Cells of the myeloid lineage, particularly monocytes and macrophages, play a key role in HIV infection by contributing to viral replication, immune response, and maintaining immune balance during suppressive therapy. We hypothesized that metabolic reprogramming and altered chemokine signaling in people living with HIV (PWH) on long-term antiretroviral therapy (ART) affect monocyte transport and polarization due to ongoing inflammation. Therefore, the present study aimed to identify the mechanism of impaired monocyte/macrophage function in PWH on well-treated ART that can lead to clinical intervention strategies to improve health. Single-cell RNA sequencing, immune-phenotyping, and metabolic modeling identified altered expression of chemokine and metabolite receptors and altered metabolic flux in PWH monocytes that decreased monocyte migration. The plasma secretome revealed a nonclassical inflammatory microenvironment in PWH. Integrative multi-omics and single-cell proteomics of differentiated monocyte-derived macrophages (MDMs) detected metabolic reprogramming orchestrated by α-ketoglutarate (AKG) that affected macrophage function and HIV infection. Increased levels of AKG in plasma were shown to occur in PWH under ART. Therefore, when differentiating MDM with serum from PWH or AKG, macrophage function was found polarized towards an M2-like state. AKG alone was shown to increase CCR5 levels and increase HIV-1 infection in MDM. Here, we utilize systems biology-driven identification and ex vivo assays to show impaired macrophage polarization, due to metabolic training, can leads to a low-grade nonclassical inflammatory environment in well-treated PWH.

Natural killer (NK) cells are critical modulators of HIV transmission and disease. Recent evidence suggests a loss of NK cell cytotoxicity during aging, yet analysis of NK cell biology and aging in people with HIV (PWH) is lacking. Herein, we perform comprehensive analyses of people aging with and without HIV to determine age-related NK phenotypic changes. Utilizing high-dimensional flow cytometry, we analyze 30 immune-related proteins on peripheral NK cells from healthy donors, PWH with viral suppression, and viremic PWH. NK cell phenotypes are dynamic across aging but change significantly in HIV and on antiretroviral drug therapy (ART). NK cells in healthy aging show increasing ⍺4β7 and decreasing CCR7 expression and a reverse phenomenon in PWH. These HIV-associated trafficking patterns could be due to NK cell recruitment to HIV reservoir formation in lymphoid tissue or failed mucosal signaling in the HIV-infected gut but appear to be tight delineators of age-related NK cell changes.
Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

The clinical outcome and disease severity in coronavirus disease 2019 (COVID-19) are heterogeneous, and the progression or fatality of the disease cannot be explained by a single factor like age or comorbidities. In this study, we used system-wide network-based system biology analysis using whole blood RNA sequencing, immunophenotyping by flow cytometry, plasma metabolomics, and single-cell-type metabolomics of monocytes to identify the potential determinants of COVID-19 severity at personalized and group levels. Digital cell quantification and immunophenotyping of the mononuclear phagocytes indicated a substantial role in coordinating the immune cells that mediate COVID-19 severity. Stratum-specific and personalized genome-scale metabolic modeling indicated monocarboxylate transporter family genes (e.g., SLC16A6), nucleoside transporter genes (e.g., SLC29A1), and metabolites such as α-ketoglutarate, succinate, malate, and butyrate could play a crucial role in COVID-19 severity. Metabolic perturbations targeting the central metabolic pathway (TCA cycle) can be an alternate treatment strategy in severe COVID-19.
Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Many mediators and regulators of extravasation by bona fide human memory-phenotype T cells remain undefined. Mucosal-associated invariant T (MAIT) cells are innate-like, antibacterial cells that we found excelled at crossing inflamed endothelium. They displayed abundant selectin ligands, with high expression of FUT7 and ST3GAL4, and expressed CCR6, CCR5, and CCR2, which played non-redundant roles in trafficking on activated endothelial cells. MAIT cells selectively expressed CCAAT/enhancer-binding protein delta (C/EBPδ). Knockdown of C/EBPδ diminished expression of FUT7, ST3GAL4 and CCR6, decreasing MAIT cell rolling and arrest, and consequently the cells' ability to cross an endothelial monolayer in vitro and extravasate in mice. Nonetheless, knockdown of C/EBPδ did not affect CCR2, which was important for the step of transendothelial migration. Thus, MAIT cells demonstrate a program for extravasastion that includes, in part, C/EBPδ and C/EBPδ-regulated genes, and that could be used to enhance, or targeted to inhibit T cell recruitment into inflamed tissue.

The major reservoirs for HIV in the CNS are in the microglia, perivascular macrophages, and to a lesser extent, astrocytes. To study the molecular events controlling HIV expression in the microglia, we developed a reliable and robust method to immortalize microglial cells from primary glia from fresh CNS tissues and commercially available frozen glial cells. Primary human cells, including cells obtained from adult brain tissue, were transformed with lentiviral vectors expressing SV40 T antigen or a combination of SVR40 T antigen and hTERT. The immortalized cells have microglia-like morphology and express key microglial surface markers including CD11b, TGFβR, and P2RY12. Importantly, these cells were confirmed to be of human origin by sequencing. The RNA expression profiles identified by RNA-seq are also characteristic of microglial cells. Furthermore, the cells demonstrate the expected migratory and phagocytic activity, and the capacity to mount an inflammatory response characteristic of primary microglia. The immortalization method has also been successfully applied to a wide range of microglia from other species (macaque, rat, and mouse). To investigate different aspects of HIV molecular regulation in CNS, the cells have been superinfected with HIV reporter viruses and latently infected clones have been selected that reactive HIV in response to inflammatory signals. The cell lines we have developed and rigorously characterized will provide an invaluable resource for the study of HIV infection in microglial cells as well as studies of microglial cell function.