Razi Journal of Medical Sciences

fa مقایسه‌ای سطح سرمی اینترلوکین-35 در عفونت همزمان HIV/HCV، عفونت HIV و کنترل‌های سالم Comparative Analysis of Interleukin-35 Levels in HIV/HCV Coinfection, HIV Monoinfection, and Healthy Controls ایمنی‌شناسی Immunology پژوهشي Research چکیده زمینه و هدف: اینترلوکین-35 (IL-35) یک سایتوکاین تازه کشف شده از خانواده IL-12 است. این سایتوکاین یک سایتوکاین مهاری بوده که  توسط سلول‌های T و B تنظیمی ترشح می‌شود. در مطالعات قبلی، نقش این سایتوکاین در پاتوژنز چندین بیماری ویروسی مورد بررسی قرار گرفته است. با این حال، اطلاعات محدودی درباره رابطه بین IL-35 و عفونت همزمان HIV/HCV وجود دارد. هدف این مطالعه مقایسه غلظت IL-35 در بیماران مبتلا به عفونت همزمان HIV/HCV، عفونت HIV و کنترل‌های سالم بود. روش‌ها و مواد: در این مطالعه، 50 بیمار ایرانی که تحت درمان ضد ویروسی (ART) بودند، انتخاب شدند. از این تعداد، 17 بیمار تنها مبتلا به عفونت HIV و 33 بیمار مبتلا به عفونت همزمان HIV/HCV بودند. این بیماران با 30  فرد سالم و منفی از نظر HIV و HCV مقایسه شدند. یافته‌ها: نتایج ما نشان داد که سطح سرمی IL-35 پلاسما در بیماران مبتلا به عفونت همزمان HIV/HCV به طور معناداری نسبت به کنترل‌های سالم (P-value=0.008) و بیماران مبتلا به عفونتHIV   (P-value=0.01)افزایش یافته است. علاوه بر این، سطح سرمی IL-35 در بیماران که تنها مبتلا به عفونت  HIV بودند، نسبت به کنترل‌های سالم به طور معناداری بالاتر بود (P-value=0.017). نتیجه‌گیری: اختلال در سیستم ایمنی یک عامل کلیدی در پاتوژنز HCV در افراد مبتلا به HIV است و به نظر می‌رسد افزایش سطح سرمی IL-35 به عنوان یک عامل مهم در این فرآیند نقش دارد. با این حال، مطالعات بیشتری برای روشن‌تر شدن این مسئله مورد نیاز است.   Comparative Analysis of Interleukin-35 Levels in HIV/HCV Coinfection, HIV Monoinfection, and Healthy Controls Mohammad Jafari1, Mostafa Hajimola Hoseini2, Majid Khoshmirsafa3, Maryam Keykhaee4, Roghayeh Babaei5, Hamid Nickho3 Alireza Najafi3 1. Cellular and Molecular Research Center, Gerash University of Medical Sciences, Gerash, Iran 2. Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3. Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 4. Department of Pharmaceutical Biomaterials and Medical Biomaterial Research Center (MBRC), Faculty of Pharmacy, Tehran University of Medical Science, Tehran, Iran. 5. Department of Nanotechnology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Introduction: Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections are major public health concerns. HIV/HCV co-infection is common due to similar transmission routes, with HIV-infected individuals being about six times more likely to have HCV infection than HIV-negative individuals[<a href="#_ENREF_1" style="color:#0563c1; text-decoration:underline" title="Modi, 2007 #4">1</a>] [<a href="#_ENREF_2" style="color:#0563c1; text-decoration:underline" title="Platt, 2016 #6">2</a>]. It is estimated that about 20-30% of HIV-positive patients are also infected with HCV[<a href="#_ENREF_3" style="color:#0563c1; text-decoration:underline" title="Sherman, 2002 #5">3</a>]. Studies have shown that HIV/HCV co-infection is associated with faster progression of liver fibrosis [<a href="#_ENREF_4" style="color:#0563c1; text-decoration:underline" title="Benhamou, 1999 #6">4</a>] and a significantly increased risk of cirrhosis [<a href="#_ENREF_5" style="color:#0563c1; text-decoration:underline" title="Graham, 2001 #7">5</a>]. While various factors contribute to liver fibrosis development in HCV-infected patients, the situation is more complex in HCV/HIV co-infection. Immune status, combined antiretroviral therapy, and endocrine and metabolic factors associated with HIV infection promote liver disease progression[<a href="#_ENREF_6" style="color:#0563c1; text-decoration:underline" title="Mandorfer, 2016 #8">6</a>]. Interleukin-35 (IL-35) is a recently discovered cytokine