Stromal down-regulation of macrophage CD4/CCR5 expression and NF-κB activation mediates HIV-1 non-permissiveness in intestinal macrophages.
ABSTRACT: Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation.
Project description:Human immunodeficiency virus type 1 (HIV-1) infection in mononuclear phagocyte lineage cells (monocytes, macrophages, and microglia) is a critical component in the pathogenesis of viral infection. Viral replication in macrophages serves as a reservoir, a site of dissemination, and an instigator for neurological sequelae during HIV-1 disease. Recent studies demonstrated that chemokine receptors are necessary coreceptors for HIV-1 entry which determine viral tropism for different cell types. To investigate the relative contribution of the beta-chemokine receptors CCR3 and CCR5 to viral infection of mononuclear phagocytes we utilized a panel of macrophage-tropic HIV-1 strains (from blood and brain tissue) to infect highly purified populations of monocytes and microglia. Antibodies to CD4 (OKT4A) abrogated HIV-1 infection. The beta chemokines and antibodies to CCR3 failed to affect viral infection of both macrophage cell types. Antibodies to CCR5 (3A9) prevented monocyte infection but only slowed HIV replication in microglia. Thus, CCR5, not CCR3, is an essential receptor for HIV-1 infection of monocytes. Microglia express both CCR5 and CCR3, but antibodies to them fail to inhibit viral entry, suggesting the presence of other chemokine receptors for infection of these cells. These studies demonstrate the importance of mononuclear phagocyte heterogeneity in establishing HIV-1 infection and persistence.
Project description:HIV-1 recognition by, interaction with, and/or infection of CD4(+)CCR5(+) tissue macrophages and dendritic cells (DCs) play important roles in HIV-1 transmission and pathogenesis. By comparison, circulating CD4(+)CCR5(+) monocytes appear relatively resistant to HIV-1, and a fundamental unresolved question involves deciphering restriction factors unique to this precursor population. Not only do monocytes, relative to macrophages, possess higher levels of the innate resistance factor APOBEC3G, but we uncovered APOBEC3A, not previously associated with anti-HIV activity, as being critical in monocyte resistance. Inversely correlated with susceptibility, silencing of APOBEC3A renders monocytes vulnerable to HIV-1. Differences in promiscuity of monocytes, macrophages, and DCs can be defined, at least partly, by disparities in APOBEC expression, with implications for enhancing cellular defenses against HIV-1.
Project description:Intestinal macrophages in healthy human mucosa are profoundly down-regulated for inflammatory responses (inflammation anergy) due to stromal TGF-? inactivation of NF-?B. Paradoxically, in cytomegalovirus (CMV) intestinal inflammatory disease, one of the most common manifestations of opportunistic CMV infection, intestinal macrophages mediate severe mucosal inflammation. Here we investigated the mechanism whereby CMV infection promotes macrophage-mediated mucosal inflammation. CMV infected primary intestinal macrophages but did not replicate in the cells or reverse established inflammation anergy. However, CMV infection of precursor blood monocytes, the source of human intestinal macrophages in adults, prevented stromal TGF-?-induced differentiation of monocytes into inflammation anergic macrophages. Mechanistically, CMV up-regulated monocyte expression of the TGF-? antagonist Smad7, blocking the ability of stromal TGF-? to inactivate NF-?B, thereby enabling MyD88 and NF-?B-dependent cytokine production. Smad7 expression also was markedly elevated in mucosal tissue from subjects with CMV colitis and declined after antiviral ganciclovir therapy. Confirming these findings, transfection of Smad7 antisense oligonucleotide into CMV-infected monocytes restored monocyte susceptibility to stromal TGF-?-induced inflammation anergy. Thus, CMV-infected monocytes that recruit to the mucosa, not resident macrophages, are the source of inflammatory macrophages in CMV mucosal disease and implicate Smad7 as a key regulator of, and potential therapeutic target for, CMV mucosal disease.
