Toll-like receptor 9 polymorphisms and Hepatitis B virus clearance in Moroccan chronic carriers

Accepted Manuscript Toll-like receptor 9 polymorphisms and Hepatitis B virus clearance in Moroccan chronic carriers

Hajar Chihab, Imane Zaidane, Abdellah Elhabazi, Fatima-Zahra Jadid, Raouia El Fihri, Mohcine Elmessaoudi-Idrissi, Mohammed Chair, Wafaa Badre, Mohamed Tahiri, Pascal Pineau, Isabelle Chemin, Sayeh Ezzikouri, Soumaya Benjelloun PII: DOI: Reference:

S0378-1119(18)31186-7 https://doi.org/10.1016/j.gene.2018.11.041 GENE 43386

To appear in:

Gene

Received date: Revised date: Accepted date:

6 July 2018 11 November 2018 15 November 2018

Please cite this article as: Hajar Chihab, Imane Zaidane, Abdellah Elhabazi, Fatima-Zahra Jadid, Raouia El Fihri, Mohcine Elmessaoudi-Idrissi, Mohammed Chair, Wafaa Badre, Mohamed Tahiri, Pascal Pineau, Isabelle Chemin, Sayeh Ezzikouri, Soumaya Benjelloun , Toll-like receptor 9 polymorphisms and Hepatitis B virus clearance in Moroccan chronic carriers. Gene (2018), https://doi.org/10.1016/j.gene.2018.11.041

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ACCEPTED MANUSCRIPT Toll-like receptor 9 polymorphisms and Hepatitis B virus clearance in Moroccan chronic carriers Hajar Chihab 1,2, Imane Zaidane 1, Abdellah Elhabazi 2, Fatima-Zahra Jadid 1, Raouia El Fihri 1

, Mohcine Elmessaoudi-Idrissi 1, Mohammed Chair 2, Wafaa Badre 3, Mohamed Tahiri 3,

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Pascal Pineau 4, Isabelle Chemin 5, Sayeh Ezzikouri 1, Soumaya Benjelloun 1# Virology Unit, Viral Hepatitis Laboratory, Institut Pasteur du Maroc, Casablanca, Maroc.

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Laboratoire de Biotechnologie, Biochimie et Nutrition - Université Chouaib Doukkali,

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Faculté des Sciences d’El Jadida, 24000, El Jadida, Maroc.

Faculté de médecine de Casablanca, CHU Ibn Rochd, Casablanca, Maroc.

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Unité “Organisation Nucléaire et Oncogenèse”, INSERM U993, Institut Pasteur, Paris,

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France.

INSERM U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon

Correspondence should be addressed to Dr. Benjelloun Soumaya, Virology Unit, Viral

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Université Claude Bernard, Lyon, France.

Hepatitis Laboratory, Institut Pasteur du Maroc, 1 Place Louis Pasteur, 20360 CasablancaTel:

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Morocco.

+212662241959;

Fax:

+212522260957;

E.mail:

[email protected]

Running Title: The impact of TLR9 polymorphisms on HBV infection Declarations of interest: The authors declare no conflicts of interest. 1

ACCEPTED MANUSCRIPT Summary Objectives Toll-like receptor 9 (TLR9) plays a crucial role in the innate immune response against viral infections. The failure of this system may result, in an attenuated immune response against HBV. Recent research has focused on the possibility of targeting the defects in TLR9 pathway

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as a novel approach for anti-HBV treatment. Our study aimed to assess the impact of both TLR9 rs5743836 and rs187084 polymorphisms on spontaneous HBV clearance in Moroccan

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chronic HBV carriers.

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Methods

spontaneously

resolved

the

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In this study, 239 individuals chronically infected with HBV (CHB) and 133 subjects who infection

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genotyped

using

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Taqman allelic discrimination assay.

(SRB)

Results/Conclusion

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Remarkably, we observed a dosage effect of both SNPs on viral loads; with a significant

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increase of circulating HBV DNA within AA, AG to GG rs5743836 genotypes, whereas the inverse phenomenon was noticed within rs187084 genotypes. There were no consistent

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association between TLR9 polymorphisms and spontaneous clearance of HBV, however, a significant association was observed between rs187084 AA genotype and HBV progression to

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advanced liver disease. Further studies on larger populations might be necessary to understand the modulating effect of TLR9 polymorphisms on HBV loads that remain a viral factor of paramount importance to predict HCC development.

