RESEARCH ARTICLE
HCV coinfection contributes to HIV pathogenesis by increasing immune exhaustion in CD8 T-cells Norma Rallo´n1,2*, Marcial Garcı´a1,2, Javier Garcı´a-Samaniego3, Noelia Rodrı´guez1,2, ´ lvarez4, Rosa Garcı´a4, Miguel Go´rgolas4, Alfonso Cabello4, Clara Restrepo1,2, Beatriz A Jose´ M. Benito1,2
a1111111111 a1111111111 a1111111111 a1111111111 a1111111111
1 IIS-Fundacio´n Jime´nez Dı´az, UAM, Madrid, Spain, 2 Hospital Universitario Rey Juan Carlos, Mo´stoles, Spain, 3 Hospital Universitario Carlos III-La Paz, Madrid, Spain, 4 Hospital Universitario Fundacio´n Jime´nez Dı´az, Madrid, Spain * [email protected]
Abstract OPEN ACCESS Citation: Rallo´n N, Garcı´a M, Garcı´a-Samaniego J, Rodrı´guez N, Cabello A, Restrepo C, et al. (2017) HCV coinfection contributes to HIV pathogenesis by increasing immune exhaustion in CD8 T-cells. PLoS ONE 12(3): e0173943. https://doi.org/ 10.1371/journal.pone.0173943 Editor: Lishomwa C. Ndhlovu, University of Hawaii System, UNITED STATES
Background There are several contributors to HIV-pathogenesis or insufficient control of the infection. However, whether HIV/HCV-coinfected population exhibits worst evolution of HIV-pathogenesis remains unclear. Recently, some markers of immune exhaustion have been proposed as preferentially upregulated on T-cells during HIV-infection. Herein, we have analyzed T-cell exhaustion together with several other contributors to HIV-pathogenesis that could be affected by HCV-coinfection.
Patients and methods
Received: November 1, 2016 Accepted: March 1, 2017 Published: March 21, 2017 Copyright: © 2017 Rallo´n et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study has been funded by projects: RD12/0017/0031 and PI14/00518 integrated in the Plan Nacional I+D+I and co-funded by ISCIIISubdireccio´n General de Evaluacio´n and European Regional Development Fund (Fondos FEDER). Norma Rallo´n is a Miguel Servet investigator from the ISCIII (CP14/00198), Madrid, Spain. M Garcı´a is a predoctoral student co-funded by CP14/00198
Ninety-six patients with chronic HIV-infection (60 HIV-monoinfected and 36 HIV/HCV-coinfected), and 20 healthy controls were included in the study. All patients were untreated for both infections. Several CD4 and CD8 T-cell subsets involved in HIV-pathogenesis were investigated. Non-parametric tests were used to establish differences between groups and associations between variables. Multivariate linear regression was used to ascertain the variables independently associated with CD4 counts.
Results HIV-patients presented significant differences compared to healthy controls in most of the parameters analyzed. Both HIV and HIV/HCV groups were comparable in terms of age, CD4 counts and HIV-viremia. Compared to HIV group, HIV/HCV group presented significantly higher levels of exhaustion (Tim3+PD1- subset) in total CD8+ T-cells (p = 0.003), and higher levels of exhaustion in CD8+HLADR+CD38+ (p = 0.04), CD8+HLADR-CD38+ (p = 0.009) and CD8+HLADR-CD38- (p = 0.006) subsets of CD8+ T-cells. Interestingly these differences were maintained after adjusting by CD4 counts and HIV-viremia.
PLOS ONE | https://doi.org/10.1371/journal.pone.0173943 March 21, 2017
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HIV pathogenesis is influenced by HCV presence
project and Intramural Research Scholarship from IIS-FJD. N Rodriguez and C Restrepo are funded by project RD12/0017/0031. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conclusions We show a significant impact of HCV-coinfection on CD8 T-cells exhaustion, an important parameter associated with CD8 T-cell dysfunction in the setting of chronic HIV-infection. The relevance of this phenomenon on immunological and/or clinical HIV progression prompts HCV treatment to improve management of coinfected patients.
Competing interests: The authors have declared that no competing interests exist.
