Objectives: Cytomegalovirus (CMV) establishes a lifelong, asymptomatic infection in immuno­competent hosts. Interleukin-17 producing CD4+ T-cells (Th-17) are a subtype of CD4+ T-cells. The precise role of Th-17 responses during cytomegalovirus replication has not been elucidated, although recent studies suggest that infections such as murine cytomegalovirus induce a Th-17 response. Th-17 cells also have been associated with allograft rejection and autoimmune diseases. In this study, we tried to find the relation of cytomegalovirus infection and interleukin 17 (IL-17) cytokine in liver-transplanted patients.

Materials and Methods: Two groups of patients were evaluated in this study. The first group consisted of 54 cytomegalovirus uninfected liver-transplanted patients, and the second group consisted of 15 cytomegalovirus-infected patients. Three ethylenediaminetetraacetic acid-treated blood samples were collected from each patient on days 1, 4 and 7 post liver transplant. For diagnosing cytomegalovirus infection antigenemia and Taq-Man real-time polymerase chain reaction protocols were used. Also, to determine the expression level of IL-17 gene, an in-house SYBR green real-time polymerase chain reaction technique was used.

Results: Using antigenemia and also Taq-Man real-time polymerase chain reaction helps find active cytomegalovirus infection, and the load of cytomegalovirus in each patient. The first group of patients showed that IL-17 expression level was down-regulated after day 4 of sampling. But in cytomegalovirus-infected patients, IL-17 expression level was increased significantly. The results between IL-17 gene expression level between the 2 groups of patients showed that IL-17 expression level significantly increased in second group during day 4 (P = .038) and 7 (P = .009) postliver transplant.

Conclusions: Significant increase of IL-17 mRNA levels in cytomegalovirus-infected group compared with the uninfected one reinforced the role of IL-17 as a proinflammatory cytokine dealing with cytomegalovirus infection in liver transplanted patients.

Key words : Viral infection, Interleukin-17, Allograft rejection

Introduction

Orthotopic liver transplant (OLT) is mentioned as a final therapeutic protocol for end-stage liver diseases.^1-3 Surveillance duration of transplanted liver is related to many factors including pathogens.⁴ Many of the pathogens can threaten the health of a transplanted liver, among them cytomegalovirus (CMV) can cause mortality and morbidity directly and/or indirectly influencing immunosuppression that make the patient susceptible to different super-infections.¹ Cytomegalovirus is an opportunistic pathogen that can infect people worldwide (from 40% to 90%), but commonly make active infection inimmunocompromised patients⁵ like organ transplant recipients and undoubtedly have negative effect on the results of transplant.^1,6,7 Active CMV infection risk in transplant recipients is around 30% to 75%, which cause mortality rate ~ 5%.⁶ Cytomegalovirus can affect an allograft outcome by interfering cellular and humoral immune responses with changing the subpopulation of CD4+ and CD8+ T lymphocytes and also cytokines.⁸ It is known that CMV infection can induce increasing of the level of several chemokines and cytokines in liver transplanted patients leading to control of infection⁷ and sometimes this struggle renders to allograft injury or rejection.⁹ There also are reports demonstrating that both chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are related to virus-induced liver inflammation, infiltration, and activation of Th17 cells, and the amount of liver damage caused by the antiviral immune response.¹⁰

After transplant, CD4+ and CD8+ T cells especially play an important role in controlling CMV replication. Recently, a new subset of CD4+ T cells, Th17 cells, were introduced to have prominent roles in the result of liver transplant.⁹ Th17 cells can activate specific transcription factors and produce IL-17 cytokine as its major cytokine product and also IL-21, IL-22, and IL-23 receptor,^11,12 and IL-23 acts as a stabilizing agent for production of Th17 cells.^13,14 Interleukin-17 has no sequence similarity to any other known cytokine and mammalian protein.¹⁵

