- Email: [email protected]
Reduced expression of TRIF in chronic HBV infected Iranian patients
Clinics and Research in Hepatology and Gastroenterology (2013) 37, 491—495
Available online at www.sciencedirect.com
ORIGINAL ARTICLE
Reduced expression of TRIF in chronic HBV infected Iranian patients Fatemeh Ayoobi a, Gholamhossein Hassanshahi b, Nahid Zainodini c, Hossein Khorramdelazad b, Mohammad Kazemi Arababadi c,∗, Derek Kennedy d a
Physiology and Pharmacology Research center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Molecular Medicine Research center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran c Immunology of Infectious Diseases Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran d School of Biomolecular and Physical Science, Eskitis Institute for Cell and Molecular Therapies, Griffith University Nathan, Queensland, Australia b
Available online 20 February 2013
Summary Background and aims: TRIF is one of the main intracellular adaptor proteins required for TLR3 and 4 signaling. Abnormal gene expression of TRIF may lead to abrogated immune responses against viral infections including hepatitis B infection. The aim of this study was to identify the mRNA levels of TRIF in PBMCs isolated from chronic HBV (CHB) infected patients. Material and methods: mRNA was isolated from 63 CHB patients and 60 healthy controls and transcript levels of TRIF were examined in parallel with beta-actin (as housekeeping gene) using Real-Time PCR techniques. Results: Our results demonstrated that expression of TRIF was significantly decreased in PBMCs isolated from CHB patients when compared to healthy controls. Conclusions: Based on the results reported here, it seems that CHB patients are unable to express appropriate levels of the TRIF gene, which may attenuate TLR3 and 4 signaling subsequent to HBV infection. Our results suggest a possible mechanism, which may explain why hepatitis B infection is stable in CHB patients. © 2012 Elsevier Masson SAS. All rights reserved.
Abbreviations: HBV, Hepatitis B virus; PBMC, peripheral blood mononuclear cell; TRIF, TIR-domain-containing adapter-inducing interferon-; TLR, Toll Like Receptor; PAMP, pathogen associated molecular patterns; MYD88, myeloid differentiation primary response; IRAK1, Interleukin-1 receptor associated kinase-1; TRAF6, TNF receptor associated factor; NF-B, Nuclear factor kappa-light-chain-enhancer of activated B cells; IRF3, Interferon regulatory transcription factor 3. ∗ Corresponding author. Immunology of Infectious Diseases Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. Tel.: +00983915234003 5; +00989139922445; fax: +00983915225209. E-mail address: [email protected] (M.K. Arababadi). 2210-7401/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.clinre.2012.11.005
492
Introduction Chronic HBV (CHB) infected patients harbor a clinical form of hepatitis B in which HBV is not completely removed either from liver hepatocytes or their serum, and this leads to an intermediate symptomatic liver disease [1,2]. Scientists have proposed that CHB could be one of the main reasons for pathogenesis of hepatocarcinoma and cirrhosis [3,4]. The main mechanism(s) responsible for the initiation and progression of pathologies caused by CHB, and other clinical manifestations associated with HBV infection, has yet to be identified. Researchers believe that multifactorial parameters, such as immunological, genetic and epigenetic characteristics of the hosts are responsible for the differences between individuals with persistent HBV infections and those who overcome the disease by clearing the virus [5—8]. TLRs are the main intra/extra-cellular innate immune cell receptors that identify PAMPs of the microbes, including those found associated with viruses [9]. Interactions between TLRs and their ligands lead to the induction of intra-cytoplasmic and nuclear signaling molecules and consequently; activate and stimulate immune cell (e.g. dendritic cells) migration [9], NADPH oxidase activation [10], phagocytosis [11] and inflammatory cytokine expression [12]. TLRs induce inflammatory cytokine expression through the recognition of pathogen PAMPs via the MYD88 and TRIF dependent pathways [13]. Assembly of TRIF (as an adaptor molecule) leads to the activation of several intracellular signaling pathways such as IRAK1, TRAF6 and NF-(B [14]. NF-(B, as a transcription factor which recognizes consensus elements present in the regulatory region of several genes, including inflammatory cytokines [14]. Therefore, reduced expression of TRIF may lead to inappropriate signal activation, and consequently, defective immune responses against viral infection. Due to the fact that CHB infected patients are unable to completely eradicate intercellular HBV from the hepatocytes, it seems that these patients may be functionally defective in some aspects of their immune system. Because of the important role that TRIF plays in the activation of downstream signaling molecules such as, TLR3 and 4, and the overall induction of immune responses against viruses, it may be speculated that the molecule is crucially important in the pathogenesis of CHB. Therefore, the main aim of this study was to investigate mRNA expression levels of TRIF in the PBMCs of CHB patients.
