TLR3 plays significant roles against hepatitis B virus

Masoud Karimi-Googheri • Mohammad Kazemi Arababadi

Received: 31 May 2013 / Accepted: 21 January 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Hepatitis B virus (HBV) as the main prevalent

infectious agent, play important roles in inducing severe

liver diseases. Previous studies demonstrated that during

prolonged forms of hepatitis B infection including chronic,

asymptomatic and occult forms, patients are unable to

eradicate HBV from hepatocytes completely. The main

mechanisms responsible for development of the forms of

hepatitis B are yet to be identified. Investigators suggested

that the various genetic and immunological parameters of

the patients may are responsible for resulting in the pro-

longed infection forms. It has been evidenced that TLRs

play key roles in inducing appropriate immune responses,

against viral infections. Therefore, these molecules can be

considered as crucial sensors for HBV detection to induce

immune responses against this virus. It has also been

documented that the TLR3 detects intracellular viral

dsRNA and subsequently activates NF-jB via the TRIF

pathway. Therefore, impaired TLR3 expression may result

in inappropriate immune responses against HBV which is

reported in prolonged forms of hepatitis B. This review

collected the recent information regarding the important

roles of TLR3 in immune responses against HBV and also

the status of TLR3 expression and its genetic variations in

prolonged forms of HBV infections.

Keywords Prolonged HBV infection � TLR3 �Polymorphism � dsRNA

Abbreviation

TLR Toll like receptor

PAMP Pathogen associated molecular patterns

HBV Hepatitis B virus

MYD88 Myeloid differentiation primary response

IRAK1 Interleukin-1 receptor associated kinase-1

TRAF6 TNF receptor associated factor

NF-jB Nuclear factor kappa-light-chain-enhancer

of activated B cells

DNMT-1 DNA methyltransferase 1

TAK1 Transforming growth factor b-activated

kinase 1

MAPK Mitogen-activated protein kinase

AP-1 Activator protein 1

PBMC Peripheral blood mononuclear cell

NADPH Nicotinamide adenine dinucleotide phosphate

MHC Major histocompatibility complex

IRF Interferon regulatory factor

Introduction

Hepatitis B virus (HBV) is the prevalent infectious agent

amongst hepatitis viruses which results in impaired func-

tions of liver in human [1–3]. Previous investigations

demonstrated that during prolonged forms of hepatitis B,

including chronic, asymptomatic and occult HBV infec-

tions, immune system is unable to clear HBV from hepa-

tocytes and serum completely [3–5].

It has been evidenced that the prolonged form of hep-

atitis B could be considered as a main reason of cirrhosis

and hepatocarcinoma [6, 7]. Previous studies were unable

to clarify the main mechanisms responsible for progression

M. Karimi-Googheri

Department of Immunology, Faculty of Medicine, Kerman

University of Medical Sciences, Kerman, Iran

M. K. Arababadi (&)

Immunology of Infectious Diseases Research Center, Rafsanjan

University of Medical Sciences, Rafsanjan, Iran

e-mail: dr.kazemi@rums.ac.ir

123

Mol Biol Rep

DOI 10.1007/s11033-014-3190-x

of the prolonged forms of hepatitis B. The researchers

hypothesized that the various genetic and immunological

parameters in patients in comparison to clearance group

may are the main causes for development of prolonged

hepatitis B forms [1, 5, 8].

Toll like receptors (TLRs) play crucial roles in recog-

nition of microbial, including viral, PAMPs and subse-

quently lead to activation of immune response against

microbial infections, including HBV [9].

It has been demonstrated that TLRs consist of 14

members which TLR1-10 are expressed on cytoplasmic

membrane (TLR1, 2, 4, 5 and 6) or inside the endosomes

(TLR3, 7, 8 and 9) of human immune cells [10]. These

molecules recognize various ranges of PAMPs including

microbial ds/ssRNA, lipopolysaccharide, bacterial flagel-

lin, lipopeptides and CpG-rich unmethylated DNA [10].