Keywords: TLR9, polymorphisms, HBV clearance, IFN

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ACCEPTED MANUSCRIPT Abbreviations Immunoglobulin M : IgM, Interleukin 6 : IL-6, Pattern recognition receptors : PRRs, Retinoic-acid-inducible gene 1 (RIG-I) -like receptors : RLRs, Nucleotide-binding oligomerization -like receptors: NOD-like receptors, Cytosine–phosphate–guanine : CpG, Myeloid differentiation protein 88 : MYD88, Interleukin-1 receptor-associated kinase :IRAK,

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Nuclear factor-kappa B : NF-κB, Activator protein 1 :AP-1, Tumor necrosis factor receptor

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(TNFR)-associated factor :TRAF6, Peripheral Blood Mononuclear Cells : PBMCs, Interferon

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Regulatory Factor 7 : IRF7, The suppressor of cytokine signaling :SOCS-1, Blood dendritic

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cell antigen 2: BDCA2

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ACCEPTED MANUSCRIPT 1. Introduction The outcome of a primary HBV infection depends primarily on the strength of the cellular and humoral host immune responses. Chronically HBV carriers are unable to develop appropriate adaptive immune responses. The mechanisms enabling the HBV to escape immune surveillance are still not clear (1). In fact, HBV infection is characterized by an undetectable

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viremia during the early phase followed by high levels of replication due to a defective

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activation of type I interferons (IFNα/β), which represent key-effectors of the antiviral response (2).

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Plasmacytoid dendritic cells (pDCs) play a central role in the host defense against HBV (3).

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Once exposed to pathogens components, the pDCs secrete type I IFN in response to the recognition of viral nucleic acids by intracellular toll-like receptors (TLRs) within the early

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endosomes (1, 4, 5). This initial trigger induces B lymphocytes proliferation with subsequent IgM and IL-6 secretion (6). The pDC cells also activate natural killer (NK) cells and T

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lymphocytes to ensure further priming and regulation of the anti-viral immunity (7-9).

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The detection of viral infections by pattern recognition receptors (PRRs), namely Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors, is an essential step

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for sensing pathogens that initiates signaling by inflammatory and antiviral cytokines, mainly type I IFN. IFN subsequently controls the spread of the infection and promotes an efficient

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adaptive immune response (10). The ten TLR present in the human genome are the most studied PRR (6). TLR9 identifies unmethylated CpG motifs in the DNA of bacteria and viruses (11).

The expression of the TLR9 receptor is restricted to pDCs and B lymphocytes (6). The TLR9 signaling is MYD88-dependent (12). Upon its interaction with its ligand, MYD88, in turn, forms a complex with IRAK family protein that engages as signaling cascade which ultimately activates AP-1 and NF-κB transcription factors (13-16). Several studies have

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ACCEPTED MANUSCRIPT reported that HBV has developed different strategies to escape immune responses including the modulation of TLR9 expression in patients with prolonged hepatitis B infection (17). The virus might inhibit the TLR9 pathway in pDCs either directly through the activity of its surface antigen (HBsAg), or indirectly through an increased interleukin 10 (IL-10) release by the monocytes (18). HBV can directly impair pDC-producing IFN by inhibiting the TLR9-

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IRF7-IFN pathway due to the SOCS-1 upregulation and to the binding of HBsAg to C-type

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its mRNA and protein expression in the total PBMC (11).

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lectin receptor BDCA-2 (1). As Vincent IE et al, HBV-mediated TLR9 inhibition targets both

Overall, the inhibition of TLR9 signaling represent a common feature in viral infections

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thought (Human Immunodeficiency Virus) HIV, (Hepatitis C Virus) HCV, Epstein Barr virus (EBV) and Human Papillomavirus (HPV), are known to interfere with TLR9 signaling (19).

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Previous investigations reported the association of the TLR9 polymorphisms with several types of persistent viral infections including HIV, HCV and HBV (20-22).

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Recently, the allelic variation located in the promoter region of the TLR9 locus, were shown to alter the function of the receptor by regulating its expression (23, 24). So far, the role of these TLR9 variations has been essentially assessed in the context of the populations in

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Eastern Asia well characterized by its vertical infection pattern, long-term tolerance phase and

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high viral loads. The place devoted to TLR9 polymorphisms in the context of infection with genotype D, in an horizontal transmission pattern has been only partially explored (25). The aim of our work is to investigate among Moroccan subjects, the impact of rs5743836 and rs187084 allelic variants located within the TLR9 promoter on the capacity of the subject to spontaneously clear HBV and on the severity of infection once the chronicity is established.

