Interactions with Commensal and Pathogenic Bacteria Induce HIV-1 Latency in Macrophages through Altered Transcription Factor Recruitment to the LTR.

Macrophages are infected by HIV-1 in vivo and contribute to both viral spread and pathogenesis. Recent human and animal studies suggest that HIV-1-infected macrophages serve as a reservoir that contributes to HIV-1 persistence during anti-retroviral therapy. The ability of macrophages to serve as persistent viral reservoirs is likely influenced by the local tissue microenvironment, including interactions with pathogenic and commensal microbes. Here we show that the sexually transmitted pathogen Neisseria gonorrhoeae (GC) and the gut-associated microbe Escherichia coli (E. coli), which encode ligands for both Toll-like receptor 2 (TLR2) and TLR4, repressed HIV-1 replication in macrophages and thereby induced a state reminiscent of viral latency. This repression was mediated by signaling through TLR4 and the adaptor protein TRIF and was associated with increased production of type I interferons. Inhibiting TLR4 signaling, blocking type 1 interferon, or knocking-down TRIF reversed LPS- and GC-mediated repression of HIV-1. Finally, the repression of HIV-1 in macrophages was associated with the recruitment of interferon regulatory factor 8 (IRF8) to the interferon stimulated response element (ISRE) downstream of the 5' HIV-1 long terminal repeat (LTR). Our data indicate that IRF8 is responsible for repression of HIV-1 replication in macrophages in response to TRIF-dependent signaling during GC and E. coli co-infection. These findings highlight the potential role of macrophages as HIV-1 reservoirs as well as the role of the tissue microenvironment and co-infections as modulators of HIV-1 persistence.IMPORTANCE The major barrier toward the eradication of HIV-1 infection is the presence of a small reservoir of latently infected cells, which include CD4+ T cells and macrophages that escape immune-mediated clearance and the effects of anti-retroviral therapy. There remain crucial gaps in our understanding of the molecular mechanisms that lead to transcriptionally silent or latent HIV-1 infection of macrophages. The significance of our research is in identifying microenvironmental factors, such as commensal and pathogenic microbes, that can contribute to the establishment and maintenance of latent HIV-1 infection in macrophages. It is hoped that identifying key processes contributing to HIV-1 persistence in macrophages may ultimately lead to novel therapeutics to eliminate latent HIV-1 reservoirs in vivo.

Reduced Monocyte Proportions and Responsiveness in Convalescent COVID-19 Patients.

The clinical manifestations of acute severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection and COVID-19 suggest a dysregulation of the host immune response that leads to inflammation, thrombosis, and organ dysfunction. It is less clear whether these dysregulated processes persist during the convalescent phase of disease or during long COVID. We investigated the effects of SARS-CoV-2 infection on the proportions of classical, intermediate, and non-classical monocytes, their activation status, and their functional properties in convalescent COVID-19 patients and uninfected control subjects. We found that the percentage of total monocytes was decreased in convalescent COVID-19 patients compared to uninfected controls. This was due to decreased intermediate and non-classical monocytes. Classical monocytes from convalescent COVID-19 patients demonstrated a decrease in activation markers, such as CD56, in response to stimulation with bacterial lipopolysaccharide (LPS). In addition, classical monocytes from convalescent COVID-19 patients showed decreased expression of CD142 (tissue factor), which can initiate the extrinsic coagulation cascade, in response to LPS stimulation. Finally, we found that monocytes from convalescent COVID-19 patients produced less TNF-α and IL-6 in response to LPS stimulation, than those from uninfected controls. In conclusion, SARS-CoV-2 infection exhibits a clear effect on the relative proportions of monocyte subsets, the activation status of classical monocytes, and proinflammatory cytokine production that persists during the convalescent phase of disease.

Reduced monocyte proportions and responsiveness in convalescent COVID-19 patients.

