Chronic immune activation and inflammation are a hallmark of HIV infection [1,2] and represent a key component of HIV pathogenesis . Such persistent immune activation has been shown to increase cardiovascular risk, favors loss of bone mineral density, neurocognitive disorders or increase incidence of malignant diseases [4–15]. Although combined antiretroviral therapy (cART) is efficient in reducing both cellular and soluble activation markers, some of them remain elevated in virologically controlled patients [16–21]. Whether a step further down in viral suppression of plasma HIV-RNA can be associated to a lesser degree of immune activation and inflammation is an important question. In this cross-sectional study, we aimed to characterize the residual immune activation and inflammation using cellular and soluble markers in HIV-infected individuals with maximally suppressed viremia (viral load ≤20 copy/ml) compared with results in healthy blood donors. We also evaluated whether residual immune activation and inflammation were different depending on having an HIV-1 plasma viral load below or above 1 copy/ml.
HIV-infected individuals under cART for at least 6 months who achieved a controlled viral load were enrolled consecutively during a 1.5-month period (from 2 February, 2010 to 19 March, 2010) in the Infectious Diseases Department of the Pitié Salpêtrière Hospital, in Paris, France, and compared with 20 HIV-negative healthy blood donors whose samples were provided by the Etablissement Français du Sang of the Pitié Salpêtrière Hospital. T cell classical activation markers were studied on fresh peripheral blood by multiparameter flow cytometry (CD4-PC7, CD8-FITC, HLA-DR-APC-Cy7, CD38-PE, CD3-APC on a FACS Canto I; Becton Dickinson, Franklin Lakes, New Jersey, USA). Monocytes were subdivided on the basis of CD14-PerCP (BD Biosciences, San Diego, California, USA) and CD16-PC7 (Beckman Coulter, Fullerton, California, USA) expression into classical (CD14+ CD16−), intermediate (CD14+ CD16+/low) and nonclassical (CD14low CD16hi) monocytes. HLA-DR-APC-Cy7, CD38-PE, and CD11b-APC (BD Biosciences) activation markers were studied on subpopulations. A large panel of soluble immune activation and inflammation markers was studied in plasma including MCP-1/CCL2, IP-10/CXCL10 and MIG/CXCL9 (BD CBA, FACS Canto I, Becton Dickinson, Franklin Lakes), and interleukin-6, interleukin-18 and sCD14 (ELISA arrays R&D Quantikine HS, R&D Systems, Minneapolis, Minnesota, USA). Plasma HIV RNA was measured using an ultrasensitive technique (threshold ≤1 copy/ml), as previously described . HIV-infected patients were classified in 2 groups according to the level of plasma viral load; viral load less than or equal to 1 copy/ml (n = 35) and 1 <viral load <20 copies/ml (n = 16). Activation and inflammation markers were compared between each of these two HIV-infected groups and the group of HIV-negative healthy blood donors and between the two HIV-infected groups using Mann–Whitney U test. Association between activation and inflammation markers and immunovirological parameters, in HIV-infected patients, was assessed using a nonparametric Spearman test. All reported P values were two-tailed, with a significance level of 0.05. Analyses were performed with SPSS version 19.0 for Windows (SPSS Inc, Chicago, Illinois, USA).
A total of 51 HIV-infected patients were enrolled, 35 males and 16 females with the following baseline characteristics: median age 47 years [interquartile range (IQR), 42–56], Nadir CD4 cell count 212 cells/mm3 (IQR, 99–313), antiretroviral therapy duration 8.9 years (IQR, 4.0–13.7), time of undetectable viral load 2.0 years (IQR, 0.9–4.4), current CD4+ cell count 593 cells/mm3 (IQR, 407–750), current CD8+ cell count 697 cells/mm3 (IQR, 537–897), current CD4+/CD8+ ratio 0.8 (IQR, 0.6–1.2). Four were HCV and two were HBV coinfected (one with HCV and HBV coinfection).