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ACCEPTED MANUSCRIPT 2. Patients and Methods 2.1. Study subjects The subjects enrolled in this study included 239 individuals chronically infected with HBV and 133 individuals who spontaneously resolved HBV infection (SRB) (HBsAg(-), anti-

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HBc(+), anti-HBs(+)). They were recruited between 2008 and 2013. All patients were interviewed and gaved their informed consent to participate to this study. They were recruited

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at the Medical Center of Biology of the Pasteur Institute of Morocco, Casablanca. The study

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protocol was in accordance with the Helsinki declaration and received an approval from the ethics Committee of the Faculty of Medicine of Casablanca. Patients selected for this study

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were classified into i) “HBeAg-negative chronic HBV infection” (n=119) with persistently

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normal serum ALT level and low HBV-DNA level (<2000UI/ml) during at least 6 months of follow-up as recommended by the European Association for the Study of the Liver (26), ii)

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patients with “HBeAg-negative chronic hepatitis B” (n=60) defined by persistent elevation of

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the level of both serum ALT and of HBV-DNA (2000UI/ml) for at least 6 months and histological signs of moderate/ severe necro-inflammation, iii) patients with advanced liver disease (AdLD, n=33) including patients with liver cirrhosis (LC, n=19) and patients with

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hepatocellular carcinoma (HCC, n=14). All the patients were negative for HCV and HIV.

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Serological markers for hepatitis B virus (HBsAg, HBeAg, anti-HBe, anti-HBc, and antiHBsAg) were analyzed using commercial kits (Axsym, Abbott Diagnostics, Germany). HBV DNA was detected by the TaqMan assay (Roche Diagnostics, Mannheim, Germany). 2.2. DNA isolation and genotyping of TLR9 polymorphism Whole blood was collected by venipuncture into an EDTA tube. PBMC samples were washed with Tris/EDTA (TE) buffer and subjected to SDS/proteinase K digestion at 37°C for 16 h, followed by phenol/chloroform extraction. Eluted DNA was then precipitated in cold ethanol 6

ACCEPTED MANUSCRIPT and re-suspended in TE buffer as described previously in (27). The purity and the concentration of genomic DNA were assessed using a NanoVue plus spectrophotometer (GE Healthcare, US). DNA samples were stored at -20°C until genotyping. The SNP rs187084 and rs5743836 were genotyped using a predesigned TaqMan SNP Genotyping Assay (Applied Biosystems). The target sequence was amplified in a 10 μl reaction volume that contained 5 μl

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of SensiFAST Genotyping Lo-ROX Mix (Bioline, London, UK), 0.125 μl of SNP Genotyping

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Assay, 1.5 μl of genomic DNA (20 ng), and 3.375 μl of DNase-free water. The thermal

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profile of the qPCR was as following : preincubation at 60°C for 1 min and then 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 15 s, and annealing/ extension at 60°C

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for 1 min, followed by post-incubation at 60°C for 1min, then by a final cooling at 40°C for 30 sec. Genotype analysis was conducted using Light Cycler 480 software by attributing a

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genotype of each sample (LightCycler® 480 Instrument, Roche). 2.3. Statistical analysis

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Statistical analysis was carried out using SPSS 21.0 software (SPSS, Chicago, Illinois, USA)

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and GraphPad Prism 7 software. The demographic data were compared using χ2 test. Viral load, ALT, AST levels were compared between genotype groups using Mann-Whitney U test

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and One Way ANOVA test. Hardy-Weinberg equilibrium was assessed by a goodness-of-fit

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χ2 test with 1 degree of freedom to compare observed and expected genotype frequencies. The association between the different groups and allelic or genotypic frequencies, measured by the odds ratio (OR) and its corresponding 95% confidence interval (CI), was estimated using multiple logistic regression and binary logistic regression models. A p-value < 0.05 was considered statistically significant. All tests were two sided. Haplotype analysis of TLR9 rs187084 and rs5743836 polymorphisms were also performed in this article using Arlequin v3.5Software.

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ACCEPTED MANUSCRIPT 3. Results 3.1. Clinical and biochemical Features The characteristics of the participants to the study including demographic data, biochemical features, and virological data are summarized in Table 1. Two hundred and thirty-nine patients with persistent HBV infection with a mean age of 41.8 ± 9.8 years, and 133

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individuals with spontaneous resolution with a mean age of 55.0 ± 10.3 years were enrolled in

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this study. Both distributions of gender and age were significantly different between CHB and

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SRB groups (p<0.0001).

The mean levels of serum ALT and AST were significantly higher in the persistently infected

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group compared to resolved group (p<0.0001). Regarding the viral genotypes, 91.2% of the patients were infected with genotype D and 8.8% of patients with genotype A.

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3.2. Association between rs5743836 and rs187084 TLR9 polymorphisms and the spontaneous clearance of HBV infection

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equilibrium (p > 0.05).

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The genotype distribution of these TLR9 SNPs was in accordance with the Hardy-Weinberg

For rs5743836 and rs187084 polymorphisms, both CHB and SRB displayed decreasing

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genotypic frequencies from AA, to GG. No significant difference in the allelic or genotypic distribution was observed between CHB and SRB for the two SNPs (table 2).