The expression of important immune system related mol-

ecules including inflammatory cytokines, major histocom-

patibility complex (MHC) and homing molecules are

dependent on TLRs/PAMPs interactions. Accordingly,

TLR3 is critical intracellular molecules which recognizes

dsRNA as viral PAMPs and activate the corresponding

immune cells in a TRIF dependent manner [11, 12]. It has

been documented that some of HBV antigens such as

HBcAg are binding to RNA and also HBV-DNA replicates

via an intermediate RNA, hence, it appears that this mol-

ecules can be recognized by TLR3 [13]. So, defected or

declined TLR3 expression may be associated with attenu-

ated immune responses against HBV. Due to the fact that

prolonged term HBV infected patients are unable to erad-

icate HBV completely [3, 14, 15], and based on the

important roles played by TLR3 in the viral PAMPs rec-

ognition [16] and induction of immune responses against

viral infection [17], it may be hypothesized that the altered

expression of TLR3 may result in persistent of HBV

infection. Therefore, this review article was designed to

address the recent information regarding the role of TLR3

in HBV infection, its expression status on the immune cells

and genetic variations of TLR3 in prolonged HBV infected

patients. This review also presents current information

regarding the plausible mechanisms of effects of HBV and

its factors on TLR3 and its signaling molecules functions.

Finally, TLR3 as a novel target for immunotherapy has

been discussed in this review article.

Introducing of TLR3

The gene of TLR3, also known as CD283, is located on

4q35 [18]. TLR3 plays important roles in PAMP and

DAMP recognition and consequently activation of

inflammatory transcription factors including IRF3, NF-kB

and AP-1 which transcript from inflammatory molecules

including cytokines, chemokines, stimulatory molecules,

selectin and integrin as adhesion molecules and others [19,

20]. This molecule is a type I transmembrane protein

which consists of three sections as follow: (1) N-termi-

nal which is outside the membrane and consists of leu-

cine-rich repeats, (2) a hydrophobic transmembrane

domain, (3) a cytoplasmic Toll/interleukin-1 receptor (TIR)

domain (Fig. 1) [21]. Interestingly, the TLR3 gene is

highly conserved in several species including mouse,

human, chimpanzee, Rhesus monkey, zebrafish, cow, dog,

rat and chicken [22]. The intracellular membrane organ-

elles such as the endoplasmic reticulum (ER), the endo-

some and the lysosome are the places of TLR3

localizations as well as other intracellular TLRs including

TLR7, 8 and 9 [23]. Several cells including a broad range

of antigen-processing cells, epithelial cells, monocytes,

dendritic cells, NK cells, mast cells and so on express

TLR3 to detect microbial dsRNA [24].

TLR3 ligands

Previous study by Alexopoulou et al. [25] in 2001, initially

demonstrated that viral or synthetic dsRNA but not homo-

polymer single stranded (ss) RNA is the specific TLR3

ligand. Furthermore, other studies revealed that in addition to

viral dsRNA, endogenous dsRNA, known as DAMP, also

activates TLR3 [26]. Interestingly, TLR3 also recognizes

cellular or in vitro mRNAs because these molecules can form

secondary structures, such as hairpins, which contain double

stranded sequences [26]. Previous studies demonstrated that

polyriboinosinic:polyribocytidylic acid [poly(I:C)], as sta-

ble synthetic dsRNA analogue, can be used as a TLR3 ligand

to mimic viral infection [27]. Interestingly, TLR3 prefer to

detect synthetic poly(I:C) rather than viral dsRNA, hypoth-

esized that TLR3 recognize a unique dsRNA structure [28].

Additionally, it has been documented that TLR3 is able to

recognize cell-associated poly(I:C) more efficient than sol-

uble dsRNA, hence, it appears that TLR3 triggers with

dsRNA derived from dying cells more than dsRNA from live

cells [29]. Based on the aforementioned sentences, it appears

that TLR3 can be considered as an important candidate to

detect HBV mRNAs in the infected cells. Therefore, several

researchers have evaluated the status and roles played by

TLR3 in hepatitis B, especially in chronic hepatitis B, which

will be discussed in the Sect. 5.

