Dopamine-induced pruning in monocyte-derived-neuronal-like cells (MDNCs) from patients with schizophrenia.

The long lapse between the presumptive origin of schizophrenia (SCZ) during early development and its diagnosis in late adolescence has hindered the study of crucial neurodevelopmental processes directly in living patients. Dopamine, a neurotransmitter consistently associated with the pathophysiology of SCZ, participates in several aspects of brain development including pruning of neuronal extensions. Excessive pruning is considered the cause of the most consistent finding in SCZ, namely decreased brain volume. It is therefore possible that patients with SCZ carry an increased susceptibility to dopamine's pruning effects and that this susceptibility would be more obvious in the early stages of neuronal development when dopamine pruning effects appear to be more prominent. Obtaining developing neurons from living patients is not feasible. Instead, we used Monocyte-Derived-Neuronal-like Cells (MDNCs) as these cells can be generated in only 20 days and deliver reproducible results. In this study, we expanded the number of individuals in whom we tested the reproducibility of MDNCs. We also deepened the characterization of MDNCs by comparing its neurostructure to that of human developing neurons. Moreover, we studied MDNCs from 12 controls and 13 patients with SCZ. Patients' cells differentiate more efficiently, extend longer secondary neurites and grow more primary neurites. In addition, MDNCs from medicated patients expresses less D1R and prune more primary neurites when exposed to dopamine. Haloperidol did not influence our results but the role of other antipsychotics was not examined and thus, needs to be considered as a confounder.

Cell-Associated HIV Cross-Presentation by Plasmacytoid Dendritic Cells Is Potentiated by Noncognate CD8+ T Cell Preactivation.

IFN-γ secretion by Ag-specific T cells is known to be tightly regulated by engagement of the TCR. Human plasmacytoid dendritic cells (pDC) can cross-present Ags from apoptotic HIV-infected cells or tumor cells to CD8+ T cells. As pDC respond to HIV virions by maturing and secreting cytokines, we hypothesized that this might affect cross-presentation from HIV-infected cells. Purified blood DC were incubated with apoptotic HIV-infected H9 cells in the presence of saquinavir, after which the activation process of HIV-specific cloned CD8+ T cells was studied. IFN-γ secretion by HIV-specific T cells was stimulated by pDC and conventional DC (cDC1) more than by cDC2 and was strictly MHC class I restricted. Surprisingly, intracellular production of IFN-γ was only partly MHC class I restricted for pDC, indicating a noncognate CD8+ T cell activation. pDC, but not cDC, matured and secreted IFN-α in the presence of apoptotic H9HIV cells. A mixture of IFN-α, IFN-ß, and TNF-α induced intracellular production of IFN-γ but not granzyme B, mimicking the noncognate mechanism. Neutralization of type I IFN signaling blocked noncognate intracellular production of IFN-γ. Moreover, cognate stimulation was required to induce IFN-γ secretion in addition to the cytokine mixture. Thus, IFN-γ secretion is tightly regulated by engagement of the TCR as expected, but in the context of virus-infected cells, pDC can trigger intracellular IFN-γ accumulation in CD8+ T cells, potentializing IFN-γ secretion once CD8+ T cells make cognate interactions. These findings may help manipulate type I IFN signaling to enhance specifically Ag-specific CD8+ T cell activation against chronic infections or tumors.

Human inflammatory dendritic cells induce Th17 cell differentiation.

Dendritic cells (DCs) are critical regulators of immune responses. Under noninflammatory conditions, several human DC subsets have been identified. Little is known, however, about the human DC compartment under inflammatory conditions. Here, we characterize a DC population found in human inflammatory fluids that displayed a phenotype distinct from macrophages from the same fluids and from steady-state lymphoid organ and blood DCs. Transcriptome analysis showed that they correspond to a distinct DC subset and share gene signatures with in vitro monocyte-derived DCs. Moreover, human inflammatory DCs, but not inflammatory macrophages, stimulated autologous memory CD4(+) T cells to produce interleukin-17 and induce T helper 17 (Th17) cell differentiation from naive CD4(+) T cells through the selective secretion of Th17 cell-polarizing cytokines. We conclude that inflammatory DCs represent a distinct human DC subset and propose that they are derived from monocytes and are involved in the induction and maintenance of Th17 cell responses.