Material and methods Subjects Peripheral blood samples were collected from 63 healthy controls and 60 CHB infected patients within Rafsanjan (located in the South-Eastern region of Iran) in 5.5 ml tubes with and without anti-coagulant. CHB infected patients with coinfection of either HCV or HIV were excluded from the study. The CHB diagnosis was performed based on the ‘‘Guide of Prevention and Treatment in Viral Hepatitis’’ [15] by an expert internal medicine specialist. Patients were entered to the study based on assessment of their previous clinical records. Controls were also selected with the same age, sex and socio-economical status. The sera, collected
F. Ayoobi et al. from blood without anti-coagulant, were stored at —20 ◦ C for a maximum of 2 months or at —70 ◦ C when longer storage periods were required for analysis. The anti-coagulant treated samples were immediately subjected to RNA extraction (to obtain either more quantity or qualitative RNA samples) following entrance to the laboratory. This study was approved by the ethical committee of the Rafsanjan University of Medical Sciences and written informed consent was obtained from all of participants prior to sample collection.
Detection of serological HBV markers The samples were screened for HBsAg and HBeAg using ELISA (Behring, Germany) techniques according to the manufacturer’s guidelines.
HBV-DNA extraction and Real-Time PCR conditions The viral DNA was purified from 200 L of plasma from CHB patients using a commercial kit (Cinnaclon, Iran) according to manufactures guidelines. The HBV-DNA amplification was also undertaken using a commercial kit (Primer Design Company, UK) following the manufacturer’s instructions.
RNA extraction, reverse transcription and quantitative Real-Time PCR Total RNA was extracted from PBMCs using an RNX extraction kit from the Cinnaclon Company (Iran). The extracted RNA quality was determined by electrophoresis on the ethidium bromide pretreated agarose gel and by measuring absorption at 260/280 nm by spectrophotometery. Subsequent cDNA synthesize was performed using a cDNA synthesis kit (Parstous, Iran) with both oligo(dT) and random hexamer primers (Aryatous, Iran). The reverse transcription step was performed using the following program: 70 ◦ C for 10 min (without reverse transcription enzymes), —20 ◦ C for 1 min (cooling), reverse transcription enzyme was added and the sample incubated at 42 ◦ C for 60 min. Finally, the sample was treated at 95 ◦ C for 10 min to inactivate the reverse transcription enzyme. Quantitative Real-Time PCR was performed by addition of a SYBR green master mix (Parstous, Iran), 200 ng of the generated cDNA and 2 pg/l of appropriate primers (Table S1). The following cycling program was set on a BIO-RAD CFX96 system (Bio-Rad Company, USA): one cycle of 95 ◦ C for 15 min, 40 cycles of 95 ◦ C for 30 s, 60 ◦ C for 30 s and 72 ◦ C for 30 s. Real-Time PCR was carried out in triplicate and the -actin was assessed as a housekeeping gene for the normalization of amplification signals of the target gene. The relative amounts of PCR product were determined using the 2—Ct formula. The dissociation stages, melting curves and quantitative analyses of the data were performed using CFX manager software version 1.1.308.111 (Bio-Rad, USA). All PCR products were screened using electrophoreses on a 1% gel containing 0.5 mg/ml ethidium bromide to check the size of PCR product.
TRIF expression in chronic HBV infection
493
HBV genotyping
5.0
Liver enzymes detection
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
pa ti e
co nt ro l
nt
Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct and total bilirubin were evaluated in the patients and controls using commercial kits (Man Ltd, Tehran-Iran) according to the manufactures guidelines.
Expression level of TRIF
4.5
HBV genotypes were determined using a gap-PCR technique according to our previous study [16].
C
H
H ea lth y
B
Data analysis and statistical methods Parametric statistical analyses were performed using the ttest in the SPSS software version 18. When a P value was less than 0.05 it was considered significant.
Results
Figure 1 mRNA levels of TRIF in PBMCs of CHB patients and healthy controls. Results are shown as mean ± standard error. The comparison between CHB and healthy controls is statistically different (P = 0.048).
Our results demonstrated that all the patients were HBsAg positive. The HBsAg titration was higher in the patients that suffered from medium (between 20,000 to 400,000) and high (greater than 1000,000) HBV copy numbers/ml. The results also demonstrated that two patients were positive for HBeAg (3.17%) and the patients carried greater than 1000,000 HBV copy numbers/ml.
