Uninfected Monocyte-derived Macrophages Research Articles

Macrophage-Derived Factors with the Potential to Contribute to Pathogenicity of HIV-1 and HIV-2: Role of CCL-2/MCP-1.

Human immunodeficiency virus type 2 (HIV-2) is known to be less pathogenic than HIV-1. However, the mechanism(s) underlying the decreased HIV-2 pathogenicity is not fully understood. Herein, we report that β-chemokine CCL2 expression was increased in HIV-1-infected human monocyte-derived macrophages (MDM) but decreased in HIV-2-infected MDM when compared to uninfected MDM. Inhibition of CCL2 expression following HIV-2 infection occurred at both protein and mRNA levels. By microarray analysis, quantitative PCR, and Western blotting, we identified that Signal Transducer and Activator of Transcription 1 (STAT1), a critical transcription factor for inducing CCL2 gene expression, was also reduced in HIV-2-infected MDM. Blockade of STAT1 in HIV-infected MDM using a STAT1 inhibitor significantly reduced the production of CCL2. In contrast, transduction of STAT1-expressing pseudo-retrovirus restored CCL2 production in HIV-2-infected MDM. These findings support the concept that CCL2 inhibition in HIV-2-infected MDM is meditated by reduction of STAT1. Furthermore, we showed that STAT1 reduction in HIV-2-infected MDM was regulated by the CUL2/RBX1 ubiquitin E3 ligase complex-dependent proteasome pathway. Knockdown of CUL2 or RBX1 restored the expression of STAT1 and CCL2 in HIV-2-infected MDM. Taken together, our findings suggest that differential regulation of the STAT1-CCL2 axis may be one of the mechanisms underlying the different pathogenicity observed for HIV-1 and HIV-2.

Dopamine May Exacerbate Neuroinflammation in the Context of HIV by Driving Inflammatory Phenotypes in Macrophages

Globally, there are around 38 million people living with HIV (PLWH). Treatment with anti‐retroviral therapy (ART) can suppress HIV replication and improve the length and quality of life, but chronic infection and ART treatment has resulted in the development of a variety of new co‐morbidities. Among these are the constellation of neuropathological, behavioral, cognitive and motor symptoms collectively known as neuroHIV. These symptoms are found in approximately 50% of the infected population, and multiple lines of evidence indicate that neuroHIV is primarily mediated by chronic CNS infection and inflammatory response in CNS myeloid populations such as macrophages and microglia. In many PLWH, these inflammatory conditions can be exacerbated by substance use disorders, which are highly comorbid with HIV infection and disproportionately common in HIV‐infected populations. Substance use disorders of all types are a major risk factor for the development of neuroHIV, suggesting a common mechanism by which substances of abuse interact with HIV to promote inflammation. All addictive substances increase extracellular dopamine in the CNS and our previous data show that dopamine promotes an inflammatory phenotype in human macrophages. Thus, we hypothesize that exposure to dopamine concentrations (10‐6M) induced by stimulant use will more robustly increase inflammation in HIV‐infected human monocyte‐derived macrophages (MDM) than in uninfected MDM. To test this, uninfected and HIV‐infected human MDM were treated with dopamine and changes in inflammatory profiles were assessed by measuring NF‐κB nuclear translocation using high content imaging and quantifying cytokine production by AlphaLISA analysis. Dopamine significantly increased the production of the inflammatory cytokines IL‐6 and IL‐1β, and these were specifically mediated via dopamine receptors and the activation of NF‐κB. These findings correlate with expression of dopaminergic receptors and nod‐like receptor (NLR) inflammasome transcripts and were altered in the presence of HIV infection. We also found that baseline macrophage activation could be attributed to culture medium type. Overall, these studies provide a broader understanding of the immunological role of dopamine, particularly in the development of neuroHIV and may suggest a potential mechanism for further pharmacological investigation to target HIV infection.

Different Patterns of HIV-1 Replication in MACROPHAGES is Led by Co-Receptor Usage.

