Infected Monocyte-derived Macrophages Research Articles

Cytosolic sulfotransferase 1A1 regulates HIV-1 minus-strand DNA elongation in primary human monocyte-derived macrophages.

BackgroundThe cellular sulfonation pathway modulates key steps of virus replication. This pathway comprises two main families of sulfonate-conjugating enzymes: Golgi sulfotransferases, which sulfonate proteins, glycoproteins, glycolipids and proteoglycans; and cytosolic sulfotransferases (SULTs), which sulfonate various small molecules including hormones, neurotransmitters, and xenobiotics. Sulfonation controls the functions of numerous cellular factors such as those involved in cell-cell interactions, cell signaling, and small molecule detoxification. We previously showed that the cellular sulfonation pathway regulates HIV-1 gene expression and reactivation from latency. Here we show that a specific cellular sulfotransferase can regulate HIV-1 replication in primary human monocyte-derived macrophages (MDMs) by yet another mechanism, namely reverse transcription.MethodsMDMs were derived from monocytes isolated from donor peripheral blood mononuclear cells (PBMCs) obtained from the San Diego Blood Bank. After one week in vitro cell culture under macrophage-polarizing conditions, MDMs were transfected with sulfotranserase-specific or control siRNAs and infected with HIV-1 or SIV constructs expressing a luciferase reporter. Infection levels were subsequently monitored by luminescence. Western blotting was used to assay siRNA knockdown and viral protein levels, and qPCR was used to measure viral RNA and DNA products.ResultsWe demonstrate that the cytosolic sulfotransferase SULT1A1 is highly expressed in primary human MDMs, and through siRNA knockdown experiments, we show that this enzyme promotes infection of MDMs by single cycle VSV-G pseudotyped human HIV-1 and simian immunodeficiency virus vectors and by replication-competent HIV-1. Quantitative PCR analysis revealed that SULT1A1 affects HIV-1 replication in MDMs by modulating the kinetics of minus-strand DNA elongation during reverse transcription.ConclusionsThese studies have identified SULT1A1 as a cellular regulator of HIV-1 reverse transcription in primary human MDMs. The normal substrates of this enzyme are small phenolic-like molecules, raising the possibility that one or more of these substrates may be involved. Targeting SULT1A1 and/or its substrate(s) may offer a novel host-directed strategy to improve HIV-1 therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0491-9) contains supplementary material, which is available to authorized users.

Metabolic profiling during HIV-1 and HIV-2 infection of primary human monocyte-derived macrophages

We evaluated cellular metabolism profiles of HIV-1 and HIV-2 infected primary human monocyte-derived macrophages (MDMs). First, HIV-2 GL-AN displays faster production kinetics and greater amounts of virus as compared to HIV-1s: YU-2, 89.6 and JR-CSF. Second, quantitative LC–MS/MS metabolomics analysis demonstrates very similar metabolic profiles in glycolysis and TCA cycle metabolic intermediates between HIV-1 and HIV-2 infected macrophages, with a few notable exceptions. The most striking metabolic change in MDMs infected with HIV-2 relative to HIV-1-infected MDMs was the increased levels of quinolinate, a metabolite in the tryptophan catabolism pathway that has been linked to HIV/AIDS pathogenesis. Third, both HIV-1 and HIV-2 infected MDMs showed elevated levels of ribose-5-phosphate, a key metabolic component in nucleotide biosynthesis. Finally, HIV-2 infected MDMs display increased dNTP concentrations as predicted by Vpx-mediated SAMHD1 degradation. Collectively, these data show differential metabolic changes during HIV-1 and HIV-2 infection of macrophages.

ATP Induces IL-1β Secretion in Neisseria gonorrhoeae-Infected Human Macrophages by a Mechanism Not Related to the NLRP3/ASC/Caspase-1 Axis.