belonging to the IL-12 family. It plays a crucial role in regulating immune responses and is secreted by regulatory T and B cells [<a href="#_ENREF_7" style="color:#0563c1; text-decoration:underline" title="Xiang, 2015 #23">7</a>]. IL-35 is expressed in response to inflammatory stimuli and inhibits the proliferation and function of immature effector T-cells. Furthermore, IL-35 converts conventional CD4+ T cells into strongly inhibitory IL-35-induced regulatory T cells, which further induce immune tolerance in hosts and promote viral infections[<a href="#_ENREF_8" style="color:#0563c1; text-decoration:underline" title="Collison, 2007 #25">8</a>, <a href="#_ENREF_9" style="color:#0563c1; text-decoration:underline" title="Shen, 2014 #26">9</a>]. Previous studies have investigated the role of IL-35 in several viral diseases. Influenza virus infections increase serum levels of IL-35, which suppresses the immune system and makes the patient susceptible to bacterial infections such as pneumococcal infections[<a href="#_ENREF_10" style="color:#0563c1; text-decoration:underline" title="Chen, 2016 #27">10</a>].The effect of IL-35 on chronic HBV infection has also been evaluated in several studies. The inhibitory effect of this interleukin on secretion of pro-inflammatory cytokines involved in the HBV pathogenesis, such as IFN-γ, IL-1β, IL-6, and IL-8 produced by PBMCs, is shown[<a href="#_ENREF_11" style="color:#0563c1; text-decoration:underline" title="Shao, 2017 #29">11</a>]. Additionally, IL-35 can inhibit HBV-specific CTL responses, affecting HBV clearance [<a href="#_ENREF_12" style="color:#0563c1; text-decoration:underline" title="Li, 2015 #30">12</a>]. The previous study indicates that IL-35 suppresses the immune system in chronic HCV infection and inhibits inflammatory responses[<a href="#_ENREF_13" style="color:#0563c1; text-decoration:underline" title="Liu, 2017 #28">13</a>]. Increased serum concentration of this cytokine, along with an increased number of regulatory T cells, may indicate the role of IL-35 in viral persistence and progression of HCV infection [<a href="#_ENREF_14" style="color:#0563c1; text-decoration:underline" title="Abd El-Ghani, 2021 #31">14</a>]. There is a lack of studies investigating the levels of IL-35 in patients with HIV and HIV/HCV co-infection. The aim of the present study is to compare the levels of IL-35 among individuals with HIV/HCV co-infection, HIV infection, and healthy controls. Materials and Methods Patient Selection The study enrolled 50 HIV-positive individuals referred to the Iran Blood Transfusion Center (IBTO). Additionally, 30 healthy subjects matched for age and gender were recruited as the control group. The study was approved by the Clinical Research Ethics Committee of Gerash University of Medical Sciences, Iran (IR.GERUMS.REC.1403.020), and written informed consent was obtained from all participants. Enzyme Linked Immunosorbent Assay (ELISA) Serum levels of IL-35 were measured using commercial ELISA kits with a sensitivity of 9.38 pg/ml and a detection range of 15.6-1000 pg/ml. Serum samples were also tested for anti-HCV antibodies using commercial ELISA kits. Flow cytometry The frequency of CD4+T cells was determined using a previously established method[<a href="#_ENREF_15" style="color:#0563c1; text-decoration:underline" title="Najafi, 2013 #32">15</a>]. CD4+and CD8+T cells in whole blood were analyzed using the CD3/CD4+and CD3/CD8+double staining method Statistical analysis Data were analyzed using GraphPad Prism version 8.1. The significance of the variables was assessed using the Mann-Whitney U test and Kruskal-Wallis test. Additionally, linear regression and Pearson's correlation coefficient were performed to evaluate the relationships