Background and objectives: To enter the target cell, HIV-1 binds not only CD4 but also a co-receptor β-chemokine receptor 5 (CCR5) or α chemokine receptor 4 (CXCR4). Limited information is available on the impact of co-receptor usage on HIV-1 replication in monocyte-derived macrophages (MDM) and on the homeostasis of this important cellular reservoir. Materials and Methods: Replication (measured by p24 production) of the CCR5-tropic 81A strain increased up to 10 days post-infection and then reached a plateau. Conversely, the replication of the CXCR4-tropic NL4.3 strain (after an initial increase up to day 7) underwent a drastic decrease becoming almost undetectable after 10 days post-infection. The ability of CCR5-tropic and CXCR4-tropic strains to induce cell death in MDM was then evaluated. While for CCR5-tropic 81A the rate of apoptosis in MDM was comparable to uninfected MDM, the infection of CXCR4-tropic NL4.3 in MDM was associated with a rate of 14.3% of apoptotic cells at day 6 reaching a peak of 43.5% at day 10 post-infection. Results: This suggests that the decrease in CXCR4-tropic strain replication in MDM can be due to their ability to induce cell death in MDM. The increase in apoptosis was paralleled with a 2-fold increase in the phosphorylated form of p38 compared to WT. Furthermore, microarray analysis showed modulation of proapoptotic and cancer-related genes induced by CXCR4-tropic strains starting from 24 h after infection, whereas CCR5 viruses modulated the expression of genes not correlated with apoptotic-pathways. Conclusions: In conclusion, CXCR4-tropic strains can induce a remarkable depletion of MDM. Conversely, MDM can represent an important cellular reservoir for CCR5-tropic strains supporting the role of CCR5-usage in HIV-1 pathogenesis and as a pharmacological target to contribute to an HIV-1 cure.

AmpliSeq transcriptome analysis of human alveolar and monocyte-derived macrophages over time in response to Mycobacterium tuberculosis infection.

Human alveolar macrophages (HAM) are primary bacterial niche and immune response cells during Mycobacterium tuberculosis (M.tb) infection, and human blood monocyte-derived macrophages (MDM) are a model for investigating M.tb-macrophage interactions. Here, we use a targeted RNA-Seq method to measure transcriptome-wide changes in RNA expression patterns of freshly obtained HAM (used within 6 h) and 6 day cultured MDM upon M.tb infection over time (2, 24 and 72 h), in both uninfected and infected cells from three donors each. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) uses primers targeting 18,574 mRNAs and 2,228 non-coding RNAs (ncRNAs) for a total of 20,802 transcripts. AmpliSeqTM yields highly precise and reproducible gene expression profiles (R2 >0.99). Taking advantage of AmpliSeq’s reproducibility, we establish well-defined quantitative RNA expression patterns of HAM versus MDM, including significant M.tb-inducible genes, in networks and pathways that differ in part between MDM and HAM. A similar number of expressed genes are detected at all time-points between uninfected MDM and HAM, in common pathways including inflammatory and immune functions, but canonical pathway differences also exist. In particular, at 2 h, multiple genes relevant to the immune response are preferentially expressed in either uninfected HAM or MDM, while the HAM RNA profiles approximate MDM profiles over time in culture, highlighting the unique RNA expression profile of freshly obtained HAM. MDM demonstrate a greater transcriptional response than HAM upon M.tb infection, with 2 to >10 times more genes up- or down-regulated. The results identify key genes involved in cellular responses to M.tb in two different human macrophage types. Follow-up bioinformatics analysis indicates that approximately 30% of response genes have expression quantitative trait loci (eQTLs in GTEx), common DNA variants that can influence host gene expression susceptibility or resistance to M.tb, illustrated with the TREM1 gene cluster and IL-10.

The Cholinergic Anti-Inflammatory Response and the Role of Macrophages in HIV-Induced Inflammation.

Macrophages are phagocytic immune cells that protect the body from foreign invaders and actively support the immune response by releasing anti- and proinflammatory cytokines. A seminal finding revolutionized the way macrophages are seen. The expression of the neuronal alpha7 nicotinic acetylcholine receptor (α7-nAChR) in macrophages led to the establishment of the cholinergic anti-inflammatory response (CAR) in which the activation of this receptor inactivates macrophage production of proinflammatory cytokines. This novel neuroimmune response soon began to emerge as a potential target to counteract inflammation during illness and infection states. Human immunodeficiency virus (HIV)-infected individuals suffer from chronic inflammation that persists even under antiretroviral therapy. Despite the CAR’s importance, few studies involving macrophages have been performed in the HIV field. Evidence demonstrates that monocyte-derived macrophages (MDMs) recovered from HIV-infected individuals are upregulated for α7-nAChR. Moreover, in vitro studies demonstrate that addition of an HIV viral constituent, gp120IIIB, to uninfected MDMs also upregulates the α7-nAChR. Importantly, contrary to what was expected, activation of upregulated α7-nAChRs in macrophages does not reduce inflammation, suggesting a CAR disruption. Although it is reasonable to consider this receptor as a pharmacological target, additional studies are necessary since its activity seems to differ from that observed in neurons.