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The investigated TLR9 polymorphism presents no association with the spontaneous clearance of HBV infection (p > 0.05). In order to ensure the roles of TLR9 rs5743836 and rs187084 polymorphisms in HBV infection, the linkage disequilibrium and haplotype were analyzed. According to table 3, a four possible haplotypes (A-A, A-G, G-A, G-G) were identified. SNPs were not in linkage disequilibrium and no haplotype was associated with HBV clearance.

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ACCEPTED MANUSCRIPT 3.3. Association between TLR9 rs5743836 and rs187084 polymorphisms and the progression of HBV infection To analyze the impact of TLR9 rs5743836 and rs187084 polymorphisms on the progression of HBV-induced liver disease, we stratified the chronically infected patients in advanced stage

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(AdLD ie LC+HCC, n=33), and non-advanced liver disease (non AdLD, n=179) (Table 4). For TLR9 rs5743836 polymorphism, multivariate logistic regression analysis did not reveal

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any significant difference between these groups. However, a significant association was

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observed between rs187084 AA carriers and HBV progression to advanced liver disease, which was pronounced by a protective effect against progression of HBV infection in patients

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carrying genotype G only in the dominant model AA vs AG+GG (OR=0.452, 95% CI 0.211-

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0.966, p= 0.03730) and (OR=0.46, 95% CI 0.21-0.99, p= 0.050), before and after gender adjustment respectively (Table 4).

3.4. Correlation of TLR9 rs5743836 and rs187084 polymorphisms with clinical

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markers in patients with chronic HBV infection Finally, to estimate the putative role of TLR9 rs5743836 and rs187084 polymorphisms on variations of clinical features of CHB patients, demographic, biochemical and virological data

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were analyzed according to SNPs genotypes. Distribution of TLR9 variations were not

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modulated by sex or age. Similarly they did not exhibit any influence on aminotransferases (AST and ALT) levels (Figure 1 and 2). Remarkably, TLR9 rs5743836 polymorphism was correlated with HBV DNA loads (p <0.0001) with an increase in viral loads from major genotype AA to minor genotype GG (Figure 1-A). Regarding TLR9 rs187084 polymorphism, a significant difference was observed as well with AA genotype presenting higher DNA loads than patients with genotype AG (Figure 2-A).

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ACCEPTED MANUSCRIPT 4. Discussion There is multiple evidence showing that the host genetic background influences the outcome of HBV infection (28, 29). This generates the hope that a better understanding of the hostvirus interaction will improve disease management (30). TLRs pathways play a critical role in innate immunity against bacterial, fungal, and viral pathogens in humans (31-33).

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TLR9, located in endosomes, can recognize unmethylated CpG dinucleotide motifs of viral

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DNA, to trigger a cascade of signaling that activates host defense against viral infection (34,

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35). It was reported that the CpG oligonucleotides (CpG-ODN), the ligands of TLR9, can inhibit HBV replication in the liver of transgenic mice (32), suggesting the involvement of

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this receptor in HBV regulation. Recently, Aillot L et al demonstrated that synthetic TLR9CpG-ODN might efficiently inhibit HBV entry by “coating” viral particles and thereby

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prevent its interaction with cellular receptor (36). In addition, the TLR9 receptor has been proven to mediate/stimulate thymosin alpha-1 signaling “a natural thymic peptide that was

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approved for anti-HBV treatment” (37).

The need to treat CHB patients and to achieve a functional cure requires the development of drugs that activate the innate immunity. To reach that goal, TLR9 agonists have been used for

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cancer and chronic infections for their ability to enhance both innate and adaptive

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antitumoral/antiviral responses. TLR9-Ligands agonists PF-3512676 and ISS-1018 have been tested in clinical trials as immune-cancer therapeutics (17, 38, 39). To the best of our knowledge, the present study is the first to assess the association between TLR9 rs187084 and rs5743836 polymorphisms with spontaneous clearance of HBV infection. However, no consistent association between the carriage of these TLR9 polymorphisms and the spontaneous clearance or the persistence of HBV was shown. Regarding TLR9 haplotype frequencies, no significant differences were observed between HBV infected patients and

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ACCEPTED MANUSCRIPT controls as well. Our most remarkable finding was the association of rs187084 AA carriers with HBV infection progression to advanced liver disease. In addition to the correlation of TLR9 SNPs with loads of HBV DNA. Indeed, for rs5743836, the genotypes AA, AG to GG were significantly associated with an increase in circulating HBV DNA whereas the opposite situation was noted with AA, AG to GG for rs187084. This suggests that GG homozygosity at

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rs5743836 and the AA genotype at rs187084 may probably participate to the establishment of

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HBV persistence, as well as the association of the latter to HBV progression. This result

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seems in line with a previous study that observed an association of TLR9 rs5743836 G with a higher susceptibility to HIV infection in European descendants, and a correlation of TLR9

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rs5743836 GA genotype with protection to HIV infection in subjects with African ancestry (21). In addition, the TLR9 rs5743836 G variant was linked to viral load, CD4+ T cell counts

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and disease progression in HIV-1 infected individuals in another report (40). In regards to rs187084 polymorphism, the protective effect of G carriers in our study, has already been

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proven by Fischer J 2016, who found that TLR9 rs187084 C allele was associated with

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spontaneous virus clearance in women, in addition, the minor G allele of rs187084 was associated with a 1.9-fold to 2.2-fold increase in likelihood of HCV spontaneous clearance in

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women (20).