Interleukin-17 is the major proinflammatory cytokine of these cells which is a part of normal host response to infection.^2,7,9,16 One study elucidated the role of Th17 in viral infection persistence by up-regulating the antiapoptotic molecules that renders survival of virus-infected cells.¹⁷ Also, another study showed the in situ existence of the Th17 cells in HBV-infected liver can worsen the liver physiology.¹⁴ Interleukin-17 can recruit neutrophils and monocytes to the site of inflammation.¹⁸

The importance of Th17 cells and its important cytokine, IL-17, is detected in different inflammatory and autoimmune diseases (eg, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and multiple sclerosis),20 and allograft rejection.^2,9,11,21 Also, IL-17 by its proinflammatory action, can induce expression of many factors dealing with inflammation.⁷ Increase of IL-17 after transplant and rejection in different transplant type in human and mice is documented.² In rat acute renal allograft rejection models, elevation of IL-17 proteins in day 2 after transplant has been shown.^22,23 In mouse heart transplant model, using IL-17 inhibitor, reduced production of inflammatory cytokines within the graft and increased graft survival.²⁴ The role of Th17 and IL-17 in liver transplant is not studied much in humans, but reported a significant increase of IL-17/IL-23 protein level in patients with acute rejection after liver transplant;^25,26 Also, our recent study4 showed the increase of IL-17 mRNA level in the first week after liver transplant in rejected ones. As IL-17 is important in organ allograft rejection, it may have capacity for being considered as a target for anti-rejection therapy, either alone or in combination with immunosuppressive agents.^26,27

Taking together, considering the role of IL-17 as a proinflammatory cytokine in liver transplanted patients and the role of CMV in causing inflammation in transplanted patients, in this study we determined the mRNA expression level of IL-17 in CMV infected liver transplanted patients.

Materials and Methods

Study groups
The patients that were enrolled in this study were composed of 2 groups of liver transplanted patients who underwent surgery at Namazi Hospital, Shiraz, Iran, between years 2011 and 2013. EDTA-treated blood samples were collected from each studied patient in days 1, 4 and 7 intervals after the liver transplant. Using Ficol, the buffy coat and plasma were isolated from each blood sample and preserved in -80°C for further analysis. Based on experiencing CMV infection or not, studied patients were divided into infected and uninfected liver transplanted. The uninfected patients consisted of 54 patients and the infected group were composed of 15 patients. The study was approved by the Ethical Committee of Shiraz University of Medical Sciences (The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki). For detecting CMV antigenemia technique was used for all of the samples and Taq-man real-time PCR were administrated for certifying the results of antigenemia. For determination of the mRNA expression level of IL-17 SYBR Green real-time PCR was used and β-actin was also considered for internal control.

The routine immunosuppression regimen consisted of tacrolimus or cyclosporine with mycophenolate mofetil and steroids. Drug dosage was adjusted to maintain target therapeutic blood levels of 200 mg/mL for CsA (5 mg/kg/d) or 10 mg/mL for tacrolimus. Donors were selected on the basis of ABO blood group compatibility. HLA matching is routinely not done for liver transplant patients.

Cytomegalovirus Quantification
Viral genome extraction
Using Invisorb Spin Virus DNA Mini Kit (Invitek, Birkenfeld, Germany) CMV-DNA was extracted from plasma samples according to the manufacturer’s protocol. Before starting the extraction procedure, the internal control was added to each sample. Determination of CMV viral load was done using gensig real-time PCR kit (Primer Design Ltd TM, Advanced kit, United Kingdom). The reaction mix for PCR was performed in 20 μL total volume and was composed of 10 μL Precision TM Master Mix (Applied Biosystems Grand Iland, NY, USA), 1 μL primers and a probe targeting the glycoprotein B (gB) sequence, 1 μL primers and a probe targeting the internal control (IC) gene, 5 μL of the DNA and 3 μL DEPS water.

The program used for this reaction was included 1 cycle 95°C for 10 minutes, followed by 50 cycles of 95°C for 5 seconds and 60°C for 60 seconds using Step One Plus Real-Time thermocycler (Applied Biosystems-Grand Iland, NY, USA). This quantitative PCR assay was sensitive enough to detect as few as 10 copy of CMV genome per milliliter of body samples.