Introduction Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) are two of the most relevant persistent infections afflicting the human population, with 37 and 150 million chronically infected individuals worldwide respectively [1]. Coinfection with both viruses is common due to the existence of shared transmission routes [2] and as much as 20–30% of HIV positive patients are coinfected with HCV [1]. Since both viruses interfere with host immune response [3, 4] and interact at the molecular level [5, 6], coinfection poses a formidable challenge to host immune system. As a consequence of this, each virus can impact on the clinical course of the other. In fact, the negative impact of HIV infection on hepatitis C pathogenesis is well described. HIV/HCV coinfection has been associated with lower rates of spontaneous HCV clearance, higher HCV viral load, accelerated liver disease progression and reduced response to interferon-based HCV therapy [1]. Since the introduction of highly active antiretroviral therapy (HAART), the clinical prognosis for HIV patients has markedly improved and, as a result, HCV infection is now a leading cause of morbidity and mortality in HIV/HCV coinfected individuals [7]. This coinfection is probably also associated with more rapid progression of HIV disease, but data on the impact of HCV coinfection on HIV disease progression and mortality are controversial [8–11]. Immune defects caused by HCV infection could impact on different markers of HIV disease progression, although this aspect of HIV/HCV interaction has been less studied, with most previous studies addressing only a few markers of HIV progression. Among the different markers, T-cell activation and T-cell apoptosis have been addressed in different studies with discordant results. Thus, some studies have found higher levels of T-cell activation [12–14] and of T-cell apoptosis [14–16] in HCV/HIV coinfection compared to HIV monoinfection, but not others [16–18]. Much more scarce are studies addressing the effect of HCV on other important markers of HIV pathogenesis, such as senescence, cell turnover and maturation stage of T cells [19]. Regarding T-cell exhaustion, which is an important mechanism of T-cell dysfunction in the setting of chronic viral infections [20], it has been explored in HIV [21–24] and HCV [25] infections, as well as in the setting of HIV/HCV coinfection [14, 26, 27]. In this setting it has been shown that HIV increases the level of exhaustion of HCV-specific T cells [26], but no study so far has evaluated the impact of HCV presence on exhaustion of HIV-specific T cells in the HIV/HCV coinfected population. In the present study, we have tested the effect of HCV infection on several aspects of HIV disease pathogenesis, performing an in-depth analysis of several markers associated with HIV progression in a large cohort of individuals including HIV/HCV coinfected and HIV monoinfected patients. The demonstration of HCV-induced impairment of any of these markers, will support early treatment of HCV in order to attenuate or halt not only the accelerated liver injury observed in HIV/HCV coinfected patients, but also the deleterious effect of HCV on HIV disease progression.
PLOS ONE | https://doi.org/10.1371/journal.pone.0173943 March 21, 2017
2 / 11
HIV pathogenesis is influenced by HCV presence
Patients and methods Study population This cross-sectional study included a total of 116 subjects, distributed as follows: 60 patients with chronic HIV infection (HIV group), 36 patients with chronic HIV infection and coinfected with HCV (HIV/HCV goup), and 20 age and sex-matched HIV and HCV seronegative healthy controls. All patients were naïve for anti-HCV treatment and for antiretroviral therapy (ART) at the moment of study. To participate in the study, written informed consent was obtained from all individuals, and the study protocol was evaluated and approved by the Ethical Committee of Hospital Carlos III-La Paz.
Cell samples All analyses were done in cryopreserved peripheral blood mononuclear cells (PBMCs). EDTAanticoagulated blood was obtained by venipuncture; PBMCs were immediately isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) and frozen in FCS plus 10% DMSO. Viability of thawed PBMCs was always greater than 85%.
Immunophenotypic analysis We used a comprehensive approach of simultaneous measurement of different T-cells parameters involved in HIV pathogenesis. For CD8 T cells the next parameters were analysed: level of exhaustion (measuring PD1 and Tim3 expression), and level of activation (measuring CD38 and HLADR expression). For CD4 T cells the next parameters were evaluated: naïve/memory subsets (using CD45RA expression), recent thymic emigrants (RTE, using CD31 expression), cell turnover (using Ki67 expression), apoptosis (using CD95 expression), senescence (using CD57 expression), and exhaustion (using PD1 and Tim3 expression). All parameters were evaluated using multiparameter flow cytometry. PBMCs from each patient were stained with proper antibodies according to the different parameters evaluated. A detailed description of staining conditions is given as S1 Text, a complete list of all monoclonal antibodies and fluorochromes used in the study is shown in S1 Table and a representative flow cytometry experiment illustrating the gating strategies for CD4 and CD8 cell subsets analyses is shown in S1 Fig.