Previous functional studies performed on these two TLR9 polymorphisms revealed that the

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minor allele (G) carriers exhibit a 25% higher gene expression compared to those with WT promoter (A). This was explained by the fact that rs187084 A>G polymorphism creates a new binding site for the specificity protein 1 (Sp-1), a transcription factor which binds to GC-rich motifs of many promoters (41). A second variant, the rs5743836 G allele generates a potential novel NF-κB-binding site that promotes TLR9 transcription more efficiently than the WT rs5743836 A, which was associated with an enhanced inflammatory state and a premalignant state of the gastric cancer in H. pylori- positive subjects (42). On the contrary, Novak et al,

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ACCEPTED MANUSCRIPT found that a higher transcriptional activity of the TLR9 was rather associated with the TT allelic variant at rs5743836 (24). In addition, rs5743836 G allele was shown to generate a novel IL-6-responsive element mediating an IL6 up-regulation (43), known to promote the hepatic inflammation and the development of liver fibrosis (44).

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Only one study was performed on the polymorphism of the TLR9 promoter rs5743836 and the HBV infection in Taiwan and found an association with early HBeAg seroconversion (22).

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By contrast, a recent study on this same SNP didn’t show any association with the evolution

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of HBV infection on Turkish population (25). On the other hand most of the studies carried out on these 2 SNPs were conducted on patients with cancers. The outcomes were

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heterogeneous, a situation somewhat expected due to the genetic diversity of the populations

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studied as well as by the variety of disease stages investigated. 5. Conclusion

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In conclusion, our study focused on the evaluation of the putative association of the rs187084

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and rs5743836 polymorphisms located in the TLR9 promoter with HBV outcomes. These SNPs do not show any association with the spontaneous clearance or the persistence of HBV

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infection. Regarding TLR9 rs187084, patients with the AA genotype are more likely to progress to advanced liver disease. Interestingly, a significant correlation was observed

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between these SNPs and HBV DNA loads. This study is the first attempt to investigate the association of the polymorphisms of genes involved in the innate immune response in the context of HBV infection in the Moroccan population. Our result with TLR9 variants is considered preliminary in the context of HBV spontaneous clearance due to the relatively low sample size with a small number of patients carrying advanced liver disease, and which focused on a Moroccan population with only one ethnic origin. Given the importance of the TLR9 receptor in the immune response to HBV infection, 12

ACCEPTED MANUSCRIPT it would be particularly interesting to investigate the association of these polymorphisms with viral load and HBV pathogenesis on a larger sample size and in a different genetic context to confirm or to infirm our results. Acknowledgments

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The authors would like to acknowledge all patients for their participation in this study. The authors would like to thank also Dr. Janet Hall from DR2 Inserm du CRCL for the revision of

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the paper.

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Funding source

This work was supported by research grant from the Actions Concertées Inter Pasteuriennes

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Ethics approval and consent to participate

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(ACIP 2014) and the Association de Lutte Contre le SIDA (ALCS, FASP 2016).

The study was approved by the Ethics Committee of the Faculté de Medicine of Casablanca in

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accordance with the ethical guidelines of the Declaration of Helsinki.

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Authors’ contributions: Chihab H, Benjelloun S. conceived, designed the experiments and wrote the paper; BenjellounS., Ezzikouri S, ElHabazi A and Pineau P contributed to the

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drafting and revision of the article; Zaidane I, Jadid F and Elfihry R helped in the collection of samples; Badre W et Tahiri M contributed to the clinical data; Elmessaoudi-Idrissi M helped

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in the statistical analysis; Chemin I and Chair M helped in the interpretation of data; Benjelloun S contributed to the final approval of the version to be submitted. All authors read and approved the final manuscript.