Type-2 diabetes alters the basal phenotype of human macrophages and diminishes their capacity to respond, internalise, and control Mycobacterium tuberculosis.

BACKGROUND Type 2 diabetes (T2D) is a risk factor for the development of tuberculosis (TB), although the associated mechanisms are not known.OBJECTIVES To study the association between T2D and the basal phenotype of macrophages, and their immune response to Mycobacterium tuberculosis (Mtb) infection.METHODS We evaluated the influence of T2D on the response of monocyte-derived macrophages (MDM) to Mtb in patients with T2D (n = 10) compared to healthy subjects (n = 9), before and after infection with Mtb clinical isolates bearing different degrees of virulence. The levels of cell surface markers for activation secreted cytokines and chemokines, bacterial association, and intracellular bacterial growth were evaluated.FINDINGS The expression levels of HLA-DR, CD80, and CD86 were low while those of of PD-L1 were high in uninfected MDMs derived from patients with diabetes; as a result of Mtb infection, changes were only observed in the expression levels of PD-L1. The levels of cytokines (e.g., IL-6, IL-1β, IL-10, and IL-12) and chemokines (e.g., MCP-1, MIG, and RANTES) are perturbed in MDMs derived from patients with diabetes, both before infection and in response to Mtb infection. In response to the more virulent Mtb strains, the levels of association and bacterial clearance were diminished in MDMs derived from patients with diabetes.CONCLUSIONS T2D affects the basal activation state of the macrophages and its capacity to respond and control Mtb infection.

Differentially expressed host long intergenic noncoding RNA and mRNA in HIV-1 and HIV-2 infection

Non-coding RNAs and mRNAs have been implicated in replication, pathogenesis and host response in HIV infection. However, the impact of long intergenic non-coding RNAs (lincRNAs) on HIV-1 and HIV-2 infection is not known. In this study, we have analyzed expression profiles of lincRNAs and mRNAs in monocyte derived macrophages (MDMs) infected with HIV-1/HIV-2 using microarrays. Our study identified many differentially expressed lincRNAs and mRNAs in MDMs infected with HIV-1/HIV-2 compared to uninfected MDMs. Genes involved in glutathione metabolism and lysine degradation were differentially regulated only in HIV-1 infected MDMs. In HIV-2 infected MDMs, CUL 2, SFRS9, and RBBP4 genes were differentially expressed. Furthermore, we found that plasma levels of lincRNA: chr2: 165509129-165519404 and lincRNA: chr12: 57761837-57762303 were better indicators of HIV-1 infection while lincRNA: chr10:128586385-128592960, XLOC_001148 and lincRNA: chr5:87580664-87583451, were better indicators of HIV-2 infection. In summary, our study has demonstrated that there is substantial alteration in lincRNA and mRNA expression in response to HIV-1/HIV-2 infection. These differentially expressed lincRNAs and mRNAs could serve as prognostic and diagnostic biomarkers of HIV infection and help in the identification of new targets for therapy.

Impact of Silver and Carbon Nanoparticle Exposures on Macrophage Responses to Mycobacterium tuberculosis (M.tb)

