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Innate immunity cell activation in virologically suppressed HIV-infected maraviroc-treated patients

Dentone, Chiaraa,b; Di Biagio, Antonioc; Parodi, Alessiab; Bozzano, Federicab,d; Fraccaro, Paoloe; Signori, Alessiof; Cenderello, Giovannig; Mantia, Eugenioh; Orofino, Giancarloi; De Maria, Andreab,c,j; Filaci, Gilbertob,k; Fenoglio, Danielab,k

doi: 10.1097/QAD.0000000000000194
Research Letter

This is a cross-sectional, case–control study analyzing the effect of antiretroviral therapy (ART) including or not maraviroc, on circulating monocytes and natural killer cells. Sixty-eight HIV-positive patients virologically suppressed receiving ART at least 6 months were subdivided as receiving (group 1) or not (group 2) maraviroc in their ART. Frequency of monocytes and natural killer cells, as well as their activation markers, were studied. Modulation of innate immune cells may be differently affected by combined ART.

aInfectious Diseases Department, Sanremo Hospital, Sanremo

bCenter of Excellence for Biomedical Research (CEBR), University of Genoa

cInfectious Diseases Department, IRCCS San Martino Hospital

dDIMES Department of Experimental Medicine

eDepartment of Informatics, Bioengineering, Robotic and System Engineering (DIBRIS)

fSection of Biostatistics, Department of Health Sciences (DISSAL), University of Genoa

gInfectious Diseases Department, Galliera Hospital, Genoa

hInfectious Diseases Department, SS. Antonio, Biagio, Cesare Arrigo Hospital, Alessandria

iInfectious Diseases Department, Amedeo di Savoia Hospital, Turin


kDepartment of Internal Medicine (DIMI), University of Genoa, Genoa, Italy.

Correspondence to Chiara Dentone, MD, Infectious Diseases Department, Sanremo Hospital, Via Borea, 36 18038 Sanremo, Imperia, Italy. Tel: +39 0184536844; e-mail:

Received 10 August, 2013

Revised 6 January, 2014

Accepted 6 January, 2014

Innate and adaptive immunity respond rapidly to HIV infection [1–4]. Although combined antiretroviral therapy (cART) is able to induce the control of HIV replication, immune activation persists in infected individuals [5–7]. During successful cART, the relationship existing among activated innate immune cells, including natural killer (NK) cells [7,8] and monocytes [9,10], has been less explored. Maraviroc (MVC) seems to exert antiinflammatory effects inducing the decrease of D-dimer and stabilizing C-reactive protein (CRP) [11]. MVC inhibits the migration of innate immune cells [12], which plays a central role in the inflammation-dependent atherosclerosis [10,13,14].

This was an observational, cross-sectional, case–control study performed in eight Infectious Diseases Units. Patients, enrolled from September 2011 to April 2012, provided written informed consent. Inclusion criteria were HIV-RNA 50 copies/ml or less, previous cART regimen not including MVC, actual cART treatment lasting for at least 6 months either including (group 1, G1) or not (group 2, G2) MVC. Patients were paired by age, gender, risk factors, race, TCD4+ cell count, and ratio CD4+/CD8+ cells. One single bleeding per patient was collected.

Exclusion criteria were HIV-2 infection, presence of opportunistic infections or cancers, use of antiinflammatory drugs, chemotherapy, or steroid administration in the previous 6 months.

All parameters were collected in a relational database connected through a web-based interface [15,16]. The normalization of laboratory value ranges applied by the different units was achieved through the z-score method, thus assuring harmonization of data [17].

Phenotypic analyses were performed on fresh blood samples (100 μl) using fluorochrome-conjugated monoclonal antibodies (mAbs). Monocytes were analyzed with CD3-PECy7, CD38-PE, CD11b Horizon V450, CCR2 APC, CD16-Horizon V500, PD-L1 FITC, CD14 APC-Cy7, and HLA-DR-PerCP-Cy 5.5 mAbs (Becton Dickinson, San Diego, California, USA). CD14+CD16++ proinflammatory (pM), CD14++CD16+ intermediate (intM), and CD14++CD16 inflammatory monocytes (iM) were identified [18]. Mean fluorescence intensity (MFI) of CD38, HLA-DR, PD-L1 [19,20], CCR2, and CD11b [21,22] antigen expression was measured. CD3 staining was used to exclude T lymphocytes.

