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) . MVC inhibits the migration of innate immune cells , 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 .
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 . 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.
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 .
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.
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).
Conflicts of interest
The study was supported by an unrestricted grant from ViiV Healthcare.
There are no conflicts of interest.
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