Neisseria gonorrhoeae (Ngo) has developed multiple immune evasion mechanisms involving the innate and adaptive immune responses. Recent findings have reported that Ngo reduces the IL-1β secretion of infected human monocyte-derived macrophages (MDM). Here, we investigate the role of adenosine triphosphate (ATP) in production and release of IL-1β in Ngo-infected MDM. We found that the exposure of Ngo-infected MDM to ATP increases IL-1β levels about ten times compared with unexposed Ngo-infected MDM (P < 0.01). However, we did not observe any changes in inflammasome transcriptional activation of speck-like protein containing a caspase recruitment domain (CARD) (ASC, P > 0.05) and caspase-1 (CASP1, P > 0.05). In addition, ATP was not able to modify caspase-1 activity in Ngo-infected MDM but was able to increase pyroptosis (P > 0.01). Notably ATP treatment defined an increase of positive staining for IL-1β with a distinctive intracellular pattern of distribution. Collectively, these data demonstrate that ATP induces IL-1β secretion by a mechanism not related to the NLRP3/ASC/caspase-1 axis and likely is acting at the level of vesicle trafficking or pore formation.

A Novel Role for the Receptor of the Complement Cleavage Fragment C5a, C5aR1, in CCR5-Mediated Entry of HIV into Macrophages.

The complement system is an ancient pattern recognition system that becomes activated during all stages of HIV infection. Previous studies have shown that C5a can enhance the infection of monocyte-derived macrophages and T cells indirectly through the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and the attraction of dendritic cells. C5a exerts its multiple biologic functions mainly through activation of C5a receptor 1 (C5aR1). Here, we assessed the role of C5aR1 as an enhancer of CCR5-mediated HIV infection. We determined CCR5 and C5aR1 heterodimer formation in myeloid cells and the impact of C5aR1 blockade on HIV entry and genomic integration. C5aR1/CCR5 heterodimer formation was identified by immunoprecipitation and western blotting. THP-1 cells and monocyte-derived macrophages (MDM) were infected by R5 laboratory strains or HIV pseudotyped for the vesicular stomatitis virus (VSV) envelope. Levels of integrated HIV were measured by quantitative PCR after targeting of C5aR1 by a C5aR antagonist, neutralizing C5aR1 monoclonal antibody (mAb) or hC5a. C5aR1 was also silenced by specific siRNA prior to viral entry. We found that C5aR1 forms heterodimers with the HIV coreceptor CCR5 in myeloid cells. Targeting C5aR1 significantly decreased integration by R5 viruses but not by VSV-pseudotyped viruses, suggesting that C5aR1 is critical for viral entry. The level of inhibition achieved with C5aR1-blocking reagents was comparable to that of CCR5 antagonists. Mechanistically, C5aR1 targeting decreased CCR5 expression. MDM from CCR5Δ32 homozygous subjects expressed levels of C5aR1 similar to CCR5 WT individuals, suggesting that mere C5aR1 expression is not sufficient for HIV infection. HIV appeared to preferentially enter THP-1 cells expressing high levels of both C5aR1 and CCR5. Targeted reduction of C5aR1 expression in such cells reduced HIV infection by ~50%. Our data thus suggest that C5aR1 acts as an enhancer of CCR5-mediated HIV entry into macrophages, the targeting of which may prove useful to reduce HIV infection by R5 strains.

Glutaminase-containing microvesicles from HIV-1-infected macrophages and immune-activated microglia induce neurotoxicity.