Limited HIV-2 reservoirs in central-memory CD4 T-cells associated to CXCR6 co-receptor expression in attenuated HIV-2 infection.

The low pathogenicity and replicative potential of HIV-2 are still poorly understood. We investigated whether HIV-2 reservoirs might follow the peculiar distribution reported in models of attenuated HIV-1/SIV infections, i.e. limited infection of central-memory CD4 T lymphocytes (TCM). Antiretroviral-naive HIV-2 infected individuals from the ANRS-CO5 (12 non-progressors, 2 progressors) were prospectively included. Peripheral blood mononuclear cells (PBMCs) were sorted into monocytes and resting CD4 T-cell subsets (naive [TN], central- [TCM], transitional- [TTM] and effector-memory [TEM]). Reactivation of HIV-2 was tested in 30-day cultures of CD8-depleted PBMCs. HIV-2 DNA was quantified by real-time PCR. Cell surface markers, co-receptors and restriction factors were analyzed by flow-cytometry and multiplex transcriptomic study. HIV-2 DNA was undetectable in monocytes from all individuals and was quantifiable in TTM from 4 individuals (median: 2.25 log10 copies/106 cells [IQR: 1.99-2.94]) but in TCM from only 1 individual (1.75 log10 copies/106 cells). HIV-2 DNA levels in PBMCs (median: 1.94 log10 copies/106 PBMC [IQR = 1.53-2.13]) positively correlated with those in TTM (r = 0.66, p = 0.01) but not TCM. HIV-2 reactivation was observed in the cells from only 3 individuals. The CCR5 co-receptor was distributed similarly in cell populations from individuals and donors. TCM had a lower expression of CXCR6 transcripts (p = 0.002) than TTM confirmed by FACS analysis, and a higher expression of TRIM5 transcripts (p = 0.004). Thus the low HIV-2 reservoirs differ from HIV-1 reservoirs by the lack of monocytic infection and a limited infection of TCM associated to a lower expression of a potential alternative HIV-2 co-receptor, CXCR6 and a higher expression of a restriction factor, TRIM5. These findings shed new light on the low pathogenicity of HIV-2 infection suggesting mechanisms close to those reported in other models of attenuated HIV/SIV infection models.

Chronic Type I IFN Is Sufficient To Promote Immunosuppression through Accumulation of Myeloid-Derived Suppressor Cells.

Failure of the immune system to eradicate viruses results in chronic viral infections, which are associated with increased susceptibility to secondary infections. Pathogenic HIV or lymphocytic choriomeningitis virus chronic infections display a persistent type I IFN signature. In chronic lymphocytic choriomeningitis virus infection, blockade of type I IFN signaling partially restores antiviral responses. In a mouse model, we tested whether chronic administration of type I IFN, at doses mimicking chronic viral infection, induced immunosuppression. Chronic exposure of mice to IFN-α alone was sufficient to strongly suppress specific CD8+ T cells responses to subsequent vaccinia virus infection. It resulted in the accumulation of Ly6Chi monocytes. These monocytes were similar, phenotypically and functionally, to the myeloid-derived suppressor cells found in cancer because they exerted a potent suppression on CD8+ T cell responses in vitro. They acted at least partly through the l-arginine pathway. In vivo, their elimination restored antiviral CD8+ T cell responses. Our work provides a specific mechanism accounting for the role of IFN-α in immunosuppression and predicts that type I IFN modulation will be pivotal to cure human chronic infections, cancer, or autoimmune diseases.

TLR3-responsive, XCR1+, CD141(BDCA-3)+/CD8α+-equivalent dendritic cells uncovered in healthy and simian immunodeficiency virus-infected rhesus macaques.