Cytomegalovirus antigenemia protocol
All of the patients were tested with antigenemia for detection of active infection. For executing antigenemia test on the samples, EDTA-treated whole blood samples were used and the procedure was followed through the package insert of the CMV Brite Turbo kit (IQ Products, Groningen, The Netherlands). Two hundred thousand cells were collected for Cyto-centrifuged preparations (Cytospin 3, Shandon Scientific, Cheshire, England). Then a cocktail of 2 fluorescein isothiocyanate-labeled monoclonal antibodies (C10/C11) were used for staining indirect immunofluorescence and directed against CMV lower matrix protein pp65. By acquiring fluorescence microscope, bright green stained nucleus of CMV infected polymorphonuclear cells were detected and finally the CMV antigen positive cells were counted and reported.

RNA isolation and cDNA synthesis
Total RNA was isolated by RNX plus (CinnaGen, Iran) from Buffy coats by using an in-house RNA extraction protocol. For certifying the purity and integrity of RNA, the optical density 260/280 and agarose gel (1%) electrophoresis measured. One microgram of each RNA sample was changed to cDNA by Reverse Transcriptase (Vivants, Subang Jaya, Malaysia) and random hexamer. This cDNA synthesis was performed in two steps. First, RNA (10 μg/μL), dNTPs (1 μL/10 mm), and random hexamer (0.2 μg/μL) were mixed and incubated at 65°C for 7 minutes and then on ice for 2 minutes. Second, M-MLV reverse transcriptase enzyme (1 μL/200 U), reverse transcriptase-buffer (2 μL/× 10), RNase inhibitor (1.3 μL/60 U), and nuclease free water were mixed and added to product of first step. Then final mix was incubated at 45°C for 90 minutes and 85°C for 5 minutes.

SYBR green real time polymerase chain reaction
For the quantitative analysis of IL-17 mRNA expression profile in both groups of studied patients, real-time PCR method was performed. After evaluation of the β-actin and GAPDH genes as internal controls, the β-actin gene was finally used as internal control for minor fluctuations. The primer was originally designed for transcripts of IL-17 (NM_002190.2) and β-actin (NM_001101.3) as the internal control. The primer sequences for amplification of IL-17 and β-actin transcripts were as follows: 5’-TCTGGGAGGCAA AGTGCCGC-3’ and IL-17R: 5’-GGGCAGTGTGG AGGCTCCCT-3’, β-actin F: 5’-GGCGGCACCACCA TGTACCC-3’, and β-actin R: 5’- GACGATGGAGGG GCCCGACT-3’. The PCR mixes were composed of SYBR green Premix (10 μL) by Ex Taq (Takara, Otsa, Shiga, Japan), SYBR green dye (0.2 μL), forward and reverse primers (3 PM), and template cDNA (2 μL). The temperature program for PCR condition was as follows: One cycle 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, and 65°C for 20 seconds using Step One Plus real-time instrument (ABI, Step One Plus, Grand Iland, NY, USA). For checking the specificity of amplification reaction melting-curve analysis was evaluated. The results for the target genes were measured as fluorescent signal intensity and normalized to the internal standard gene β-actin.

Statistical analyses
For evaluating the IL-17 expression pattern between 2 sampling collection intervals between CMV infected and uninfected liver transplant patient groups using Livak method. Statistical analysis was performed using SPSS, version 16 for Windows (SPSS, Chicago, IL, USA). The t test, Mann-Whitney U test, and ANOVA test were used to evaluate statistical association and/or difference inside and between 2 studied groups. P < .05 was considered as statistically significant.