BackgroundHIV-1-infected and/or immune-activated microglia and macrophages are pivotal in the pathogenesis of HIV-1-associated neurocognitive disorders (HAND). Glutaminase, a metabolic enzyme that facilitates glutamate generation, is upregulated and may play a pathogenic role in HAND. Our previous studies have demonstrated that glutaminase is released to the extracellular fluid during HIV-1 infection and neuroinflammation. However, key molecular mechanisms that regulate glutaminase release remain unknown. Recent advances in understanding intercellular trafficking have identified microvesicles (MVs) as a novel means of shedding cellular contents. We posit that during HIV-1 infection and immune activation, microvesicles may mediate glutaminase release, generating excessive and neurotoxic levels of glutamate.ResultsMVs isolated through differential centrifugation from cell-free supernatants of monocyte-derived macrophages (MDM) and BV2 microglia cell lines were first confirmed in electron microscopy and immunoblotting. As expected, we found elevated number of MVs, glutaminase immunoreactivities, as well as glutaminase enzyme activity in the supernatants of HIV-1 infected MDM and lipopolysaccharide (LPS)-activated microglia when compared with controls. The elevated glutaminase was blocked by GW4869, a neutral sphingomyelinase inhibitor known to inhibit MVs release, suggesting a critical role of MVs in mediating glutaminase release. More importantly, MVs from HIV-1-infected MDM and LPS-activated microglia induced significant neuronal injury in rat cortical neuron cultures. The MV neurotoxicity was blocked by a glutaminase inhibitor or GW4869, suggesting that the neurotoxic potential of HIV-1-infected MDM and LPS-activated microglia is dependent on the glutaminase-containing MVs.ConclusionsThese findings support MVs as a potential pathway/mechanism of excessive glutamate generation and neurotoxicity in HAND and therefore MVs may serve as a novel therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0058-z) contains supplementary material, which is available to authorized users.

ID: 240: HCMV infected human monocyte-derived macrophages and DC produce type I IFN in a cGAS-dependent manner

The majority of the human population is latently infected with human cytomegalovirus (HCMV). In the immunocompromised host HCMV reactivation can lead to serious symptoms and vertical infection can cause severe disabilities in the infant. We addressed the susceptibility of primary human antigen presenting cells to HCMV infection and their capacity to mount type I interferon (IFN-I) responses. HCMV treated plasmacytoid dendritic cells (pDC) did not efficiently support viral gene expression, while abundant to intermediate viral gene expression was detected in M-CSF macrophages (M-CSF M Φ ) , monocyte-derived DC (moDC), and GM-CSF macrophages (GM-CSF M Φ ) in descending order. Even though HCMV encodes multiple evasion mechanisms, several of which exploit the interferon system, the magnitude of IFN-I responses mounted by the different monocyte-derived subsets correlated with the degree of infection. In contrast, pDC that were largely resistant to HCMV infection showed the highest IFN-I expression. These data implied that pDC sensed HCMV in a different manner than monocyte-derived cells. Since microbial dsDNA can activate cyclic GMP/AMP synthase (cGAS) to produce cyclic GMP-AMP dinucleotides (cGAMP), which trigger the stimulator of interferon genes (STING) that in turn induces IFN-I, we analyzed cGAS-dependent IFN-I responses in pDC, moDC, GM-CSF M Φ , and M-CSF M Φ . We found that upon HCMV infection monocyte-derived macrophages and DC, but not pDC, showed enhanced cGAMP production as a measure of cGAS activity. Moreover, monocyte-derived cells devoid of cGAS showed impaired IFN-I responses. Thus, HCMV stimulated primary monocyte-derived macrophages and DC, but not pDC, showed cGAS dependent cGAMP formation that triggered IFN-I.

HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

BackgroundMiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM).MethodsThe incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs.Results and ConclusionPAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

Thymosin α 1 potentiates the release by CD8+ cells of soluble factors able to inhibit HIV-1 and human T lymphotropic virus 1 infection in vitro

