Short communication: Nitazoxanide inhibits HIV viral replication in monocyte-derived macrophages.

We document the anti-HIV activity of nitazoxanide (NTZ), the first member of the thiazolide class of antiinfective drugs, originally effective against enteritis caused by Cryptosporidium parvum and Giardia lamblia. NTZ has been administered extensively worldwide, with no severe toxicities associated with its use. Here, we show for the first time that NTZ decreases HIV-1 replication in monocyte-derived macrophages (MDM) if present before or during HIV-1 infection. This NTZ effect is associated with downregulation of HIV-1 receptors CD4 and CCR5, and increasing gene expression of host cell anti-HIV resistance factors APOBEC3A/3G and tetherin. As NTZ is already in clinical use for other conditions, this newly described anti-HIV activity in MDM may facilitate innovative intensification strategies against HIV-1 when combined with current antiretroviral drug regimens.

Constitutive gene expression in monocytes from chronic HIV-1 infection overlaps with acute Toll-like receptor induced monocyte activation profiles.

Elevated TLR expression/signalling in monocyte/macrophages has been shown to mediate systemic immune activation, a hallmark of progressive HIV-1 infection. Here we show, via differential gene expression comparisons, the presence of a constitutive in vivo TLR-like gene activation signature in steady-state circulating monocytes from chronically HIV-1 infected subjects. The TLR2-like gene signature was defined as an 82 gene subset of the 376 genes constitutively modulated in in vivo HIV-1 monocytes, based on their overlap with de novo TLR2-induced genes in uninfected subjects' monocytes following acute ex vivo stimulation with Staphylococcus Aureus Cowan (SAC). Additional comparison of in vivo gene networks with available datasets from acute TLR activations in M/M expanded the overlap to 151-gene concordance among the 376 differential genes with emphasis on ERK/MAPK, TNF/IL6 (NFκB) and p53 gene networks. TLR2 stimulation of monocytes from HIV-1 infected subjects resulted in further upregulation of inflammatory genes indicative of a sustained transcriptional potential upon stimulation. In summary, our data support the presence of a sustained TLR-like gene activation profile in circulating monocyte from steady-state viremia in HIV-1 infected subjects.

Retinoblastoma protein induction by HIV viremia or CCR5 in monocytes exposed to HIV-1 mediates protection from activation-induced apoptosis: ex vivo and in vitro study.

We have previously described an antiapoptotic steady-state gene expression profile in circulating human monocytes from asymptomatic viremic HIV(+) donors, but the mechanism associated with this apoptosis resistance remains to be fully elucidated. Here, we show that Rb1 activation is a dominant feature of apoptosis resistance in monocytes exposed to HIV-1 in vivo (as measured ex vivo) and in vitro. Monocytes from asymptomatic viremic HIV(+) individuals show a positive correlation between levels of hypophosphorylated (active) Rb1 and VL in conjunction with increases in other p53-inducible proteins associated with antiapoptosis regulation, such as p21 and PAI-1 (SERPINE1), when compared with circulating monocytes from uninfected donors. Monocytes exposed in vitro to HIV-1 R5 isolates but not X4 isolates showed lower caspase-3 activation after apoptosis induction, indicating a role for the CCR5 signaling pathway. Moreover, monocytes exposed to R5 HIV-1 or MIP-1 ß induced Rb1 and p21 expression and an accumulation of autophagy markers, LC3 and Beclin. The inhibition of Rb1 activity in HIV-1 R5 viral-exposed monocytes using siRNA led to increased apoptosis sensitivity, thereby confirming a central role for Rb1 in the antiapoptotic phenotype. Our data identify Rb1 induction in chronic asymptomatic HIV-1 infection as a mediator of apoptosis resistance in monocytes in association with protective autophagy and contributing to monocyte survival during immune activation and/or HIV-1 viremia.

Increased metallothionein gene expression, zinc, and zinc-dependent resistance to apoptosis in circulating monocytes during HIV viremia.

Circulating monocytes exhibit an apoptotic resistance phenotype during HIV viremia in association with increased MT expression. MTs are known to play an important role in zinc metabolism and immune function. We now show, in a cross-sectional study using peripheral monocytes, that expression of MT1 isoforms E, G, H, and X is increased significantly in circulating monocyte cells from HIV+ subjects during chronic viremic episodes as compared with uninfected subjects. This increase in expression is also observed during acute viremia following interruption of suppressive ART. Circulating monocytes from HIV+ donors were also found to have elevated zinc importer gene Zip8 expression in conjunction with elevated intracellular zinc levels in contrast to CD4(+)T-lymphocytes. In vitro HIV-1 infection studies with elutriated MDM confirm a direct relation between HIV-1 infection and increased MDM MT1 (isoform G) gene expression and increased intracellular zinc levels. A direct link between elevated zinc levels and apoptosis resistance was established using a cell-permeable zinc chelator TPEN, which reversed apoptosis resistance effectively in monocytes from HIV-infected to levels comparable with uninfected controls. Taken together, increases in MT gene expression and intracellular zinc levels may contribute directly to maintenance of an immune-activated monocyte by mediating an increased resistance to apoptosis during active HIV-1 viremia.

