmembrane domain, and an intracellular Toll-interleukin 1 receptor domain required for downstream signal transduction [33,34]. TLR signaling is involved in the regulation of both pro- and anti-inflammatory cytokines and links early innate and adaptive immunity [35]. Several studies have indicated the association of TLR-mediated signaling with antiviral mechanisms and suppression of HBV replication [36,37], as well as TLR response suppression with HBV persistence [38]. 

Therefore, activation of innate immune response by TLR agonists may play a significant role in modulating the outcome of chronic HBV infection. Moreover, TLRs are important triggering molecules in activating trained immunity and can be used as vaccine adjuvants [39–42]. In a previous study, the phenomenon of trained immunity in newborn infants of HBV-infected mothers was reported [43]. Thus, the use of TLR agonists as HBV-specific therapeutics/vaccines could be investigated to enhance the immune response against chronic infections. In this study, we have discussed our current understanding of HBV–host interactions by focusing on TLR–HBV interactions during chronic infections in human and animal models. In addition, we have also addressed the immunomodulatory potential of TLR agonists for improving host innate immune responses during chronic HBV infection.

2. TLR Response to HBV Infection

Although innate immune response during the early phase of HBV infection is considered to be negligible, an increasing number of studies have indicated that HBV infection modulates innate immune responses, including TLR response [38]. HBV has a restricted host range, naturally infecting humans and chimpanzees, and to some extent, tree shrews [44–46], which also limits the early immune response study of HBV infection. In a previous study, Isogawa et al. reported the induction of IFN-α/β in the liver of HBV transgenic mice within 24 h of a single intravenous injection of TLR3, 4, 5, 7, and 9 agonists, which inhibited HBV replication [37], suggesting the anti-HBV role of these TLRs. TLRmediated control of HBV replication using TLR agonists has also been demonstrated in mice non-parenchymal liver cells [47]. 

Markedly low expression of TLR transcripts, including TLR1, 2, 4, and 6, was observed in peripheral blood mononuclear cells (PBMCs) obtained from patients with chronic hepatitis B (CHB). The cells also showed an impaired cytokine response after stimulation with TLR2 and TLR4 agonists, which correlated with the plasma HBsAg level of the patients [48], suggesting a possible interaction between HBsAg and TLR signaling. Another study showed that after stimulation with ligands for TLR2, TLR3, and TLR9 on PBMCs obtained from children with chronic HBV infection, there was an increase in the production of IL-6, CCL3, and CXCL10 [49], indicating the induction of TLR-mediated inflammatory response. 

However, on stimulation with ligands for TLR2, TLR3, and TLR4, the PBMCs from children with CHB showed a significantly lower IFN-α production than in those from healthy children [49], suggesting a suppressed IFN response. A significantly decreased expression of TLR signaling molecules, including IRAK4, TRAF3, and IRF7, was found in PBMCs of patients with CHB, as compared to that in those of healthy controls [50], suggesting an impaired immune response in chronic HBV infection. Genetic variations in the TLR3 gene were found to affect the outcome of HBV infection [51,52], highlighting the involvement of TLR3. Decreased expression of both TLR7 mRNA and protein was observed in PBMCs obtained from patients with CHB, as compared to healthy controls; however, a decreased TLR9 mRNA expression but an increased TLR9 protein level was observed in patients with CHB, which correlated with their serum HBV DNA, suggesting a possible link between TLR9 protein expression and HBV replication [53]. In another study, the expression of both TLR9 mRNA and protein was found to be downregulated in PBMCs from patients with CHB [54], highlighting the association of TLR9 with HBV infection. 

A previous study reported a lowered production of IFN-α by pDCs from patients with CHB, which was in response to loxoribine, a ligand for TLR7, and CpG ODN, a ligand for TLR9 [55]. Moreover, several studies have reported an inverse correlation between the number of pDCs and the expression of TLR9 in pDCs with the serum HBV load [55,56], suggesting the antiviral role of TLR9 in HBV infection. A previous study reported the suppression of TLR9-induced IFN-α production by plasmacytoid DCs obtained from HBeAg-positive patients with CHB [57]. In addition, a recent study indicated the predictive value of pDCs and TLR9 in HBeAg-positive patients with CHB; after IFN-α treatment, pDC-mediated expression of blood dendritic cell antigen 2 (BDCA-2), and expression of immunoglobulin-like transcript 7 (ILT7) and TLR9 mRNA were significantly increased in the response group compared with that in the non-responders group [58].

Similar to that of patients with CHB, the transcriptomic analysis of the woodchuck model of CHB also revealed a limited intrahepatic type I IFN response [59]. In the woodchuck model of chronic hepatitis, a suppressive trend of TLR expression was reported in the hepatocytes, compared to that in healthy animals, suggesting an impairment of the innate immune response in chronic infection [60]. However, a recent study indicated the role of TLR2 in the resolution of HBV infection in a woodchuck model of hepatitis [61]. In our previous study, we found significant suppression of IFN-β response at 31 weeks post-infection in the tree shrew model [62], which might have contributed to the chronicity. 