Results

Patients’ profile
The CMV-infected liver transplant patients who termed CMV+ consisted of patients with positive results of antigenemia and also Taq-Man real-time PCR methods. The uninfected patients consisted of 54 patients whose age range was between 1 and 74 years with a mean of 34.33 ± 21.26 years old. The 36 of 54 uninfected patients (68%) were male, and 17 of 54 were female (32%). The infected patients consisted of 15 patients who experienced CMV infection. The age range in these patients was between 1.5 and 62 with a mean of 30.25 ± 15.3 years old. The infected group was composed of 7 of 15 men (46.6%) and 8 of 15 women (53.4%). The distribution frequency of the underlying disease in both uninfected and infected liver transplant patients was as follows: Hepatitis B virus infection 15 in uninfected (27.8%), and 2 infected (13.3%), auto immune hepatitis 9 in uninfected (16.7%) and 3 infected (20%), primary sclerosing cholangitis 9 in uninfected (16.7%) and 2 were infected (13.3%), cryptogenic cirrhosis 7 in uninfected (13%) and 3 infected (20%), Wilson disease 3 uninfected (5.5%) and 1 infected (6.7%), biliary atresia 3 uninfected (5.5%) and 1 infected (6.7%), hypertyrosinemia 2 uninfected (3.7%) and 1 infected (6.7%), hepatitis C virus infection 1 uninfected (1.85%), and 2 infected (13.3%), and other diseases 5 uninfected (9.25%) and 0 infected groups. The highest frequency of the underlying diseases was hepatitis B virus infection and auto immune hepatitis in uninfected and infected transplant patients. The most frequent ABO blood group also was O+ in uninfected and A+ in infected transplant patients. The details of ABO blood groups are shown in Table 1.

IL-17 gene expression in uninfected and infected liver transplant patients
To identify whether IL-17 had affected liver transplant outcomes, 69 liver transplant patients were selected. The analysis of results in uninfected patients showed that IL-17 expression was down-regulated after day 4 of follow-up (Figure 1). In infected patients, IL-17 expression was significantly and steadily increased during all days of follow-up (Figure 2).

Time dependent IL-17 gene expression between infected and uninfected liver transplant patients
The results of comparing the interleukin-17 gene between uninfected and infected liver transplant patients are presented in Figure 3. Interleukin-17 expression level decreased in infected patients in the first day sampling time and increased significantly, on the second (day 4) and third (day 7) compared with uninfected during sampling days post-transplant (Figure 3A-3B), but this increase was significant in the fourth and seventh day of sampling (P = .038, 95% CI: 0.00-0.05 and P = .009, 95% CI: 0.00-0.09) (Figure 3B and 3C). Interleukin-17 expression level also was increased but not significantly in uninfected patients during the first (P = .215, 95%CI: 0.189-0.408) sampling day post-transplant. The IL-17 gene expression was increased around 140 times in infected patients compared with uninfected ones during posttransplant follow-up.

Discussion

Liver transplant should be mentioned as a costly and critical therapy for many irreversible liver damages and should be preserved not to be threatened by any harmful agent.³ It is a fact that organ transplant can make an inflammatory environment that many inflammatory cytokines like IL-17 are released.¹³ Th17 cells are a specific subset of CD4+ T cells, which control tissue-inflammation and proinflammatory cytokines.¹²

Viral infections like CMV can be troublesome for liver transplanted patients. Little is known about the role of Th17 cells and IL-17 in CMV-infected patients; therefore, by considering these data, in this study, the possible association of the mRNA expression level of IL-17 gene with CMV pathogenesis was evaluated in liver transplanted patients. The rate of IL-17 expression in uninfected group showed a statistically significant increase in second sampling day (fourth day), which is followed by a decrease in the next sampling time. This expression pattern is a result of ischemic reperfusion occurring early after transplant and by removing of the inflammatory factors, the IL-17 expression reduces in day 7 of sampling.4 The other group (CMV+) showed a detectable increase in the last sampling day, which was also statistically significant.

The comparison of IL-17 mRNA expression level between both studied groups and in all 3 sampling intervals also showed a no significant increase in uninfected group in the first sampling time, which was followed by 2 statistically significant increases in second and third sampling time, which highlights the previously mentioned role of Th17 cells and its cytokine (IL-17) in the time of CMV infection in liver transplanted patients.

