1. Introduction. Keywords Hepatitis C virus, Toll like receptor, Regulatory T cell

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1 American Journal of Biochemistry 2015, 5(3): DOI: /j.ajb Study of Expression of Toll-like Receptor (TLR)2 on Monocytes and Frequency of CD4 + CD25 + CD127 low/- (Treg) in Egyptian Patients with Chronic Hepatitis C Entsar R. Mokhtar 1,*, Amany M. El Saeed 1, Eman E. Ahmad 2, Ghada F. El-Mohasseb 3 1 Department of Clinical Pathology, Faculty of Medicine (for girls), Al-Azhar University, Cairo, Egypt 2 Department of Tropical, Faculty of Medicine (for girls), Al-Azhar University, Cairo, Egypt 3 Department of Internal Medicine, Faculty of Medicine (for girls), Al-Azhar University, Cairo, Egypt Abstract Background: Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. TLR2 and regulatory T cells (Tregs) play a pivotal roles in the persistence of HCV infection. Objectives: To investigate the expression of TLR2 and frequency of Tregs and their roles in patients with chronic hepatitis C (CHC). Methods: The present study included 30 patients with CHC, 13 male (43.3%) and 17 female (56.7%); their age ranged from with mean (36±10.2). They were compared with 30 age and sex matched apparently healthy individuals as control group. TLR2 expression on peripheral blood monocytes and the number of Tregs were examined by flow cytometric analysis. Results: TLR2 expression in patient group was significantly higher than control group (P<0.001). Tregs frequency showed highly significant increase in patient group as compared with control group (P<0.001). There was a significant positive correlation between frequency of Tregs and viral load as detected by RT-PCR (r=0.667, P<0.001) and between TLR2 expression and ALT (r=0.543, P=0.002) in patients group. No significant correlation was found between TLR2 and Tregs. Conclusions: These results suggest that TLR2 and Tregs might contribute to the immune response failure during HCV infection. Keywords Hepatitis C virus, Toll like receptor, Regulatory T cell 1. Introduction Chronic hepatitis C virus (HCV) infection is a major cause of chronic liver disease leading to progressive hepatic fibrosis with long term progression to cirrhosis and hepatocellular carcinoma (HCC) [10]. After HCV infection, interactions between the innate and adaptive immune responses and immune regulation may play pivotal roles in the perpetuation or clearance of HCV infection [18]. Although it is firmly established that an impaired specific effector T cell response is the main cause of the development of chronic HCV infection, little is known about other contributing factors [5]. Toll like receptor (TLRs) and CD4 + CD25 + regulatory T cell (Tregs) have been given much attention in their roles in chronic HCV infection [8, 11]. Toll like receptors (TLRs) have emerged as some of the major components of the immune system that play a critical role in innate immune responses [13]. TLRs recognize conserved molecular patterns expressed * Corresponding author: sararaafat_1973@yahoo.com (Entsar R. Mokhtar) Published online at Copyright 2015 Scientific & Academic Publishing. All Rights Reserved by microbial pathogens and trigger inflammatory responses including activation of transcription factors and production of cytokines and chemokines which control the activation of adaptive immune responses [10]. Cell surface expressed TLRs such as TLR2 play an important role in human host defences against some viruses [6]. HCV proteins have been shown to up regulate TLR2 expression on peripheral blood mononuclear cells, thereby modulating the cell's pro-inflammatory response. Moreover, many studies have provided evidence that HCV core protein can activate TLR2 [6, 10]. Tregs have been identified as a specialized subset of T cells that can suppress auto reactive immune response to maintain immunological tolerance and inhibit autoimmunity [16]. There are two main subsets of Tregs, thymically selected natural Tregs (n Tregs), and inducible Tregs that develops during immune response in the periphery [3]. Although these Tregs are CD4 + cells with high expression of CD25 +, the most reliable marker is the transcription factor fork head box P3 (FOXP3) [16]. FOXP3 is an intracellular molecule, detection of which requires fixation and permeabilization of cells and the fixed cells cannot be used in studies of Treg function and its