Both persistent abnormal CD4+ and CD8+ T cell activation with regard to HLA-DR expression was observed in the viral load less than or equal to 1 copy/ml group. Abnormal CD4+ but not CD8+ T cell activation was observed in the 1< viral load ≤20 cp/mL group, when compared with HIV-negative healthy blood donors (Fig. 1a and b). Overall, the CD38 expression was normal in both HIV+ groups (data not shown). The results were similar when excluding the five patients with HCV and/or HBV co-infection (data not shown). Monocyte subpopulations (classical, intermediate, and nonclassical) did not differ between HIV-infected patients and HIV-negative healthy blood donors, as well as the expression of activation markers CD38, HLA-DR and CD11b (data not shown). However, IP-10, MIG and sCD14 plasma levels were significantly higher in both groups of HIV-infected patients when compared with HIV-negative subjects (Fig. 1c, d, and e), whereas other plasma inflammation markers such as interleukin-6, interleukin-18 and MCP-1 (monocyte chemoattractant protein-1) were not different between groups (data not shown). When considering the whole HIV+ group, the CD4 nadir (ρ = −0.379, P = 0.006 Fig. 1F) and the current CD4 cell counts (ρ = −0.464; P = 0.0006 Fig. 1G) were found to be significantly associated with the proportion of HLA-DR+CD4 cells.
Taken together, these results show for the first time that an immune CD4 and CD8 T-cell activation characterized by an increased HLA-DR expression but not by an elevated CD38 expression persists despite an optimal virological control supported by the ultrasensitive viral load less than or equal to 1 copy/ml in HIV-infected patients on cART for nearly 10 years. This discrepancy without CD38 expression on CD8 T cells may be linked to undetectable viral loads below 1 copy/ml [23,24], whereas persisting low grade antigenic presentation by defective viruses could lead to HLA-DR expression on T cells [25,26]. Here we demonstrate that the current CD4 activation measured by HLA-DR expression is driven by both the CD4 T cell depletion at time of nadir and the current CD4 T cell depletion. In multivariable linear regression analysis, CD4+HLA-DR+ cells were independently associated with these two parameter (β = –0.14, P = 0.05 for nadir CD4 T cell count and β = –0.77, P = 0.001 for current CD4 T cell count). Noteworthy, the current CD4 T cell count was most strongly associated with the CD4 T cell activation. Both immune parameters are hallmarks of immune reconstitution, suggesting the residual CD4 activation observed here might reflect ongoing immune restoration. This T cell activation goes along with innate activation markers, which remain elevated independently of the CD4 nadir or counts, that is the interferon-γ-inducible chemokines, IP-10 (interferon-γ-inducible protein 10), MIG (monokine induced by γ interferon), and the sCD14 level, despite the absence of monocytic activation.
These results also show some dissociation between activation and inflammatory markers in context of full viral suppression even below 1 copy/ml, and raise the hypothesis that undetectable virus production, current lymphoid homeostatic proliferation and some residual gut mucosal lymphoid alterations differentially influence this residual immune activation. Finally, these results emphasize more than ever the need to treat HIV infection as soon as possible to avoid the establishment of the permanent scar of the initial CD4+ T cell depletion.
Conflicts of interest
This work has been partially presented to the CROI 2011 International Conference in Boston, Massachusetts, USA (Abstract #D-198).
There are no conflict of interest.
Authors Contributions: A.G., C.D., C.K., B.A., D.C. and G.C. designed the study and wrote the article; A.G., C.D., S.S., R.C., C.S., A.G.M. and V.C. selected patients; C.P., C.D. and A.G. performed immunological assays; C.S., A.G.M. and V.C. did the virological assays. L.A., C.D. and D.C. performed the statistical analysis.