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Differential influence in individuals with distinct ethnic background. Human immunology. 2017 2017;78:221–6. 22. Wu JF, Chen CH, Ni YH, Lin YT, Chen HL, Hsu HY, et al. Toll-like receptor and hepatitis B virus clearance in chronic infected patients: a long-term prospective cohort study in Taiwan. J Infect Dis. 2012 Sep 1;206(5):662-8. PubMed PMID: 22740716. 23. Mockenhaupt FP, Hamann L, Gaertner CV, Bedu-Addo G, Kleinsorgen CV, Schumann RR, et al. Common Polymorphisms of Toll-Like Receptors 4 and 9 Are Associated with the Clinical Manifestation of Malaria during Pregnancy. The Journal of infectious diseases. 2006 2006;194. 24. Novak N, Yu CF, Bussmann C, Maintz L, Peng WM, Hart J, et al. Putative association of a TLR9 promoter polymorphism with atopic eczema. Allergy. 2007 2007;62:766–72. 25. Katrinli S, Nigdelioglu A, Ozdil K, Dinler-Doganay G, Doganay L. The association of variations in TLR genes and spontaneous immune control of hepatitis B virus. Clinics and research in hepatology and gastroenterology. 2017 Dec 21. PubMed PMID: 29276096. 26. Liver EAftSot. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol. 2017;67(2):370-98. 27. Ezzikouri S, Essaid EFA, Afifi R, Benazzouz M, Hassar M, Pineau P, et al. Impact of TP53 codon 72 and MDM2 promoter 309 allelic dosage in a Moroccan population with hepatocellular carcinoma. Int J Biol Markers. 2011 2011;26:229–33. 28. Lin TM, Chen CJ, Wu MM, Yang CS, Chen JS, Lin CC, et al. Hepatitis B virus markers in Chinese twins. Anticancer Res. 1989 1989;9:737–41. 29. Thursz M. Genetic susceptibility in chronic viral hepatitis. Antiviral research. 2001 2001;52:113–6. 30. Fattovich G, Bortolotti F, and Donato F. Natural history of chronic hepatitis B: special emphasis on disease progression and prognostic factors. Journal of hepatology. 2008 2008;48:335– 52. 31. Hoebe K, Du X, Georgel P, Janssen E, Tabeta K, Kim SO, et al. Identification of Lps2 as a key transducer of MyD88-independent TIR signalling. Nature. 2003 2003;424. 32. Isogawa M, Robek MD, Furuichi Y, and Chisari FV. Toll-like receptor signaling inhibits hepatitis B virus replication in vivo. Journal of virology. 2005 2005;79:7269–72. 33. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, Tripp RA, et al. Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nature immunology. 2000 2000;5. 34. Latz E, Franko J, Golenbock DT, and Schreiber JR. Haemophilus influenzae Type b-Outer Membrane Protein Complex Glycoconjugate Vaccine Induces Cytokine Production by Engaging Human Toll-Like Receptor 2 (TLR2) and Requires the Presence of TLR2 for Optimal Immunogenicity. The Journal of Immunology. 2004 2004;172:2431–8. 35. Vaidya SA, and Cheng G. Toll-like receptors and innate antiviral responses. Current Opinion in Immunology. 2003 2003;15:402–7. 36. Aillot L, Bonnin M, Ait-Goughoulte M, Bendriss-Vermare N, Maadadi S, Dimier L, et al. Interaction between TLR9-CpG-ODNs and HBV virions leads to entry inhibition in hepatocytes and reduction of IFNalpha production by pDC. Antimicrobial agents and chemotherapy. 2018 Feb 12. PubMed PMID: 29439958. 37. Song EY, Shin Y, Roh EY, Sue S, Park MH, Kim BH, et al. Serum HBsAg levels during peginterferon alpha-2a treatment with or without thymosin alpha-1 in HBeAg-positive chronic hepatitis B patients. Journal of medical virology. 2011 2011;83:88–94. 38. Krieg AM. Therapeutic potential of Toll-like receptor 9 activation. Nature reviews Drug discovery. 2006 Jun;5(6):471-84. PubMed PMID: 16763660. 39. Wittig B, Schmidt M, Scheithauer W, Schmoll HJ. MGN1703, an immunomodulator and tolllike receptor 9 (TLR-9) agonist: from bench to bedside. Critical reviews in oncology/hematology. 2015 Apr;94(1):31-44. PubMed PMID: 25577571.

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40. Pine SO, McElrath MJ, and Bochud PY. Polymorphisms in toll-like receptor 4 and toll-like receptor 9 influence viral load in a seroincident cohort of HIV-1-infected individuals. Aids. 2009 2009;23:2387–95. 41. Hamann L, Glaeser C, Hamprecht A, Gross M, Gomma A, and Schumann RR. Toll-like receptor (TLR)-9 promotor polymorphisms and atherosclerosis. Clinica chimica acta; international journal of clinical chemistry. 2006 2006;364:303–7. 42. Ng MT, Van't Hof R, Crockett JC, Hope ME, Berry S, Thomson J, et al. Increase in NF-kappaB binding affinity of the variant C allele of the toll-like receptor 9 -1237T/C polymorphism is associated with Helicobacter pylori-induced gastric disease. Infection and immunity2010 2010. 1345–52 p. 43. Carvalho A, Osorio NS, Saraiva M, Cunha C, Almeida AJ, Teixeira-Coelho M, et al. The C Allele of rs5743836 Polymorphism in the Human TLR9 Promoter Links IL-6 and TLR9 Up-Regulation and Confers Increased B-Cell Proliferation. PLoS ONE. 2011 2011;6:28256. 44. Scheller J, Chalaris A, Schmidt-Arras D, and Rose-John S. The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochimica et biophysica. acta. 2011 2011;1813:878–88.