In mice, CD8α(+) myeloid dendritic cells (mDC) optimally cross-present Ags to CD8(+) T cells and respond strongly to TLR3 ligands. Although equivalent DC have been identified by comparative genomic analysis and functional studies in humans as XCR1(+)CD141 (BDCA-3)(+)Clec9A(+)cell adhesion molecule 1(+) mDC, and in sheep as CD26(+) mDC, these cells remained elusive in nonhuman primates. To remedy this situation, we delineated precisely DC and monocyte populations by 12-color flow cytometry and transcriptomic analyses in healthy rhesus macaques. We identified a new mDC population, with strong phenotypic and transcriptional homology to human CD141(+) and murine CD8α(+) mDC, including XCR1 membrane expression as a conserved specific marker. In contrast, high CD11c expression was not characteristic of mDC in macaques, but of CD16(+) monocytes. Like their human and murine homologs, simian XCR1(+) mDC had much stronger responses to TLR3 stimulation than other myeloid cells. The importance of this new mDC population was tested in SIV(mac251) infection, the most relevant animal model for pathogenic HIV-1 infection and vaccination. This population increased sharply and transiently during acute infection, but was reduced in blood and spleen during advanced disease. The identification of XCR1(+) mDC in rhesus macaques opens new avenues for future preclinical vaccinal studies and highlights XCR1 as a prime candidate for targeted vaccine delivery.

Apoptotic cell capture by DCs induces unexpectedly robust autologous CD4+ T-cell responses.

Apoptotic cells represent an important source of self-antigens and their engulfment by dendritic cells (DCs) is usually considered to be related to tolerance induction. We report here an unexpectedly high level of human CD4(+) T-cell proliferation induced by autologous DCs loaded with autologous apoptotic cells, due to the activation of more than 10% of naive CD4(+) T cells. This proliferation is not due to an increase in the costimulatory capacity of DCs, but is dependent on apoptotic cell-associated material processed through an endo-lysosomal pathway and presented on DC MHC class II molecules. Autologous CD4(+) T cells stimulated with apoptotic cell-loaded DCs exhibit suppressive capacities. However, in the presence of bacterial lipopolysaccharide, apoptotic cell-loaded DCs induce the generation of IL-17-producing cells. Thus, apoptotic cell engulfment by DCs may lead to increased autologous responses, initially generating CD4(+) T cells with suppressive capacities able to differentiate into Th17 cells in the presence of a bacterial danger signal such as LPS.

Pivotal role of M-DC8⁺ monocytes from viremic HIV-infected patients in TNFα overproduction in response to microbial products.

HIV infects activated CD4⁺ T cells and induces their depletion. Progressive HIV infection leading to AIDS is fueled by chronic immune hyperactivation, mediated by inflammatory cytokines like TNFα. This has been related to intestinal epithelial damage and microbial LPS translocation into the circulation. Using 11-color flow cytometry, cell sorting, and cell culture, we investigated the numbers and TNFα production of fully defined circulating dendritic cell and monocyte populations during HIV-1 infection. In 15 viremic, untreated patients, compared with 8 treated, virologically suppressed patients or to 13 healthy blood donors, circulating CD141 (BDCA-3)⁺ and CD1c (BDCA-1)⁺ dendritic cell counts were reduced. Conversely, CD14⁺ CD16⁺⁺ monocyte counts were increased, particularly those expressing M-DC8, while classical CD14⁺⁺ CD16⁻ M-DC8⁻ monocyte numbers were unchanged. Blood mononuclear cells from viremic patients produced more TNFα in response to LPS than those from virologically suppressed patients. M-DC8⁺ monocytes were mostly responsible for this overproduction. Moreover, M-DC8⁺ monocytes differentiated in vitro from classical monocytes using M-CSF and GM-CSF, which is increased in viremic patient's plasma. This M-DC8⁺ monocyte population, which is involved in the pathogenesis of chronic inflammatory diseases like Crohn disease, might thus be considered as a major actor in the immune hyperactivation fueling HIV infection progression.