Expression levels of TRIF
2.5
Detection of HBsAg and HBeAg
2.0
1.5
1.0
0.5
Our results showed that all the patients had detectable HBV-DNA. Amongst the patients, 40, 12 and 11 carried less than 20,000 (low), between 20,000 to 400,000 (medium) and greater than 1000,000 (high) HBV copy numbers/ml, respectively. Two of the patients with greater than 1000,000 HBV copy numbers/ml carried HBeAg.
H ig h
ed M
Lo w
iu
m
0.0
Quantification of HBV-DNA copy numbers
Figure 2 mRNA levels of TRIF in PBMCs of CHB patients with different HBV-DNA copy numbers. The figure illustrates that expression levels of TRIF were not affected by different HBV-DNA copy number (Low: < 20,000, medium: between 20,000—400,000 and high: > 1,000,000 HBV-DNA copy numbers/ml). Results are shown as mean ± standard error.
Expression of TRIF
Detection of liver enzymes
Our results showed that expression of TRIF in the PBMCs of CHB patients was reduced by 3.75 fold when compared with healthy controls. Statistical analysis of the data revealed that the difference was significant (P = 0.048) (Fig. 1). Our results also revealed that the expression levels of TRIF were not different among CHB patients with less than 20,000, between 20,000 to 400,000 and greater than 1000,000 HBV copy numbers/ml (P = 0.475) (Fig. 2).
Our results identified that all the patients carrying; low, medium and high HBV-DNA copy numbers/ml had normal serum levels of ALT, AST, ALP as well as direct and total bilirubin.
HBV genotyping The results demonstrated that all HBV positive patients carried the HBV D genotype.
Discussion Initiation of immune responses against viral infections starts with innate immunity via the production of cytokines and chemokines. One of the mechanisms that leads to the expression of these molecules is the activation of TLRs and consequently, the activation of intracellular signaling molecules via adaptor proteins including TRIF [17,18]. Our results demonstrated that the mRNA expression levels of
494 TRIF were significantly decreased in the CHB patients in comparison to healthy controls. Therefore, based on our results, it can presumably be concluded that CHB patients are unable to express adequate values of TRIF, however, this would need to be validated by measuring TRIF protein levels. Patients with reduced TRIF expression are probably unable to activate the downstream signaling pathways of TLR3 and also 4 in response to HBV infection. Subsequently, these patients would be unable to completely eradicate the infection. Additionally, our results revealed that the mRNA expression levels of TRIF were not affected by HBV-DNA copy numbers or possibly HBV replication, hence, it may be concluded that HBV factors did not appear to affect the TRIF expression leading us to conclude that host genetic or epigenetic factors may be the main reasons for this defectiveness. Our results also demonstrated that all of the patients were infected with the HBV D genotype but had normal serum levels of liver markers. Therefore, it can be concluded that HBV-DNA copy numbers did not affect or regulate the expression levels of TRIF and was unable to influence serum levels of ALT, AST, ALP, as well as direct and total bilirubin. Due to the fact that, during hepatitis B, hepatocytes function disorders are results of immune responses, hence, downregulation of TRIF, as a marker of immune response, can describe normal serum levels of liver markers. All of the patients carried the HBV D genotype, hence, it also can be concluded that if infection was able to influence expression levels of TRIF this could be correlated to the HBV D genotype, however, we cannot make any conclusions in reference to other genotypes. In addition, we cannot exclude the possibility that other factors, such as the translational control of TRIF or the stability of TRIF may influence signal transduction pathways. To the best of our knowledge, this is the first study to report down-regulation of TRIF in the CHB patients. However, Li et al., showed that the expression levels of TLR3 and also its related cytokine (IFN-beta) were decreased in the CHB and acute HBV infected patients [19]. An et al., have stimulated dendritic cells (DC) of CHB patients with poly I:C and then evaluated the expression levels of TLR3 [20]. They reported that DCs of CHB patients express lower levels of TLR3 than healthy controls [20]. Interestingly, Chen et al., revealed that TLR4 expression is decreased in CHB patients and that cytokine production was impaired in PBMCs of CHB patients after challenge with TLR4 ligands [21]. Therefore, it seems that TLR4 signaling is also defected in their evaluated patients. Interestingly, Gue et al., demonstrated that up-regulation of TRIF in infected human hepatocyte-derived cells leads to the decrease of both HBV-mRNA and DNA levels [22]. Furthermore, Tjwa et al., showed that activation of myeloid DC via TLR3 leads to improvement of natural killer cell function in CHB patients [23]. Another study demonstrated that expression of TLR3 was up-regulated in the active phases of CHB infection [24]. It has also been shown that in contrast to PBMCs, TLR4 expression in hepatocytes was increased in CHB patients in comparison to healthy controls [25]. Thus, it seems that the expression and roles of TLRs in the hepatocytes are different from immune cells. Additionally, studies have demonstrated that HBV factors can alter expression and function of TLR3 signaling molecules [26]. Based on our results it seems that the replication rate of HBV did not affect TRIF expression but, HBV factors may affect other signaling molecules including IRF3
F. Ayoobi et al. which has been reported previously [26]. The combined results from these groups highlight the complex nature of HBV infection and reinforce the notion that the pathology of infection is most likely the result of multigenic factors in the host. Our data suggests that TRIF expression can now be added to the growing list of causes that allow persistent HBV infection. Finally, based on our results and the other mentioned studies, it seems that down-regulation of TRIF and TLRs including TLR3 and 4 in CHB patients may attenuate immune responses due to a deficit in pro-inflammatory cytokines production. Our findings may also open an avenue for the investigators of this field to focus on designing therapeutic reagents to overcome this defect in CHB patients. Furthermore, results of the current study suggest a model for future therapy in which up-regulation of these molecules is of paramount importance.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgments Authors of this article would like to take this opportunity to thank all of the CHB patients and healthy controls, which warmly attended and co-operated in this research program. This project was supported by a grant from the Rafsanjan University of Medical Sciences.