between the measured interleukins and CD4+percentage. Results Out of the 50 patients examined, 33 were found to have both HIV and HCV infections, while 17 were found to have only HIV infection. The average age of the healthy control group, HIV-mono-infected group, and HIV/HCV-coinfected patients were 35, 34, and 40 years, respectively. The percentage of males was 47% in the HIV-mono-infected group, 81.8% in the HIV/HCV-coinfected group, and 50% in the healthy control group. Serum Levels of IL-35 in HIV-Positive Individuals and Controls The average concentration of IL-35 among HIV/HCV-coinfected patients, HIV-mono-infected individuals, and healthy controls were 46.9, 23, and 16.5 pg/ml, respectively. Statistical analysis revealed a significant increase in IL-35 levels in both HIV/HCV-coinfected patients (p-value=0.008) and HIV-mono-infected cases (p-value=0.017) compared to healthy controls. Additionally, IL-35 levels were significantly higher in HIV/HCV-coinfected patients compared to HIV-mono-infected individuals (p-value=0.01). Comparison of IL-35 concentration with frequency of CD4+ The serum level of IL-35 was lower in patients with CD4+/CD8+>1 than in patients with CD4+/CD8+<1, however, this difference was not statistically significant. No correlation was observed between IL-35 concentration and T CD4+frequency. Discussion: Previous studies have investigated HIV co-infections in the Iranian population[<a href="#_ENREF_16" style="color:#0563c1; text-decoration:underline" title="Dehghani-Dehej, 2020 #37">16</a>, <a href="#_ENREF_17" style="color:#0563c1; text-decoration:underline" title="Asiyabi, 2016 #38">17</a>]. However, in this study, we aim to investigate the serum levels of IL-35 in HIV-positive patients, both with and without HCV co-infection, as well as in healthy controls. Our results showed a significant elevation in the average concentration of IL-35 in both HIV/HCV-coinfected patients and HIV-mono-infected cases compared to healthy controls. Moreover, we found that IL-35 levels were significantly higher in HIV/HCV-coinfected patients than in the HIV-mono-infected group. Previous research has explored the role of IL-35 in several viral infections, including influenza, hepatitis B, and hepatitis C. For instance, Chan et al. demonstrated that IL-35 is upregulated in response to influenza virus infection[<a href="#_ENREF_10" style="color:#0563c1; text-decoration:underline" title="Chen, 2016 #27">10</a>], while Liu et al. found that IL-35 levels increase during HCV infections and are positively associated with the RNA load of the virus [<a href="#_ENREF_13" style="color:#0563c1; text-decoration:underline" title="Liu, 2017 #28">13</a>]. Similarly, Shao et al. reported increased levels of IL-35 in patients with chronic hepatitis B and asymptomatic HBV carriers[<a href="#_ENREF_11" style="color:#0563c1; text-decoration:underline" title="Shao, 2017 #29">11</a>].  The increase in IL-35 levels in HIV/HCV-coinfected patients compared to HIV-mono-infected cases suggests that HCV co-infection may have an additive effect on the elevation of IL-35 in HIV-positive patients. The mechanisms underlying the relationship between HIV and HCV co-infection and the elevation of IL-35 levels are not fully understood. However, it has been suggested that chronic viral infections may induce the expansion of regulatory T cells (Tregs) and the production of anti-inflammatory cytokines such as IL-35, which may contribute to the immune suppression observed in these patients[<a href="#_ENREF_8" style="color:#0563c1; text-decoration:underline" title="Collison, 2007 #25">8</a>, <a href="#_ENREF_18" style="color:#0563c1; text-decoration:underline" title="Abo-Elnour, 2020 #36">18</a>]. We also investigated