NK cells were analyzed with CD3-APC, CD19-APC, CD14-APC, CD16-PE, and FITC-conjugated (Biolegend, San Diego, California, USA), CD56-PC7 (Immuno-tech-Coulter Marseille, France), anti-NKG2C, (R&D Systems, Minneapolis, Minnesota, USA), anti-NKp46 (IgG1) BAB281, anti-NKp30 (IgG1) 7A6, and anti HLA-DR (IgG2a) D1.12 (gift from Professor R. Accolla, Italy) mAbs. The analyses were performed by a FACS Canto II flow cytometer using FACS Diva (Becton Dickinson) and FlowJo (Tree Star Inc., Ashland, Oregon, USA) software.

Differences between groups for continuous clinical parameters were assessed by Mann–Whitney test and correlations by Spearman's test. To assess the differences between groups, univariate logistic regression analysis with group of treatment as binary dependent variable was adopted and subsequently clinical characteristics with a P value lower or equal to 0.10 were included in a multivariate logistic regression model. Odds ratios (OR) with 95% confidence intervals (CI) were reported.

P values ≤0.05 were considered statistically significant. Analyses were performed using SPSS 18.0 software (SPSS Inc., Chicago, Illinois, USA).

We enrolled 68 patients: 43 (63%) in G1 and 25 (37%) in G2. Main patient's characteristics in G1 and G2 were 28 (65%) and 18 (72%) men, respectively; median age in both groups 49 years; median (interquartile range) BMI 24.4 (21.1–25.5) and 23 (19.7–24.9; P = 0.42), respectively; median nadir CD4+/μl 231 (99–317) and 141 (34–289; P = 0.92), respectively; median HIV-RNA copies/ml prior to the last cART 1630 (98–49 525) and 50 (50–462; P <0.001), respectively; median TCD4+/μl 475 (350–605) and 539 (435–606; P = 0.026), respectively; and median months of last cART 31.8 (20.2–41.3) and 36.3 (24.5–36.6; P = 0.016).

Monocyte analysis showed that iM frequency was decreased in G1 compared with G2 (P = 0.04; Fig. 1a) and no differences in intM, pM, and NK cell frequencies were observed (Fig. 1b–d). CD38 MFI was reduced, whereas PD-L1 MFI was increased on iM of G1 than G2 group (P = 0.03 and P = 0.05; Fig. 1e and f). A negative correlation between CD38 and PD-L1 and CD11b MFI on iM (P <0.0001 and P = 0.0004; Fig. 1g and h) was found.

Fig. 1

Fig. 1

A positive correlation between NK cell and pM frequencies was observed in both groups and in total population. Only in G1 was NK-cell frequency negatively correlated with that of iM (P = 0.03); CD69+ NK-cell percentage negatively correlated with CCR2 and PD-L1 MFI on pM (P = 0.04 and P = 0.03).

In all patients, HLA-DR+NK-cell percentage correlated positively with CD38 and HLA-DR MFI (both P = 0.03), but negatively with PD-L1 MFI on iM (P = 0.006).

In G1, D-dimer was correlated with HLA-DR MFI on iM and pM (P = 0.05 and P = 0.04). Negative correlations were detected between total cholesterol and HLA-DR+NK-cell percentage (P = 0.03), and between triglycerides and CD11b MFI on pM (P = 0.04). Positive correlations were detected between minimum arterial pressure and HLA-DR MFI on iM (P = 0.01) and between BMI and frequency of iM (P = 0.03).

Minimum arterial pressure levels were significantly reduced in G1 compared to G2 at univariate (P = 0.002, odds ratio 0.87, 95% confidence interval 0.79–0.96) and multivariate (P = 0.004, odds ratio 0.76, 95% confidence interval 0.63–0.91) analyses.

In G2, CRP positively correlated with CD69+CD56+NK and NKp30+ cell percentage (P = 0.03 and P = 0.02); D-dimer negatively correlated with HLA-DR+NK-cell percentage and CD11b MFI on iM (P = 0.05 and P = 0.04); BMI correlated positively with CD69+ NK cell (P = 0.04) and negatively with the intM (P = 0.001) percentages.