Appendix A. Supplementary data Supplementary data (Table S1) associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.clinre.2012.11.005.
References [1] Assar A, Arababadi MK, Mohit M, Ahmadabadi BN, Pumpens P, Khorramdelazad H, et al. T helper and B cell escape mutations within the HBc gene in patients with asymptomatic HBV infection: a study from the South-Eastern region of Iran. Clin Lab 2012;58:53—60. [2] Chan HL, Jia J. Chronic hepatitis B in Asia-new insights from the past decade. J Gastroenterol Hepatol 2011;26(Suppl 1):131—7. [3] Mendy ME, Welzel T, Lesi OA, Hainaut P, Hall AJ, Kuniholm MH, et al. Hepatitis B viral load and risk for liver cirrhosis and hepatocellular carcinoma in The Gambia West Africa. J Viral Hepat 2009;27:27. [4] Michielsen P, Ho E. Viral hepatitis B and hepatocellular carcinoma. Acta Gastroenterol Belg 2011;74(1):4—8. [5] Ahmadabadi BN, Hassanshahi G, Arababadi MK, Leanza C, Kennedy D. The IL-10 promoter polymorphism at position 592 is correlated with susceptibility to occult HBV infection. Inflammation 2012;35(3):818—21. [6] Arababadi MK, Pourfathollah AA, Jafarzadeh A, Hassanshahi G, Rezvani ME. Association of exon 9 but not intron 8 VDR polymorphisms with occult HBV infection in south-eastern Iranian patients. J Gastroenterol Hepatol 2009;25(1):90—3. [7] Feitelson MA. Parallel epigenetic and genetic changes in the pathogenesis of hepatitis virus-associated hepatocellular carcinoma. Cancer Lett 2006;239(1):10—20.
TRIF expression in chronic HBV infection [8] Arababadi MK, Ahmadabadi BN, Kennedy D. Current information on the immunological status of occult hepatitis B infection. Transfusion 2012;52(8):1819—26. [9] Nguyen-Pham TN, Lim MS, Nguyen TA, Lee YK, Jin CJ, Lee HJ, et al. Type I and II interferons enhance dendritic cell maturation and migration capacity by regulating CD38 and CD74 that have synergistic effects with TLR agonists. Cell Mol Immunol 2011;8(4):341—7. [10] Bae YS, Lee JH, Choi SH, Kim S, Almazan F, Witztum JL, et al. Macrophages generate reactive oxygen species in response to minimally oxidized low-density lipoprotein: toll-like receptor 4- and spleen tyrosine kinase-dependent activation of NADPH oxidase 2. Circ Res 2009;104(2):210—8, 21p following 8. [11] Hirayama T, Tamaki Y, Takakubo Y, Iwazaki K, Sasaki K, Ogino T, et al. Toll-like receptors and their adaptors are regulated in macrophages after phagocytosis of lipopolysaccharide-coated titanium particles. J Orthop Res 2011;29(7):984—92. [12] Imani Fooladi AA, Mousavi SF, Seghatoleslami S, Yazdani S, Nourani MR. Toll-like receptors: role of inflammation and commensal bacteria. Inflamm Allergy Drug Targets 2011;10(3):198—207. [13] Jia N, Xie Q, Lin L, Gui H, Wang H, Jiang S, et al. Common variants of the TLR9 gene influence the clinical course of HBV infection. Mol Med Report 2009;2(2):277—81. [14] Takeda K, Akira S. Toll-like receptors. Curr Protoc Immunol 2007:2. Chapter 14(14): Unit 14 2. [15] Tili E, Michaille JJ, Cimino A, Costinean S, Dumitru CD, Adair B, et al. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock. J Immunol 2007;179(8):5082—9. [16] Eftekhari Y, Arababadi MK, Hakimi H, Zarandi ER. Common HBV genotype in southeastern Iranian patients. Arch Iran Med 2010;13(2):147—9. [17] Cheng XY, Zhang LL. The significance of CD14 and TLR4 expressions in severe hepatitis B induced by endotoxin. Zhonghua Gan Zang Bing Za Zhi 2007;18(6):428—32.
