PACAP key interactions with PAC1, VPAC1, and VPAC2 identified by molecular dynamics simulations.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) belongs to the glucagon/secretin family. PACAP interacts with the pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) and vasoactive intestinal peptide receptors 1 and 2 (VPAC1 and VPAC2), exhibiting functions in the immune, endocrine, and nervous systems. This peptide is upregulated in numerous instances of brain injury, acting as a neuroprotective agent. It can also suppress HIV-1 and SARS-CoV-2 viral replication in vitro. This work aimed to identify, in each peptide-receptor system, the most relevant residues for complex stability and interaction energy communication via Molecular Dynamics (MD), Free Energy calculations, and Protein-energy networks, thus revealing in detail the underlying mechanisms of activation of these receptors. Hydrogen bond formation, interaction energies, and computational alanine scanning between PACAP and its receptors showed that His1, Asp3, Arg12, Arg14, and Lys15 are crucial to the peptide's stability. Furthermore, several PACAP interactions with structurally conserved positions deemed necessary in GPCR B1 activation, including Arg2.60, Lys2.67, and Glu7.42, were significant for the peptide's stability within the receptors. According to the protein-energy network, the connection between Asp3 of PACAP and the receptors' conserved Arg2.60 represents a critical energy communication hub in all complexes. Additionally, the ECDs of the receptors were also found to function as energy communication hubs for PACAP. Although the overall binding mode of PACAP in the three receptors was found to be highly conserved, Arg12 and Tyr13 of PACAP were more prominent in complex with PAC1, while Ser2 of PACAP was with VPAC2. The detailed analyses performed in this work pave the way for using PACAP and its receptors as therapeutic targets.Communicated by Ramaswamy H. Sarma.

Interleukin-27 Promotes Divergent Effects on HIV-1 Infection in Peripheral Blood Mononuclear Cells through BST-2/Tetherin.

Interleukin-27 (IL-27) is able to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs), macrophages, and dendritic cells. Here, we identify that IL-27 can produce opposing effects on HIV-1 replication in PBMCs and that the HIV-1 restriction factor BST-2/Tetherin is involved in both inhibitory and enhancing effects on HIV-1 infection induced by IL-27. IL-27 inhibited HIV-1 replication when added to cells 2 h after infection, promoting the prototypical BST-2/Tetherin-induced virion accumulation at the cell membrane of HIV-1-infected PBMCs. BST-2/Tetherin gene expression was significantly upregulated in the IL-27-treated PBMCs, with a simultaneous increase in the number of BST-2/Tetherin+ cells. The silencing of BST-2/Tetherin diminished the anti-HIV-1 effect of IL-27. In contrast, IL-27 increased HIV-1 production when added to infected cells 4 days after infection. This enhancing effect was prevented by BST-2/Tetherin gene knockdown, which also permitted IL-27 to function again as an HIV-1 inhibitory factor. These contrasting roles of IL-27 were associated with the dynamic of viral production, since the IL-27-mediated enhancement of virus replication was prevented by antiretroviral treatment of infected cells, as well as by keeping cells under agitation to avoid cell-to-cell contact. Likewise, inhibition of CD11a, an integrin associated with HIV-1 cell-to-cell transmission, abrogated the IL-27 enhancement of HIV-1 production. Our findings illustrate the complexity of the HIV-1-host interactions and may impact the potential therapeutic use of IL-27 and other soluble mediators that induce BST-2/Tetherin expression for HIV-1 infection. IMPORTANCE Here, we describe new findings related to the ability of the cytokine IL-27 to regulate the growth of HIV-1 in CD4+ T lymphocytes. IL-27 has long been considered a potent inhibitor of HIV-1 replication, a notion based on several reports showing that this cytokine controls HIV-1 infection in peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages, and dendritic cells. However, our present results are contrary to the current knowledge that IL-27 acts only as a powerful downregulator of HIV-1 replication. We observed that IL-27 can either prevent or enhance viral growth in PBMCs, an outcome dependent on when this cytokine is added to the infected cells. We detected that the increase of HIV-1 dissemination is due to enhanced cell-to-cell transmission with the involvement of the interferon-induced HIV-1 restriction factor BST-2/Tetherin and CD11a (LFA-1), an integrin that participates in formation of virological synapse.

HIV-1 Gag and Vpr impair the inflammasome activation and contribute to the establishment of chronic infection in human primary macrophages.