Statistical analysis The main characteristics of the study population, and the different parameters evaluated are expressed as median [interquartile range]. Comparisons between groups were done using Mann-Whitney U-test. Correlations between quantitative parameters were explored using Pearson correlation coefficient, and between qualitative variables using the Chi-square test or Fishers’s exact test as appropriate. All statistical analyses were performed using the SPSS software version 15 (SPSS Inc., Chicago, IL, USA). All p-values were two-tailed, and were considered as significant only when lower than 0.05.
Results Patient’s characteristics The main characteristics of patients at the time of inclusion in the study are shown in Table 1. All patients were naïve for both anti-HCV and ART at the moment of inclusion in the study. There were no significant differences between HIV and HIV/HCV groups in terms of age, CD4 counts, and plasma HIV-RNA load. However, proportion of males was significantly higher in HIV group and the distribution of risk factors for HIV acquisition was also
PLOS ONE | https://doi.org/10.1371/journal.pone.0173943 March 21, 2017
3 / 11
HIV pathogenesis is influenced by HCV presence
Table 1. Characteristics of patients included in the study. Characteristic
HIV group(n = 60)
HIV/HCV group(n = 36)
p-value
Median age (years)
46 [40–51]
48 [45–51]
0.61
Gender (% of males)
93%
69%
0.002
436 [302–702]
411 [321–546]
0.53 0.19
Median CD4 count (cells/μl) Median HIV-RNA (log copies/mL)
4.3 [3.9–4.7]
4.1 [3.4–4.6]
Median HCV-RNA (log copies/mL)
-
6.1 [5.0–6.7]
HCV coinfection contributes to HIV pathogenesis by increasing immune exhaustion in CD8 T-cells Norma Rallo´n1,2*, Marcial Garcı´a1,2, Javier Garcı´a-Samaniego3, Noelia Rodrı´guez1,2, ´ lvarez4, Rosa Garcı´a4, Miguel Go´rgolas4, Alfonso Cabello4, Clara Restrepo1,2, Beatriz A Jose´ M. Benito1,2
a1111111111 a1111111111 a1111111111 a1111111111 a1111111111
1 IIS-Fundacio´n Jime´nez Dı´az, UAM, Madrid, Spain, 2 Hospital Universitario Rey Juan Carlos, Mo´stoles, Spain, 3 Hospital Universitario Carlos III-La Paz, Madrid, Spain, 4 Hospital Universitario Fundacio´n Jime´nez Dı´az, Madrid, Spain * [email protected]
Abstract OPEN ACCESS Citation: Rallo´n N, Garcı´a M, Garcı´a-Samaniego J, Rodrı´guez N, Cabello A, Restrepo C, et al. (2017) HCV coinfection contributes to HIV pathogenesis by increasing immune exhaustion in CD8 T-cells. PLoS ONE 12(3): e0173943. https://doi.org/ 10.1371/journal.pone.0173943 Editor: Lishomwa C. Ndhlovu, University of Hawaii System, UNITED STATES
Background There are several contributors to HIV-pathogenesis or insufficient control of the infection. However, whether HIV/HCV-coinfected population exhibits worst evolution of HIV-pathogenesis remains unclear. Recently, some markers of immune exhaustion have been proposed as preferentially upregulated on T-cells during HIV-infection. Herein, we have analyzed T-cell exhaustion together with several other contributors to HIV-pathogenesis that could be affected by HCV-coinfection.
