Cytokine Response Associated with Hepatitis C Virus Clearance in HIV Coinfected Patients Initiating Peg Interferon-α Based Therapy

Truong Tam Nguyen^1,2,  Reihani Niloofar¹, Pierre-Alain Rubbo¹, Kuster Nils¹, Karine Bollore¹, Jacques Ducos⁵, Georges-Philippe Pageaux^1,3, Jacques Reynes^1,4, Philippe Van de Perre^1,5 and Edouard Tuaillon^1,5 

¹ Université Montpellier 1, INSERM U 1058, , 34394 Montpellier, France.
² Pham Ngoc Thach University of Medicine, Thanh Thai, Ho Chi Minh City, Vietnam.
³ CHU Montpellier, Département d'Hépato-Gastro-Entérologie, 34295 Montpellier, France.
⁴ CHU Montpellier, Département des Maladies Infectieuses et Tropicales, 34295, France.
⁵ CHU Montpellier, Département de Bactériologie - Virologie, 34295 Montpellier, France. 

Published: January 1, 2016
Received: May 19, 2015
Accepted: November 9, 2015
Mediterr J Hematol Infect Dis 2016, 8(1): e2016003, DOI 10.4084/MJHID.2016.003
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Introduction

Infection by hepatitis C virus (HCV) and HIV shared common routes of transmission and thus confections with the two viruses are frequent. HCV infection is seen in 15–30% of HIV-infected patients in Western Europe, with almost 20 000 HIV-infected individuals chronically infected with HCV in France.[1,2] Since the introduction of antiretroviral therapy (ART), HCV-related liver diseases have become leading causes of morbidity and mortality in HIV-infected individuals.[3,4]
Faster progression to cirrhosis has been observed in patients coinfected with HCV and HIV by comparison with subjects infected with HCV alone.[5] HIV/HCV coinfection is also associated with higher HCV viral levels in serum.[6] Finally, ART-related adverse events are more frequent in HCV/HIV coinfected individuals, with increased difficulties in optimally treating HIV infection.[7,8]
Until recently, a combination of pegIFN and ribavirin has been the standard-of-care for treatment of chronic HCV patients. In coinfected individuals pegINFα-ribavirin therapy is less efficacious than in single HCV infection.[9,10] Approximately 30% of patients receiving a combination of ribavirin with pegIFN have a sustained virological response when infected with HCV genotype 1 and 4.[9] This rate increases to 60% when genotype 2 and 3 are involved.[10] Most of the current anti-HCV therapeutic options remained based on pegIFN and/or ribavirin in addition to protease inhibitors or NS5A or NS5B inhibitors.[11]
PegIFN constitutes an active immunotherapy triggering innate immune response and T cell activation. The cytokine response to pegIFNα-based regimens plays probably an important role in treatment outcome. IFNα stimulates Th1 cells and limits secretion of Th2 related cytokines such as IL-4 or IL-10.[12] IFNα signals through Janus kinase signal transducer, an activator of transcription pathway, and induction of IFN-stimulated genes.[13] Ribavirin synergies this effect by reducing IL-10 production, enhancing autocrine IFN-β and IL-8 secretion.[14-16] Several immune factors are predictive of virological response on pegIFNα-ribavirin-based therapy: pretreatment concentration of IFNα-inducible protein 10 (IP-10, also called CXCL-10), polymorphisms near the interleukin-28B gene coding for the IFNα-3, and CD4⁺ T cell count in HIV/HCV coinfected individuals.[18,19] Progressive impairment of immune functions and T cell exhaustion induced by prolonged exposure to virus antigens are fundamental features of HIV infection. Inhibitory networks, such as the programmed cell death protein-1 (PD-1) and IL-10 play a key role in this process.[20,21] PD-1 is upregulated on CD8⁺ and CD4⁺ T cells and mediates a dysfunction partially reversible on ART.[22-24] PD-1 signalling impacts the expression of both Th1 and Th2 cytokines but limits particularly the capacity of Th1 and IFNγ secretion by CD4⁺ and CD8⁺ T cells.[21]
Although HCV clearance depends on immune response induced by pegIFNα-ribavirin therapy, the dynamics of cytokine response and T cell activation in individuals coinfected with HIV and HCV remain poorly characterized.[25] In this study, we monitor serum concentration of 25 cytokines alongside with the level of CD8⁺ T cell activation in HIV/HCV coinfected patients initiating anti-HCV therapy.