495 [18] Khvalevsky E, Rivkin L, Rachmilewitz J, Galun E, Giladi H. TLR3 signaling in a hepatoma cell line is skewed towards apoptosis. J Cell Biochem 2007;100(5):1301—12. [19] Li N, Li Q, Qian Z, Zhang Y, Chen M, Shi G. Impaired TLR3/IFN-beta signaling in monocyte-derived dendritic cells from patients with acute-on-chronic hepatitis B liver failure: relevance to the severity of liver damage. Biochem Biophys Res Commun 2009;390(3):630—5. [20] An BY, Xie Q, Lin LY, Shen HC, Jia NN, Wang H, et al. Expression of Toll-like receptor 3 on peripheral blood dendritic cells in HBeAg positive patients with chronic hepatitis B. Zhonghua Gan Zang Bing Za Zhi 2007;15(10):729—33. [21] Chen Z, Cheng Y, Xu Y, Liao J, Zhang X, Hu Y, et al. Expression profiles and function of Toll-like receptors 2 and 4 in peripheral blood mononuclear cells of chronic hepatitis B patients. Clin Immunol 2008;128(3):400—8. [22] Guo H, Jiang D, Ma D, Chang J, Dougherty AM, Cuconati A, et al. Activation of pattern recognition receptor-mediated innate immunity inhibits the replication of hepatitis B virus in human hepatocyte-derived cells. J Virol 2009;83(2):847—58. [23] Tjwa ET, van Oord GW, Biesta PJ, Boonstra A, Janssen HL, Woltman AM. Restoration of TLR3-activated myeloid dendritic cell activity leads to improved natural killer cell function in chronic hepatitis B. J Virol 2011;2012:8. [24] Wang K, Liu H, He Y, Chen T, Yang Y, Niu Y, et al. Correlation of TLR1-10 expression in peripheral blood mononuclear cells with chronic hepatitis B and chronic hepatitis B-related liver failure. Hum Immunol 2010;71(10):950—6. [25] Wei XQ, Guo YW, Liu JJ, Wen ZF, Yang SJ, Yao JL. The significance of Toll-like receptor 4 (TLR4) expression in patients with chronic hepatitis B. Clin Invest Med 2008;31(3):E123—30. [26] Yu S, Chen J, Wu M, Chen H, Kato N, Yuan Z. Hepatitis B virus polymerase inhibits RIG-I- and Toll-like receptor 3-mediated beta interferon induction in human hepatocytes through interference with interferon regulatory factor 3 activation and dampening of the interaction between TBK1/IKKepsilon and DDX3. J Gen Virol 2011;91(Pt 8):2080—90.
Available online at www.sciencedirect.com
ORIGINAL ARTICLE
Reduced expression of TRIF in chronic HBV infected Iranian patients Fatemeh Ayoobi a, Gholamhossein Hassanshahi b, Nahid Zainodini c, Hossein Khorramdelazad b, Mohammad Kazemi Arababadi c,∗, Derek Kennedy d a
Physiology and Pharmacology Research center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Molecular Medicine Research center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran c Immunology of Infectious Diseases Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran d School of Biomolecular and Physical Science, Eskitis Institute for Cell and Molecular Therapies, Griffith University Nathan, Queensland, Australia b
Available online 20 February 2013
Summary Background and aims: TRIF is one of the main intracellular adaptor proteins required for TLR3 and 4 signaling. Abnormal gene expression of TRIF may lead to abrogated immune responses against viral infections including hepatitis B infection. The aim of this study was to identify the mRNA levels of TRIF in PBMCs isolated from chronic HBV (CHB) infected patients. Material and methods: mRNA was isolated from 63 CHB patients and 60 healthy controls and transcript levels of TRIF were examined in parallel with beta-actin (as housekeeping gene) using Real-Time PCR techniques. Results: Our results demonstrated that expression of TRIF was significantly decreased in PBMCs isolated from CHB patients when compared to healthy controls. Conclusions: Based on the results reported here, it seems that CHB patients are unable to express appropriate levels of the TRIF gene, which may attenuate TLR3 and 4 signaling subsequent to HBV infection. Our results suggest a possible mechanism, which may explain why hepatitis B infection is stable in CHB patients. © 2012 Elsevier Masson SAS. All rights reserved.