TLR3 signaling

It has been documented that intracellular signaling of

TLR3, in contrast with other TLRs, is only in TRIF

dependent manner [2]. Following TLR3/ligand interaction,

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Fig. 1 Intracellular signaling of TLR3 and the probable mechanisms

for down-regulation f TLR3 and its molecular signaling during

prolonged hepatitis B infections. The figure demonstrates that HBsAg

induce monocytes to secret IL-10 and JAK/STAT3 pathway in IL-10

bearing cells. JAK/STAT3 pathway activation results in inhibition of

TLR3 signaling molecules. Prevalent polymorphisms within TLR3

and signaling molecules genes as well as epigenetic factors may also

affect their expression. HBV DNA polymerase also leads to

suppression of IRF3, hence, inhibits type 1 interferon production

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TIR domain of TLR3 and TRIF interact with each other

[30] and result in TRAF6, RIP-1 and TBK1 activation [31].

This event activates several inflammatory transcription

factors including IRF3, AP-1 and NF-jB (Fig. 1) [32, 33].

IRF3, AP-1 and NF-jB are responsible factors which

transcript from several genes including inflammatory

cytokines, chemokines, co-stimulatory molecules and

addressing as well as homing molecules [34]. Previous

studies reported that type I interferons play important roles

in induction of immune responses against viral infections

including HBV [35].

TLR3 and hepatitis B

Previous sections have been described that viral dsRNA is

the main target for TLR3 [26]. Based on the fact that HBV

RNA is produced in infected cells, hence, this virus is a

candidate for recognition by TLR3. Previous investigations

also demonstrated that TLR3 knockout mouse are unable to

express IL-8 and other necessary molecules for activation

of immune responses against HBV [36]. Additionally,

studies demonstrated that TLR3/HBV-RNA interaction is a

crucial way to activation of immune responses. For

example, Lai et al. [13] reported that HBV-RNA signifi-

cantly induces immune responses when have been used

with double-strand RNA-binding proteins. Additionally,

another study by Yu et al. [37] demonstrated that HBV

polymerase inhibits type 1 interferon production in human

hepatocytes via interference with IRF3 as TLR 3-mediated

transcription factor. Therefore, it appears that TLR3/

ligands interaction is a main mechanism to HBV recogni-

tion and activation of immune cells. Based on the fact that,

prolonged HBV infected patients are unable to clear the

virus from host cells completely, so, impaired TLR3

expression and function may be a main reason for persis-

tence of infection. Interestingly, previous investigations

approved this hypothesis which An et al. [38] revealed that

the elevation of TLR3 expression is slower in the patients

with chronic HBV infection than healthy controls. Li et al.

identified that patients with chronic hepatitis B express

lower TLR3 in monocyte-derived dendritic cells (MoDCs)

than normal controls [39]. A previous study also demon-

strated that mRNA levels of TRIF, as unique adaptor

protein for TLR3, were decreased in the patients with

chronic hepatitis B when compared to healthy controls [2].

Interestingly, the investigators have also reported that the

expression of TLR3 was significantly increased in MoDCs

of acute-on-chronic hepatitis B liver failure (ACHBLF)

surviving patients in compare to non-surviving ACHBLF

patients [39]. Additionally, another study by Tjwa et al.

[40] demonstrated that activation of myeloid dendritic cell

using TLR3 ligands leads to improvement of natural killer

cell function in chronic HBV infection. This result was also

confirmed by Wang et al. [41], which revealed that mRNA

levels of TLR3 were increased in active stage of chronic

HBV infection, suggested that elevated expression of

TLR3 is associated with stronger immune responses

against HBV.