Methods

Patient samples: Patients coinfected by HIV/HCV followed at Montpellier University Hospital and initiating anti-HCV therapy were included consecutively between January 2007 and January 2010 after providing written informed consent. The study was approved by the local institution ethical committee (DC-2011-1405). Chronic hepatitis was proved by the presence of serum HCV antibodies and detectable viral RNA. HCV genotype, HCV and HIV-1 viral loads, CD4⁺ T cell count, and liver enzyme levels were all determined using standard procedures. All patients received pegIFNα2a 180 microg/week plus ribavirin 800 to 1,200 mg daily; patients infected with HCV genotype 2 and 3 received 800 mg daily and genotype 1 and 4 received a weight based dose: 1,000 mg/day for persons less than 75 kg and 1200 mg/day over 75 kg.
Subjects were ranged into two distinct groups based on HCV response to therapy:
i)    Sustained virological responders (SVR) when serum HCV RNA was undetectable 24 weeks after completing therapy, and
ii)  non responders (NR) when HCV RNA decrease was < 2 log copies/mL at week 12 or when HCV RNA was detectable at the end of treatment.
iii)   Patient relapsing in the 24 weeks period after therapeutic cessation were excluded from the study.
Routine laboratory testing: Serum HCV RNA was quantified using COBAS AmpliPrep/COBAS TaqMan HCV assay (Roche Diagnostic Systems). HCV genotypes were determined using INNO-LiPA HCV II test (Innogenetics). Alanine aminotransferase (ALT) dosages were executed using standard methods.
Quantitation of serum cytokine concentrations: Cytokines were quantified in serum samples obtained before initiation of treatment, and four weeks after the start of PegIFNα-based therapy. A multiplexed microbead assay was used according to manufacturer's instructions (cytokine twenty-five-plex kit, Life Technologies Ltd, Paisley, UK) and an FIDIS™ apparatus (BMD). Twenty five cytokines were quantified in duplicate: IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40/70, IL-13, IL-15, IL-17, eotaxin, GM-CSF, IFN-α, IFN-γ, IP-10, MCP-1, MIG, MIP-1α, MIP-1β, RANTES, and TNF-α. Data were analyzed using the MLX-Booster program (BMD). Mean concentrations (pg/ml) of cytokines were all superior to the detection limits, defined as the mean background value plus 2 SD.
Analysis of CD8⁺/CD38^bright T cells: Activation of CD8⁺ T cells was assessed by flow cytometry analysis on EDTA-treated fresh whole blood using FC 500 apparatus (Beckman Coulter, Miami, Florida). T cell activation was explored at day 0, week 4 and week 12 after initiation of pegIFNα-ribavirin therapy. The expression of CD38^bright on CD8⁺ T cells was analyzed as previously described using a two-colour staining with anti-CD8 and anti-CD38 conjugated to fluorescein isothiocyanate (FITC) and phycoerythrin (PE), respectively (Beckman Coulter).[26] The positive threshold for CD38^bright analysis was established using the CellQuant CD38/CD8 kit for quantitation of CD38 cell surface expression (BioCytex, Marseille, France) and was defined as 8,500 CD38 binding sites/cells. The CD8⁺/CD38^bright values were expressed as the percentage of CD38^bright cells from the CD8⁺ T cell populations.
Statistical Analysis: The Mann–Whitney U test and Kruskal–Wallis test were used to analyze continuous variables where appropriate. The Friedman test was used to evaluate changes in serum cytokine levels over time. A mixed model was used to analyze the association between T cell activation during anti-HCV therapy and therapeutic response. Statistical analyses were done using SPSS software version 18.0J.