Background: Thymosin α-1 (Tα1) exploits a specific action on lymphoid cells and is able to induce in peripheral blood mononuclear cells (PBMCs) a strong transcriptional response. CD8 antiviral factor activity plays a role in the control or prevention of HIV-1 infection by a non-cytolytic mechanism. The ability of Tα1 to modulate the release of antiretroviral soluble factors by CD8+ cells was investigated.Methods: Supernatants from lipopolysaccharide (LPS) stimulated CD8+-isolated cells treated with Tα1 were screened on in vitro infection of human monocyte-derived macrophages (MDMs) and PBMCs with HIV-1, and of PBMCs with human T lymphotropic virus 1 (HTLV-1). In CD8+ cells, as well as in PBMCs of healthy donors as from HIV+ individuals, a microarray analysis to assess the transcriptional response after treatment was performed.Results: Tα1 potentiates the release, in LPS-stimulated CD8+ cells, of soluble factors able to inhibit both in vitro HIV-1 infection of MDMs and PBMCs and in vitro HTLV-1 infection of PBMCs. A distinctive transcriptional profile was induced by Tα1 in PBMCs from HIV+ donors.Conclusions: These findings suggest that Tα1 would represent a re-evaluated approach to antiretroviral therapy in combination with innovative treatments and with vaccine administration.

FcRγ-chain, Grb2 and SHP-1 are invovled in mannose receptor (CD206)-mediated signaling in relation to tuberculosis (INC4P.329)

Abstract The mannose receptor (MR, CD206) is highly expressed on human macrophages. It is a major pattern recognition receptor for pathogens, mediates phagocytosis and endocytosis, and regulates inflammatory signaling pathways. We have shown that the MR mediates Mycobacterium tuberculosis (M.tb) phagocytosis by human macrophages which leads to up-regulation of PPARγ expression and a delay in phagosome-lysosome (P-L) fusion. However, the associated receptor-specific downstream signaling molecules are unknown. We assayed for MR-associated proteins by MALDI-TOF and Western blot using a pull down assay with macrophage lysates and phosphorylated and un-phosphorylated MR cytoplasmic tail peptides. We have identified that FcRg-chain interacts with the MR and this interaction is necessary for MR surface exposure on human monocyte-derived macrophages (MDMs). Furthermore, M.tb infection of MDMs leads to phosphorylation of MR tyrosine residues and recruitment of Grb2 which initiates the Rac/Pak/Cdc-42 signaling cascade required for phagocytosis, and activation of MAP kinases and the PI3k/Akt pathway. MR activation also recruits and activates SHP-1. SHP-1 blockade increases M.tb P-L fusion and inflammatory cytokines. In conclusion, we have identified key MR signaling molecules and regulatory pathways which initiate phagocytosis and other immune-related functions in human macrophages.

Viral infection of human lung macrophages increases PDL1 expression via IFNβ.

Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM) and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNβ mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNβ by siRNA, resulted in a 37.5% reduction in IFNβ gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNβ, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNβ production.

HIV-1 reprograms the migration of macrophages

AbstractMacrophages are motile leukocytes, targeted by HIV-1, thought to play a critical role in host dissemination of the virus. However, whether infection impacts their migration capacity remains unknown. We show that 2-dimensional migration and the 3-dimensional (3D) amoeboid migration mode of HIV-1–infected human monocyte-derived macrophages were inhibited, whereas the 3D mesenchymal migration was enhanced. The viral protein Nef was necessary and sufficient for all HIV-1–mediated effects on migration. In Nef transgenic mice, tissue infiltration of macrophages was increased in a tumor model and in several tissues at steady state, suggesting a dominant role for mesenchymal migration in vivo. The mesenchymal motility involves matrix proteolysis and podosomes, cell structures constitutive of monocyte-derived cells. Focusing on the mechanisms used by HIV-1 Nef to control the mesenchymal migration, we show that the stability, size, and proteolytic function of podosomes are increased via the phagocyte-specific kinase Hck and Wiskott-Aldrich syndrome protein (WASP), 2 major regulators of podosomes. In conclusion, HIV-1 reprograms macrophage migration, which likely explains macrophage accumulation in several patient tissues, which is a key step for virus spreading and pathogenesis. Moreover, Nef points out podosomes and the Hck/WASP signaling pathway as good candidates to control tissue infiltration of macrophages, a detrimental phenomenon in several diseases.

