Altered Monocyte Phenotype in HIV-1 Infection Tends to Normalize with Integrase-Inhibitor-Based Antiretroviral Therapy

Abstract

Background: Monocytes are increasingly implicated in the inflammatory consequences of HIV-1 disease, yet their phenotype following antiretroviral therapy (ART) initiation is incompletely defined. Here, we define more completely monocyte phenotype both prior to ART initiation and during 48 weeks of ART.

Methods: Cryopreserved peripheral blood mononuclear cells (PBMCs) were obtained at baseline (prior to ART initiation) and at weeks 12, 24, and 48 of treatment from 29 patients participating in ACTG clinical trial A5248, an open label study of raltegravir/emtricitibine/tenofovir administration. For comparison, cryopreserved PBMCs were obtained from 15 HIV-1 uninfected donors, each of whom had at least two cardiovascular risk factors. Thawed samples were stained for monocyte subset markers (CD14 and CD16), HLA-DR, CCR2, CX3CR1, CD86, CD83, CD40, CD38, CD36, CD13, and CD163 and examined using flow cytometry.

Results: In untreated HIV-1 infection there were perturbations in monocyte subset phenotypes, chiefly a higher frequency and density (mean fluorescence intensity-MFI) of HLA-DR (%-p = 0.004, MFI-p = .0005) and CD86 (%-p = 0.012, MFI-p = 0.005) expression and lower frequency of CCR2 (p = 0.0002) expression on all monocytes, lower CCR2 density on inflammatory monocytes (p = 0.045) when compared to the expression and density of these markers in controls' monocytes. We also report lower expression of CX3CR1 (p = 0.014) on patrolling monocytes at baseline, compared to levels seen in controls. After ART, these perturbations tended to improve, with decreasing expression and density of HLA-DR and CD86, increasing CCR2 density on inflammatory monocytes, and increasing expression and density of CX3CR1 on patrolling monocytes.

Conclusions: In HIV-1 infected patients, ART appears to attenuate the high levels of activation (HLA-DR, CD86) and to increase expression of the chemokine receptors CCR2 and CX3CR1 on monocyte populations. Circulating monocyte phenotypes are altered in untreated infection and tend to normalize with ART; the role of these cells in the inflammatory environment of HIV-1 infection warrants further study.

Fig 1. Gating strategy for flow cytometry and comparison between fresh and cryopreserved monocyte surface marker expression.

(A) Shown are isotype control dot plots and CD14 and CD16 expression in freshly obtained and cryopreserved PBMC from the same healthy volunteer and the expression of CX3CR1 on each monocyte subset in the cryopreserved sample. Monocyte subsets were gated using the isotype staining as a guide, as seen in the farthest left panel. Traditional monocytes are in purple, inflammatory monocytes are in pink, patrolling monocytes are in green. Gates are drawn based on negative isotype staining using Rat IgG2b, in the case of CX3CR1. (B) Summary surface marker expression, both proportion and MFI, in fresh and cryopreserved PBMCs on total monocytes of healthy control subjects, with medians, are shown. Each shape (triangle, square, diamond, and circle) represents an individual healthy control subject. (C) Summary surface marker expression, both proportion and MFI, in fresh and cryopreserved PBMCs on total monocytes of virologically suppressed HIV-infected subjects, with medians, are shown. Each shape (triangle, square, diamond, and circle) represents an individual HIV-infected subject.

Fig 2. Monocyte subset proportions at baseline and after ART initiation compared to proportions among controls.

(A) Jitterplot comparing the subset proportions in HIV-1-infected individuals prior to ART initiation and subset proportions in controls. Medians are shown, and p values were determined using Mann Whitney U tests. Figs B-D display Tukey boxplots of medians and interquartile ranges. Outliers are shown as open circles. Tukey boxplots show the proportions of traditional monocytes (B), inflammatory monocytes (C) and patrolling monocytes (D) in controls (red) and in HIV-1-infected subjects at baseline and over the course of 48 weeks of ART.

Fig 3. Expression and density (MFI) of HLA-DR on patient monocytes at baseline and after ART initiation compared to values among controls.

Values for frequency and density of HLA-DR on monocyte subsets in untreated HIV-1 infection were compared to levels among controls (in red) using Mann Whitney U tests, and baseline values among patients were compared to values on ART using GEE and Signed Rank test (see boxed legend). Figures show Tukey boxplots of medians and interquartile ranges, outliers are shown as open circles. HLA-DR densities and proportions were increased at baseline when compared to the levels seen in the controls (A-G). Tukey boxplots show the proportions of HLA-DR+ total monocytes (A), traditional monocytes (C), inflammatory monocytes (E), and patrolling monocytes (G) in controls’ samples (red) and in patient samples at baseline and after ART initiation. Tukey boxplots show the density of HLA-DR on total monocytes (B), traditional monocytes (D), inflammatory monocytes (F), and patrolling monocytes (H) in controls’ samples (red) and in patient samples at baseline and after ART initiation.

Fig 4. Expression and density (MFI) of CD86 on patient monocytes at baseline and after ART initiation compared to values among controls.

Values for frequency and density of CD86 on monocyte subsets in untreated HIV-1 infection were compared to levels among controls (in red) using Mann Whitney U tests, and baseline values among patients were compared to values on ART using GEE and Signed Rank test (see boxed legend). Figures show Tukey boxplots of medians and interquartile ranges; outliers are shown as open circles. Tukey boxplots show the proportion of CD86+ total monocytes (A), traditional monocytes (C), inflammatory monocytes (E), and patrolling monocytes (G) in control samples (red) and in patient samples at baseline and after ART initiation. Tukey boxplots show the density of CD86 on total monocytes (B), traditional monocytes (D), inflammatory monocytes (F), and patrolling monocytes (H) in control samples (red) and in patient samples at baseline and after ART initiation.

Fig 5. Expression and density (MFI) of CCR2 on patient monocytes at baseline and after ART initiation compared to values among controls.

Values for frequency and density of CCR2 on monocyte subsets in untreated HIV-1 infection were compared to levels among controls using Mann Whitney U tests, and baseline values among patients were compared to values after ART initiation using GEE and Signed Rank test (see boxed legend). Figures show boxplots of medians and interquartile ranges; outliers are shown as open circles. Tukey boxplots show the proportion of CCR2+ total monocytes (A), traditional monocytes (C), inflammatory monocytes (E), and patrolling monocytes (G) in control samples (red) and in patient samples at baseline and after ART initiation. Tukey boxplots show the density of CCR2 on total monocytes (B), traditional monocytes (D), inflammatory monocytes (F), and patrolling monocytes (H) in control samples (red) and in patient samples at baseline and after ART initiation.

Fig 6. Expression and density (MFI) of CX3CR1 on patient monocytes at baseline and after ART initiation compared to values among controls.

Values for frequency and density of CX3CR1 on monocytes in untreated HIV-1 infection were compared to levels among controls using the Mann Whitney U test, and baseline values were compared to values after ART initiation using GEE and Signed Rank test. Figures show boxplots of medians and interquartile ranges; outliers are shown as open circles. Tukey boxplots show the proportion of CX3CR1+ total monocytes (A), traditional monocytes (C), inflammatory monocytes (E), and patrolling monocytes (G) in control samples (red) and in patient samples at baseline and after ART initiation. Tukey boxplots show the density of CX3CR1 on total monocytes (B), traditional monocytes (D), inflammatory monocytes (F), and patrolling monocytes (H) in control samples (red) and in patient samples at baseline and after ART initiation.