Abbreviations: HBV, Hepatitis B virus; PBMC, peripheral blood mononuclear cell; TRIF, TIR-domain-containing adapter-inducing interferon-; TLR, Toll Like Receptor; PAMP, pathogen associated molecular patterns; MYD88, myeloid differentiation primary response; IRAK1, Interleukin-1 receptor associated kinase-1; TRAF6, TNF receptor associated factor; NF-B, Nuclear factor kappa-light-chain-enhancer of activated B cells; IRF3, Interferon regulatory transcription factor 3. ∗ Corresponding author. Immunology of Infectious Diseases Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. Tel.: +00983915234003 5; +00989139922445; fax: +00983915225209. E-mail address: [email protected] (M.K. Arababadi). 2210-7401/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.clinre.2012.11.005
492
Introduction Chronic HBV (CHB) infected patients harbor a clinical form of hepatitis B in which HBV is not completely removed either from liver hepatocytes or their serum, and this leads to an intermediate symptomatic liver disease [1,2]. Scientists have proposed that CHB could be one of the main reasons for pathogenesis of hepatocarcinoma and cirrhosis [3,4]. The main mechanism(s) responsible for the initiation and progression of pathologies caused by CHB, and other clinical manifestations associated with HBV infection, has yet to be identified. Researchers believe that multifactorial parameters, such as immunological, genetic and epigenetic characteristics of the hosts are responsible for the differences between individuals with persistent HBV infections and those who overcome the disease by clearing the virus [5—8]. TLRs are the main intra/extra-cellular innate immune cell receptors that identify PAMPs of the microbes, including those found associated with viruses [9]. Interactions between TLRs and their ligands lead to the induction of intra-cytoplasmic and nuclear signaling molecules and consequently; activate and stimulate immune cell (e.g. dendritic cells) migration [9], NADPH oxidase activation [10], phagocytosis [11] and inflammatory cytokine expression [12]. TLRs induce inflammatory cytokine expression through the recognition of pathogen PAMPs via the MYD88 and TRIF dependent pathways [13]. Assembly of TRIF (as an adaptor molecule) leads to the activation of several intracellular signaling pathways such as IRAK1, TRAF6 and NF-(B [14]. NF-(B, as a transcription factor which recognizes consensus elements present in the regulatory region of several genes, including inflammatory cytokines [14]. Therefore, reduced expression of TRIF may lead to inappropriate signal activation, and consequently, defective immune responses against viral infection. Due to the fact that CHB infected patients are unable to completely eradicate intercellular HBV from the hepatocytes, it seems that these patients may be functionally defective in some aspects of their immune system. Because of the important role that TRIF plays in the activation of downstream signaling molecules such as, TLR3 and 4, and the overall induction of immune responses against viruses, it may be speculated that the molecule is crucially important in the pathogenesis of CHB. Therefore, the main aim of this study was to investigate mRNA expression levels of TRIF in the PBMCs of CHB patients.
Material and methods Subjects Peripheral blood samples were collected from 63 healthy controls and 60 CHB infected patients within Rafsanjan (located in the South-Eastern region of Iran) in 5.5 ml tubes with and without anti-coagulant. CHB infected patients with coinfection of either HCV or HIV were excluded from the study. The CHB diagnosis was performed based on the ‘‘Guide of Prevention and Treatment in Viral Hepatitis’’ [15] by an expert internal medicine specialist. Patients were entered to the study based on assessment of their previous clinical records. Controls were also selected with the same age, sex and socio-economical status. The sera, collected
F. Ayoobi et al. from blood without anti-coagulant, were stored at —20 ◦ C for a maximum of 2 months or at —70 ◦ C when longer storage periods were required for analysis. The anti-coagulant treated samples were immediately subjected to RNA extraction (to obtain either more quantity or qualitative RNA samples) following entrance to the laboratory. This study was approved by the ethical committee of the Rafsanjan University of Medical Sciences and written informed consent was obtained from all of participants prior to sample collection.
Detection of serological HBV markers The samples were screened for HBsAg and HBeAg using ELISA (Behring, Germany) techniques according to the manufacturer’s guidelines.