Opposing regulation of endolysosomal pathways by long-acting nanoformulated antiretroviral therapy and HIV-1 in human macrophages.

BackgroundLong-acting nanoformulated antiretroviral therapy (nanoART) is designed to improve patient regimen adherence, reduce systemic drug toxicities, and facilitate clearance of human immunodeficiency virus type one (HIV-1) infection. While nanoART establishes drug depots within recycling and late monocyte-macrophage endosomes, whether or not this provides a strategic advantage towards viral elimination has not been elucidated.ResultsWe applied quantitative SWATH-MS proteomics and cell profiling to nanoparticle atazanavir (nanoATV)-treated and HIV-1 infected human monocyte-derived macrophages (MDM). Native ATV and uninfected cells served as controls. Both HIV-1 and nanoATV engaged endolysosomal trafficking for assembly and depot formation, respectively. Notably, the pathways were deregulated in opposing manners by the virus and the nanoATV, likely by viral clearance. Paired-sample z-scores, of the proteomic data sets, showed up- and down- regulation of Rab-linked endolysosomal proteins. NanoART and native ATV treated uninfected cells showed limited effects. The data was confirmed by Western blot. DAVID and KEGG bioinformatics analyses of proteomic data showed relationships between secretory, mobility and phagocytic cell functions and virus and particle trafficking.ConclusionsWe posit that modulation of endolysosomal pathways by antiretroviral nanoparticles provides a strategic path to combat HIV infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0133-5) contains supplementary material, which is available to authorized users.

HIV-1 subtype C unproductively infects human cardiomyocytes in vitro and induces apoptosis mitigated by an anti-Gp120 aptamer.

HIV-associated cardiomyopathy (HIVCM) is of clinical concern in developing countries because of a high HIV-1 prevalence, especially subtype C, and limited access to highly active antiretroviral therapy (HAART). For these reasons, we investigated the direct and indirect effects of HIV-1 subtype C infection of cultured human cardiomyocytes and the mechanisms leading to cardiomyocytes damage; as well as a way to mitigate the damage. We evaluated a novel approach to mitigate HIVCM using a previously reported gp120 binding and HIV-1 neutralizing aptamer called UCLA1. We established a cell-based model of HIVCM by infecting human cardiomyocytes with cell-free HIV-1 or co-culturing human cardiomyocytes with HIV-infected monocyte derived macrophages (MDM). We discovered that HIV-1 subtype C unproductively (i.e. its life cycle is arrested after reverse transcription) infects cardiomyocytes. Furthermore, we found that HIV-1 initiates apoptosis of cardiomyocytes through caspase-9 activation, preferentially via the intrinsic or mitochondrial initiated pathway. CXCR4 receptor-using viruses were stronger inducers of apoptosis than CCR5 utilizing variants. Importantly, we discovered that HIV-1 induced apoptosis of cardiomyocytes was mitigated by UCLA1. However, UCLA1 had no protective effective on cardiomyocytes when apoptosis was triggered by HIV-infected MDM. When HIV-1 was treated with UCLA1 prior to infection of MDM, it failed to induce apoptosis of cardiomyocytes. These data suggest that HIV-1 causes a mitochondrial initiated apoptotic cascade, which signal through caspase-9, whereas HIV-1 infected MDM causes apoptosis predominantly via the death-receptor pathway, mediated by caspase-8. Furthermore the data suggest that UCLA1 protects cardiomyocytes from caspase-mediated apoptosis, directly by binding to HIV-1 and indirectly by preventing infection of MDM.