The results show that frequency and activation of iM was lower in G1 than in G2; activation markers of NK cells correlated positively with activation and negatively with antiinflammatory markers on monocytes in G1; and clinical parameters of inflammation correlated with activation parameters of innate immunity cells.

Indeed, cART has generally poor efficiency in downregulating the activation of monocytes [9,23]. However, lower frequency of iM and lower expression of activation markers were observed in G1 than in G2. These findings suggest that different cARTs may have an impact on innate immunity cells and in modulation of iM.

Our data show that iM and NK-cell frequencies, as well as their expression of inflammatory markers, correlated with cholesterol and triglyceride levels, BMI, and minimal arterial pressure in both groups. Data on BMI corroborate observations linking obesity with inflammation [24].

In conclusion, this study provides original information on how the interaction of HIV infection and effective therapeutic protocols may have a different impact on the innate immune system. Future studies could aim to verify the importance of innate immunity parameters.

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The authors would like to thank all of the patients and collaborators who participated in this study.

Chiara Dentone and Antonio Di Biagio contributed equally to the writing of the article. C.D., A.D.B., D.F, A.D.M, and G.F. designed and performed research, analyzed, interpreted data, and wrote the article; D.F. and A.P. performed monocytes analysis, F.B. performed NK analysis, A.S. performed statistical analysis, P.F. created the database managed through a web-based interface, C.D., A.D.B., G.C., E.M., and G.O. provided blood samples of HIV-infected patients and collected data. Furthermore, the authors want to appoint as collaborators: F. Kalli and F. Battaglia (Center of Excellence for Biomedical Research, Genoa), F. Marras (Gaslini Institute, Genoa), G. Ferrea (Sanremo Hospital, Infectious Diseases Department), M. Giacomini (Department of Informatics, Bioengineering, Robotic and System Engineering DIBRIS, University of Genoa), C. Viscoli (IRCCS San Martino Hospital, Infectious Diseases Department, DISSAL University of Genoa), E. Firpo (IRCCS San Martino Hospital, Infectious Diseases Department), R. Piscopo and G. Cassola (Galliera Hospital, Infectious Diseases Department), V. Bartolacci and G. Casalino Finocchio (Albenga Hospital, Infectious Diseases Department), P. De Leo (Savona Hospital, Infectious Diseases Department), M. Zoppi (Alessandria Hospital, Infectious Diseases Department), M. Guerra (La Spezia Hospital, Infectious Diseases Department), B. Bruzzone (DISSAL, Section of Virology, IRCCS San Martino Hospital), M.P. Sormani (DISSAL, Section of Biostatistics, University of Genoa).

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Conflicts of interest

The study was supported by an unrestricted grant from ViiV Healthcare.

There are no conflicts of interest.

