Small alveolar macrophages are infected preferentially by HIV and exhibit impaired phagocytic function

Abstract

HIV-1-infected persons are at higher risk of lower respiratory tract infections than HIV-1-uninfected individuals. This suggests strongly that HIV-infected persons have specific impairment of pulmonary immune responses, but current understanding of how HIV alters pulmonary immunity is incomplete. Alveolar macrophages (AMs), comprising small and large macrophages, are major effectors of innate immunity in the lung. We postulated that HIV-1 impairs pulmonary innate immunity through impairment of AM physiological functions. AMs were obtained by bronchoalveolar lavage from healthy, asymptomatic, antiretroviral therapy-naive HIV-1-infected and HIV-1-uninfected adults. We used novel assays to detect in vivo HIV-infected AMs and to assess AM functions based on the HIV infection status of individual cells. We show that HIV has differential effects on key AM physiological functions, whereby small AMs are infected preferentially by the virus, resulting in selective impairment of phagocytic function. In contrast, HIV has a more generalized effect on AM proteolysis, which does not require direct viral infection. These findings provide new insights into how HIV alters pulmonary innate immunity and the phenotype of AMs that harbors the virus. They underscore the need to clear this HIV reservoir to improve pulmonary immunity and reduce the high incidence of lower respiratory tract infections in HIV-1-infected individuals.

Figure 1

Definition of cell populations in bronchoalveolar lavage (BAL) by flow cytometry. The major cell populations in whole BAL were defined initially by their forward scatter (FSC) and side scatter (SSC) characteristics. The identities of the cell populations were then confirmed by demonstrating the gated cell populations labeled with anti-CD206 (macrophage marker) and anti-CD3 (T-lymphocyte marker), respectively. (a) Representative pseudo-color plot showing the gating strategy for macrophage and lymphocyte populations. (b) Representative dot plot showing the major cell populations in whole BAL (macrophages=blue and lymphocytes=red). (c) Labeling of gated cell populations with macrophage and T-lymphocyte markers, and (d) the proportion of small and large alveolar macrophages (AMs) in BAL from HIV-1-infected and HIV-1-uninfected individuals. Data were analyzed using the Mann–Whitney U-test; black horizontal bars represent medians (HIV−, n=19; HIV+, n=22).

Figure 2

Surface marker expression by small and large alveolar macrophages (AMs) from healthy HIV-1-uninfected participants. Bronchoalveolar lavage (BAL) cells were stained with anti-CD206 fluorescein isothiocyanate (FITC), anti-CD71 PE-Cy5, anti-HLA-DR Alexa fluor 700, anti-CD16 PE, anti-CD14 PE-Cy7, anti-CD11C PE, and anti-CD123 PE-Cy5, and analyzed by flow cytometry. (a) Proportions of small and large AMs expressing these surface markers. (b) The magnitude of surface marker expression by large and small AMs as measured by the geometric mean fluorescence intensity. Data were analyzed using the Wilcoxon matched-pairs signed-ranked test; black horizontal bars represent medians (HIV−, n=10).

Figure 3

Detection of HIV-infected human cells by fluorescence in situ hybridization (FISH). The presence of HIV mRNA in human monocyte-derived macrophages (hMDMs) infected experimentally with the macrophage-tropic strain BaL and in bronchoalveolar lavage (BAL) cells isolated from healthy HIV-infected individuals was detected by flow cytometry using FISH probes against HIV-gag mRNA. (a) Representative pseudo-color plots from experiments to validate the FISH assay for label specificity using uninfected hMDMs and hMDMs infected in vitro with HIV. (b) Whole BAL cells were colabeled with HIV-gag Quasar 670 FISH probes and anti-CD3 antibody, and analyzed by flow cytometry. Alveolar macrophages (AMs) and lymphocyte populations were identified by their forward scatter (FSC) and side scatter (SSC) characteristic properties. T cells were confirmed by CD3 positivity. (c) Detection of HIV-infected cells in AMs and T cells in whole BAL from the same individual (color coded). Data were analyzed using the Wilcoxon matched-pairs signed-rank test; black horizontal bars represent medians (n=6).

Figure 4

Small alveolar macrophages (AMs) are the dominant macrophage population harboring HIV in the alveolar space. Adherent AMs isolated from bronchoalveolar lavage (BAL) cells of healthy, HIV-infected individuals were stained with fluorescence in situ hybridization (FISH) probes against HIV-gag mRNA and analyzed by flow cytometry. AMs were identified by their forward scatter (FSC) and side scatter (SSC) characteristic properties and HIV-infected cells were identified by their HIV-gag mRNA positivity. (a) Representative flow cytometry pseudo-color plots of adherent AMs from HIV-1-uninfected and HIV-1-infected participants. (b) Proportions of HIV-infected AMs in adherent BAL cells from individuals with chronic HIV infection Data were analyzed using the Mann–Whitney U-test; black horizontal bars represent medians (HIV−, n=6; HIV+, n=14).