Project description:Using identical (ID) twins, we have previously demonstrated that host cell genes exert a significant impact on productive human immunodeficiency virus (HIV) infection of monocytes and macrophages (J. Chang et al., J. Virol. 70:7792-7803, 1996). Therefore, the stage in the replication cycle at which these host genetic influences act was investigated in a study using 8 pairs of ID twins and 10 pairs of sex- and age-matched unrelated donors (URDs). In the first phase of the study, blood monocytes and monocyte-derived macrophages (MDM) of ID twins and URDs were infected with 15 HIV type 1 strains. Four well-characterized primary isolates and HIV-BaL were then examined in more detail. The host cell genetic effect in MDM was exerted predominantly prior to complete reverse transcription, as the HIV DNA level and p24 antigen levels were concordant (r = 0.91, P = 0.0001) and similar between the pairs of ID twin pairs (r = 0.96, P = 0.0001) but discordant between URD pairs (r = 0.11, P = 0.3) in both phases of the study. To further examine genetic influence on viral entry, we examined the proportion of CCR5 membrane expression on MDM. As expected, there was wide variability in proportion of MDM expressing CCR5 among URDs (r = 0. 58, P = 0.2); however, this variability was significantly reduced between ID twin pairs (r = 0.81, P = 0.01). Differences in viral entry did not necessarily correlate with CCR5 expression, and only very low levels of CCR5 expression restricted HIV entry and production. In summary, the host cell genetic effect on HIV replication in macrophages appears to be exerted predominantly pre-reverse transcription. Although CCR5 was necessary for infection, other unidentified host genes are likely to limit productive infection.
Project description:Most HIV strains that enter the brain are macrophage-tropic and use the CCR5 receptor to bind and infect target cells. Because the cytoskeleton is a network of protein filaments involved in cellular movement and migration, we investigated whether CCR5 and the cytoskeleton are involved in endothelial-mononuclear phagocytes interactions, adhesion, and HIV-1 infection.Using a cytoskeleton phospho-antibody microarray, we showed that after co-culture with human brain microvascular endothelial cells (HBMEC), HIV-1 infected monocytes increased expression and activation of cytoskeleton-associated proteins, including Rac1/cdc42 and cortactin, compared to non-infected monocytes co-cultured with HBMEC. Analysis of brain tissues from HIV-1-infected patients validated these findings, and showed transcriptional upregulation of Rac1 and cortactin, as well as increased activation of Rac1 in brain tissues of HIV-1-infected humans, compared to seronegative individuals and subjects with HIV-1-encephalitis. Confocal imaging showed that brain cells expressing phosphorylated Rac1 were mostly macrophages and blood vessels. CCR5 antagonists TAK-799 and maraviroc prevented HIV-induced upregulation and phosphorylation of cytoskeleton-associated proteins, prevented HIV-1 infection of macrophages, and diminished viral-induced adhesion of monocytes to HBMEC. Ingenuity pathway analysis suggests that during monocyte-endothelial interactions, HIV-1 alters protein expression and phosphorylation associated with integrin signaling, cellular morphology and cell movement, cellular assembly and organization, and post-translational modifications in monocytes. CCR5 antagonists prevented these HIV-1-induced alterations.HIV-1 activates cytoskeletal proteins during monocyte-endothelial interactions and increase transcription and activation of Rac1 in brain tissues. In addition to preventing macrophage infection, CCR5 antagonists could diminish viral-induced alteration and phosphorylation of cytoskeletal proteins, monocyte adhesion to the brain endothelium and viral entry into the central nervous system.
Project description: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.
Project description:A hallmark of persistent HIV-1 infection in the central nervous system is increased activation of mononuclear phagocytes and surrounding astrogliosis, conferring persistent HIV-induced inflammation. This inflammation is believed to result in neuronal dysfunction and the clinical manifestations of HIV-associated neurocognitive disorders (HAND). The Jak/STAT pathway is activated in macrophages/myeloid cells upon HIV-1 infection, modulating many pro-inflammatory pathways that result in HAND, thereby representing an attractive cellular target. Thus, the impact of ruxolitinib, a Janus Kinase (Jak) 1/2 inhibitor that is FDA approved for myelofibrosis and polycythemia vera, was assessed for its potential to inhibit HIV-1 replication in macrophages and HIV-induced activation in monocytes/macrophages in culture. In addition, a murine model of HIV encephalitis (HIVE) was used to assess the impact of ruxolitinib on histopathological features of HIVE, brain viral load, as well as its ability to penetrate the blood-brain-barrier (BBB). Ruxolitinib was found to inhibit HIV-1 replication in macrophages, HIV-induced activation of monocytes (CD14/CD16) and macrophages (HLA-DR, CCR5, and CD163) without apparent toxicity. In vivo, systemically administered ruxolitinib was detected in the brain during HIVE in SCID mice and markedly inhibited astrogliosis. Together, these data indicate that ruxolitinib reduces HIV-induced activation and infiltration of monocytes/macrophages in vitro, reduces the replication of HIV in vitro, penetrates the BBB when systemically administered in mice and reduces astrogliosis in the brains of mice with HIVE. These data suggest that ruxolitinib will be useful as a novel therapeutic to treat humans with HAND.
