Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4&lt;sup&gt;+&lt;/sup&gt; T-cell Immunophenotyping and HIV Reservoir Assessment

Salahuddin, Syim; Thomson, Elaine; Méziane, Oussama; Farnós, Omar; Pagliuzza, Amélie; Chomont, Nicolas; Olivenstein, Ron; Costiniuk, Cecilia T.; Jenabian, Mohammad-Ali

doi:10.3791/59427

Cited by 8 publications

(10 citation statements)

References 19 publications

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“…To ascertain a more physiological sense of miR-103/107 in tissue-resident macrophages, we examined the levels of miR-103, miR-107, and p53 mRNA in colon macrophages obtained from healthy donors ( 24 ) as well as in lung alveolar macrophages (ALVMs) recovered from either HIV-negative or HIV-infected individuals under long-term antiretroviral therapy (>9 years) ( 45 , 46 ) and compared their expression levels to those in blood monocytes obtained from the same individuals. First, as we previously described for the CD4-targeting miR-221 ( 24 ), miR-103/107 and p53 mRNA were greatly upregulated in colon macrophages (3 donors) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Individuals were recruited at the McGill University Health Centre and the CR-CHUM (Montreal, Canada). Bronchoalveolar lavage (BAL) specimens were collected from 2 HIV-negative individuals and 5 HIV-positive individuals under suppressive ART (undetectable plasma viral load and CD4 count higher than 350 cells/mm 3 ) for at least 3 years and without any respiratory symptoms or active infections and processed as previously described ( 45 ). Matched peripheral blood was also collected from the participants, and CD14 + blood monocytes were isolated using an Influx cell sorter and recovered directly in RLT lysis buffer (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

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Interleukin-1β Triggers p53-Mediated Downmodulation of CCR5 and HIV-1 Entry in Macrophages through MicroRNAs 103 and 107

Lodge

Bellini

Laporte

et al. 2020

mBio

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Interleukin-1β Triggers p53-Mediated Downmodulation of CCR5 and HIV-1 Entry in Macrophages through MicroRNAs 103 and 107

Lodge

Bellini

Laporte

et al. 2020

mBio

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“…(C2, S2, OII, V97%) • For cellular analyses, we recommend centrifugation at 250g for 10 minutes, as higher speeds may not be optimal for preserving viable cells (statement 4.2.2). The BAL pellet can be resuspended and cryopreserved in a 10% final concentration of dimethyl sulfoxide in fetal calf serum 75 (C1, S2, OI, V92%). Centrifugation forces (in g or rcf) should be reported rather than speeds (rpm) because rpm corresponds to different centrifugal forces based on the rotor size.…”

Section: Statementsmentioning

confidence: 99%

International Society for Heart and Lung Transplantation consensus statement for the standardization of bronchoalveolar lavage in lung transplantation

Martinu

Koutsokera

Benden

et al. 2020

The Journal of Heart and Lung Transplantation

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Bronchoalveolar lavage (BAL) is a key clinical and research tool in lung transplantation (LTx). However, BAL collection and processing are not standardized across LTx centers. This International Society for Heart and Lung Transplantation–supported consensus document on BAL standardization aims to clarify definitions and propose common approaches to improve clinical and research practice standards. The following 9 areas are covered: (1) bronchoscopy procedure and BAL collection, (2) sample handling, (3) sample processing for microbiology, (4) cytology, (5) research, (6) microbiome, (7) sample inventory/tracking, (8) donor bronchoscopy, and (9) pediatric considerations. This consensus document aims to harmonize clinical and research practices for BAL collection and processing in LTx. The overarching goal is to enhance standardization and multicenter collaboration within the international LTx community and enable improvement and development of new BAL-based diagnostics.

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“…Bronchoscopies were performed to obtain up to 100 ml of BAL fluid. BAL specimen cells and matched peripheral blood mononuclear cells (PBMCs) were then isolated as we previously reported ( 7 , 73 ). Of note, due to the limited numbers of purified BAL specimen cells, interindividual variations, and low DN T-cell frequencies, we were not able to perform all study measures as described below in all individuals, and we prioritized the measures to be assessed based on the available cell number for each study individual.…”

Section: Methodsmentioning

confidence: 99%

“…and BV605 anti-CD8. CD4+ and DN T-cells were FACS-sorted using a BD FACSAria, as we previously described (73).…”

Section: Downloaded Frommentioning

confidence: 99%

HIV Infection and Persistence in Pulmonary Mucosal Double Negative T Cells In Vivo

Méziane

Salahuddin

Pham

et al. 2020

J Virol

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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.

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Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4<sup>+</sup> T-cell Immunophenotyping and HIV Reservoir Assessment

Cited by 8 publications

References 19 publications

Interleukin-1β Triggers p53-Mediated Downmodulation of CCR5 and HIV-1 Entry in Macrophages through MicroRNAs 103 and 107

Interleukin-1β Triggers p53-Mediated Downmodulation of CCR5 and HIV-1 Entry in Macrophages through MicroRNAs 103 and 107

International Society for Heart and Lung Transplantation consensus statement for the standardization of bronchoalveolar lavage in lung transplantation

HIV Infection and Persistence in Pulmonary Mucosal Double Negative T Cells In Vivo

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