Abstract To assess biological risks of nanoparticle (NP) exposures, we examined the effects of silver (Ag) core Ag20-citrate (Ag20) and carbon black (CB) on human host innate immune responses to M.tb, a respiratory pathogen that causes tuberculosis. Human peripheral blood monocyte-derived macrophages (MDMs) were exposed to 1, 10, 25 and 50 μg/mL of Ag20 and CB for 4 and 24 h. At 4 h no significant cytotoxicity was observed with neither of the NPs at none of the concentrations examined relative to unexposed MDMs. At 24 h, viability of the MDMs was reduced by 60–70% and 20% following Ag20 and CB exposure, respectively, at doses ≥25 μg/mL. M.tb-induced host immune responses are mediated by the activation of TLR signalling pathways that culminate in NF-κB activation. Effects of Ag20 and CB (10μg/mL for 4 h, a nontoxic dose) on TLR signalling in M.tb-infected and uninfected MDMs were compared using a human TLR signaling pathway-specific profiler array. Ag20 exposure suppressed the M.tb-induced expression of a subset of NF-κB-mediated target genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A). Ag20-mediated suppression of M.tb-induced activation of the TLR-signalling pathway was not due to decreased uptake of M.tb by MDMs or mycobacteriocidal effects of Ag20. Ag20 exposure increased the expression of HSPA1A mRNA that encodes for the stress-induced Hsp72. In contrast, exposure to CB resulted in little effects on M.tb-induced host gene expression indicating that immunosuppressive effects of Ag20 are NP-specific. Since Hsp72 has been shown to suppress NF-κB activation, we propose that Ag20-mediated Hsp72 upregulation contributes to the suppression of M.tb-induced NF-κB activation and host immune responses. Funding NIEHS 5U19 ES019536

β2 Integrin Adhesion Complexes Maintain the Integrity of HIV‐1 Assembly Compartments in Primary Macrophages

In human monocyte-derived macrophages (MDM), human immunodeficiency virus type 1 (HIV-1) assembly takes place primarily on complex intracellular plasma membrane domains connected to the cell surface by closely apposed membrane sheets or narrow channels. Some of the membranes associated with these compartments are decorated by thick (≈30 nm), electron-dense, cytoplasmic coats. Here we show by immunolabelling of ultrathin cryosections that the β2 integrin CD18, together with the αM and αX integrins (CD11b and CD11c), is clustered at these coated domains, and that the coats themselves contain the cytoskeletal linker proteins talin, vinculin and paxillin that connect the integrin complexes to the actin cytoskeleton. Intracellular plasma membrane-connected compartments (IPMC) with CD18-containing focal adhesion-like coats are also present in uninfected MDM. These compartments become more prominent as the cells mature in tissue culture and their appearance correlates with increased expression of CD18, CD11b/c and paxillin. Depletion of CD18 by RNA interference leads to parallel down-regulation of CD11b and CD11c, as well as of paxillin, and the disappearance of the adhesion-like coats. In addition, CD18 knockdown alters the appearance of virus-containing IPMC in HIV-infected MDM, indicating that the β2 integrin/focal adhesion-like coat structures are involved in the organization of these compartments.

Human Cytomegalovirus Paralyzes Macrophage Motility through Down-Regulation of Chemokine Receptors, Reorganization of the Cytoskeleton, and Release of Macrophage Migration Inhibitory Factor

Macrophages contribute to host defense and to the maintenance of immune homeostasis. Conversely, they are important targets of human cytomegalovirus (HCMV), a herpesvirus that has evolved many strategies to modulate the host immune response. Because an efficient macrophage trafficking is required for triggering an adequate immune response, we investigated the effects exerted by HCMV infection on macrophage migratory properties. By using endotheliotropic strains of HCMV, we obtained high rates of productively infected human monocyte-derived macrophages (MDM). Twenty-four hours after infection, MDM showed reduced polar morphology and became unable to migrate in response to inflammatory and lymphoid chemokines, bacterial products and growth factors, despite being viable and metabolically active. Although chemotactic receptors were only partially affected, HCMV induced a dramatic reorganization of the cytoskeleton characterized by rupture of the microtubular network, stiffness of the actin fibers, and collapse of the podosomes. Furthermore, supernatants harvested from infected MDM contained high amounts of macrophage migration inhibitory factor (MIF) and were capable to block the migration of neighboring uninfected MDM. Because immunodepletion of MIF from the conditioned medium completely restored MDM chemotaxis, we could show for the first time a functional role of MIF as an inhibitor of macrophage migration in the context of HCMV infection. Our findings reveal that HCMV uses different mechanisms to interfere with movement and positioning of macrophages, possibly leading to an impairment of antiviral responses and to an enhancement of the local inflammation.

Increased Mycobacterium tuberculosis growth in HIV-1-infected human macrophages: role of tumour necrosis factor-alpha.