TIP47 is required for the production of infectious HIV-1 particles from primary macrophages.

Macrophages are among the major targets of HIV-1 infection and play a key role in viral pathogenesis. Identification of the cellular cofactors involved in the production of infectious HIV-1 from macrophages is thus crucial. Here, we investigated the role of the cellular cofactor TIP47 in HIV-1 morphogenesis in primary macrophages. Using siRNA approach, we show that TIP47 is essential for HIV-1 infectivity and propagation. TIP47 silencing disrupts Gag and Env colocalization in macrophages. Moreover, mutations in HIV-1 Gag or Env, which abolish interaction with TIP47, impair HIV-1 propagation and infectivity preventing colocalization of Gag and Env, Gag and Env coimmunoprecipitation. Interestingly, disruption of Gag-TIP47 interaction by matrix mutation or TIP47 depletion also causes Gag to localize in scattered dots in the vicinity of the plasma membrane of macrophages. Therefore, TIP47 is required for the encounter between Gag and Env, and thus for the generation of infectious HIV-1 particles from primary macrophages.

Cross-presentation by dendritic cells from live cells induces protective immune responses in vivo.

Cross-presentation is an essential mechanism that allows dendritic cells (DCs) to efficiently present exogenous antigens to CD8(+) T cells. Among cellular antigen sources, apoptotic cells are commonly considered as the best for cross-presentation by DCs. However, the potential of live cells as a source of antigen has been overlooked. Here we explored whether DCs were able to capture and cross-present antigens from live cells. DCs internalized cytosolic and membrane material into vesicles from metabolically labeled live cells. Using time-lapse confocal microscopy in whole spleens, we showed that DCs internalized material from live cells in vivo. After ovalbumin uptake from live cells, DCs cross-primed ovalbumin-specific naive OT-I CD8(+) T cells in vitro. Injected into mice previously transferred with naive OT-I T cells, they also cross-primed in vivo, even in the absence of endogenous DCs able to present the epitope in the recipient mice. Interestingly, DCs induced stronger natural CD8(+) T-cell responses and protection against a lethal tumor challenge after capture of antigens from live melanoma cells than from apoptotic melanoma cells. The potential for cross-presentation from live cells uncovers a new type of cellular intercommunication and must be taken into account for induction of tolerance or immunity against self, tumors, grafts, or pathogens.

A tribute to Nilabh Shastri and a special issue on antigen processing and presentation in Paris (APP10, Paris 2019).

The Special Issue is dedicated to the 10th Antigen Processing and Presentation Workshop, which took place at Institut Cochin in Paris from May 28th to June 2nd, 2019. It contains several reviews or original articles from contributors to this workshop. It is also a vibrant Tribute to Nilabh Shastri, founder of the APP Workshops, who untimely passed away in 2021 and is deeply missed by his colleagues and friends.

Plasmacytoid dendritic cells accumulate in spleens from chronically HIV-infected patients but barely participate in interferon-alpha expression.

We characterized the localization, phenotype, and some functions of plasmacytoid dendritic cells (pDCs) in the human spleen. pDCs were localized in the marginal zone and the periarteriolar region. Some were also found in the red pulp. pDCs were immature by phenotypic labeling, consistently with their capacity to internalize Dextran in a functional assay. In spleens from HIV-infected patients with thrombocytopenic purpura, these characteristics were unaffected. However, an accumulation of pDCs, but not myeloid dendritic cells (mDCs), was observed in some HIV+ patients, correlating with high proviral loads. Moreover, although undetectable in most HIV- patients, interferon-alpha (IFN-alpha) production was evidenced in situ and by flow cytometry in most HIV+ patients. IFN-alpha was located in the marginal zone. Surprisingly, IFN-alpha colocalized only with few pDCs, but rather with other cells, including T and B lymphocytes, mDCs, and macrophages. Therefore, pDCs accumulated in spleens from HIV+ patients with high proviral loads, but they did not seem to be the main IFN-alpha producers.