Introduction: The clinical manifestations of acute severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) suggest a dysregulation of the host immune response that leads to inflammation, thrombosis, and organ dysfunction. It is less clear whether these dysregulated processes persist during the convalescent phase of disease or during long COVID. We sought to examine the effects of SARS-CoV-2 infection on the proportions of classical, intermediate, and nonclassical monocytes, their activation status, and their functional properties in convalescent COVID-19 patients. Methods: Peripheral blood mononuclear cells (PBMCs) from convalescent COVID-19 patients and uninfected controls were analyzed by multiparameter flow cytometry to determine relative percentages of total monocytes and monocyte subsets. The expression of activation markers and proinflammatory cytokines in response to LPS treatment were measured by flow cytometry and ELISA, respectively. Results: We found that the percentage of total monocytes was decreased in convalescent COVID-19 patients compared to uninfected controls. This was due to decreased intermediate and non-classical monocytes. Classical monocytes from convalescent COVID-19 patients demonstrated a decrease in activation markers, such as CD56, in response to stimulation with bacterial lipopolysaccharide (LPS). In addition, classical monocytes from convalescent COVID-19 patients showed decreased expression of CD142 (tissue factor), which can initiate the extrinsic coagulation cascade, in response to LPS stimulation. Finally, we found that monocytes from convalescent COVID-19 patients produced less TNF-α and IL-6 in response to LPS stimulation, than those from uninfected controls. Conclusion: SARS-CoV-2 infection exhibits a clear effect on the relative proportions of monocyte subsets, the activation status of classical monocytes, and proinflammatory cytokine production that persists during the convalescent phase of disease.

Nuclear receptor signaling inhibits HIV-1 replication in macrophages through multiple trans-repression mechanisms.

Sexually transmitted pathogens activate HIV-1 replication and inflammatory gene expression in macrophages through engagement of Toll-like receptors (TLRs). Ligand-activated nuclear receptor (NR) transcription factors, including glucocorticoid receptor (GR), peroxisome proliferator-activated receptor gamma (PPARγ), and liver X receptor (LXR), are potent inhibitors of TLR-induced inflammatory gene expression. We therefore hypothesized that ligand-activated NRs repress both basal and pathogen-enhanced HIV-1 replication in macrophages by directly repressing HIV-1 transcription and by ameliorating the local proinflammatory response to pathogens. We show that the TLR2 ligand PAM3CSK4 activated virus transcription in macrophages and that NR signaling repressed both basal and TLR-induced HIV-1 transcription. NR ligand treatment repressed HIV-1 expression when added concurrently with TLR ligands and in the presence of cycloheximide, demonstrating that they act independently of new cellular gene expression. We found that treatment with NR ligands inhibited the association of AP-1 and NF-κB subunits, as well as the coactivator CBP, with the long terminal repeat (LTR). We show for the first time that the nuclear corepressor NCoR is bound to HIV-1 LTR in unstimulated macrophages and is released from the LTR after TLR engagement. Treatment with PPARγ and LXR ligands, but not GR ligands, prevented this TLR-induced clearance of NCoR from the LTR. Our data demonstrate that both classical and nonclassical trans-repression mechanisms account for NR-mediated HIV-1 repression. Finally, NR ligand treatment inhibited the potent proinflammatory response induced by PAM3CSK4 that would otherwise activate HIV-1 expression in infected cells. Our findings provide a rationale for studying ligand-activated NRs as modulators of basal and inflammation-induced HIV-1 replication.

PPARgamma and LXR signaling inhibit dendritic cell-mediated HIV-1 capture and trans-infection.