Synergism between Mycobacterium tuberculosis (M. tuberculosis) and HIV-1 infections was demonstrated in several in vitro models and clinical studies. Here, we investigated their reciprocal effects on growth in chronically HIV-1-infected promonocytic U1 cells and in acutely infected monocyte-derived macrophages (MDM). Phagocytosis of M. tuberculosis induced HIV-1 expression in U1 cells, together with increased TNF-alpha production. M. tuberculosis growth, evaluated by competitive PCR, was greater in HIV-1-infected MDM compared to uninfected cells. M. tuberculosis phagocytosis induced greater TNF-alpha and IL-10 production in HIV-1-infected MDM than in uninfected cells. In uninfected MDM, addition of TNF-alpha and IFN-gamma decreased, whereas IL-10 increased M. tuberculosis growth. On the contrary, in HIV-1-infected MDM, addition of TNF-alpha and IFN-gamma increased, whereas IL-10 has no effect on M. tuberculosis growth. TNF-alpha seems to play a pivotal role in the enhanced M. tuberculosis growth observed in HIV-1-infected MDM, being unable to exert its physiological antimycobacterial activity. Here, for the first time we demonstrated an enhanced M. tuberculosis growth in HIV-1-infected MDM, in line with the observed clinical synergism between the two infections.

HIV-1 Activates Proinflammatory and Interferon-Inducible Genes in Human Brain Microvascular Endothelial Cells: Putative Mechanisms of Blood—Brain Barrier Dysfunction

The mechanisms underlying blood-brain barrier (BBB) dysfunction seen in human immunodeficiency virus 1 (HIV-1) infection are poorly understood; however, they are believed to be caused by interactions of human brain microvascular endothelial cells (HBMEC) with virus-infected macrophages. Using a transwell system and Affymetrix arrays, we investigated HIV-1-induced genomic changes in HBMEC after coculture with HIV-1-infected or -uninfected monocyte-derived macrophages (MDM). Differentially expressed genes were determined by linear modeling and then were grouped by hierarchical clustering. Compared to HBMEC cocultured with noninfected MDM, 184 probe sets corresponding to 84 genes were differentially expressed in HBMEC cocultured with HIV-infected MDM. Genes activated in HIV-1 MDM-exposed HBMEC included proinflammatory cytokines and chemokines, tumor necrosis factor-alpha-induced proteins, interferon (IFN)-inducible genes, intercellular adhesion molecule-1, transcription factors of the nuclear factor-kappaB family, and signal transducer and activator of transcription 1. Analysis of molecular networks and canonical pathways associated with differentially expressed genes suggest that HIV-1 causes BBB impairment by mechanisms involving inflammation, cytokine, and IFN signaling in HBMEC.

TRAIL-Mediated Apoptosis in HIV-1-Infected Macrophages Is Dependent on the Inhibition of Akt-1 Phosphorylation

HIV-1 uses mononuclear phagocytes (monocytes, tissue macrophages, and dendritic cells) as a vehicle for its own dissemination and as a reservoir for continuous viral replication. The mechanism by which the host immune system clears HIV-1-infected macrophages is not understood. TRAIL may play a role in this process. TRAIL is expressed on the cell membrane of peripheral immune cells and can be cleaved into a soluble, secreted form. The plasma level of TRAIL is increased in HIV-1-infected patients, particularly those with high viral loads. To study the effect of elevated TRAIL on mononuclear phagocytes, we used recombinant human (rh) TRAIL and human monocyte-derived macrophages (MDM) as an in vitro model. Our results demonstrated rhTRAIL-induced apoptosis in HIV-1-infected MDM and inhibited viral replication, while having a reduced effect on uninfected MDM. HIV-1 infection significantly decreased Akt-1 phosphorylation; rhTRAIL exposure further decreased Akt-1 phosphorylation. Infection with a dominant-negative Akt-1 adenovirus potentiated rhTRAIL-induced apoptosis, while constitutively active Akt-1 blocked rhTRAIL-induced apoptosis in HIV-1-infected MDM. From this data we conclude the death ligand TRAIL preferentially provokes apoptosis of HIV-1-infected MDM, and the mechanism is reliant upon the inhibition of Akt-1 phosphorylation. Understanding this mechanism may facilitate the elimination of HIV-1-infected macrophages and lead to new therapeutic avenues for treatment of HIV-1 infection.