2 American Journal of Biochemistry 2015, 5(3): staining is hardly usable on a daily clinical routine basis in large series of samples (several steps of incubation, washing and long incubation times) [14]. The expression of CD127 inversely correlates with FOXP3 expression and suppressive activity of Tregs [15]. CD4 + CD25 + CD127 low/- isolated Tregs showed the best reached Treg population regarding purity, function, stability and in vitro expansion capacity, promising isolation of pure Treg populations with high suppressive functionality [17]. Many studies have investigated the relationship between Tregs and CHC infection and showed that Tregs are increased in patients with CHC which may contribute to the sustained suppression of HCV specific T cell responses in a cell- cell contact-dependent manner and viral persistence in HCV infected individuals [19]. In the light of these data, the aim of the present study was to investigate the expression of TLR2 as a component of innate immunity and frequencies of CD4 + CD25 + CD127 low/- Tregs as a regulator of adaptive immunity in CHC patients as well as their roles in HCV infection. 2. Patients and Methods This study included 30 patients diagnosed as having CHC, 13 males (43.3%) and 17(56.7%) females, selected from out patients clinics of tropical and internal medicine departments of Al-Zahraa University Hospital during the period from April 2014 to Dec Their age ranged from (23-46) with mean (36±10.2) years. They were diagnosed as HCV patients by the presence of HCV antibodies which detected by HCV ELISA kits and confirmed by viral load detection using real time PCR. Thirty healthy volunteers were chosen as controls. They were 14 males and 16 females. Their age ranged from (20-45) years. None of them had a history of systemic disease. Exclusion criteria included patients who have co-infection with human immunodeficiency virus or hepatitis B virus or diagnosed with autoimmune, alcoholic or metabolic hepatitis or current immune suppression or antiviral therapy. An informed written consent was obtained from the subjects before their contribution in this study. Six ml of venous blood were withdrawn from each subject and divided into three portions: 1.8ml was taken in Na citrate tube for PT assay, 2.5 ml of whole blood was collected in evacuated tube containing EDTA as anticoagulant which was sent immediately for the lab for flow cytometric analysis. The remaining part was centrifuged and serum was obtained for ALT, AST and albumin quantitation. They were quantified using chemistry analyser Cobas C 311(Roche Diagnostics) Flow Cytometric Analysis of Expression of TLR-2 on CD14 + Monoctes Flow cytometric analysis was performed in fresh samples within 1 hour of blood collection, 100 µl of EDTA anti coagulated blood was stained with combination of 10 µl conjugated phycoerythrin (PE) labeled monoclonal antibodies for human TLR-2 and 10 µl conjugated fluoresceneisothiocynate (FITC) labelled monoclonal antibodies for CD14 +. The mixture was incubated in dark room for 30 minutes then add 1 ml of fix and lyses mixture, vortex the tubes immediately for one second and incubation of the tube was done again for 10 minutes in dark at room temperature. Centrifugation of the tubes at low speed was done for 5 minutes followed by aspiration of supernatant and resuspension of pellet residual fluid. Two ml of phosphate buffer saline (PBS) were added to each tube, the suspension was centrifuged at low speed. The supernatant was discarded, and then the residual suspension was passed through the flow cytometer. Monoclonal antibodies for TLR-2 and CD14 + were supplied by R&M systems Inc (Minneopolis, Minnesota, USA). Data acquisition and analysis were performed on cell quest program of the coulter EPICS XL flow cytometry. Gating on monocytes, events were acquired and statistical analysis was done by cell quest software. Results were expressed as percentage (%) and mean florescence intensity (MFI) Flow Cytometric Analysis of Tregs It was done on Epics XL flow cytometry using system 11 version 3 software with a standard 3-color filter configuration using R&D system kits. (carboxyluorescien (CFS) conjugated monoclonal antihuman CD4 with lot number LKNO4, phycoerythrin (PE) conjugated monoclonal antihuman CD25 with lot number LLBo4, peridininchiorophyll protein complex (percp) conjugated antihuman CD127 with lot number ABUWO Statistical Analysis Data were collected, revised, coded and entered to the statistical package for social science SPSS version 20. The qualitative data were presented as number and percentages. The comparison between two groups with qualitative data were done by using Chi-square test, while the quantitative data were presented as means and standard deviations. The comparison between two independent groups with quantitative data were done by using Independent t-test. Spearman correlation coefficients were used to assess the relation between two quantitative parameters in the same group. The confidence interval was set of 95% and the margin of error accepted was set to 5%. So, the p-value was considered non-significant at the level of >0.05; significant at the level of <0.05 and highly significant at the level of < Results The results of this study showed highly significant over expression of TLR2 in monocytes of patients group as compared to control (P<0.001), also there was highly significant increase in the frequency of Tregs in patients

