Background:The lungs were historically identified as one of the major anatomic sites for HIV replication in the pre-antiretroviral therapy (ART) era. However, their contribution to HIV persistence in individuals under suppressive ART remains understudied.Design:We assessed HIV persistence and comprehensively characterized pulmonary mucosal CD4+ T cells in HIV-infected (HIV+) individuals receiving long-term suppressive ART versus uninfected participants.Methods:Bronchoalveolar lavage (BAL), bronchial biopsies, and matched peripheral blood were obtained from n = 24 HIV-infected adults receiving long-term suppressive ART (median: 9 years) and n = 8 healthy volunteers without respiratory symptoms. HIV-DNA and cell-associated HIV-RNA were quantified by ultra-sensitive PCR, and lung mucosal CD4+ T-cell subsets were characterized by multiparameter flow cytometry.Results:The levels of HIV-DNA were 13-fold higher in total BAL cells compared to blood. Importantly, FACS-sorted CD4+ T cells from BAL contained greater levels of HIV-DNA compared to peripheral CD4+ T cells. BAL CD4+ T cells in HIV+ individuals were characterized mostly by an effector memory phenotype, whereas naive and terminally differentiated cells were underrepresented compared to blood. Furthermore, BAL CD4+ T cells expressed higher levels of immune activation (HLA-DR/CD38) and senescence (CD57) markers. Importantly, BAL was enriched in T-cell subsets proposed to be preferential cellular HIV reservoirs, including memory CD4+CCR6+, Th1Th17 (CD4+CCR6+CCR4−CXCR3+), CD4+CCR6+CXCR3−CCR4−, and CD4+CD32a+ T cells.Conclusion:The pulmonary mucosa represents an important immunological effector site highly enriched in activated and preferential CD4+ T-cell subsets for HIV persistence during long-term ART in individuals without respiratory symptoms. Our findings raise new challenges for the design of novel HIV eradication strategies in mucosal tissues.

No abstract

No abstract

Macrophages are a target of human immunodeficiency virus type 1 (HIV-1) and may serve as a viral reservoir during antiretroviral therapy (ART). Their susceptibility to HIV-1 infection is subject to variations from permissiveness to resistance depending on their origin, tissue localization, and polarization profile. This is in part due to the expression of regulatory microRNAs. Here, we identify two microRNA paralogs, microRNA 103 (miR-103) and miR-107, as regulators of CCR5 expression that are upregulated in noninfected bystander cells of HIV-1-infected-monocyte-derived macrophage (MDM) cultures. Transfection of microRNA 103 mimics in MDMs reduced CCR5 expression levels and inhibited CCR5-dependent HIV-1 entry, whereas the corresponding antagomirs enhanced virus spread in HIV-infected MDMs. Treatment of MDMs with interleukin-1β (IL-1β) enhanced microRNA 103 expression, a condition that we found contributed to the reduction of CCR5 mRNA in IL-1β-exposed MDMs. Interestingly, we show that the induction of miR-103/107 expression is part of a tumor suppressor p53 response triggered by secreted IL-1β that renders macrophages refractory to HIV-1 entry. In a more physiological context, the levels of microRNAs 103 and 107 were found enriched in tissue-resident colon macrophages of healthy donors and alveolar macrophages of individuals under antiretroviral therapy, conceivably contributing to their relative resistance to HIV-1 infection. Overall, these findings highlight the role of p53 in enforcing proinflammatory antiviral responses in macrophages, at least in part, through miR-103/107-mediated downmodulation of CCR5 expression and HIV-1 entry. IMPORTANCE Macrophages are heterogeneous immune cells that display varying susceptibilities to HIV-1 infection, in part due to the expression of small noncoding microRNAs involved in the posttranscriptional regulation of gene expression and silencing. Here, we identify microRNAs 103 and 107 as important p53-regulated effectors of the antiviral response triggered by the proinflammatory cytokine IL-1β in macrophages. These microRNAs, which are enriched in colon macrophages of healthy donors and alveolar macrophages of HIV-infected individuals under antiretroviral therapy, act as inhibitors of HIV-1 entry through their capacity to downregulate the CCR5 coreceptor. These results highlight the important role played by miR-103/107 in modulating CCR5 expression and HIV-1 entry in macrophages. They further underscore a distinct function of the tumor suppressor p53 in enforcing proinflammatory antiviral responses in macrophages, thus providing insight into a cellular pathway that could be targeted to limit the establishment of viral reservoirs in these cells.