Impaired Complement-Mediated Phagocytosis by HIV Type-1-Infected Human Monocyte-Derived Macrophages Involves a cAMP-Dependent Mechanism

HIV-1 infection of cells of macrophage lineage impairs a number of effector functions performed by these cells, including phagocytosis of opsonized pathogens. In this study we investigate the effects of HIV-1 on the mechanism of complement (C')-mediated phagocytosis by human monocyte-derived macrophages (MDM). Using C'-opsonized sheep red blood cells (sRBC) as targets, we demonstrate that phagocytosis is inhibited by HIV-1 infection in vitro. Inhibition is not due to downregulation of surface C' receptors (R) or altered binding of C'-opsonized targets to HIV-1-infected MDM, suggesting a postreceptor-mediated mechanism of suppression. Having shown that increased levels of intracellular cAMP in uninfected MDM inhibit phagocytosis, we demonstrate that HIV-1 infection of MDM is associated with increased intracellular cAMP. Using the adenylate cyclase inhibitors 2',5'-dideoxyadenosine and MDL-12,330A, we show that phagocytosis by HIV-1- infected MDM can be restored by inhibition of cAMP production. Defective phagocytosis by HIV-1-infected MDM did not correlate with prostaglandin secretion, and was less in uninfected MDM within the HIV-1-infected cell culture suggesting a minimal bystander effect. Inhibition required viral entry but not active viral replication, as shown by use of the antiretroviral drug lamivudine. Hence, our study suggests that HIV-1 impairs C'R-mediated phagocytosis in MDM by elevating intracellular cAMP levels, independent of prostaglandin secretion, and contributes to our understanding of how HIV-1 impairs cell-mediated immunity.

Adverse Effects of Antiretroviral Drugs on HIV-1-Infected and -Uninfected Human Monocyte-Derived Macrophages

Antiretroviral drugs approved for treatment of HIV-1 infection include nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). Use of these drugs in combinations (highly active antiretroviral therapy) has delayed disease progression. However, long-term therapy is associated with potentially serious adverse effects. NRTIs are thought to contribute to these adverse effects via depletion of mtDNA. Inasmuch as macrophages (major targets for HIV-1) are highly metabolically active with large numbers of mitochondria, we investigated the effects of NRTIs (didanosine, stavudine, lamivudine, and zidovudine) on the viability and function of HIV-1-infected and -uninfected human monocyte-derived macrophages (MDMs). We demonstrate that the combinations didanosine/stavudine and lamivudine/zidovudine decrease mtDNA content in MDMs, with HIV-1-infected MDMs displaying a greater reduction than uninfected cells. This decrease correlated with decreased complement-mediated phagocytosis (C'MP) by MDMs, a process dependent on mitochondrial function. Inasmuch as PIs have previously been reported to interact with cellular proteases and given that cellular proteases are involved in the phagocytic process, we investigated the effects of the PI indinavir on C'MP. We demonstrate that indinavir augments C'MP by uninfected MDMs, but not HIV-1-infected MDMs. This study provides additional understanding on the effects of commonly used antiretroviral drugs on cellular immune function.

The nature of extracellular iron influences iron acquisition by Mycobacterium tuberculosis residing within human macrophages.

We have reported that Mycobacterium tuberculosis residing within the phagosomes of human monocyte-derived macrophages (MDM) can acquire Fe from extracellular transferrin (TF) and sources within the MDM. In the lung, Fe is also bound to lactoferrin (LF) and low-molecular-weight chelates. We therefore investigated the ability of intraphagosomal M. tuberculosis to acquire Fe from these sources. M. tuberculosis acquired 30-fold and 3-fold more Fe from LF and citrate, respectively, compared to TF, in spite of similar MDM-associated Fe. M. tuberculosis infection decreased MDM-associated Fe relative to uninfected MDM as follows: TF (38.7%), citrate (21.1%), and LF (15.3%). M. tuberculosis Fe acquisition from extracellular chelates (exogenous source) and from endogenous MDM Fe initially acquired from the three chelates (endogenous source) was compared. M. tuberculosis Fe acquisition was similar from exogenous and endogenous sources supplied as Fe-TF. In contrast, there was much greater intracellular M. tuberculosis Fe uptake from LF and citrate from the exogenous than endogenous source. Gamma interferon (IFN-gamma) reduced MDM Fe uptake from each chelate by approximately 50% and augmented the M. tuberculosis-induced decrease in MDM Fe uptake from exogenous TF, but not from LF or citrate. IFN-gamma minimally decreased intracellular M. tuberculosis Fe acquisition from exogenous Fe-TF but significantly increased Fe uptake from LF and citrate. Intraphagosomal M. tuberculosis Fe acquisition from both exogenous and endogenous MDM sources, and the effect of IFN-gamma on this process, is influenced by the nature of the extracellular Fe chelate. M. tuberculosis has developed efficient mechanisms of acquiring Fe from a variety of Fe chelates that it likely encounters within the human lung.