Figure 5

HIV-infected alveolar macrophages (AMs) do not adhere well to plastic. Whole bronchoalveolar lavage (BAL) and adherent BAL cells were stained with HIV-gag Quasar 670 fluorescence in situ hybridization (FISH) probes and analyzed by flow cytometry. On the basis of forward scatter (FSC) and side scatter (SSC) characteristics of the cells, AMs were identified and the frequency of HIV-infected cells determined. (a) Representative pseudo-color plots showing the gating strategy and the frequency of HIV-infected AMs in whole BAL and adherent BAL cells from the same asymptomatic HIV-infected individual. (b) Comparison of the frequencies of HIV-infected AMs in whole BAL and adherent BAL cells from the same individuals. Data were analyzed using the paired Student's t-test (n=5).

Figure 6

Surface marker expression by HIV-infected alveolar macrophages (AMs) in whole bronchoalveolar lavage (BAL) from HIV-1-infected participants. Whole BAL cells stained with anti-CD206 fluorescein isothiocyanate (FITC), anti-HLA-DR AF700, anti-CD45 PE, and anti-CD71 PE-Cy5 for surface marker expression, and HIV-gag Quasar 670 fluorescence in situ hybridization (FISH) probes to detect HIV-infected cells were analyzed by flow cytometry. AMs were identified by their forward scatter (FSC) and side scatter (SSC) characteristics and the expression of CD206, HLA-DR, CD45 and CD71 by HIV-infected and HIV-uninfected AMs from the same individual was determined. (a) Representative histograms showing expression of CD206, HLA-DR, CD45 and CD71 by HIV-infected and HIV-uninfected AMs. (b) Expression of CD206, HLA-DR, CD45 and CD71 by HIV-infected and HIV-uninfected AMs. Data were analyzed by the Wilcoxon matched-pairs signed-ranked test (n=6).

Figure 7

Assessment of alveolar macrophage (AM) function based on the HIV infection status of participants. Adherent AMs were incubated with reporter beads to measure phagocytosis, superoxide burst, and bulk proteolysis, and analyzed by flow cytometry. (a) Phagocytosis of reporter beads by large and small AMs (HIV−, n=25; HIV+, n=14). (b) Phagosomal superoxide burst activity in AMs from HIV-1-infected compared with HIV-1-uninfected participants (HIV−, n=25; HIV+, n=14). (c) Phagosomal proteolytic activity in AMs from HIV-1-infected compared with HIV-1-uninfected participants (HIV−, n=18; HIV+, n=11). The data in panels b and c are expressed as the Activity Index, which is the ratio of relative fluorescence units of the substrate fluorescence divided by the calibration fluorescence to ensure dosage correction for minor variations in bead number. Data were analyzed by Mann–Whitney U–test (HIV+ vs. HIV−) and Wilcoxon matched-pairs signed-ranked test (small vs. large AMs) (a), or unpaired (HIV+ vs. HIV−) and paired Student's t-test (small vs. large AMs) on log-transformed data (b,c); black horizontal bars represent medians (a) and means (b,c).

Figure 8

Assessment of alveolar macrophage (AM) function based on the HIV infection status of individual cells. Adherent AMs were incubated with reporter beads to measure phagocytosis, superoxide burst, and bulk proteolysis. The cells were then stained with HIV-gag Quasar 670-labeled probes by fluorescence in situ hybridization (FISH) to detect HIV-infected AMs and analyzed by flow cytometry. (a) Representative flow cytometry pseudo-color plots showing phagocytosis of beads by small and large, HIV-infected, and HIV-uninfected AMs. The plots show four different cell populations: HIV-infected cells without beads (Q1), HIV-infected cells with beads (Q2), HIV-uninfected cells with beads (Q3), and HIV-uninfected cells without beads (Q4). (b) Phagocytosis of reporter beads by HIV-infected and HIV-uninfected AMs from the same HIV-infected individuals (n=14). (c,d) Phagosomal superoxide burst and bulk proteolytic activities, respectively, in HIV-infected compared with HIV-uninfected AMs (n=10). The data in panels c and d are expressed as the Activity Index, which is the ratio of relative fluorescence units of the substrate fluorescence divided by the calibration fluorescence to ensure dosage correction for minor variations in bead number. Data were analyzed using matched-pair one-way analysis of variance with Bonferroni correction; black horizontal bars represent medians (b) and means (c,d).