Patients and methods
Received: November 1, 2016 Accepted: March 1, 2017 Published: March 21, 2017 Copyright: © 2017 Rallo´n et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study has been funded by projects: RD12/0017/0031 and PI14/00518 integrated in the Plan Nacional I+D+I and co-funded by ISCIIISubdireccio´n General de Evaluacio´n and European Regional Development Fund (Fondos FEDER). Norma Rallo´n is a Miguel Servet investigator from the ISCIII (CP14/00198), Madrid, Spain. M Garcı´a is a predoctoral student co-funded by CP14/00198
Ninety-six patients with chronic HIV-infection (60 HIV-monoinfected and 36 HIV/HCV-coinfected), and 20 healthy controls were included in the study. All patients were untreated for both infections. Several CD4 and CD8 T-cell subsets involved in HIV-pathogenesis were investigated. Non-parametric tests were used to establish differences between groups and associations between variables. Multivariate linear regression was used to ascertain the variables independently associated with CD4 counts.
Results HIV-patients presented significant differences compared to healthy controls in most of the parameters analyzed. Both HIV and HIV/HCV groups were comparable in terms of age, CD4 counts and HIV-viremia. Compared to HIV group, HIV/HCV group presented significantly higher levels of exhaustion (Tim3+PD1- subset) in total CD8+ T-cells (p = 0.003), and higher levels of exhaustion in CD8+HLADR+CD38+ (p = 0.04), CD8+HLADR-CD38+ (p = 0.009) and CD8+HLADR-CD38- (p = 0.006) subsets of CD8+ T-cells. Interestingly these differences were maintained after adjusting by CD4 counts and HIV-viremia.
PLOS ONE | https://doi.org/10.1371/journal.pone.0173943 March 21, 2017
1 / 11
HIV pathogenesis is influenced by HCV presence
project and Intramural Research Scholarship from IIS-FJD. N Rodriguez and C Restrepo are funded by project RD12/0017/0031. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conclusions We show a significant impact of HCV-coinfection on CD8 T-cells exhaustion, an important parameter associated with CD8 T-cell dysfunction in the setting of chronic HIV-infection. The relevance of this phenomenon on immunological and/or clinical HIV progression prompts HCV treatment to improve management of coinfected patients.
Competing interests: The authors have declared that no competing interests exist.
Introduction Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) are two of the most relevant persistent infections afflicting the human population, with 37 and 150 million chronically infected individuals worldwide respectively [1]. Coinfection with both viruses is common due to the existence of shared transmission routes [2] and as much as 20–30% of HIV positive patients are coinfected with HCV [1]. Since both viruses interfere with host immune response [3, 4] and interact at the molecular level [5, 6], coinfection poses a formidable challenge to host immune system. As a consequence of this, each virus can impact on the clinical course of the other. In fact, the negative impact of HIV infection on hepatitis C pathogenesis is well described. HIV/HCV coinfection has been associated with lower rates of spontaneous HCV clearance, higher HCV viral load, accelerated liver disease progression and reduced response to interferon-based HCV therapy [1]. Since the introduction of highly active antiretroviral therapy (HAART), the clinical prognosis for HIV patients has markedly improved and, as a result, HCV infection is now a leading cause of morbidity and mortality in HIV/HCV coinfected individuals [7]. This coinfection is probably also associated with more rapid progression of HIV disease, but data on the impact of HCV coinfection on HIV disease progression and mortality are controversial [8–11]. Immune defects caused by HCV infection could impact on different markers of HIV disease progression, although this aspect of HIV/HCV interaction has been less studied, with most previous studies addressing only a few markers of HIV progression. Among the different markers, T-cell activation and T-cell apoptosis have been addressed in different studies with discordant results. Thus, some studies have found higher levels of T-cell activation [12–14] and of T-cell apoptosis [14–16] in HCV/HIV coinfection compared to HIV monoinfection, but not others [16–18]. Much more scarce are studies addressing the effect of HCV on other important markers of HIV pathogenesis, such as senescence, cell turnover and maturation stage of T cells [19]. Regarding T-cell exhaustion, which is an important mechanism of T-cell dysfunction in the setting of chronic viral infections [20], it has been explored in HIV [21–24] and HCV [25] infections, as well as in the setting of HIV/HCV coinfection [14, 26, 27]. In this setting it has been shown that HIV increases the level of exhaustion of HCV-specific T cells [26], but no study so far has evaluated the impact of HCV presence on exhaustion of HIV-specific T cells in the HIV/HCV coinfected population. In the present study, we have tested the effect of HCV infection on several aspects of HIV disease pathogenesis, performing an in-depth analysis of several markers associated with HIV progression in a large cohort of individuals including HIV/HCV coinfected and HIV monoinfected patients. The demonstration of HCV-induced impairment of any of these markers, will support early treatment of HCV in order to attenuate or halt not only the accelerated liver injury observed in HIV/HCV coinfected patients, but also the deleterious effect of HCV on HIV disease progression.