Based on the presented data by the aforementioned

studies, it seems that the patients infected with prolonged

forms of hepatitis B infection (chronic asymptomatic and

occult) are suffering from impaired TLR3 expression or

functions which this may explain how infection is persis-

tent in the patients. Previous studies suggested that several

factors influence TLR3 expression in the patients suffering

from prolonged forms of hepatitis B including; (1) inhibi-

tory effects of HBV, (2) genetic variations and (3) epige-

netic factors (Fig. 1). For instance, Yu et al. [37]

demonstrated that HBV polymerase inhibits TLR3 intra-

cellular signaling in human hepatocytes via interference

with IRF3 activation. An et al. [42] also reported that

patients with chronic HBV infection were unable to acti-

vate IRF3 following the virus infection. Shi et al. [43]

demonstrated that HBsAg suppressed TLR3 signaling in

plasmacytoid dendritic cells by induction of monocytes to

release IL-10 (Fig. 1). Another study reported that IL-10

down-regulates several inflammatory molecules via JAK1/

STAT3 signaling pathway [44], thus, this mechanism may

also be responsible for down-regulation of TLR3 in the

patients with prolonged form of hepatitis B (Fig. 1).

Therefore, it appears that HBV interfere with TLR3 path-

way to regulate immune responses. Moreover, it appears

that host genetic can be considered as another potential

candidate for declined expression of TLR3 during pro-

longed infection of hepatitis B. For example, Al-Qahtani

et al. [45] reported that the differences between chronic

HBV infected patient and healthy controls regarding

rs1879026 (G/T) polymorphism within TLR3 gene was

significant. Their results also demonstrated that GCGA

(rs1879026, rs5743313, rs5743314, and rs5743315,

respectively) haplotype was significantly associated with

chronic HBV infection [45]. Rong et al. [46] also revealed

that the polymorphisms within ?1,234 region of TLR3

gene are significantly associated with the development of

chronic HBV infection. Interestingly, Li et al. [47] also

reported that TLR3 ?1234C/T polymorphism were sig-

nificantly associated with HBV-related hepatocellular car-

cinoma (HCC). Additionally, it seems that other factors,

including environmental and epigenetical factors, may also

contribute in down-regulation of TLR3 in the patients with

prolonged HBV infected. For instance, previous investi-

gations revealed that micro-RNAs play key roles in the

regulation of gene expression [48], hence, it can be

hypothesized that HBV or host encoded micro-RNAs may

alter TLR3 and its signaling molecules expression in

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patients suffering from prolonged forms of hepatitis B.

Interestingly, our unpublished studies revealed that miR-

155, 1, 21 and 125 were significantly upregulated in Iranian

chronic HBV infected patients. Therefore, this miRNAs

may alter expression of immune related molecules

including TLRs. Future studies regarding the status of

miRNAs which target mRNA of TLR3 in patients with

prolonged forms of hepatitis B can be helpful to clear the

main mechanisms of hepatitis B persistence. Furthermore,

it has been evidenced that immune tolerance results in

down-regulation of inflammatory molecules including

TLRs [49], due to the fact that patients with prolonged

forms of hepatitis B suffering from immune tolerance to

HBV antigens [50], hence, it may be concluded that HBV

antigens dependent immune tolerance results in TLR3

down-regulation in the patients. Therefore, based on the

aforementioned studies it appears that TLR3 plays key

roles in activation of immune responses against HBV, and

the viral, environmental and epigenetical factors affect

TLR3 expression in the patients suffering from prolonged

forms of hepatitis B.

Type-I interferons and hepatitis B

It has been documented that IRF transcription factors are

activated following TLR3/ligand interactions [51] and

according to the fact that type-I interferons (type-I IFNs),

including a and b IFNs, are transcripted by these factors

[51], hence, it appears that these cytokines actively par-

ticipate in induction of immune responses against hepatitis

B. Interestingly, several investigations confirm the signifi-

cant roles played by type-I IFNs in stimulation of immune

responses versus HBV. For example, several cytokines

perform their anti-HBV functions via up-regulation or

activation of type-I IFNs signaling pathway [52]. Up-reg-

ulation of type-I IFNs was also reported previously [53].