3 68 Entsar R. Mokhtar et al.: Study of Expression of Toll-like Receptor (TLR)2 on Monocytes and Frequency of CD4 + CD25 + CD127 low/- (Treg) in Egyptian Patients with Chronic Hepatitis C group as compared to control (P<0.001). There was a significant positive correlation between TLR2 and ALT (r=0.543, P=0.002) and a significant positive correlation between Tregs and HCV-viral load (r=0.667, P<0.001) in patients group. Table 1. Comparison between patients and control group regarding demographic data Sex Age Patient group Chi square test No. % No. % X 2 /t* P-value Female Male Mean ±SD ± ±10.2 Range Table 2. Comparison between patients and control group regarding laboratory data Patients group Independent t-test Mean ± SD Range Mean ± SD Range t P-value ALT (u/l) ± ± <0.001 AST (u/l) ± ± <0.001 ALB (gm/dl) 3.88 ± ± PT (sec) ± ± <0.001 PC (%) ± ± <0.001 Table 3. Results of HCV-viral load in patients group Range Mean ± SD (HCV) viral load (IU/ML) ± Table 4. Comparison between patients and control group regarding TLR2 and Tregs Patients group Independent t-test Mean ± SD Range Mean ± SD Range t P-value TLR2 MFI 2.73 ± ± <0.001 Tregs % 3.93 ± ± < Patients group 0 MFI2 TLR % Tregs Figure 1. Comparison between patients and control group regarding TLR2 and Tregs

4 American Journal of Biochemistry 2015, 5(3): Figure 2. Example of flow cytometric analysis of Tregs; in this patient with CHC (CD4 + CD25 + CD127 low/- represent 7.4% of CD4 + cells) Figure 3. Example of flowcytometric analysis of Tregs in control group (CD4 + CD25 + CD127 low/- represent 5% of CD4 + cells)

5 70 Entsar R. Mokhtar et al.: Study of Expression of Toll-like Receptor (TLR)2 on Monocytes and Frequency of CD4 + CD25 + CD127 low/- (Treg) in Egyptian Patients with Chronic Hepatitis C Figure 4. Example of flow cytometric analysis of TLR2 in patients with CHC; MFI of TLR2 expression represent 10.7 Figure 5. Example of flow cytometric analysis of TLR2 in control; MFI of TLR2 expression represent 3.8