PLOS ONE | https://doi.org/10.1371/journal.pone.0173943 March 21, 2017
2 / 11
HIV pathogenesis is influenced by HCV presence
Patients and methods Study population This cross-sectional study included a total of 116 subjects, distributed as follows: 60 patients with chronic HIV infection (HIV group), 36 patients with chronic HIV infection and coinfected with HCV (HIV/HCV goup), and 20 age and sex-matched HIV and HCV seronegative healthy controls. All patients were naïve for anti-HCV treatment and for antiretroviral therapy (ART) at the moment of study. To participate in the study, written informed consent was obtained from all individuals, and the study protocol was evaluated and approved by the Ethical Committee of Hospital Carlos III-La Paz.
Cell samples All analyses were done in cryopreserved peripheral blood mononuclear cells (PBMCs). EDTAanticoagulated blood was obtained by venipuncture; PBMCs were immediately isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) and frozen in FCS plus 10% DMSO. Viability of thawed PBMCs was always greater than 85%.
Immunophenotypic analysis We used a comprehensive approach of simultaneous measurement of different T-cells parameters involved in HIV pathogenesis. For CD8 T cells the next parameters were analysed: level of exhaustion (measuring PD1 and Tim3 expression), and level of activation (measuring CD38 and HLADR expression). For CD4 T cells the next parameters were evaluated: naïve/memory subsets (using CD45RA expression), recent thymic emigrants (RTE, using CD31 expression), cell turnover (using Ki67 expression), apoptosis (using CD95 expression), senescence (using CD57 expression), and exhaustion (using PD1 and Tim3 expression). All parameters were evaluated using multiparameter flow cytometry. PBMCs from each patient were stained with proper antibodies according to the different parameters evaluated. A detailed description of staining conditions is given as S1 Text, a complete list of all monoclonal antibodies and fluorochromes used in the study is shown in S1 Table and a representative flow cytometry experiment illustrating the gating strategies for CD4 and CD8 cell subsets analyses is shown in S1 Fig.
Statistical analysis The main characteristics of the study population, and the different parameters evaluated are expressed as median [interquartile range]. Comparisons between groups were done using Mann-Whitney U-test. Correlations between quantitative parameters were explored using Pearson correlation coefficient, and between qualitative variables using the Chi-square test or Fishers’s exact test as appropriate. All statistical analyses were performed using the SPSS software version 15 (SPSS Inc., Chicago, IL, USA). All p-values were two-tailed, and were considered as significant only when lower than 0.05.
Results Patient’s characteristics The main characteristics of patients at the time of inclusion in the study are shown in Table 1. All patients were naïve for both anti-HCV and ART at the moment of inclusion in the study. There were no significant differences between HIV and HIV/HCV groups in terms of age, CD4 counts, and plasma HIV-RNA load. However, proportion of males was significantly higher in HIV group and the distribution of risk factors for HIV acquisition was also
PLOS ONE | https://doi.org/10.1371/journal.pone.0173943 March 21, 2017
3 / 11
HIV pathogenesis is influenced by HCV presence
Table 1. Characteristics of patients included in the study. Characteristic
HIV group(n = 60)
HIV/HCV group(n = 36)
p-value
Median age (years)
46 [40–51]
48 [45–51]
0.61
Gender (% of males)
93%
69%
0.002
436 [302–702]
411 [321–546]
0.53 0.19
Median CD4 count (cells/μl) Median HIV-RNA (log copies/mL)
4.3 [3.9–4.7]
4.1 [3.4–4.6]
Median HCV-RNA (log copies/mL)
-
6.1 [5.0–6.7]