The successful establishment of HIV-1 infection is related to inflammasome blocking or inactivation, which can result in the viral evasion of the immune responses and formation of reservoirs in several tissues. In this sense, we aimed to evaluate the viral and cellular mechanisms activated during HIV-1 infection in human primary macrophages that allow an effective viral replication in these cells. We found that resting HIV-1-infected macrophages, but not those activated in classical or alternative patterns, released IL-1ß and other pro-inflammatory cytokines, and showed increased CXCL10 expression, without changes in the NLRP3, AIM2 or RIG-I inflammasome pathways. Also, similar levels of Casp-1, phosphorylated NF-κB (p65) and NLRP3 proteins were found in uninfected and HIV-1-infected macrophages. Likewise, no alterations were detected in ASC specks released in the culture supernatant after HIV-1 infection, suggesting that macrophages remain viable after infection. Using in silico prediction studies, we found that the HIV-1 proteins Gag and Vpr interact with several host proteins. Comparable levels of trans-LTB4 were found in the supernatants of uninfected and HIV-1-infected macrophages, whereas ROS production was impaired in infected cells, which was not reversed after the PMA stimulus. Immunofluorescence analysis showed structural alterations in the mitochondrial architecture and an increase of BIM in the cytoplasm of infected cells. Our data suggest that HIV-1 proteins Gag and Vpr, through interacting with cellular proteins in the early steps of infection, preclude the inflammasome activation and the development of effective immune responses, thus allowing the establishment of the infection.

VIP plasma levels associate with survival in severe COVID-19 patients, correlating with protective effects in SARS-CoV-2-infected cells.

Infection by SARS-CoV-2 may elicit uncontrolled and damaging inflammatory responses. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the inflammatory reaction. Here, we show that the plasma levels of the immunoregulatory neuropeptide VIP are elevated in patients with severe COVID-19, correlating with reduced inflammatory mediators and with survival on those patients. In vitro, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), highly similar neuropeptides, decreased the SARS-CoV-2 RNA content in human monocytes and viral production in lung epithelial cells, also reducing cell death. Both neuropeptides inhibited the production of proinflammatory mediators in lung epithelial cells and in monocytes. VIP and PACAP prevented in monocytes the SARS-CoV-2-induced activation of NF-kB and SREBP1 and SREBP2, transcriptions factors involved in proinflammatory reactions and lipid metabolism, respectively. They also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF-kB. Specific inhibition of NF-kB and SREBP1/2 reproduced the anti-inflammatory, antiviral, and cell death protection effects of VIP and PACAP. Our results support further clinical investigations of these neuropeptides against COVID-19.

Unlike Chloroquine, Mefloquine Inhibits SARS-CoV-2 Infection in Physiologically Relevant Cells.

Despite the development of specific therapies against severe acute respiratory coronavirus 2 (SARS-CoV-2), the continuous investigation of the mechanism of action of clinically approved drugs could provide new information on the druggable steps of virus-host interaction. For example, chloroquine (CQ)/hydroxychloroquine (HCQ) lacks in vitro activity against SARS-CoV-2 in TMPRSS2-expressing cells, such as human pneumocyte cell line Calu-3, and likewise, failed to show clinical benefit in the Solidarity and Recovery clinical trials. Another antimalarial drug, mefloquine, which is not a 4-aminoquinoline like CQ/HCQ, has emerged as a potential anti-SARS-CoV-2 antiviral in vitro and has also been previously repurposed for respiratory diseases. Here, we investigated the anti-SARS-CoV-2 mechanism of action of mefloquine in cells relevant for the physiopathology of COVID-19, such as Calu-3 cells (that recapitulate type II pneumocytes) and monocytes. Molecular pathways modulated by mefloquine were assessed by differential expression analysis, and confirmed by biological assays. A PBPK model was developed to assess mefloquine's optimal doses for achieving therapeutic concentrations. Mefloquine inhibited SARS-CoV-2 replication in Calu-3, with an EC50 of 1.2 µM and EC90 of 5.3 µM. It reduced SARS-CoV-2 RNA levels in monocytes and prevented virus-induced enhancement of IL-6 and TNF-α. Mefloquine reduced SARS-CoV-2 entry and synergized with Remdesivir. Mefloquine's pharmacological parameters are consistent with its plasma exposure in humans and its tissue-to-plasma predicted coefficient points suggesting that mefloquine may accumulate in the lungs. Altogether, our data indicate that mefloquine's chemical structure could represent an orally available host-acting agent to inhibit virus entry.