HBV-DNA extraction and Real-Time PCR conditions The viral DNA was purified from 200 L of plasma from CHB patients using a commercial kit (Cinnaclon, Iran) according to manufactures guidelines. The HBV-DNA amplification was also undertaken using a commercial kit (Primer Design Company, UK) following the manufacturer’s instructions.
RNA extraction, reverse transcription and quantitative Real-Time PCR Total RNA was extracted from PBMCs using an RNX extraction kit from the Cinnaclon Company (Iran). The extracted RNA quality was determined by electrophoresis on the ethidium bromide pretreated agarose gel and by measuring absorption at 260/280 nm by spectrophotometery. Subsequent cDNA synthesize was performed using a cDNA synthesis kit (Parstous, Iran) with both oligo(dT) and random hexamer primers (Aryatous, Iran). The reverse transcription step was performed using the following program: 70 ◦ C for 10 min (without reverse transcription enzymes), —20 ◦ C for 1 min (cooling), reverse transcription enzyme was added and the sample incubated at 42 ◦ C for 60 min. Finally, the sample was treated at 95 ◦ C for 10 min to inactivate the reverse transcription enzyme. Quantitative Real-Time PCR was performed by addition of a SYBR green master mix (Parstous, Iran), 200 ng of the generated cDNA and 2 pg/l of appropriate primers (Table S1). The following cycling program was set on a BIO-RAD CFX96 system (Bio-Rad Company, USA): one cycle of 95 ◦ C for 15 min, 40 cycles of 95 ◦ C for 30 s, 60 ◦ C for 30 s and 72 ◦ C for 30 s. Real-Time PCR was carried out in triplicate and the -actin was assessed as a housekeeping gene for the normalization of amplification signals of the target gene. The relative amounts of PCR product were determined using the 2—Ct formula. The dissociation stages, melting curves and quantitative analyses of the data were performed using CFX manager software version 1.1.308.111 (Bio-Rad, USA). All PCR products were screened using electrophoreses on a 1% gel containing 0.5 mg/ml ethidium bromide to check the size of PCR product.
TRIF expression in chronic HBV infection
493
HBV genotyping
5.0
Liver enzymes detection
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
pa ti e
co nt ro l
nt
Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct and total bilirubin were evaluated in the patients and controls using commercial kits (Man Ltd, Tehran-Iran) according to the manufactures guidelines.
Expression level of TRIF
4.5
HBV genotypes were determined using a gap-PCR technique according to our previous study [16].
C
H
H ea lth y
B
Data analysis and statistical methods Parametric statistical analyses were performed using the ttest in the SPSS software version 18. When a P value was less than 0.05 it was considered significant.
Results
Figure 1 mRNA levels of TRIF in PBMCs of CHB patients and healthy controls. Results are shown as mean ± standard error. The comparison between CHB and healthy controls is statistically different (P = 0.048).
Our results demonstrated that all the patients were HBsAg positive. The HBsAg titration was higher in the patients that suffered from medium (between 20,000 to 400,000) and high (greater than 1000,000) HBV copy numbers/ml. The results also demonstrated that two patients were positive for HBeAg (3.17%) and the patients carried greater than 1000,000 HBV copy numbers/ml.
Expression levels of TRIF
2.5
Detection of HBsAg and HBeAg
2.0
1.5
1.0
0.5
Our results showed that all the patients had detectable HBV-DNA. Amongst the patients, 40, 12 and 11 carried less than 20,000 (low), between 20,000 to 400,000 (medium) and greater than 1000,000 (high) HBV copy numbers/ml, respectively. Two of the patients with greater than 1000,000 HBV copy numbers/ml carried HBeAg.
H ig h
ed M
Lo w
iu
m
0.0
Quantification of HBV-DNA copy numbers
Figure 2 mRNA levels of TRIF in PBMCs of CHB patients with different HBV-DNA copy numbers. The figure illustrates that expression levels of TRIF were not affected by different HBV-DNA copy number (Low: < 20,000, medium: between 20,000—400,000 and high: > 1,000,000 HBV-DNA copy numbers/ml). Results are shown as mean ± standard error.
Expression of TRIF
Detection of liver enzymes
Our results showed that expression of TRIF in the PBMCs of CHB patients was reduced by 3.75 fold when compared with healthy controls. Statistical analysis of the data revealed that the difference was significant (P = 0.048) (Fig. 1). Our results also revealed that the expression levels of TRIF were not different among CHB patients with less than 20,000, between 20,000 to 400,000 and greater than 1000,000 HBV copy numbers/ml (P = 0.475) (Fig. 2).
Our results identified that all the patients carrying; low, medium and high HBV-DNA copy numbers/ml had normal serum levels of ALT, AST, ALP as well as direct and total bilirubin.