Primary infection with simian immunodeficiency virus: plasmacytoid dendritic cell homing to lymph nodes, type I interferon, and immune suppression.

Plasmacytoid dendritic cells (pDCs) are antigen-presenting cells that develop into type-I interferon (IFN-I)-producing cells in response to pathogens. Their role in human immunodeficiency virus (HIV) pathogenesis needs to be understood. We analyzed their dynamics in relation to innate and adaptive immunity very early during the acute phase of simian immunodeficiency virus (SIV) infection in 18 macaques. pDC counts decreased in blood and increased in peripheral lymph nodes, consistent with early recruitment in secondary lymphoid tissues. These changes correlated with the kinetic and intensity of viremia and were associated with a peak of plasma IFN-I. IFN-I and viremia were positively correlated with functional activity of the immune suppression associated enzyme indoleamine-2,3-dioxygenase (IDO) and FoxP3(+)CD8(+) T cells, which both negatively correlated with SIV-specific T-cell proliferation and CD4(+) T-cell activation. These data suggest that pDCs and IFN-I play a key role in shaping innate and adaptive immunity toward suppressive pathways during the acute phase of SIV/HIV primary infection.

Monitoring antigen cross-presentation by human dendritic cells purified from peripheral blood.

Tumor immunosurveillance, regression and therapy require most often the action of CD8+ T cells. These cells are primed by dendritic cells (DC), which are the only antigen presenting cells able to stimulate naive T cells. Tumor antigen presentation requires cross-presentation of antigens from the tumor cells by DC. Dendritic cells capture antigens from tumor cells into endosomal compartments and process them. Then they expose at their cell surface their own MHC class I molecules complexed with tumor cell epitopes, which are recognized by the T cell receptors of specific CD8+ T cells. This allows the activation of anti-tumoral functions of these specific CD8+ T cells, mediated by cytokines and by cytotoxic mechanisms. Here, we describe in detail the delicate methods required to (1) prepare antigen donor cells, (2) prepare CD8+ T cells specific for these antigens, (3) purify human DC from peripheral blood, (4) preincubate purified DC and antigen donor cells for antigen capture, (5) incubate these DC with antigen-specific CD8+ T cells for cross-presentation and (6) assess cross-presentation to specific CD8+ T cells. These methods allowed our laboratory to characterize in detail cross-presentation from cells containing viral antigens and can be applied to study cross-presentation from tumor antigens.

Conventional Dendritic Cells and Slan+ Monocytes During HIV-2 Infection.

HIV-2 infection is characterized by low viremia and slow disease progression as compared to HIV-1 infection. Circulating CD14++CD16+ monocytes were found to accumulate and CD11c+ conventional dendritic cells (cDC) to be depleted in a Portuguese cohort of people living with HIV-2 (PLWHIV-2), compared to blood bank healthy donors (HD). We studied more precisely classical monocytes; CD16+ inflammatory (intermediate, non-classical and slan+ monocytes, known to accumulate during viremic HIV-1 infection); cDC1, important for cross-presentation, and cDC2, both depleted during HIV-1 infection. We analyzed by flow cytometry these PBMC subsets from Paris area residents: 29 asymptomatic, untreated PLWHIV-2 from the IMMUNOVIR-2 study, part of the ANRS-CO5 HIV-2 cohort: 19 long-term non-progressors (LTNP; infection ≥8 years, undetectable viral load, stable CD4 counts≥500/µL; 17 of West-African origin -WA), and 10 non-LTNP (P; progressive infection; 9 WA); and 30 age-and sex-matched controls: 16 blood bank HD with unknown geographical origin, and 10 HD of WA origin (GeoHD). We measured plasma bacterial translocation markers by ELISA. Non-classical monocyte counts were higher in GeoHD than in HD (54 vs. 32 cells/µL, p = 0.0002). Slan+ monocyte counts were twice as high in GeoHD than in HD (WA: 28 vs. 13 cells/µL, p = 0.0002). Thus cell counts were compared only between participants of WA origin. They were similar in LTNP, P and GeoHD, indicating that there were no HIV-2 related differences. cDC counts did not show major differences between the groups. Interestingly, inflammatory monocyte counts correlated with plasma sCD14 and LBP only in PLWHIV-2, especially LTNP, and not in GeoHD. In conclusion, in LTNP PLWHIV-2, inflammatory monocyte counts correlated with LBP or sCD14 plasma levels, indicating a potential innate immune response to subclinical bacterial translocation. As GeoHD had higher inflammatory monocyte counts than HD, our data also show that specific controls are important to refine innate immunity studies.