The Role of Extracellular Vesicles from Human Macrophages on Host-Pathogen Interaction.

The nano-sized membrane enclosed extracellular vesicles (EVs) released by virtually all cell types play an essential role in intercellular communication via delivering bio-molecules, such as nucleic acids, proteins, lipids, and other molecules to recipient cells. By mediating an active and steady-state cell-to-cell communication, EVs contribute to regulating and preserving cellular homeostasis. On the other hand, EVs can also spread pathogen-derived molecules during infections, subverting the host immune responses during infections and thus worsening pathophysiological processes. In recent years, the biological functioning of EVs has become a widespread research field in basic and clinical branches of medical sciences due to their potential role in therapeutic applications for several diseases. This review aims to summarize the main recent findings regarding the implication of EVs shed by human macrophages (MΦ-EVs) and how they can modulate the host immune response to control or increase the damage caused by infectious agents. We will also present the methods used to describe MΦ-EVs, as well as the potential of these EVs as disease diagnostic tools for some human pathogens. We believe that an in-depth understanding of the host-pathogen interactions mediated by MΦ-EVs may trigger the development of innovative therapeutic strategies against infectious diseases.

Neutrophil extracellular traps from healthy donors and HIV-1-infected individuals restrict HIV-1 production in macrophages.

Neutrophils release extracellular traps (NETs) after interaction with microorganisms and physiological or synthetic products. NETs consist of decondensed chromatin complexed with proteins, some of them with microbicidal properties. Because NETs can modulate the functioning of HIV-1 target cells, we aimed to verify whether they modify HIV-1 replication in macrophages. We found that exposure of HIV-1-infected macrophages to NETs resulted in significant inhibition of viral replication. The NET anti-HIV-1 action was independent of other soluble factors released by the activated neutrophils, but otherwise dependent on the molecular integrity of NETs, since NET-treatment with protease or DNase abolished this effect. NETs induced macrophage production of the anti-HIV-1 ß-chemokines Rantes and MIP-1ß, and reduced the levels of integrated HIV-1 DNA in the macrophage genome, which may explain the decreased virus production by infected macrophages. Moreover, the residual virions released by NET-treated HIV-1-infected macrophages lost infectivity. In addition, elevated levels of DNA-elastase complexes were detected in the plasma from HIV-1-infected individuals, and neutrophils from these patients released NETs, which also inhibited HIV-1 replication in in vitro infected macrophages. Our results reveal that NETs may function as an innate immunity mechanism able to restrain HIV-1 production in macrophages.

Characterization and internalization of small extracellular vesicles released by human primary macrophages derived from circulating monocytes.

Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes.

Neutrophil extracellular trap-enriched supernatants carry microRNAs able to modulate TNF-α production by macrophages.

Neutrophil extracellular traps (NETs) emerge from the cell as a DNA scaffold associated with cytoplasmic and granular proteins, able to immobilize and kill pathogens. This association occurs following nuclear and granular membrane disintegration, allowing contact with the decondensed chromatin. Thus, it is reasonable to speculate that the DNA can also mix with miRNAs and carry them in NETs. Here, we report for the first time the presence of the miRNA carriers associated with NETs and miRNAs present in NET-enriched supernatants (NET-miRs), thus adding a novel class of molecules and new proteins that can be released and transported in the NET platform. We observed that the majority of NET-miRs were common to all four stimuli used (PMA, interleukin-8, amyloid fibrils and Leishmania), and that miRNA-142-3p carried by NETs down-modulates protein kinase Cα and regulates TNF-α production in macrophages upon NET interaction with these cells. Our findings unveil a novel role for NETs in the cell communication processes, allowing the conveyance of miRNA from neutrophils to neighboring cells.

IL-1ß Blockade Attenuates Thrombosis in a Neutrophil Extracellular Trap-Dependent Breast Cancer Model.