The transplant procedure by its own can have direct effects on expression of cytokines related to graft.³ After transplant, periods of ischemia and then reperfusion renders the release of various inflammatory mediators,^23,28 which are important in T-cell differentiation and even graft destructive immune cells.^28,29 One study group reported that the factors, which are released by endothelial cells during ischemia-reperfusion injury, can increase production of some cytokines like IL-17 and IFN-γ by CD4+ T cells.³⁰ They also reported that these factors can result in increase of Th17 activity within the graft.

Liver injuries are followed by inflammatory responses, which are composed of infiltration of innate immune cells. Infiltration of monocytes also as a result of liver injury is a critical cellular mechanism which cause inflammation and activation of profibrogenic hepatic satellite cells in humans and mice.^31,32 In mentioned situation, adaptive immune cells like CD8+ and CD4+ T-cells are involved in inflammation of the liver.^33,34 Activation of CD4+ T-cells is followed by their differentiation into various subsets of cells like Th1, Th2, Tregs, and Th17, depending on the cytokines present in the local environment.^2,28 Th17 cells and their important cytokine, IL-17, deal with inflammation and even rejection in liver transplanted patients.¹¹ It is believed that Th17 cells have more potential for changing situations, rather Th1 and Th2 cells.²⁸ Hanidziar and associates believed that Th17 cells present in the allograft, are more a biomarker of tissue-inflammatory state rather than a single mechanism, which is responsible for rejection.²⁸

Research shows that IL-17 is involved in defense against bacterial infections and autoimmune diseases of the host and also its role is mentioned in transplant.^15,20 IL-17 increase is detected in human lung allograft rejection.²⁵ Also, in mouse studies, the role of Th17 and IL-17 is mentioned. In bone marrow transplant, patients experiencing acute graft versus host disease Th17 cells increased in peripheral blood.²⁶ Furthermore, Fabrega and associates showed the increase of IL-17/IL-23 protein level in human with rejected liver transplant. Recently, our previous study, also confirmed this data.⁴

Viral infections are another threat for a newly transplanted liver and among them, CMV mortality and morbidity.8 We know both of the innate and adaptive immunity are responsible in viral control. In immunocompromised patients like liver transplanted ones, CD8+ T cells that are virus-specific act against CMV-related disease, which their maintenance is done by uninfected specific CD4+T cells. Around 4% to 5% of all CD4+ and CD8+ T cells are specific for CMV in seropositive humans.⁵ It is suggested that the Th17 responses happen during CMV replication, but the details are unclear.⁹ One study as the first research on the role of IL-17 HBV-infected patients showed that Th17 population and IL-17 cytokine significantly increase.¹² They believed that significant elevation of Th17 cells percentage express that Th17 cells play a role in HBV infection. At the same time, another study confirmed the findings of Ge and associates and showed that in the damage that is caused by HBV in liver, Th17 cells have prominent role. These findings elucidated that the population of Th17 cells, inside liver, and in the peripheral environment are elevated in HBV patients and these cells can activate dendritic cells and monocytes and make them to produce inflammatory cytokines during HBV infection.¹⁴ Also, in immune responses during Murine CMV (MCMV) infection models, IL-17 producing CD4+ T cells have important roles.⁵ Recently, Egli and associates demonstrated that Th17 cells are harmful in making inflammatory autoimmune diseases, and IL-17 can lead to immunopathology responses of CMV-infected hosts.9 Furthermore, another study claimed that lung lesions that are made by MCMV infection in animal models render to production of IL-17 cytokine that can control immune responses and make lung lesions either. Also they proposed that IL-17 may be responsible for developing MCMV pneumonia after allogenic transplant. They believed that in studying the IL-17 dynamic evolution, the most prominent part is the time that is selected for collecting of samples, in pneumonia induced by MCMV.7

Putting this data together, this study on the role of Th17 in CMV infection in liver transplanted patients is barely studied. This project highlights the importance of IL-17 cytokine in immune responses against CMV infection in transplanted livers, which also needs more studies to elucidate this mechanism in details.

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