Mannose receptor (CD206)-mediated signaling in human macrophages in the context of tuberculosis (INC7P.418)

Abstract The mannose receptor (MR, CD206) is abundantly expressed on alveolar and monocyte-derived macrophages (MDMs), recognizes several pathogens, mediates phagocytosis and endocytosis, and regulates inflammatory signaling pathways. It is a major human macrophage phagocytic receptor for Mycobacterium tuberculosis (M.tb). We have shown that MR-mediated phagocytosis of M.tb by MDMs up-regulates PPARγ expression, a negative regulator of NF-kB activation. However, the associated receptor-specific downstream signaling molecules are unknown. Thus, we investigated the signaling molecules involved in MR-mediated phagocytosis and other immune functions in MDMs. M.tb infection of MDMs leads to phosphorylation of tyrosine residues in the MR cytoplasmic tail that enable the recruitment of tyrosine kinases (e.g. Syk) which transmit the signals downstream. To identify MR-mediated signaling proteins, we assayed for MR-associated proteins by MALDI-TOF and Western blot using a pull down assay with macrophage lysates and phosphorylated and un-phosphorylated MR cytoplasmic tail peptides. We have identified several proteins bound to the phosphorylated peptides and linked to signaling pathways involved in MR-mediated phagocytosis and related immune funtions. In conclusion, we are identifying key signaling molecules and pathways used by the MR to transmit signals that initiate phagocytosis and other immune-related functions in human macrophages.

Epstein-Barr Virus Infection Induces Indoleamine 2,3-Dioxygenase Expression in Human Monocyte-Derived Macrophages through p38/Mitogen-Activated Protein Kinase and NF-κB Pathways: Impairment in T Cell Functions

Epstein-Barr virus (EBV) infection has been observed in tumor-infiltrated macrophages, but its infection effects on macrophage immune functions are poorly understood. Here, we showed that some macrophages in the tumor stroma of nasopharyngeal carcinoma (NPC) tissue expressed the immunosuppressive protein indoleamine 2,3-dioxygenase (IDO) more strongly than did tumor cells. EBV infection induced mRNA, protein, and enzymatic activity of IDO in human monocyte-derived macrophages (MDMs). Infection increased the production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), whereas the neutralizing antibodies against TNF-α and IL-6 inhibited IDO induction. EBV infection also activated the mitogen-activated protein kinase (MAPK) p38 and NF-κB, and the inhibition of these two pathways with SB202190 and SN50 almost abrogated TNF-α and IL-6 production and inhibited IDO production. Moreover, the activation of IDO in response to EBV infection of MDMs suppressed the proliferation of T cells and impaired the cytotoxic activity of CD8(+) T cells, whereas the inhibition of IDO activity with 1-methyl-l-tryptophan (1-MT) did not affect T cell proliferation and function. These findings indicate that EBV-induced IDO expression in MDMs is substantially mediated by IL-6- and TNF-α-dependent mechanisms via the p38/MAPK and NF-κB pathways, suggesting that a possible role of EBV-mediated IDO expression in tumor stroma of NPC may be to create a microenvironment of suppressed T cell immune responses. CD8(+) cytotoxic T lymphocytes (CTLs) play an important role in the control of viral infections and destroy tumor cells. Activation of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) in cancer tissues facilitates immune escape by the impairment of CTL functions. IDO expression was observed in some macrophages of the tumor stroma of nasopharyngeal carcinoma (NPC) tissue, and IDO could be induced in Epstein-Barr virus (EBV)-infected human monocyte-derived macrophages (MDMs). NPC cells and macrophages have been found to produce IDO in a gamma interferon (IFN-γ)-dependent manner. Instead, EBV-induced IDO expression in MDMs is substantially mediated by IL-6- and TNF-α-dependent mechanisms via the p38/MAPK and NF-κB pathways, which suppressed the proliferation of T cells and impaired the cytotoxic activity of CD8(+) T cells. This finding provides a new interpretation of the mechanism of immune escape of EBV and shows the immunosuppressive role of EBV-mediated IDO expression in tumor stroma of NPC.