Therefore, it may be concluded that up-regulation of type-I

IFNs via TLR3/ligand interactions may be an important

responsible mechanisms to eradicate HBV. Additionally,

based on the key roles played by type-I IFNs against HBV,

they are used for treatment of hepatitis B [54–56]. It

appears that, administration of type-I IFNs induces cellular

immunity in hepatitis B infected patients [57]. Accord-

ingly, it seems that in prolonged hepatitis B infected

patients, HBV inhibits type-I IFNs effects to escape from

immune responses [58]. For instance, Cho et al. [58]

reported that HBX protein down-regulates type-I IFN

receptors which leads to suppression of extracellular IFN-

a-mediated signal transduction. HB9Ag also can inhibit

IRF3 activation via disrupting the virus-induced signaling

adaptor (VISA) complex [59]. Additionally, previous

investigations revealed that sex can participate in induction

of immune responses via TLRs, for instance, Roberts et al.

[60] showed that Coxsackievirus B3 infection leads to

increased expression of TLR2 and TLR4 in female and

male mice, respectively. Interestingly, the study demon-

strated that females was resistance to Coxsackievirus B3

infection, while, male were susceptible. Although, there is

not a study which evaluated the roles of sex in the TLRs

dependent immune responses against HBV, but it may play

important roles and more study can shed light on the

understanding of the main mechanisms lead to various

immune responses against HBV.

TLR3 ligands as a novel target for immunotherapy

against HBV

Based on important roles played by TLR3 in activation of

immune responses against HBV, several investigations

have focused on using TLR3 ligands as novel immune

therapy against hepatitis B. For instance, Isogawa et al.

[61] reported that using TLR3 ligands in animal model

result in inhibition of HBV replication. Heiberg et al. [62]

demonstrated that using TLR3 ligands led to production of

IL-6, CCL3 and CXCL10, as inflammatory cytokine and

chemokines in CHB children patients. Chen et al. [63]

reported that TLR3 agonist, dsRNA, inhibits the HBV

proliferation, invasion and secretion from HepG2.2.15 cell

lines. Furthermore, it has been evidenced that TLR3

ligands led to increased cytotoxic T lymphocyte responses

in prolonged HBV infected patients [64]. Wu et al. [65]

also revealed that supernatants from TLR3 stimulated

Kupffer cells and sinusoidal endothelial cells inhibited

HBV replication. Therefore, it appears that TLR3 and its

molecular signaling is a main pathway to stimulate immune

responses and further investigations shed light on the

understanding of significant roles played by this receptor in

recognition of HBV and induction of appropriate immune

responses. Additionally, due to the aforementioned studies

it seems that TLR3 ligands are plausibly the important

promising candidates for the development of novel

immunotherapy against HBV.

Conclusion remarks

Due to the results presented by aforementioned studies a

hypothesize may be arisen which firstly, TLR3 signifi-

cantly participates in induction of appropriate immune

responses against HBV. Secondly, TLR3 and its signaling

molecules expression are suppressed in prolonged HBV

infected patients by several factors including viral, host

genetic and epigenetic factors. Thirdly, previous studies

demonstrated that the infected patients with prolonged

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form of hepatitis B are unable to produce pro-inflammatory

cytokines, chemokines and co-stimulatory molecules [3,

15, 66–69] which are crucial for eradications of the HBV

infection. Based on the collected information in this review

article, it may be concluded that down-regulation of TLRs,

especially, TLR3, is responsible for the impaired immune

responses. Thus, it seems that future treatments could focus

on immunotherapy, which can up-regulate the expression

of TLR3 and its signaling molecules as well as using TLR3

agonists which are crucial way to re-activation the immune

responses and HBV clearance from prolonged HBV

infected patients.

Acknowledgments This project was granted by the Rafsanjan

University of Medical Sciences.

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