Project description:Human immunodeficiency virus type-1 (HIV-1) infection of monocyte/macrophages is modulated by the levels of entry receptors cluster of differentiation 4 (CD4) and C-C chemokine receptor type 5 (CCR5), as well as by host antiviral restriction factors, which mediate several post-entry blocks. We recently identified two microRNAs, miR-221 and miR-222, which limit HIV-1 entry during infection of monocyte-derived macrophages (MDMs) by down-regulating CD4 expression. Interestingly, CD4 is also down-regulated during the differentiation of monocytes into macrophages. In this study, we compared microRNA expression profiles in primary monocytes and macrophages by RNAseq and found that miR-221/miR-222 are enhanced in macrophages. We took advantage of the monocytic THP-1 cell line that, once differentiated, is poorly susceptible to HIV-1. Accordingly, we found that CD4 levels are very low in THP-1 differentiated cells and that this down-regulation of the virus receptor is the result of miR-221/miR-222 up-regulation during differentiation. We thus established a THP-1 cell line stably expressing a modified CD4 (THP-1-CD4R) that is not modulated by miR-221/miR-222. We show that in contrast to parental THP-1, this line is productively infected by HIV-1 following differentiation, sustaining efficient HIV-1 CD4-dependent replication and spread. This new THP-1-CD4R cell line represents a useful tool for the study of HIV-1-macrophage interactions particularly in contexts where spreading of viral infection is necessary.
Project description:Infiltrating monocytes and macrophages play a crucial role in the progression of HIV-1 infection in the CNS. Previous studies showed that activation of the CB? can attenuate inflammatory responses and affect HIV-1 infectivity in T cells and microglia. Here, we report that CB? agonists can also act as immunomodulators on HIV-1-infected macrophages. First, our findings indicated the presence of elevated levels of CB? expression on monocytes/macrophages in perivascular cuffs of postmortem HIV-1 encephalitic cases. In vitro analysis by FACS of primary human monocytes revealed a step-wise increase in CB? surface expression in monocytes, MDMs, and HIV-1-infected MDMs. We next tested the notion that up-regulation of CB? may allow for the use of synthetic CB? agonist to limit HIV-1 infection. Two commercially available CB? agonists, JWH133 and GP1a, and a resorcinol-based CB? agonist, O-1966, were evaluated. Results from measurements of HIV-1 RT activity in the culture media of 7 day-infected cells showed a significant decrease in RT activity when the CB? agonist was present. Furthermore, CB? activation also partially inhibited the expression of HIV-1 pol. CB? agonists did not modulate surface expression of CXCR4 or CCR5 detected by FACS. We speculate that these findings indicate that prevention of viral entry is not a central mechanism for CB?-mediated suppression in viral replication. However, CB? may affect the HIV-1 replication machinery. Results from a single-round infection with the pseudotyped virus revealed a marked decrease in HIV-1 LTR activation by the CB? ligands. Together, these results indicate that CB? may offer a means to limit HIV-1 infection in macrophages.
Project description:Classic Hodgkin lymphoma tumor cells express a functional CCR5 receptor, and tumor tissues express high CCL5 levels, suggesting that CCL5-CCR5 signaling is involved in tumor-microenvironment formation and tumor growth. Using the CCR5 antagonist, maraviroc, and a neutralizing anti-CCL5 antibody, we found that CCL5 secreted by classic Hodgkin lymphoma cells recruited mesenchymal stromal cells and monocytes. The "education" of mesenchymal stromal cells by tumor cell-conditioned medium enhanced mesenchymal stromal cells' proliferation and CCL5 secretion. In turn, educated mesenchymal stromal cell-conditioned medium increased the clonogenic growth of tumor cells and monocyte migration, but these effects were reduced by maraviroc. Monocyte education by tumor cell-conditioned medium induced their growth and reprogrammed them towards immunosuppressive tumor-associated macrophages that expressed IDO and PD-L1 and secreted IL-10, CCL17 and TGF-β. Educated monocyte-conditioned medium slowed the growth of phytohemagglutinin-activated lymphocytes. Maraviroc decreased tumor cell growth and synergized with doxorubicin and brentuximab vedotin. A three-dimensional heterospheroid assay showed that maraviroc counteracted both the formation and viability of heterospheroids generated by co-cultivation of tumor cells with mesenchymal stromal cells and monocytes. In mice bearing tumor cell xenografts, maraviroc reduced tumor growth by more than 50% and inhibited monocyte accumulation, without weight loss. Finally, in classic Hodgkin lymphoma human tumor tissues, CCL5 and CD68 expression correlated positively, and patients with high CCL5 levels had poor prognosis. In conclusion, since the present challenges are to find molecules counteracting the formation of the immunosuppressive tumor microenvironment or new, less toxic drug combinations, the repurposed drug maraviroc may represent a new opportunity for classic Hodgkin lym phoma treatment.