Inability of plasmacytoid dendritic cells to directly lyse HIV-infected autologous CD4+ T cells despite induction of tumor necrosis factor-related apoptosis-inducing ligand.

The function of plasmacytoid dendritic cells (PDC) in chronic human immunodeficiency virus type 1 (HIV-1) infection remains controversial with regard to its potential for sustained alpha interferon (IFN-alpha) production and induction of PDC-dependent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity of HIV-infected cells. We address these areas by a study of chronically HIV-1-infected subjects followed through antiretroviral therapy (ART) interruption and by testing PDC cytolytic function against autologous HIV-infected CD4(+) T cells. Rebound in viremia induced by therapy interruption showed a positive association between TRAIL and viral load or T-cell activation, but comparable levels of plasma IFN-alpha/beta were found in viremic ART-treated and control subjects. While PDC from HIV-infected subjects expressed less interferon regulator factor 7 (IRF-7) and produced significantly less IFN-alpha upon Toll-like receptor 7/9 (TLR7/9) engagement than controls, membrane TRAIL expression in PDC from HIV(+) subjects was increased. Moreover, no significant increase in death receptor 5 (DR5) expression was seen in CD4(+) T cells from viremic HIV(+) subjects compared to controls or following in vitro infection/exposure to infectious and noninfectious virus or exogenous IFN-alpha, respectively. Although activated PDC killed the DR5-expressing HIV-infected Sup-T1 cell line, PDC did not lyse primary autologous HIV(+) CD4(+) T cells yet could provide accessory help for NK cells in killing HIV-infected autologous CD4(+) T cells. Taken together, our data show a lack of sustained high levels of soluble IFN-alpha in chronic HIV-1 infection in vivo and document a lack of direct PDC cytolytic activity against autologous infected or uninfected CD4(+) T cells.

Circulating monocytes in HIV-1-infected viremic subjects exhibit an antiapoptosis gene signature and virus- and host-mediated apoptosis resistance.

Mechanisms that may allow circulating monocytes to persist as CD4 T cells diminish in HIV-1 infection have not been investigated. We have characterized steady-state gene expression signatures in circulating monocytes from HIV-infected subjects and have identified a stable antiapoptosis gene signature comprised of 38 genes associated with p53, CD40L, TNF, and MAPK signaling networks. The significance of this gene signature is indicated by our demonstration of cadmium chloride- or Fas ligand-induced apoptosis resistance in circulating monocytes in contrast to increasing apoptosis in CD4 T cells from the same infected subjects. As potential mechanisms in vivo, we show that monocyte CCR5 binding by HIV-1 virus or agonist chemokines serves as independent viral and host modulators resulting in increased monocyte apoptosis resistance in vitro. We also show evidence for concordance between circulating monocyte apoptosis-related gene expression in HIV-1 infection in vivo and available datasets following viral infection or envelope exposure in monocyte-derived macrophages in vitro. The identification of in vivo gene expression associated with monocyte resistance to apoptosis is of relevance to AIDS pathogenesis since it would contribute to: 1) maintaining viability of infection targets and long-term reservoirs of HIV-1 infection in the monocyte/macrophage populations, and 2) protecting a cell subset critical to host survival despite sustained high viral replication.

Signal transduction induced by apoptotic cells inhibits HIV transcription in monocytes/macrophages.

The primary targets of HIV are CD4(+) T cells and macrophages. HIV infection is associated with an increase in apoptosis of infected and uninfected CD4(+) T cells, and these infected cells undergo apoptosis and produce HIV virions with phosphatidylserine (PS) on their surface. During phagocytosis of apoptotic cells, macrophages, using an array of receptors, are able to perceive various surface changes on apoptotic cells. The engagement of phagocytic receptors by ligands on the apoptotic cell surface results in the activation of signaling cascades, which facilitate engulfment. In this study, we examined how PS associated with virions and apoptotic cells influences HIV replication. We demonstrate that virus-associated PS is required for HIV infection of macrophages at a step prior to integration but following strong-stop, indicating that PS-initiated signals alter the establishment of HIV provirus. Conversely, apoptotic cells inhibited HIV transcription in infected macrophages, although this ability to suppress transcription was independent of PS. Furthermore, we show that ELMO, a key signaling molecule that participates in the phagocytosis of apoptotic cells, inhibited HIV transcription; however, knocking down endogenous ELMO expression in infected U937 cells rescued HIV transcription when these cells were coincubated with apoptotic targets. Taken together, these data show that apoptotic cells and the signals, which they initiate upon recognition by macrophages, influence the successful establishment of HIV infection and provirus transcription.