the correlation between IL-35 levels and the frequency of CD4+T cells, which are a key target of HIV. However, we did not find any significant correlation between IL-35 concentration and CD4+T cell frequency. Furthermore, we did not observe a statistically significant difference in IL-35 levels between patients with different CD4+/CD8+ratios, although our findings suggest a possible trend towards lower IL-35 levels in patients with a higher CD4+/CD8+ratio. One limitation of our study was the inability to investigate lymphocyte subtypes, particularly Treg cells, which play a crucial role in the synthesis and secretion of IL-35. Since Treg cells are also involved in the pathogenesis of HIV infection[<a href="#_ENREF_19" style="color:#0563c1; text-decoration:underline" title="Kleinman, 2018 #23">19</a>], it is necessary to conduct further studies to explore the correlation between IL-35 and effector T lymphocyte subclasses, including Treg cells. Such investigations could provide valuable insights into the mechanisms underlying the immune response to HIV infection and aid in the development of more effective treatments for this disease. In conclusion, this study provides evidence that IL-35 levels are elevated in HIV-positive patients with or without HCV co-infection compared to healthy controls. The findings suggest that HCV co-infection may have an additive effect on the elevation of IL-35 in HIV-positive patients. These results contribute to the growing body of literature on the role of IL-35 in viral infections and underscore the importance of investigating the mechanisms underlying the relationship between chronic viral infections and the immune system. Keywords: HIV, HIV/HCV-coinfection, Interleukin-35   HIV, عفونت همزمان HIV/HCV, اینترلوکین-35 HIV, HIV/HCV-coinfection, Interleukin-35 0 0 http://rjms.iums.ac.ir/browse.php?a_code=A-10-8455-1&slc_lang=fa&sid=1 Mohammad Jafari محمد جعفری jafari@gerums.ac.ir 3900319475328460088095 3900319475328460088095 No Cellular and Molecular Research Center, Gerash University of Medical Sciences, Gerash, Iran مرکز تحقیقات سلولی و مولکولی، دانشکده علوم پزشکی گراش، گراش، ایران Mostafa Hajimola Hoseini مصطفی حاجی‌ملا حسینی m.mollahoseini@sbmu.ac.ir 3900319475328460088096 3900319475328460088096 No Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran گروه ایمنی‌شناسی، دانشکده پزشکی، دانشگاه علوم پزشکی شهید بهشتی، تهران، ایران Majid Khoshmirsafa مجید حوش میرصفا Khoshmirsafa.m@iums.ac.ir 3900319475328460088097 3900319475328460088097 No Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran گروه ایمنی‌شناسی، دانشکده پزشکی، دانشگاه علوم پزشکی ایران، تهران، ایران Maryam Keykhaee مریم کیخایی m.keykhaee1360@gmail.com 3900319475328460088098 0009-0009-2400-5459 No Department of Pharmaceutical Biomaterials and Medical Biomaterial Research Center (MBRC), Faculty of Pharmacy, Tehran University of Medical Science, Tehran, Iran. گروه زیست مواد دارویی و مرکز تحقیقات زیست مواد در پزشکی (MBRC)، دانشکده داروسازی، دانشگاه علوم پزشکی تهران، تهران، ایران Roghayeh Babaei رقیه بابایی R.babaei1986@gmail.com 3900319475328460088099 3900319475328460088099 No Department of Nanotechnology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran گروه نانوتکنولوژی، دانشکده علوم و فناوری‌های پیشرفته، دانشگاه علوم پزشکی آزاد اسلامی تهران، تهران، ایران Hamid Nickho حمید نیکخو Nickho.h@iums.ac.ir 3900319475328460088100 3900319475328460088100 No Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran گروه ایمنی‌شناسی، دانشکده پزشکی، دانشگاه علوم پزشکی ایران، تهران، ایران Alireza Najafi علیرضا نجفی najafi.ar@iums.ac.ir 3900319475328460088101 3900319475328460088101 Yes Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran گروه ایمنی‌شناسی، دانشکده پزشکی، دانشگاه علوم پزشکی ایران، تهران، ایران