Selective inhibition of HIV replication in primary macrophages but not T lymphocytes by macrophage-derived chemokine.

Macrophage-derived chemokine (MDC) has been reported to inhibit different HIV-1 strains in activated peripheral blood mononuclear cells (T cell blasts), although other investigators have not confirmed these findings. Here we demonstrate that MDC inhibits the replication of CCR5-dependent (R5) HIV-1(BaL) in monocyte-derived macrophages (MDM), but not in T cell blasts, although with variable potency depending on donor variability. Analysis of HIV-1(BaL) proviral DNA synthesis in MDM indicated that the suppressive effect of MDC did not involve inhibition of early events such as entry or reverse transcription. Finally, an inverse correlation was observed between the levels of endogenous MDC secreted by uninfected MDM of different donors and the efficiency of different HIV strains, including two primary isolates with different coreceptor usage, to replicate in these cells. Thus, MDC represents an example of a chemokine inhibiting HIV replication in macrophages acting at one or more postentry levels in the virus life cycle.

Effects of HIV-1 on the surface expression of LFA-1 on cultured monocytes.

CD11a, the alpha chain of LFA-1, which is a member of the LeuCAM family of integrins, has been implicated in the formation of HIV-induced syncytia and may contribute to the depletion of CD4-positive lymphocytes seen in patients with HIV infection. In this study, we examined the effects of HIV-1 infection on the expression of CD11a on cultured monocyte-derived macrophages (MDMs). Monocytes isolated from peripheral blood and maintained in suspension culture were infected in vitro with a monocytotropic variant of HIV-1 (Ba-L). Surface expression of CD11a, measured by indirect immunofluorescence and flow cytometry, was significantly higher on HIV-infected cells than on mock-infected cells from the same donor. Upregulation of CD11a expression was unaffected by the HIV reverse transcriptase inhibitor, zidovudine, indicating that it did not depend on reverse transcription. A step before reverse transcription, such as viral binding, appears sufficient to trigger an increase in CD11a expression. This hypothesis is supported by our findings of soluble recombinant CD4 inhibition of HIV-induced CD11a upregulation. It is possible that induction of a cytokine network by HIV underlies this effect, given our findings that exposure of uninfected MDMs to granulocyte-macrophage colony-stimulating factor (GM-CSF) specifically increased CD11a expression and that HIV-infected MDMs secreted more GM-CSF than mock-infected cells.

Vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages.

HIV infection of monocyte-derived macrophages in vitro reduces phagocytosis of Candida albicans.

HIV-1 infection of peripheral blood monocyte-derived macrophages (MDMs) is unrelated to the level of CD4 expression on the surface of the cell, is associated with considerable donor variability, causes minimal cytopathology, and results in peak viral antigen production after 2 weeks of infection. Phagocytosis of opsonized Candida albicans by MDMs infected in vitro with several strains of HIV was compared with that of uninfected cells from the same donors; the proportion of MDMs containing the fluorescein isothiocyanate-labeled yeast was determined by flow cytometry and phase contrast microscopy. The intracellular localization of C. albicans was confirmed by confocal microscopy. Using paired MDMs from nine donors, 81% of uninfected and 53% of HIV-infected MDMs phagocytosed C. albicans. In addition, the number of yeast per cell was significantly higher in uninfected MDMs than in HIV-infected cells (mean 6.1 versus 2.5). These findings may partially explain the high incidence of mucocutaneous candidiasis in HIV-infected patients with advanced disease.