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1. Borrow P, Lewicki H, Hahn BH, Shaw GM, Oldstone MB. Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J Virol 1994; 68:6103–6110.
2. Levy JA. The importance of the innate immune system in controlling HIV infection and disease. Trends Immunol 2001; 22:312–316.
3. Fogli M, Costa P, Murdaca G, Setti M, Mingari MC, Moretta L, et al. Significant NK cell activation associated with decreased cytolytic function in peripheral blood of HIV-1-infected patients. Eur J Immunol 2004; 34:2313–2321.
4. Naranbhai V, Altfeld M, Karim SS, Ndung’u T, Karim QA, Carr WH. Changes in natural killer cell activation and function during primary HIV-1 infection. PLoS One 2013; 8:e53251.
5. Hunt PW, Martin JN, Sinclair E, Bredt B, Hagos E, Lampiris H, et al. T cell activation is associated with lower CD4+ T cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy. J Infect Dis 2003; 187:1534–1543.
6. Costa P, Rusconi S, Mavilio D, Fogli M, Murdaca G, Pende D, et al. Differential disappearance of inhibitory natural killer cell receptors during HAART and possible impairment of HIV-1-specific CD8 cytotoxic T lymphocytes. AIDS 2001; 15:965–974.
7. Lichtfuss GF, Cheng WJ, Farsakoglu Y, Paukovics G, Rajasuriar R, Velayudham P, et al. Virologically suppressed HIV patients show activation of NK cells and persistent innate immune activation. J Immunol 2012; 189:1491–1499.
8. Bisio F, Bozzano F, Marras F, Di Biagio A, Moretta L, De Maria A. Successfully treated HIV-infected patients have differential expression of NK cell receptors (NKp46 and NKp30) according to AIDS status at presentation. Immunol Lett 2013; 152:16–24.
9. Hearps AC, Maisa A, Cheng WJ, Angelovich TA, Lichtfuss GF, Palmer CS, et al. HIV infection induces age-related changes to monocytes and innate immune activation in young men that persist despite combination antiretroviral therapy. AIDS 2012; 26:843–853.
10. Martin GE, Gouillou M, Hearps AC, Angelovich TA, Cheng AC, Lynch F, et al. Age-associated changes in monocyte and innate immune activation markers occur more rapidly in HIV infected women. PLoS One 2013; 8:e55279.
11. Funderburg N, Kalinowska M, Eason J, Goodrich J, Heera J, Mayer H, et al. Effects of maraviroc and efavirenz on markers of immune activation and inflammation and associations with CD4+ cell rises in HIV-infected patients. PloS One 2010; 5:e13188.
12. Rossi R, Lichtner M, De Rosa A, Sauzullo I, Mengoni F, Massetti AP, et al. In vitro effect of antihuman immunodeficiency virus CCR5 antagonist maraviroc on chemotactic activity of monocytes, macrophages and dendritic cells. Clin Exp Immunol 2011; 166:184–190.
13. Cipriani S, Francisci D, Mencarelli A, Renga B, Schiaroli E, D’Amore C, et al. Efficacy of the CCR5 antagonist maraviroc in reducing early, ritonavir-induced atherogenesis and advanced plaque progression in mice. Circulation 2013; 127:2114–2124.
14. Crowe SM, Westhorpe CL, Mukhamedova N, Jaworowski A, Sviridov D, Bukrinsky M. The macrophage: the intersection between HIV infection and atherosclerosis. J Leukoc Biol 2010; 87:589–598.
15. Fraccaro P, Dentone C, Fenoglio D, Giacomini M. Multicentre clinical trials’ data management: a hybrid solution to exploit the strengths of electronic data capture and electronic health records systems. Inform Health Soc Care 2013; 38:313–329.
16. Fraccaro P, Pupella V, Gazzarata R, Dentone C, Cenderello G, De Leo P, et al. The Ligurian Human Immunodeficiency Virus Clinical Network: a web tool to manage patients with human immunodeficiency virus in primary care and multicenter clinical trials. Medicine 2.0 2013; 2:e5 .
17. Chuang-Stein C. Summarizing laboratory data with different reference ranges in multi-center clinical trials. Drug Inform J 1992; 26:77–84.
18. Ziegler-Heitbrock L, Ancuta P, Crowe S, Dalod M, Grau V, Hart DN, et al. Nomenclature of monocytes and dendritic cells in blood. Blood 2010; 116:e74–e80.
19. Musso T, Deaglio S, Franco L, Calosso L, Badolato R, Garbarino G, et al. CD38 expression and functional activities are up-regulated by IFN-gamma on human monocytes and monocytic cell lines. J Leukoc Biol 2001; 69:605–612.
20. Cheng X, Veverka V, Radhakrishnan A, Waters LC, Muskett FW, Morgan SH, et al. Structure and interactions of the human programmed cell death 1 receptor. J Biol Chem 2013; 288:11771–11785.
21. Weber C, Belge KU, von Hundelshausen P, Draude G, Steppich B, Mack M, et al. Differential chemokine receptor expression and function in human monocyte subpopulations. J Leukoc Biol 2000; 67:699–704.
22. Dosquet C, Weill D, Wautier JL. Molecular mechanism of blood monocyte adhesion to vascular endothelial cells. Nouv Rev Fr Hematol 1992; 34 (Suppl):S55–S59.
23. Burdo TH, Lentz MR, Autissier P, Krishnan A, Halpern E, Letendre S, et al. Soluble CD163 made by monocyte/macrophages is a novel marker of HIV activity in early and chronic infection prior to and after antiretroviral therapy. J Infect Dis 2011; 204:154–163.
24. Calder PC, Ahluwalia N, Brouns F, Buetler T, Clement K, Cunningham K, et al. Dietary factors and low-grade inflammation in relation to overweight and obesity. Br J Nutr 2011; 106 (Suppl 3):S5–S78.
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