1. Paiardini M, Müller-Trutwin M. HIV-associated chronic immune activation
. Immunol Rev
2. Hunt PW. HIV and inflammation: mechanisms and consequences
. Curr HIV/AIDS Rep
3. Hunt PW. Role of immune activation in HIV pathogenesis
. Curr HIV/AIDS Rep
4. Kaplan RC, Sinclair E, Landay AL, Lurain N, Sharrett AR, Gange SJ, et al. T cells activation predicts carotid artery stiffness among HIV infected women
5. Deeks SG. HIV infection, inflammation, immunosenescence, and aging
. Ann Rev Med
6. Lau B, Sharrett AR, Kingsley LA, Post W, Palella FJ, Visscher B, Gange SJ. C-reactive protein is a marker for human immunodeficiency virus disease progression
. Arch Inter Med
7. Kuller LH, Tracy R, Belloso W, De Wit S, Drummond F, Lane HC, et al. INSIGHT SMART Study Group. Inflammatory and coagulation biomarkers and mortality in patients with HIV infection
. PLoS Med
8. Deeks SG. Immune dysfunction, inflammation, and accelerated aging in patients on antiretroviral therapy
. Top HIV Med
9. Deeks SG, Phillips AN. HIV infection, antiretroviral treatment, ageing, and non-AIDS related morbidity
10. Dauby N, De Wit S, Delforge M, Necsoi VC, Clumeck N. Characteristics of non-AIDS-defining malignancies in the HAART era: a clinico-epidemiological study
. J Int AIDS Soc
11. Ouedraogo DE, Makinson A, Kuster N, Nagot N, Rubbo PA, Bollore K, et al. Tuaillon. Increased T-cell activation and Th1 cytokine concentrations prior to the diagnosis of B-cell lymphoma in HIV infected patients
. EJ Clin Immunol
12. d’Ettorre G, Paiardini M, Ceccarelli G, Silvestri G, Vullo V. HIV-associated immune activation: from bench to bedside
. AIDS Res Hum Retroviruses
13. Longenecker CT, Jiang Y, Orringer CE, Gilkeson RC, Debanne S, Funderburg NT, et al. Soluble CD14 is independently associated with coronary calcification and extent of subclinical vascular disease in treated HIV infection
14. Funderburg NT, Zidar DA, Shive C, Lioi A, Mudd J, Musselwhite LW, et al. Shared monocyte subset phenotypes in HIV-1 infection and in uninfected subjects with acute coronary syndrome
15. Erlandson KM, O’Riordan M, Labbato D, McComsey GA. Relationships between inflammation, immune activation, and bone health among HIV-infected adults on stable antiretroviral therapy
. J Acquir Immune Defic Syndr
16. Méndez-Lagares G, Romero-Sánchez MC, Ruiz-Mateos E, Genebat M, Ferrando-Martínez S, Muñoz-Fernández MÁ, et al. Long-term suppressive combined antiretroviral treatment does not normalize the serum level of soluble CD14
. J Infect Dis
17. Dentone C, Di Biagio A, Parodi A, Bozzano F, Fraccaro P, Signori A, et al. Innate immunity cell activation in virologically suppressed HIV-infected maraviroc-treated patients
18. Timmons T, Shen C, Aldrovandi G, Rollie A, Gupta SK, Stein JH, Dubé MP. Microbial translocation and metabolic and body composition measures in treated and untreated HIV infection
. AIDS Res Hum Retroviruses
19. Hattab S, Guihot A, Guiguet M, Fourati S, Carcelain G, Caby F, et al. Comparative impact of antiretroviral drugs on markers of inflammation and immune activation during the first two years of effective therapy for HIV-1 infection: an observational study
. BMC Infect Dis
20. Noel N, Boufassa F, Lécuroux C, Saez-Cirion A, Bourgeois C, Dunyach-Remy C, et al. ANRS C021 CODEX Study Group. Elevated IP10 levels are associated with immune activation and low CD4+ T-cell counts in HIV controller patients
21. Bastard JP, Soulié C, Fellahi S, Haïm-Boukobza S, Simon A, Katlama C, et al. Circulating interleukin-6 levels correlate with residual HIV viraemia and markers of immune dysfunction in treatment-controlled HIV-infected patients
. Antivir Ther
22. Palmer S, Wiegand AP, Maldarelli F, Bazmi H, Mican JM, Polis M, et al. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma
. J Clin Microbiol
23. Campillo-Gimenez L, Assoumou L, Valantin MA, Pajanirassa P, Villemonteix J, Soulié C, et al. ROCnRAL ANRS 157 Study Group. Switch to maraviroc/raltegravir dual therapy leads to an unfavorable immune profile with low-level HIV viremia
24. Benito JM, López M, Lozano S, Martinez P, González-Lahoz J, Soriano V. CD38 expression on CD8 T lymphocytes as a marker of residual virus replication in chronically HIV-infected patients receiving antiretroviral therapy
. AIDS Res Hum Retroviruses
25. Moriya N, Sanjoh K, Yokoyama S, Hayashi T. Mechanisms of HLA-DR antigen expression in phytohemagglutinin-activated T cells in man Requirement of T cell recognition of self HLA-DR antigen expressed on the surface of monocytes
. J Immunol
26. Fourati S, Lambert-Niclot S, Soulie C, Malet I, Valantin MA, Descours B, et al. HIV-1 genome is often defective in PBMCs and rectal tissues after long-term HAART as a result of APOBEC3 editing and correlates with the size of reservoirs
. J Antimicrob Chemother