Plasmacytoid dendritic cell dynamics and alpha interferon production during Simian immunodeficiency virus infection with a nonpathogenic outcome.

We addressed the role of plasmacytoid dendritic cells (PDC) in protection against AIDS in nonpathogenic simian immunodeficiency virus (SIVagm) infection in African green monkeys (AGMs). PDC were monitored in blood and lymph nodes (LNs) starting from day 1 postinfection. We observed significant declines in blood during acute infection. However, PDC then returned to normal levels, and chronically infected AGMs showed no decrease of PDC in blood. There was a significant increase of PDC in LNs during acute infection. Blood PDC displayed only weak alpha interferon (IFN-alpha) responses to TLR9 agonist stimulation before infection. However, during acute infection, both blood and LN PDC showed a transiently increased propensity for IFN-alpha production. Bioactive IFN-alpha was detected in plasma concomitant with the peak of viremia, though levels were only low to moderate in some animals. Plasma interleukin 6 (IL-6) and IL-12 were not increased. In conclusion, PDC were recruited to the LNs and displayed increased IFN-alpha production during acute infection. However, increases in IFN-alpha were transient. Together with the lack of inflammatory cytokine responses, these events might play an important role in the low level of T-cell activation which is associated with protection against AIDS in nonpathogenic SIVagm infection.

Plasmodium falciparum exposure in utero, maternal age and parity influence the innate activation of foetal antigen presenting cells.

BACKGROUND: Malaria in pregnancy is associated with immunological abnormalities in the newborns, such as hampered T-helper 1 responses and increased T-regulatory responses, while the effect of maternal Plasmodium falciparum infection on foetal innate immunity is still controversial. MATERIALS AND METHODS: The immunophenotype and cytokine release by dendritic cells (DC) and monocytes were evaluated in cord blood from 59 Beninese women with or without malaria infection by using flow cytometry. RESULTS: Accumulation of malaria pigment in placenta was associated with a partial maturation of cord blood myeloid and plasmacytoid DC, as reflected by an up-regulated expression of the major histocompatibility complex class II molecules, but not CD86 molecules. Cells of newborns of mothers with malaria pigment in their placenta also exhibited significantly increased cytokine responses upon TLR9 stimulation. In addition, maternal age and parity influenced the absolute numbers and activation status of cord blood antigen-presenting cells. Lastly, maternal age, but not parity, influenced TLR3, 4 and 9 responses in cord blood cells. DISCUSSION: Our findings support the view that placental parasitization, as indicated by the presence of malaria pigment in placental leukocytes, is significantly associated with partial maturation of different DC subsets and also to slightly increased responses to TLR9 ligand in cord blood. Additionally, other factors, such as maternal age and parity should be taken into consideration when analysing foetal/neonatal innate immune responses. CONCLUSION: These data advocate a possible mechanism by which PAM may modulate foetal/neonatal innate immunity.

Stimulation of the primary anti-HIV antibody response by IFN-alpha in patients with acute HIV-1 infection.