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ACCEPTED MANUSCRIPT Figures legends: Figure 1: Virological and Biochemical data according to rs5743836 TLR9 genotypes. (A) Viral loads according to rs5743836 TLR9 polymorphism. (B) and (C) AST and ALT levels

PT

according to rs5743836 TLR9 polymorphism, respectively.

Figure 2: Virological and Biochemical data according to rs187084 TLR9 genotypes. (A) Viral

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loads according to rs187084 TLR9 polymorphism. (B) and (C) AST and ALT levels according

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to rs187084 TLR9 polymorphism, respectively.

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ACCEPTED MANUSCRIPT Table 1: Demographic and clinical characteristics of the subjects enrolled in this study.

Age (mean ± SD, years) Gender (%) Female Male ALT (mean ± SD, IU/L) AST (mean ± SD, IU/L) Viral genotypes (%) D A Viral load (median [range], log10 copies/mL) Clinical status HBeAg-negative chronic HBV infection (%) HBeAg-negative chronic hepatitis B (%) LC (%) HCC (%) na (%)

D E

T P E

CHB (n=239)

SRB (n=133)

41.8 ± 9.80

55 ± 10.35

36.4 63.6 62.8 ± 27.38 54 ± 27.21

57.25 42.75 34.9 ± 11.95 30.9 ± 10.27

M

N A

49.6 25.4 7.6 6 11.4

CHB: chronic infection B, SRB: spontaneously resolved B, LC: liver cirrhosis, HCC: hepatocellular carcinoma.

C C

ALT, alanine aminotransferase, normal values: 7-56U/L; AST, aspartate aminotransferase, normal values: 5-35U/L. na: non available data for 27 patients.

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A

T P

I R

C S U

91.2 8.8 3.4 [1.1-9]

p-value

<0.0001 <0.0001 <0.0001 <0.0001

ACCEPTED MANUSCRIPT Table 2: Impact of rs5743836 and rs187084 TLR9 polymorphisms on HBV outcomes Genotypes

SRB (n=134)

CHB (n=239)

OR (95%CI)

p-value

TLR9 rs5743836 (n, %) AA AG GG

98/132 (74.24%) 30/132 (22.73%) 4/132 (3.03%)

174/239 (72.80%) 53/239 (22.18%) 12/239 (5.02%)

1 0.99 [0.60-1.66] 1.69 [0.53-5.38]

---0.985 0.370

Recessive model (n, %) AA+AG GG

128/132 (97%) 4/132 (3%)

227/239 (95%) 12/239 (5%)

1 0.59 [0.19-1.87]

Dominant model (n, %) AA AG+GG

98/132 (74.24%) 34/132 (25.76%)

174/239 (72.80%) 65/239 (27.2%)

1 1.08 [0.66-1.74]

Allelic frequency A allele G allele

0.86 ± 0.02 0.14 ± 0.02

0.84 ± 0.02 0.16 ±0.02

TLR9 rs187084 (n, %) AA AG GG

63/133 (47.37%) 54/133 (40.60%) 16/133 (12.03%)

Recessive model (n, %) AA+AG GG Dominant model (n, %) AA AG+GG Allelic frequency A allele G allele

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Ad OR (95%CI)

T P

Ad p-value

1 1.14 [0.66-1.94] 1.64 [0.49-5.42]

---0.635 0.414

---0.366

1 0.63 [0.19-2.05]

---0.441

---0.764

1 0.83 [0.50-1.38]

---0.475

1 1.14 [0.75-1.74]

---0.537

1 0.803 [0.43-1.50]

---0.489

106/238 (44.54%) 103/238 (43.28%) 29/238 (12.18%)

1 1.13 [0.72-1.78] 1.08 [0.54-2.14]

---0.588 0.831

1 1.08 [0.68-1.73] 0.99 [0.49-2.01]

---0.741 0.998

117/133(87.96%) 16/133(12.04%)

209/238 (87.81%) 29/238 (12.19%)

1 0.99 [0.51-1.89]

---0.965

1 1.04 [0.53-2.02]

---0.909

63/133 (47.36%) 70/133 (52.64%)

106/238 (44.53%) 132/238 (55.47%)

1 1.12 [0.73-1.71]

---0.599

1 0.94 [0.60-1.46]

---0.786

0.68 ± 0.03 0.32 ± 0.03

0.662 ± 0.02 0.338 ± 0.02

1 1.07 [0.78-1.47]

---0.679

1 0.99 [0.62-1.57]

---0.968

D E

T P E

C C

A

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C S U

N A

M

ACCEPTED MANUSCRIPT Ad: Adjusted P value and OR for gender.

