HIV infection impacts immune cells in the lung leading to pulmonary complications which persist with antiretroviral therapy (ART). Alveolar macrophages (AM) are principle immune cells type in bronchoalveolar compartment and as such play a pivotal role in host defense against pathogenic microorganisms and tissue remodeling. Examination of the effect of HIV on AM is complicated by the high prevalence of smoking in HIV infected subjects from the United States. Smoking increases auto-fluorescence of AMs, inhibiting the reliability and resolution of traditional flow cytometry. Cytometry by Time of Flight (CyTOF) utilizes pure metal conjugated antibodies and detection by mass cytometry, which effectively bypasses auto-fluorescence. Here, we utilize CyTOF to comprehensively evaluate the effect of HIV infection and smoking on AM.
Bronchoalveolar lavage (BAL) cells from 10 untreated HIV-infected non-smokers, 9 untreated HIV-infected smokers, 10 HIV-seronegative non-smokers and 9-HIV-seronegative smokers was subjected to traditional flow cytometry and CyTOF. Our CyTOF panel consisted of 34 unique markers and phenotypic analysis was performed using traditional methods and three unbiased clustering algorithms.
Compared to those without HIV we found a decrease in CD206 (p=0.0002), CD71 (p=0.03) and CD164 (p=0.002) positive cells, indicating a loss of alternatively activated AMs (M2) caused by HIV infection. The loss of M2 macrophages indicates an increased inflammatory environment. Smoking increased AM expression of CCR2 (p=0.007) which is a marker of inflammatory macrophages. Together, compared to healthy non-smokers, smoking and HIV increased CXCR4 expression on AM (p=0.004) demonstrating increased susceptibility to X4 tropic HIV infection.
While the aim of characterizing alveolar macrophages during HIV infection and smoking was our primary goal, this study also demonstrates the sensitivity of mass cytometry, and its ability to detect significant differences between patient groups which would have otherwise been masked by auto-fluorescence. Overall, these findings indicate that HIV and smoking drive alveolar macrophages toward an inflammatory state, leading to an overall more inflammatory environment in the lung.