Dendritic cells (DCs) contribute to human immunodeficiency virus type 1 (HIV-1) transmission and dissemination by capturing and transporting infectious virus from the mucosa to draining lymph nodes, and transferring these virus particles to CD4+ T cells with high efficiency. Toll-like receptor (TLR)-induced maturation of DCs enhances their ability to mediate trans-infection of T cells and their ability to migrate from the site of infection. Because TLR-induced maturation can be inhibited by nuclear receptor (NR) signaling, we hypothesized that ligand-activated NRs could repress DC-mediated HIV-1 transmission and dissemination. Here, we show that ligands for peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor (LXR) prevented proinflammatory cytokine production by DCs and inhibited DC migration in response to the chemokine CCL21 by preventing the TLR-induced upregulation of CCR7. Importantly, PPARgamma and LXR signaling inhibited both immature and mature DC-mediated trans-infection by preventing the capture of HIV-1 by DCs independent of the viral envelope glycoprotein. PPARgamma and LXR signaling induced cholesterol efflux from DCs and led to a decrease in DC-associated cholesterol, which has previously been shown to be required for DC capture of HIV-1. Finally, both cholesterol repletion and the targeted knockdown of the cholesterol transport protein ATP-binding cassette A1 (ABCA1) restored the ability of NR ligand treated cells to capture HIV-1 and transfer it to T cells. Our results suggest that PPARgamma and LXR signaling up-regulate ABCA1-mediated cholesterol efflux from DCs and that this accounts for the decreased ability of DCs to capture HIV-1. The ability of NR ligands to repress DC mediated trans-infection, inflammation, and DC migration underscores their potential therapeutic value in inhibiting HIV-1 mucosal transmission.

HIV-1-induced type I IFNs promote viral latency in macrophages.

Macrophages chronically infected with HIV-1 serve as a reservoir that contributes to HIV-1 persistence during antiretroviral therapy; however, the mechanisms governing the establishment and maintenance of this virus reservoir have not been fully elucidated. Here, we show that HIV-1 enters a state reminiscent of latency in monocyte-derived macrophages (MDMs), characterized by integrated proviral DNA with decreased viral transcription. This quiescent state is associated with decreased NF-κB p65, RNA polymerase II, and p-TEFb recruitment to the HIV-1 promoter as well as maintenance of promoter chromatin in a transcriptionally nonpermissive state. MDM transition to viral latency is mediated by type I IFN signaling, as inhibiting type I IFN signaling or blocking type 1 IFN prevents the establishment of latent infection. Knockdown studies demonstrate that the innate immune signaling molecule mitochondrial antiviral signaling protein (MAVS) is required for the transition to latency. Finally, we demonstrate a role for the viral accessory protein Vpr in the establishment of HIV-1 latency in macrophages. Our data indicate that HIV-1-induced type I IFN production is responsible for the establishment of viral latency in MDMs and identify possible therapeutic targets for the prevention or elimination of this important HIV-1 reservoir.

HIV-1 Accessory Proteins Impart a Modest Interferon Response and Upregulate Cell Cycle-Related Genes in Macrophages.

HIV-1 infection of myeloid cells is associated with the induction of an IFN response. How HIV-1 manipulates and subverts the IFN response is of key interest for the design of therapeutics to improve immune function and mitigate immune dysregulation in people living with HIV. HIV-1 accessory genes function to improve viral fitness by altering host pathways in ways that enable transmission to occur without interference from the immune response. We previously described changes in transcriptomes from HIV-1 infected and from IFN-stimulated macrophages and noted that transcription of IFN-regulated genes and genes related to cell cycle processes were upregulated during HIV-1 infection. In the present study, we sought to define the roles of individual viral accessory genes in upregulation of IFN-regulated and cell cycle-related genes using RNA sequencing. We observed that Vif induces a set of genes involved in mitotic processes and that these genes are potently downregulated upon stimulation with type-I and -II IFNs. Vpr also upregulated cell cycle-related genes and was largely responsible for inducing an attenuated IFN response. We note that the induced IFN response most closely resembled a type-III IFN response. Vpu and Nef-regulated smaller sets of genes whose transcriptomic signatures upon infection related to cytokine and chemokine processes. This work provides more insight regarding processes that are manipulated by HIV-1 accessory proteins at the transcriptional level.

Dasatinib: effects on the macrophage phospho proteome with a focus on SAMHD1 and HIV-1 infection.