The lungs are relatively unexplored anatomical HIV reservoirs in the antiretroviral therapy (ART) era. Double negative (DN) T-cells are a subset of T-cells which lack expression of CD4 and CD8 (CD4-CD8-) and may play both regulatory and effector functions during HIV infection. Notably, circulating DN T-cells were previously described as cellular HIV reservoirs. Here, we undertook a thorough analysis of pulmonary versus blood DN T-cells of people living with HIV (PLWH) under ART. Bronchoalveolar lavage (BAL) fluid and matched peripheral blood were collected from n=35 PLWH on ART and n=16 uninfected volunteers without respiratory symptoms. Both PLWH and HIV- adults displayed higher frequencies of DN T-cells in BAL versus blood, and these cells mostly exhibited an effector memory phenotype. In PLWH, pulmonary mucosal DN T-cells expressed higher levels of HLA-DR and several cellular markers associated with HIV persistence (CCR6, CXCR3 and PD-1) compared to blood. We also observed that DN T-cells were less senescent (CD28-CD57+) and expressed less immunosuppressive ectonucleotidase (CD73/CD39), granzyme B and perforin in the BAL compared to the blood of PLWH. Importantly, FACS-sorted DN T-cells from the BAL of PLWH under suppressive ART harbored HIV-DNA. Using the humanized bone marrow-liver-thymus (hu-BLT) mouse model of HIV infection, we observed higher infection frequencies of lung DN T-cells compared to blood and spleen in both early and late HIV infection. Overall, our findings show that HIV is seeded in pulmonary mucosal DN T-cells early following infection and persists in these potential cellular HIV reservoirs even during long-term ART. Importance Reservoirs of HIV during ART are the primary reasons why HIV/AIDS remains an incurable disease. Indeed, HIV remains latent and unreachable by antiretrovirals in cellular and anatomical sanctuaries, preventing its eradication. The lungs have received very little attention compared to other anatomical reservoirs, despite being immunological effector sites exhibiting characteristics ideal for HIV persistence. Furthermore, PLWH suffer from a high burden of pulmonary non-opportunistic infections suggesting impaired pulmonary immunity despite ART. Meanwhile, various immune cell populations have been proposed to be cellular reservoirs in blood, including CD4-CD8- DN T-cells, a subset that may originate from CD4 downregulation by HIV proteins. The present study aims to describe DN T-cells in human and humanized mice lungs in relation to intrapulmonary HIV burden. The characterization of DN T-cells as cellular HIV reservoirs and the lungs as an anatomical HIV reservoir will contribute towards the development of targeted HIV eradication strategies.

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

People living with HIV have high burdens of chronic lung disease, lung cancers, and pulmonary infections despite antiretroviral therapy (ART). The rates of tobacco smoking by people living with HIV vastly exceed that of the general population. Furthermore, we showed that HIV can persist within the lung mucosa despite long-term ART. As CD8 T cell cytotoxicity is pivotal for controlling viral infections and eliminating defective cells, we explored the phenotypic and functional features of pulmonary versus peripheral blood CD8 T cells in ART-treated HIV + and uninfected controls. Bronchoalveolar lavage fluid and matched blood were obtained from asymptomatic ART-treated HIV + smokers (n = 11) and nonsmokers (n = 15) and uninfected smokers (n = 7) and nonsmokers (n = 10). CD8 T cell subsets and phenotypes were assessed by flow cytometry. Perforin/granzyme B content, degranulation (CD107a expression), and cytotoxicity against autologous Gag peptide-pulsed CD4 T cells (Annexin V + ) following in vitro stimulation were assessed. In all groups, pulmonary CD8 T cells were enriched in effector memory subsets compared with blood and displayed higher levels of activation (HLA-DR + ) and exhaustion (PD1 + ) markers. Significant reductions in proportions of senescent pulmonary CD28 2 CD57 + CD8 T cells were observed only in HIV + smokers. Pulmonary CD8 T cells showed lower perforin expression ex vivo compared with blood CD8 T cells, with reduced granzyme B expression only in HIV + nonsmokers. Bronchoalveolar lavage CD8 T cells showed significantly less in vitro degranulation and CD4 killing capacity than blood CD8 T cells. Therefore, pulmonary mucosal CD8 T cells are more differentiated, activated, and exhausted, with reduced killing capacity in vitro than blood CD8 T cells, potentially contributing to a suboptimal anti-HIV immune response within the lungs.

Objectives The reported prevalence of chronic obstructive pulmonary disease ( COPD ) in people living with HIV ( PLWHIV ) varies widely. Our objective was to estimate the prevalence of airflow obstruction and COPD in unselected PLWHIV and identify characteristics that increase the risk of nonreversible airflow obstruction in order to guide case finding strategies for COPD . Methods All adults attending the Chronic Viral Illness Service were invited to participate in the study, regardless of smoking status or history of known COPD /asthma. Individuals underwent spirometric testing both before and after use of a salbutamol bronchodilator. Airflow obstruction was defined as forced expiratory volume in 1 s ( FEV 1 )/forced vital capacity ( FVC ) < 0.7 post‐bronchodilation, whereas COPD was defined as FEV 1 / FVC < 0.7 post‐bronchodilation and Medical Research Council ( MRC ) score > 2. Multivariate logistic regression was used to evaluate risk factors associated with airflow obstruction, reported as adjusted odds ratios ( aORs ). Results Five hundred and three participants successfully completed spirometry testing. The median (Q1; Q3) age was 52 (44; 58) years. The median (Q1; Q3) CD 4 count was 598 (438; 784) cells/μL and the median (Q1; Q3) nadir CD 4 count was 224 (121; 351) cells/μL. There were 119 (24%) current smokers and 145 (29%) former smokers. Among those screened, 54 (11%) had airflow obstruction whereas three (1%) of the participants had COPD . Factors that were associated with airflow obstruction included a history of smoking [ aOR 2.2; 95% confidence interval ( CI ) 1.1; 4.7], older age (aOR 1.6; 95% CI 1.2; 2.2), and lower CD 4 count (aOR 0.8; 95% CI 0.7; 1.0). Conclusions Airflow obstruction was relatively uncommon. Our findings suggest that PLWHIV who are ≥50 years old, smokers and those with nadir CD 4 counts ≤ 200 cells/μL could be targeted to undergo spirometry to diagnose chronic airflow obstruction.