Type I IFNs are needed for the production of antiviral antibodies in mice; whether they also stimulate primary antibody responses in vivo during human viral infections is unknown. This was assessed in patients acutely infected with HIV-1 and treated with IFN-alpha2b. Patients with acute HIV-1 infection were randomized to receive antiretroviral therapy alone (Group A, n=60) or combined for 14 weeks with pegylated-IFN-alpha2b (Group B, n=30). Emergence of anti-HIV antibodies was monitored during 32 weeks by Western blot (WB) analyses of serum samples. IFN-alpha2b treatment stimulated the production of anti-HIV antibodies. On Week 32, 19 weeks after the last IFN-alpha2b administration, there were 8.5 (6.5-10.0) HIV WB bands (median, interquartile range) in Group B and 7.0 (5.0-10.0) bands in Group A (P=0.054), and band intensities were stronger in Group B (P<0.05 for p18, p24, p34, p40, and p55 HIV antigens). IFN-alpha2b treatment also increased circulating concentrations of the B cell-activating factor of the TNF family (P<0.001) and ex vivo production of IL-12 (P<0.05), reflecting its effect on innate immune cells. Withdrawal of antiretroviral treatment on Week 36 resulted in a lower rebound of HIV replication in Group B than in Group A (P<0.05). Therefore, type I IFNs stimulate the emerging anti-HIV immune response in patients with acute HIV-1 infection, resulting in an improved control of HIV replication. Type I IFNs are thus critical in the development of efficient antiviral immune responses in humans, including the production of antiviral antibodies.

HIV-1 reservoirs in urethral macrophages of patients under suppressive antiretroviral therapy.

Human immunodeficiency virus type 1 (HIV-1) eradication is prevented by the establishment on infection of cellular HIV-1 reservoirs that are not fully characterized, especially in genital mucosal tissues (the main HIV-1 entry portal on sexual transmission). Here, we show, using penile tissues from HIV-1-infected individuals under suppressive combination antiretroviral therapy, that urethral macrophages contain integrated HIV-1 DNA, RNA, proteins and intact virions in virus-containing compartment-like structures, whereas viral components remain undetectable in urethral T cells. Moreover, urethral cells specifically release replication-competent infectious HIV-1 following reactivation with the macrophage activator lipopolysaccharide, while the T-cell activator phytohaemagglutinin is ineffective. HIV-1 urethral reservoirs localize preferentially in a subset of polarized macrophages that highly expresses the interleukin-1 receptor, CD206 and interleukin-4 receptor, but not CD163. To our knowledge, these results are the first evidence that human urethral tissue macrophages constitute a principal HIV-1 reservoir. Such findings are determinant for therapeutic strategies aimed at HIV-1 eradication.

CCR5 signaling through phospholipase D involves p44/42 MAP-kinases and promotes HIV-1 LTR-directed gene expression.

The chemokine receptor CCR5 plays an important role as an entry gate for the human immunodeficiency virus-1 (HIV-1) and for viral postentry events. Among signal transducers used by chemoattractant receptors, the phosphatidylcholine-specific phospholipase D (PLD) produces large amounts of second messengers in most cell types. However, the relevance of PLD isoforms to CCR5 signaling and HIV-1 infection process remains unexplored. We show here that CCR5 activation by MIP-1beta in HeLa-MAGI cells triggered a rapid and substantial PLD activity, as assessed by mass choline production. This activity required the activation of ERK1/2-MAP kinases and involved both PLD1 and PLD2. MIP-1beta also promoted the activation of an HIV-1 long terminal repeat (LTR) by the transactivator Tat in HeLa P4.2 cells through a process involving ERK1/2. Expression of wild-type and catalytically inactive PLDs dramatically boosted and inhibited the LTR activation, respectively, without altering Tat expression. Wild-type and inactive PLDs also respectively potentiated and inhibited HIV-1(BAL) replication in MAGI cells. Finally, in monocytic THP-1 cells, antisense oligonucleotides to both PLDs dramatically inhibited the HIV-1 replication. Thus, PLD is activated downstream of ERK1/2 upon CCR5 activation and plays a major role in promoting HIV-1 LTR transactivation and virus replication, which may open novel perspectives to anti-HIV-1 strategies.