Moreover, TLR3 was not induced and TLR9 was suppressed [62]. Notably, a previous study also reported decreased expression of TLR9 in peripheral CD14+ monocytes collected from patients with CHB [63]. Although the mechanism remains unknown, a previous study reported that responders to pegylated IFN and those under ETV treatment showed restoration of TLR9 expression, suggesting the role of TLR9 in HBV inhibition [63]. Similarly, an earlier study demonstrated a reduction in TLR3 expression in PBMCs and the liver cells of patients with CHB compared to healthy controls, which was restored by IFN therapy, suggesting the role of TLR3 in HBV inhibition [64]. TLR2 and 4 may inhibit HBV replication in an IFN-independent manner by activating MAPK and PI-3 K/Akt pathways in hepatocytes [36]. Notably, a recent study showed the sensing of HBV and induction of anti-HBV immune response through TLR2 signaling after infection in primary human hepatocytes (PHH) [25], a verified in vitro model of HBV infection [65]. Suppressed expression of TLR2 has been observed in chronic HBV infections [66,67], with significantly decreased expression of TNF-α and IL-6 production [66]. 

In addition, a recent study demonstrated the association of TLR2 in the regulation of T helper 17 (Th17) cell response in HBV infection [68]. Using the HBV mouse model, other recent studies have demonstrated the role of TLR signaling in enhancing HBV-specific CD8+ T-cell responses, which control the infection [25,69]. Another study reported a reduced expression of TLR7 in HBV-replicating HepG2.2.15 cells and the liver biopsy samples from patients with CHB; an inverse relationship between HBV DNA load and TLR7 expression in biopsy samples was observed [70], highlighting the antiviral role of TLR7 in HBV infection, which was further confirmed by the suppression of HBV replication in HepG2.2.15 cells by TLR7 agonist, R837 [70]. In HBV-transgenic mice, saturated fatty acids (SFAs), potential ligands for TLR4, were shown to accelerate TLR4 signaling, which inhibited HBV replication in CHB infection with non-alcoholic fatty liver disease [71], suggesting an antiviral role of TLR4 against HBV infection. The role of the innate immune response in inhibiting HBV replication has also been shown in an HBV hydrodynamic mouse model, where an intraperitoneal inoculation of STING agonist, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), induced type I IFNs that reduced HBV DNA replication intermediates in the mouse livers [72]. 

In mouse hepatocytes supporting HBV replication, the activation of STING with either cGAMP or STING agonist significantly reduced the viral DNA in a STING- and Janus kinase-1-dependent manner [73]. Overall, these findings suggest that innate immunity plays a significant role in the inhibition of HBV replication; however, further studies are required to explore the mechanisms. The interaction of TLRs in different models of HBV infection is shown in Figure 1 (Figure 1A–F), which suggests the crucial role of TLR response in inhibiting HBV infection. In this regard, we also have shown the association of TLRs with other molecules, including IRAK4, TRAF3, and IRF7 in the induction of IL-6, CCL3, and CXCL10 [36,49,74] (Figure 1G). Therefore, a proper understanding of TLR responses in HBV infection is critical for successful therapeutic or preventive interventions. Also, the potentiality of TLR agonists, particularly agonists for TLR7, 8, and 9 are under investigation in several clinical trials, which appear promising as immunomodulators to enhance host immune response [74], will be discussed in the later part of the study.

Figure 1. An overview of TLR response in different models of HBV infection. (A) Suppression of TLRs and its adaptor components in patients with chronic HBV infection. (B) Restoration and the anti-HBV response of TLRs in patients with chronic HBV infection after Peg-IFN or ETV therapy. (C) Induction of TLR responses by using TLR agonists and inhibition of HBV replication in HBV-transgenic mice. (D) Inhibition of HBV by TLR2 and downstream signaling components in HBV-infected primary human hepatocytes (PHH). (E) Anti-HBV response of TLR2 and TLR3 after using TLR1/2 and TLR3 agonists in HBV-infected PHH. (F) Suppression of TLR9 expression and downstream cytokine during chronic HBV infection in tree shrew model. (G) Cartoon showing the relationship of TLRs with other signaling molecules, including IRAK4, TRAF3, and IRF7 in the induction of IL-6, TNF-α, CCL3, and CXCL10. Upon activation of TLRs by respective TLR ligands, adaptor molecules Figure 1. 

An overview of TLR response in different models of HBV infection. (A) Suppression of TLRs and its adaptor components in patients with chronic HBV infection. (B) Restoration and the anti-HBV response of TLRs in patients with chronic HBV infection after Peg-IFN or ETV therapy. (C) Induction of TLR responses by using TLR agonists and inhibition of HBV replication in HBV-transgenic mice. (D) Inhibition of HBV by TLR2 and downstream signaling components in HBV-infected primary human hepatocytes (PHH). (E) Anti-HBV response of TLR2 and TLR3 after using TLR1/2 and TLR3 agonists in HBV-infected PHH. (F) Suppression of TLR9 expression and downstream cytokine during chronic HBV infection in tree shrew model. (G) Cartoon showing the relationship of TLRs with other signaling molecules, including IRAK4, TRAF3, and IRF7 in the induction of IL-6, TNF-α, CCL3, and CXCL10. Upon activation of TLRs by respective TLR ligands, adaptor molecules are recruited, and through downstream signaling pathways proinflammatory cytokines, chemokines, IFNs, and ISGs are activated and produced.

For more information:1950477648nn@gmail.com