Macrophages are one of the main cellular targets of human immunodeficiency virus type 1 (HIV-1). Macrophage infection by HIV-1 is inefficient due to the presence of the viral restriction factor sterile alpha motif and histidine aspartic acid domain containing protein 1 (SAMHD1). Ex vivo human monocyte-derived macrophages (MDMs) express SAMHD1 in an equilibrium between active (unphosphorylated) and inactive (phosphorylated) states. We and others have shown that treatment of MDMs with the FDA-approved tyrosine kinase inhibitor, dasatinib, ablates SAMHD1 phosphorylation, thus skewing the balance towards a cellular state that is refractory to HIV-1 infection. We hypothesized that dasatinib inhibits a putative tyrosine kinase that is upstream of SAMHD1. In search for this tyrosine kinase, we probed several candidates and were unable to identify a single target that, when inhibited, was sufficient to explain the dephosphorylation of SAMHD1 we observe upon treatment with dasatinib. On the other hand, we probed the ability of dasatinib to directly inhibit the serine/threonine cyclin dependent kinases 1, 2, 4 and 6 and confirmed that dasatinib directly inhibits these kinases. Therefore, our results show that inhibition of the proximal CDKs 1, 2, 4 and 6 by dasatinib is clearly detectable, leads to blockade of infection by HIV-1, and may be sufficient to explain the activity of dasatinib against SAMHD1 phosphorylation.

MicroRNA 155 and viral-induced neuroinflammation.

MicroRNA (miRNA) regulation of gene expression is becoming an increasingly recognized mechanism by which host immune responses are governed following microbial infection. miRNAs are short, non-coding RNAs that repress translation of target genes, and have been implicated in a number of activities that modulate host immune responses, including the regulation of immune cell proliferation, survival, expansion, differentiation, migration, polarization, and effector function. This review highlights several examples in which mammalian-encoded miR-155 influences immune responses following viral infection of the CNS.

Decapping protein 1 phosphorylation modulates IL-8 expression during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a negative-strand RNA virus that is an important cause of bronchiolitis and pneumonia. We investigated the effect of RSV infection on the expression patterns of cellular proteins involved in regulating mRNA translation and degradation, and found that a processing-body protein involved in mRNA degradation, decapping protein 1a (DCP1), was phosphorylated rapidly following infection. UV-inactivated and sucrose-purified RSV were sufficient to mediate DCP1 phosphorylation, indicating that it occurs as a consequence of an early event in RSV infection. Analysis using kinase inhibitors showed that RSV-induced DCP1 phosphorylation occurred through the ERK1/2 pathway. The DCP1 phosphorylation sites were limited to serine 315, serine 319, and threonine 321. Overexpression of wt DCP1 led to a decrease in RSV-induced IL-8 production, but this effect was abrogated in cells overexpressing phosphorylation-deficient DCP1 mutants. These results suggest that DCP1 phosphorylation modulates the host chemokine response to RSV infection.

Interleukin 1 beta and interleukin 6 potentiate retinoic acid-mediated repression of human immunodeficiency virus type 1 replication in macrophages.

All-trans-retinoic acid (RA) has been shown either to activate or repress human immunodeficiency virus type 1 (HIV-1) replication in primary monocyte-derived-macrophages (MDMs). We systematically investigated the contribution that cell donor and virus differences make to this variability. We found that the effect of RA was cell donor dependent. In addition, the ability of RA to repress HIV-1 replication varied between different virus stocks. In no case did RA affect either virus entry or integration but instead affected the accumulation of viral mRNAs in infected cells. Despite the complex variability in RA responsiveness in untreated cells, we found that RA consistently repressed virus replication when the MDMs were treated with concentrations of interleukin 1 beta (IL-1 beta) and IL-6 that are expected at local sites of infection, where HIV-1-infected macrophages reside in vivo.

Retinoid-dependent restriction of human immunodeficiency virus type 1 replication in monocytes/macrophages.

Vitamin A deficiency has been correlated with increased severity of human immunodeficiency virus type 1 (HIV-1)-associated disease. Moreover, vitamin A supplementation can reduce AIDS-associated morbidity and mortality. Our group and others have shown that retinoids, the bioactive metabolites of vitamin A, repress HIV-1 replication in monocytic cell lines and primary macrophages by blocking long-terminal-repeat (LTR)-directed transcription. Based on these studies, we hypothesize that retinoids are natural repressors of HIV-1 in vivo. We show here that all-trans-retinoic acid (RA)-mediated repression of HIV-1 activation requires pretreatment for at least 12 h and is blocked by the protein synthesis inhibitors cycloheximide and puromycin. Studies of the kinetics of RA-mediated repression in U1 cells and primary monocyte-derived macrophages (MDMs) reveal that the repressive effects of RA on HIV-1 expression are long-lasting but reversible. We demonstrate that HIV-1 expression is activated when U1 cells or MDMs are cultured in retinoid-free synthetic medium and show that physiological concentrations of RA repress this activation. In addition, the synthetic pan-retinoic acid receptor antagonist BMS-204 493 activates HIV-1 replication in U1 cells in a dose-dependent manner, suggesting that RA-induced transactivation of cellular gene expression is required for HIV-1 repression. Together, these data support the hypothesis that retinoids present in tissue culture media in vitro and serum in vivo maintain HIV-1 in a transcriptionally repressed state in monocytes/macrophages.