Primary Human Macrophages Serve as Vehicles for Vaccinia Virus Replication and Dissemination

Human monocytic and professional antigen-presenting cells have been reported only to exhibit abortive infections with vaccinia virus (VACV). We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, but not human AB serum-derived cells, were permissive to VACV replication. The titers of infectious virions in both cell-free supernatants and cellular lysates of infected M1 and M2 markedly increased in a time-dependent manner. The majority of virions produced in permissive MDMs were extracellular enveloped virions (EEV), a secreted form of VACV associated with long-range virus dissemination, and were mainly found in the culture supernatant. Infected MDMs formed VACV factories, actin tails, virion-associated branching structures, and cell linkages, indicating that MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. VACV replication was sensitive to inhibitors against the Akt and Erk1/2 pathways that can be activated by VACV infection and M-CSF stimulation. Classical activation of MDMs by lipopolysaccharide (LPS) plus gamma interferon (IFN-γ) stimulation caused no effect on VACV replication, while alternative activation of MDMs by interleukin-10 (IL-10) or LPS-plus-IL-1β treatment significantly decreased VACV production. The IL-10-mediated suppression of VACV replication was largely due to Stat3 activation, as a Stat3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data demonstrate that primary human macrophages are permissive to VACV replication. After infection, these cells produce EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread. Our results provide critical information to the burgeoning fields of cancer-killing (oncolytic) virus therapy with vaccinia virus (VACV). One type of macrophage (M2) is considered a common presence in tumors and is associated with poor prognosis. Our results demonstrate a preference for VACV replication in M2 macrophages and could assist in designing treatments and engineering poxviruses with special considerations for their effect on M2 macrophage-containing tumors. Additionally, this work highlights the importance of macrophages in the field of vaccine development using poxviruses as vectors. The understanding of the dynamics of poxvirus-infected foci is central in understanding the effectiveness of the immune response to poxvirus-mediated vaccine vectors. Monocytic cells have been found to be an important part of VACV skin lesions in mice in controlling the infection as well as mediating virus transport out of infected foci.

Monocyte‐derived macrophages as an active HIV‐1 viral reservoir in patients receiving suppressive cART (145.4)

Macrophages play a crucial role in HIV‐1 latency and persistent viremia, especially in the establishment of viral reservoir, even when the patients are receiving suppressive cART. Macrophages are long‐lived cells that are resistant to the cytopathic effects of viral replication. Due to their abundant distribution and capacity to migrate into tissues we hypothesized that they can contribute to the maintenance of known viral reservoirs such as that in CD4+ T cells. We differentiated the infected monocytes of four HIV‐ infected patients taking anti‐retroviral therapy to create macrophages. Infected PBMC (peripheral blood mononuclear cells) were cultured for ten days in presence of macrophage colony stimulating factor. After differentiation, the monocyte‐derived macrophages (MDM) were co‐cultured with phytohemagglutinin (PHA)‐activated CD8+‐depleted PBMC’s from healthy patients for four weeks. Supernatants were harvested and analyzed for viral RNA using a sensitive reverse‐transcriptase PCR assay using HIV‐1 gag gene primers. Sequencing of the C2V3 region of the env gene from each patient revealed a 99.6% match with WT HXB2 using HIV Sequence Database (Los Alamos National Laboratory). Results demonstrate that after differentiation into macrophages the infected MDM can still harbor the proviral DNA and were able to release replicate competent virions in the supernatant. Future directions include performing cell to cell infection assay. These findings will be employed in an ex‐vivo model to further analyze HIV‐1 infection and transmission in order to demonstrate whether tissue macrophages are a true viral reservoir in HIV‐1 patients under suppressive cART.Grant Funding Source: NIH Grant R25‐096955

Single Nucleotide Polymorphic Macrophage Cytokine Regulation by Mycobacterium tuberculosis and Drug Treatment