6 American Journal of Biochemistry 2015, 5(3): Table 5. Correlation between TLR2, Tregs and other studied parameters TLR2 Tregs r P-value r P-value TLR T-regs ALT (u/l) AST (u/l) (HCV) viral load (IU/ML) <0.001 ALB (gm/dl) PT (sec) PC (%) Figure 6. Correlation between TLR2 and ALT Figure 7. Correlation between Tregs and HCV-viral load 4. Discussion Toll- Like receptors are a family of non clonal, germline encoded pattern recognition receptors (PRRs) that give the innate immune system considerable specificity for a large range of pathogen classes [7]. It has been documented that intracellular TLRs are involved in recognition of viral nucleotides and induction of antiviral interferon responses. Cell surface expressed TLRs such as TLR2, play an important role in human host defences against viruses [6]. HCV core and non-structural protein 3 (NS3) stimulate TLR2 when associated with TLR1 and TLR6 in peripheral blood mononuclear cells particularly monocytes and macrophages.tlr2 stimulation leads to production of TNF alpha, IL6 and IL 8,via NFκB, c-jun-n-terminal kinase (JNK)/AP-1,P38 and extracellular signal regulator proteins (ERK) pathways with ERK being the dominant pathway for TNF alpha secretion thereby modulating cell's pro-inflammatory response which contribute to inflammation in HCV infection [7]. In the present study, we analyzed the expression of TLR2 on monocytes of patient with chronic HCV infection and we found an increase in its expression as compared with control subjects; this is matched with Wang et al. [18] who found an increase in the expression of TLR2 and TLR4 during HCV infection and concluded that, this elevation could increase the production of down-stream cytokines that may alleviate the antiviral activity of PBMCs, causing excessive inflammation and tissue injury. Our data is matched also with Ronitet al. [13] who reported an increase in TLR2 expression on monocytes in HCV-infected patients when compared to control and concluded that TLR2 may contribute to disease progression in chronic HCV infection. Coenen et al. [2] demonstrated a direct effect of HCV core protein on activation of hepatic stellate cells (HSCs) toward a pro-fibrogenic state, which is mediated via TLR2 pathway. In HCV associated liver disease, the immune system is unable to clear the viral infection and previous studies have raised the possibility of an involvement of Tregs [1]. In the present study we analysed the frequency of CD4 + CD25 + CD127 low/- Tregs and we found an increase in its frequency in HCV patients as compared with control subjects. This is in accordance with Wang et al. and Hashempoor et al. [18, 5], they reported a significant increase in Tregs in chronic hepatitis C patients. These data support the contribution of Tregs to the impaired virus specific T cells responses in chronic HCV infection. Zhai et al. [19] reported also an increase in Tregs in CHC and concluded in their study that HCV core protein and TLR2 ligands trigger CD4 + CD25 + Treg activation and expansion to inhibit host immune responses which may play a critical role in viral persistence in HCV-infected patients. Our data is matched also with Tseng et al. [16] who concluded that CHC patients irrespective of liver function had higher frequencies of Tregs than healthy subjects. All the previous studies suggested that Tregs contribute to persistent HCV by suppressing HCV specific T cell responses. Losikoff et al. [9] suggested that elevated Tregs in HCV infected patients can suppress the activity of other immune cell types including Teff cells directly by contact

7 72 Entsar R. Mokhtar et al.: Study of Expression of Toll-like Receptor (TLR)2 on Monocytes and Frequency of CD4 + CD25 + CD127 low/- (Treg) in Egyptian Patients with Chronic Hepatitis C dependent and independent mechanisms, or indirectly by inhibiting dendritic cell (DC) maturation and immuno stimulatory capacity and indicate that, immature DCS are critical factor in the pathogenesis of chronic hepatitis C by failing to induce adequately naïve Teff cell function and conversely, promoting the development and activity of Treg cells. Langhans et al. [8] provide another role of Treg in HCV infected patients based on the fact that NK cells regulate liver fibrosis by killing activated HSCs and are controlled themselves by immune cells and/or soluble factors. They concluded that Tregs interfere with NK cell regulation of fibrogenesis via both direct cell-contact dependent inhibition of NK cells and release of soluble factors, down-regulating activating NK cell receptor ligands on HSCs. In this study, we found a significant positive correlation between Tregs and serum HCV RNA load (r=0.667, P<0.001) in patients group. This is matched with Wang et al. and Hao et al. [18, 4] and this results suggest that an increased frequency of Tregs may be associated with a negative immune response, leading to poor viral clearance in HCV-infected patients. A significant positive correlation was found between TLR2 expression and ALT (r=0.543, P=0.002) in patients group and this is matched with Riordan et al. [12] who reported the same result and concluded that increased monocytes expression of TLR2 correlates with the hepatic necro-inflammatory activity inchc. 5. Conclusions In accordance with other studies, we concluded that CHC patients had higher expression of TLR2 and higher frequency of Tregs than healthy subjects which might contribute to the immune response failure during HCV infection and suggest that TLR2 and Tregs are related to chronic HCV infection. 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