Results

Clinical characteristics at baseline: Thirty HCV/HIV coinfected patients receiving pegIFNα-ribavirin therapy were included in the study. Nineteen (63%) were classified as SVR and eleven (37%) as NR. Patient characteristics are shown in Table 1.
Pretreatment levels of soluble and cellular inflammatory markers: IP-10 levels were higher in the NR group than in the SVR group, P=0.021 (Table 2). Individuals from the NR group also had higher pretreatment concentrations of MCP-1, P=0.0009. A trend for a higher serum concentration of IL-8 was also observed in patients of the NR group compared to the SVR group, P=0.106. 

Table 1. Patient characteristics

Table 2. Pre-treatment Cytokine concentrations in serum from SVR and NR patients

Cytokines change after therapeutic initiation of HCV treatment: Initiation of anti-HCV treatment elicited IFNα rise in both SVR and NR patients (Figure 1 and Supplemental Table 1). This enhancement of IFNα in serum was similar in the two groups. Besides IFNα, the anti-HCV therapy induced a significant increase in the serum levels of eleven cytokines in SVR patients, including cytokines related to Th1-dominant immune responses (IL-12p40/70, IP-10), Th2-type cytokines (IL-4, IL-5, and IL-13), and pro-inflammatory cytokines (IL-1β, IL-1RA, IL-6, IL-7). By comparison, levels of only six cytokines rose in the NR group (Supplemental Table 1). Finally, when results were analyzed without consideration of the HCV therapeutic response (combining SVR and NR patients), we also observed an increase of IL-2, IL-10, and RANTES concentrations four weeks after initiation of anti-HCV treatment (Supplemental Table 1).   

Figure 1. Impact of anti-HCV therapy on cytokine concentrations in serum of NR and SVR. Fold-changes in cytokine levels following pegIFNα-ribavirin therapy initiation in SVR (white bars) and NR (gray bars). Results are expressed as fold changes in cytokine concentrations between week 0 (pretreatment) and week 4 (four weeks of pegIFNα-ribavirin therapy). *p=0.052, **p=0.029, ***p=0.0024.

Supplemental Table 1. Fold changes in serum cytokines level four week after initiation of pegIFN-ribavirin therapy.

Comparison of the cytokine level changes induced by pegIFN-ribavirin between SVR and NR group: Changes in cytokines were compared among SVR and NR using the ratio between baseline and four weeks of treatment (Figure 1). A sharp increase of MCP-1 and MIP-1β concentration was observed after initiation of anti-HCV therapy in the SVR group whereas the value decreased or remained stable in the NR group (p=0.0024 and p=0.029, respectively). Furthermore, the IL-8 concentration remains stable after a one-month period of pegIFNα-ribavirin therapy in most of the patients responding to anti-HCV treatment, whereas this concentration declined in NR patients (p=0.052).  
Changes in T cell activation over the three-months period of therapy initiation: The impact of pegIFNα-ribavirin on T cell activation was explored at day 0, week 4 and week 12 (Figure 2). CD8⁺ T cell activation before the treatment tends to be higher in NR patients compared to the SVR patients, (p=0.066, Figure 2). Anti-HCV therapy induced a major increase in CD38^bright expression on CD8 T⁺ cells in the two groups of patients (p<0.001). No significant differences were observed between the two groups in the slope of CD38 cell surface expression of CD8⁺ T cells suggesting that the over time changes of this activation marker not be associated with a better immune response to therapy.

Figure 2. Changes in T cell activation over a 3-month period of pegIFN-ribavirin therapy. Fold-changes in CD38bright expression on CD8 T cells following pegIFNα-ribavirin therapy initiation in SVR (white bars) and NR (gray bars). A significant increase of CD38bright expression between two consecutive time periods is indicated: *p<0.001.

Figure 3