To investigate the survival of Mycobacterium tuberculosis in primary macrophages with SNPs affecting cytokine secretion under treatment with drugs in solution or microparticles. Volunteers were typed for TNF (-308G/A), IL-10 (-1082A/G) and IL-4 (-590C/T). Monocyte-derived macrophages (MDMs) were infected in vitro. Cytokine secretion and survival of intracellular bacilli were estimated. IL-10 AG associated with high secretion in uninfected and infected MDMs (p < 0.05) and was reduced more effectively by microparticles than drugs, irrespective of genotype (p < 0.05). Differences were observed between IL-4 secretion by MDMs of CC and TT genotypes (p = 0.1). Bacteria proliferated more in MDMs from volunteers with higher IL-4 levels (p = 0.05). Microparticles showed higher efficacy (p = 0.05) than drugs. IL-4 and IL-10 SNPs affect the ability of macrophages to counter infection with M. tuberculosis. Microparticles elicit favorable macrophage cytokines regardless of SNPs.

Active Replication of Middle East Respiratory Syndrome Coronavirus and Aberrant Induction of Inflammatory Cytokines and Chemokines in Human Macrophages: Implications for Pathogenesis

Middle East respiratory syndrome coronavirus (MERS-CoV) infection caused severe pneumonia and multiorgan dysfunction and had a higher crude fatality rate (around 50% vs 10%) than SARS coronavirus (SARS-CoV) infection. To understand the pathogenesis, we studied viral replication, cytokine/chemokine response, and antigen presentation in MERS-CoV–infected human monocyte–derived macrophages (MDMs) versus SARS-CoV–infected MDMs. Only MERS-CoV can replicate in MDMs. Both viruses were unable to significantly stimulate the expression of antiviral cytokines (interferon α [IFN-α] and IFN-β) but induced comparable levels of tumor necrosis factor α and interleukin 6. Notably, MERS-CoV induced significantly higher expression levels of interleukin 12, IFN-γ, and chemokines (IP-10/CXCL-10, MCP-1/CCL-2, MIP-1α/CCL-3, RANTES/CCL-5, and interleukin 8) than SARS-CoV. The expression of major histocompatibility complex class I and costimulatory molecules were significantly higher in MERS-CoV–infected MDMs than in SARS-CoV–infected cells. MERS-CoV replication was validated by immunostaining of infected MDMs and ex vivo lung tissue. We conclusively showed that MERS-CoV can establish a productive infection in human macrophages. The aberrant induction of inflammatory cytokines/chemokines could be important in the disease pathogenesis.

High multiplicity HIV-1 cell-to-cell transmission from macrophages to CD4+ T cells limits antiretroviral efficacy.

Few studies have examined the efficacy of antiretroviral therapy (ART) in the context of cell-to-cell transmission. We aimed to determine whether the activity of ART is limited by the mode of HIV-1 spread between cells and the type of immune cell implicated in transmission, or is independent of these variables. ART activity was evaluated in primary cells using in-vitro cell-free and cell to-cell HIV-1 infection systems. HIV-1 cell-free or cell-to-cell transmission between infected monocyte-derived macrophages (MDMs) and autologous target CD4+ T cells was measured in the presence or absence of reverse transcriptase and integrase inhibitors. Viral infection was evaluated using luciferase-reporter infectious molecular HIV-1 clones carrying macrophage-tropic envelope glycoproteins (Envs). Cell-free HIV-1 was titrated to yield different multiplicities of CD4+ T-cell infection. Whereas cell-free infection of CD4+ T cells was substantially reduced by all inhibitors, cell-to-cell spread from macrophages to CD4+ T cells was largely resistant to inhibition. However, when multiplicity of infection was controlled for, we observed no difference in antiretroviral inhibition of cell-to-cell or cell-free infection. Cell-to-cell spread of HIV-1 reduces the probability of antiretroviral inhibition, but it is the number of infectious viruses transferred between cells rather than the specific mode of viral spread or transmitting cell type that governs antiretroviral activity. High multiplicity infection in vivo is more likely to occur by cell-to-cell transmission, and these data will inform use of ART against viral reservoirs.