HIV Infection Is Associated with Loss of Anti-Inflammatory Alveolar Macrophages

Abstract

HIV type 1 is associated with pulmonary dysfunction that is exacerbated by cigarette smoke. Alveolar macrophages (AM) are the most prominent immune cell in the alveolar space. These cells play an important role in clearing inhaled pathogens and regulating the inflammatory environment; however, how HIV infection impacts AM phenotype and function is not well understood, in part because of their autofluorescence and the absence of well-defined surface markers. The main aim of this study was to evaluate the impact of HIV infection on human AM and to compare the effect of smoking on their phenotype and function. Time-of-flight mass cytometry and RNA sequencing were used to characterize macrophages from human bronchoalveolar lavage of HIV-infected and -uninfected smokers and nonsmokers. We found that the frequency of CD163⁺ anti-inflammatory AM was decreased, whereas CD163^-CCR7⁺ proinflammatory AM were increased in HIV infection. HIV-mediated proinflammatory polarization was associated with increased levels of inflammatory cytokines and macrophage activation. Conversely, smoking heightened the inflammatory response evident by change in the expression of CXCR4 and TLR4. Altogether, these findings suggest that HIV infection, along with cigarette smoke, favors a proinflammatory macrophage phenotype associated with enhanced expression of inflammatory molecules. Further, this study highlights time-of-flight mass cytometry as a reliable method for immunophenotyping the highly autofluorescent cells present in the bronchoalveolar lavage of cigarette smokers.

Figure 1.. CyTOF reveals that differential expression of CD163, CD206, CCR7 and HLA-DR on AM is mediated by HIV.

BAL from 19 HIV-infected and 19 HIV-seronegative subjects was barcoded and analyzed for 33 markers using CyTOF. (A) Representative dot plots of the gating strategy for alveolar macrophages identified by DNA content, viability, the lack of expression of CD3, CD19 and CD56 and the expression of CD45, CD11b and CD71. (B) Differential analysis between HIV-infected and seronegative subjects of individual macrophage makers on AM. Left frequency, right mean metal intensity (MMI). Statistical significance was calculated by Mann Whitney test. * = p < 0.05, ** = p < 0.005, p = < 0.0005.

Figure 2.. HIV infection is associated with a decrease in CD163⁺CD206⁺ and an increase in CD163⁻CCR7⁺ AM.

Frequency of CD163⁺CD206⁺ anti-inflammatory cells and Mo-AM identified as CD163⁻CCR7⁺ we evaluated in HIV-infected vs. seronegative subjects and correlated with HLA-DR expression on AM (A) Representative dot plots and cumulative data showing frequency and absolute number of CD163⁺CD206⁺ AM cells in uninfected and HIV-infected subjects. (B) Representative dot plots and cumulative data of frequency and absolute number of CD163⁻CCR7⁺ AM cells in uninfected and HIV-infected subjects. (C) Correlation of CD163⁺CD206⁺ frequency and absolute number with mean metal intensity (MMI) of HLA-DR on AM. (D) Correlation of CD163⁻CCR7⁺ frequency and absolute number with MMI of HLA-DR on AM. Statistical significance was calculated by Mann Whitney test and Pearson correlation analysis.

Figure 3.. AM gene expressions confirms anti- and pro-inflammatory phenotype correlate with decreased lung function.

RNAseq was performed on CD71⁺ purified AM from a subset of HIV-infected (10) and uninfected subjects (5). (A) Targeted genes of AM, including CD163, CD204, and CD206, were correlated with the frequency of CD163⁺CD206⁺ AM identified by CyTOF. (B) Cathespin-L gene expression correlated with the frequency of CD163⁺CD206⁺ AM and CD163⁻CCR7⁺ AM. (C) Matrix metalloproteinase protein 9 and 24 gene expression correlated with the frequency of CD163⁺CD206⁺ AM in HIV infected subjects. (D) Forced expiratory flow at 25–27% (FEF 25–75%) correlated with the frequency of CD163+CD206+ AM. Statistical significance was calculated by Pearson correlation analysis

Figure 4.. Correlations of CD163⁻CCR7⁺ and CD163⁺CD206⁺ AM frequencies with TNFα and IL-8 gene expression levels.

The gene expression levels of inflammatory cytokines, TNF-α (ENSG0000232810) and IL-8 (ENSG0000169429), from CD71⁺ purified AM were measured by RNAseq. (A) Differential comparisons of TNF-α and IL-8 gene transcription levels in HIV-uninfected and infected subjects. (B) Correlation of TNF-α gene transcription levels with the frequency of CD163⁺CD206⁺ AM, CD163⁻CCR7⁺ AM and HLA-DR mean metal intensity (MMI) on AM. (C) Correlation IL-8 gene transcriptions levels with the MMI of HLA-DR of AM. Statistical significance was calculated by Mann Whitney test and Pearson correlation analysis.

Figure 5.. Smoking increase CCR2, TLR4, PD-L1 and CXCR4 expression on AM.

(A) HIV-infected smokers, HIV-infected non-smokers, HIV-uninfected smokers and HIV-uninfected nonsmokers were compared for the expression of various surface receptors on AM determined by the mean metal intensity (MMI) and frequency. (B) Representative dot plots of C-C chemokine receptor 2 (CCR2) and Toll like receptor 4 (TLR4) for each cohort. (C) Differential comparison of CCR2, TLR4, Programed-death receptor ligand 1 (PD-L1), and C-X-C chemokine receptor type 4 (CXCR4). Statistical significance was calculated by one-way Anova with multiple comparisons.

Figure 6.. CyTOF analysis identifies common HIV-associated phenotypes in the blood which were associated with the frequency of CD163⁺CD206⁺ AM.

PBMC from 19 HIV-infected and 19 HIV-seronegative subjects was barcoded and analyzed for 35 markers using CyTOF. (A) Analysis between HIV-infected and seronegative subjects of individual makers on CD4+ T cells. Left frequency, right mean metal intensity (MMI). Statistical significance was calculated by Mann Whitney test. * = p < 0.05, ** = p < 0.005, p = < 0.0005. (B) Correlation of the frequency of CD4⁺ T cells, CD4⁺ T cells or CD8⁺ T cells in the blood with CD163⁺CD206⁺ AM in the lung. Statistical significance was calculated by Pearson correlation analysis.