HBV genotyping The results demonstrated that all HBV positive patients carried the HBV D genotype.
Discussion Initiation of immune responses against viral infections starts with innate immunity via the production of cytokines and chemokines. One of the mechanisms that leads to the expression of these molecules is the activation of TLRs and consequently, the activation of intracellular signaling molecules via adaptor proteins including TRIF [17,18]. Our results demonstrated that the mRNA expression levels of
494 TRIF were significantly decreased in the CHB patients in comparison to healthy controls. Therefore, based on our results, it can presumably be concluded that CHB patients are unable to express adequate values of TRIF, however, this would need to be validated by measuring TRIF protein levels. Patients with reduced TRIF expression are probably unable to activate the downstream signaling pathways of TLR3 and also 4 in response to HBV infection. Subsequently, these patients would be unable to completely eradicate the infection. Additionally, our results revealed that the mRNA expression levels of TRIF were not affected by HBV-DNA copy numbers or possibly HBV replication, hence, it may be concluded that HBV factors did not appear to affect the TRIF expression leading us to conclude that host genetic or epigenetic factors may be the main reasons for this defectiveness. Our results also demonstrated that all of the patients were infected with the HBV D genotype but had normal serum levels of liver markers. Therefore, it can be concluded that HBV-DNA copy numbers did not affect or regulate the expression levels of TRIF and was unable to influence serum levels of ALT, AST, ALP, as well as direct and total bilirubin. Due to the fact that, during hepatitis B, hepatocytes function disorders are results of immune responses, hence, downregulation of TRIF, as a marker of immune response, can describe normal serum levels of liver markers. All of the patients carried the HBV D genotype, hence, it also can be concluded that if infection was able to influence expression levels of TRIF this could be correlated to the HBV D genotype, however, we cannot make any conclusions in reference to other genotypes. In addition, we cannot exclude the possibility that other factors, such as the translational control of TRIF or the stability of TRIF may influence signal transduction pathways. To the best of our knowledge, this is the first study to report down-regulation of TRIF in the CHB patients. However, Li et al., showed that the expression levels of TLR3 and also its related cytokine (IFN-beta) were decreased in the CHB and acute HBV infected patients [19]. An et al., have stimulated dendritic cells (DC) of CHB patients with poly I:C and then evaluated the expression levels of TLR3 [20]. They reported that DCs of CHB patients express lower levels of TLR3 than healthy controls [20]. Interestingly, Chen et al., revealed that TLR4 expression is decreased in CHB patients and that cytokine production was impaired in PBMCs of CHB patients after challenge with TLR4 ligands [21]. Therefore, it seems that TLR4 signaling is also defected in their evaluated patients. Interestingly, Gue et al., demonstrated that up-regulation of TRIF in infected human hepatocyte-derived cells leads to the decrease of both HBV-mRNA and DNA levels [22]. Furthermore, Tjwa et al., showed that activation of myeloid DC via TLR3 leads to improvement of natural killer cell function in CHB patients [23]. Another study demonstrated that expression of TLR3 was up-regulated in the active phases of CHB infection [24]. It has also been shown that in contrast to PBMCs, TLR4 expression in hepatocytes was increased in CHB patients in comparison to healthy controls [25]. Thus, it seems that the expression and roles of TLRs in the hepatocytes are different from immune cells. Additionally, studies have demonstrated that HBV factors can alter expression and function of TLR3 signaling molecules [26]. Based on our results it seems that the replication rate of HBV did not affect TRIF expression but, HBV factors may affect other signaling molecules including IRF3
F. Ayoobi et al. which has been reported previously [26]. The combined results from these groups highlight the complex nature of HBV infection and reinforce the notion that the pathology of infection is most likely the result of multigenic factors in the host. Our data suggests that TRIF expression can now be added to the growing list of causes that allow persistent HBV infection. Finally, based on our results and the other mentioned studies, it seems that down-regulation of TRIF and TLRs including TLR3 and 4 in CHB patients may attenuate immune responses due to a deficit in pro-inflammatory cytokines production. Our findings may also open an avenue for the investigators of this field to focus on designing therapeutic reagents to overcome this defect in CHB patients. Furthermore, results of the current study suggest a model for future therapy in which up-regulation of these molecules is of paramount importance.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgments Authors of this article would like to take this opportunity to thank all of the CHB patients and healthy controls, which warmly attended and co-operated in this research program. This project was supported by a grant from the Rafsanjan University of Medical Sciences.
Appendix A. Supplementary data Supplementary data (Table S1) associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.clinre.2012.11.005.
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