Table 3: Haplotype frequencies of rs5743836 and rs187084 TLR9 polymorphisms

Haplotypes

CHB

SRB

site1-site2

(2n=476) (%)

(2n=264) (%)

A-A

255 (53.58)

149 (56.29)

A-G

144 (30.25)

77 (29.32)

G-A

60 (12.60)

29 (11.14)

G-G

17 (3.57)

9 (3.25)

D E

Site 1: rs5743836, site 2: rs187084

T P E

C C

A 20

p-value

---

U N

T P

I R

SC

0.612

A M

OR (95% CI)

1

1.09 (0.78-1.54)

0.445

1.21 (0.74-1.97)

0.816

1.10 (0.48-2.54)

ACCEPTED MANUSCRIPT Table 4: Impact of rs5743836 and rs187084 TLR9 polymorphisms on the progression of HBV infection non AdLD (n=179)

AdLD (n=33)

TLR9 rs5743836 (n, %) AA AG GG

131 (73.2) 39 (21.8) 9 (5)

25 (75.8) 6 (18.2) 2 (6)

1 0.81 [0.31-2.11] 1.16 [0.24-5.71]

Recessive model (n, %) AA+AG GG

170 (95) 9 (5)

31 (94) 2 (6)

1 0.82 [0.17-3.98]

Dominant model (n, %) AA AG+GG

131 (73.2) 48 (26.8)

25 (75.8) 8 (24.2)

1 0.87 [0.37-2.07]

Allelic frequency A allele G allele

0.84 ± 0.02 0.16 ± 0.02

0.85 ± 0.05 0.15 ± 0.05

TLR9 rs187084 (n, %) AA AG GG

73 (41) 81 (45.5) 24 (13.5)

Recessive model (n, %) AA+AG GG Dominant model (n, %) AA AG+GG Allelic frequency A allele G allele

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Ad OR (95%)

p-value

Ad p-value

OR (95%CI)

T P

1 0.69 [0.25-1.95] 1.22 [0.25-6.05]

---0.492 0.805

---0.806

1 0.76 [0.15-3.72]

---0.736

---0.758

1 1.26 [0.51-3.11]

---0.619

1 0.94 [0.45-1.96]

---0.874

1 1.25 [0.40-3.87]

---0.695

20 (60.6) 10 (30.3) 3 (9.1)

1 0.45 [0.20-1.03] 0.46 [0.12-1.67]

---0.053 0.227

1 0.46 [0.20-1.06] 0.45 [0.12-1.70]

---0.070 0.242

154 (86.5) 24 (13.5)

30 (90.9) 3 (9.1)

1 1.56 [0.44-5.51]

---0.488

1 1.55 [0.43-5.54]

---0.493

73 (41) 105 (59)

20 (60.6) 13 (39.4)

1 0.45 [0.21-0.97]

---0.037

1 0.46 [0.21-0.99]

---0.050

0.64 ± 0.02 0.36 ± 0.02

0.76 ± 0.06 0.24 ± 0.06

1 0.56 [0.31-1.03]

---0.059

1 0.44 [0.17-1.14]

---0.092

PT

E C

C A

D E

---0.659 0.851

I R

C S U

N A

M

ACCEPTED MANUSCRIPT Ad: Adjusted P value and OR for gender.

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C S U

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ACCEPTED MANUSCRIPT

Abbreviations Immunoglobulin M : IgM, Interleukin 6 : IL-6, Pattern recognition receptors : PRRs, Retinoic-acid-inducible gene 1(RIG-I) -like receptors : RLRs, Nucleotide-binding oligomerization -like receptors: NOD-like receptors, Cytosine–phosphate–guanine : CpG,

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Myeloid differentiation protein 88 : MYD88, Interleukin-1 receptor-associated kinase :IRAK,

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Nuclear factor-kappa B : NF-κB, Activator protein 1 :AP-1, Tumor necrosis factor receptor (TNFR)-associated factor :TRAF6, Peripheral Blood Mononuclear Cells : PBMCs, Interferon

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Regulatory Factor 7 : IRF7, The suppressor of cytokine signaling :SOCS-1, Blood dendritic

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cell antigen 2: BDCA2

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ACCEPTED MANUSCRIPT Highlights: TLR9 promoter polymorphisms are not associated with spontaneous HBV clearance,

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The TLR9 polymorphisms may influence the circulating HBV DNA,

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TLR9 rs187084 AA HBV carriers are more likely to progress to advanced liver disease

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Figure 1

Figure 2