Retinoic acid inhibition of chromatin remodeling at the human immunodeficiency virus type 1 promoter. Uncoupling of histone acetylation and chromatin remodeling.

All-trans retinoic acid (RA) represses HIV-1 transcription and replication in cultured monocytic cells and in primary monocyte-derived macrophages. Here we examine the role of histone acetylation and chromatin remodeling in RA-mediated repression. RA pretreatment of latently infected U1 promonocytes inhibits HIV-1 expression in response to the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). TSA is thought to activate HIV-1 transcription by inducing histone hyperacetylation within a regulatory nucleosome, nuc-1, positioned immediately downstream from the transcription start site. Acetylation of nuc-1 is thought to be a critical step in activation that precedes nuc-1 remodeling and, subsequently, transcriptional initiation. Here we demonstrate that TSA treatment induces H3 and H4 hyperacetylation and nuc-1 remodeling. Although RA pretreatment inhibits nuc-1 remodeling and HIV-1 transcription, it has no effect on histone acetylation. This suggests that acetylation and remodeling are not obligatorily coupled. We also show that growth of U1 cells in retinoid-deficient medium induces nuc-1 remodeling and HIV-1 expression but does not induce histone hyperacetylation. These findings suggest that remodeling, not histone hyperacetylation, is the limiting step in transcriptional activation in these cells. Together, these data suggest that RA signaling maintains the chromatin structure of the HIV-1 promoter in a transcriptionally non-permissive state that may contribute to the establishment of latency in monocyte/macrophages.

Activation of autoreactive B cells by CpG dsDNA.

The proliferative response of autoreactive rheumatoid factor (RF) B cells to mammalian chromatin-containing immune complexes (ICs) results from the sequential engagement of the B cell receptor (BCR) and Toll-like receptor 9 (TLR9). We have used ICs constructed from anti-hapten antibodies and defined haptenated dsDNA fragments to determine the form of mammalian DNA that mediates this process. Despite their relatively low abundance in mammalian DNA, we found that inclusion of hypomethylated CpG motifs in these ICs was necessary for effective activation. In the absence of antibody, the same fragments could efficiently stimulate low-affinity hapten-specific and DNA-reactive 3H9 B cells, but not RF B cells. These results extend the BCR/TLR9 coengagement paradigm to a second major class of autoreactive B cells, further confirm the critical role of the BCR in chromatin ligand delivery to TLR9, and implicate hypomethylated CpG motifs as ligand elements necessary for the initiation of systemic autoimmune disease.

Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry.

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors. Concentrations of inhibitors that prevent this reduction and inhibit the cleavage of surface-bound disulfide conjugate prevent infection at the level of HIV-1 entry. The entry of HIV-1 strains differing in their coreceptor specificities is similarly inhibited, and so is the reduction of gp120 bound to CD4 of coreceptor-negative cells. PDI inhibitors also prevent HIV envelope-mediated cell-cell fusion but have no effect on the entry of HIV-1 pseudo-typed with murine leukemia virus envelope. Importantly, PDI coprecipitates with both soluble and cellular CD4. We propose that a PDI.CD4 association at the cell surface enables PDI to reach CD4-bound virus and to reduce disulfide bonds present in the domain of gp120 that binds to CD4. Conformational changes resulting from the opening of gp120-disulfide loops may drive the processes of virus-cell and cell-cell fusion. The biochemical events described identify new potential targets for anti-HIV agents.