Cancer patients are at increased risk of developing thrombosis, comorbidity that has been associated with increased neutrophil counts and the formation of neutrophil extracellular traps (NETs). Interleukin-1ß (IL-1ß) modulates the expression of granulocyte colony-stimulating factor (G-CSF), a cytokine that promotes cancer-associated neutrophilia and NET generation. Herein, we combined a murine breast cancer model with a flow-restriction thrombosis model to evaluate whether the IL-1ß blockade could interfere with cancer-associated thrombosis. Mice bearing metastatic 4T1 tumors exhibited high neutrophil counts as well as elevated expression of G-CSF and IL-1ß in their tumors. On the other hand, mice bearing non-metastatic 67NR tumors showed no elevation in neutrophil counts and displayed low expression levels of G-CSF and IL-1ß in their tumors. 4T1 tumor-bearing mice but not 67NR tumor-bearing mice exhibited a NET-dependent prothrombotic state. Pharmacological blockade of IL-1 receptor (IL-1R) decreased the primary growth of 4T1 tumors and reduced the systemic levels of myeloperoxidase, cell-free DNA (cfDNA) and G-CSF, without interfering with the neutrophil counts. Most remarkably, the blockade of IL-1R abolished the prothrombotic state observed in 4T1 tumor-bearing mice. Overall, our results demonstrate that IL-1ß might be a feasible target to attenuate cancer-associated thrombosis, particularly in cancer types that rely on increased G-CSF production and involvement of NET formation.

Neuroendocrine Control of Macrophage Development and Function.

Macrophages carry out numerous physiological activities that are essential for both systemic and local homeostasis, as well as innate and adaptive immune responses. Their biology is intricately regulated by hormones, neuropeptides, and neurotransmitters, establishing distinct neuroendocrine axes. The control is pleiotropic, including maturation of bone marrow-derived myeloid precursors, cell differentiation into functional subpopulations, cytotoxic activity, phagocytosis, production of inflammatory mediators, antigen presentation, and activation of effector lymphocytes. Additionally, neuroendocrine components modulate macrophage ability to influence tumor growth and to prevent the spreading of infective agents. Interestingly, macrophage-derived factors enhance glucocorticoid production through the stimulation of the hypothalamic-pituitary-adrenal axis. These bidirectional effects highlight a tightly controlled balance between neuroendocrine stimuli and macrophage function in the development of innate and adaptive immune responses. Herein, we discuss how components of neuroendocrine axes impact on macrophage development and function and may ultimately influence inflammation, tissue repair, infection, or cancer progression. The knowledge of the crosstalk between macrophages and endocrine or brain-derived components may contribute to improve and create new approaches with clinical relevance in homeostatic or pathological conditions.

The Neuropeptides Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Control HIV-1 Infection in Macrophages Through Activation of Protein Kinases A and C.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are highly similar neuropeptides present in several tissues, endowed with immunoregulatory functions and other systemic effects. We previously reported that both neuropeptides reduce viral production in HIV-1-infected primary macrophages, with the participation of ß-chemokines and IL-10, and now we describe molecular mechanisms engaged in this activity. Macrophages exposed to VIP or PACAP before HIV-1 infection showed resistance to viral replication, comparable to that observed when the cells were treated after infection. Also, multiple treatments with a suboptimal dose of VIP or PACAP after macrophage infection resulted in a decline of virus production similar to the inhibition promoted by a single exposure to the optimal inhibitory concentration. Cellular signaling pathways involving cAMP production and activation of protein kinases A and C were critical components of the VIP and PACAP anti-HIV-1 effects. Analysis of the transcription factors and the transcriptional/cell cycle regulators showed that VIP and PACAP induced cAMP response element-binding protein activation, inhibited NF-kB, and reduced Cyclin D1 levels in HIV-1-infected cells. Remarkably, VIP and PACAP promoted G-to-A mutations in the HIV-1 provirus, matching those derived from the activity of the APOBEC family of viral restriction factors, and reduced viral infectivity. In conclusion, our findings strengthen the antiretroviral potential of VIP and PACAP and point to new therapeutic approaches to control the progression of HIV-1 infection.

HTLV-1-infected thymic epithelial cells convey the virus to CD4+ T lymphocytes.

The human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4+T cells are the main target of HTLV-1, but other cell types are known to be infected, including immature lymphocytes. Developing T cells undergo differentiation in the thymus, through migration and interaction with the thymic microenvironment, in particular with thymic epithelial cells (TEC) the major component of this three dimensional meshwork of non-lymphoid cells. Herein, we show that TEC express the receptors for HTLV-1 and can be infected by this virus through cell-cell contact and by cell-free virus suspensions. The expression of anti-apoptosis, chemokine and adhesion molecules genes are altered in HTLV-1-infected TEC, although gene expression of antigen presentation molecules remained unchanged. Furthermore, HTLV-1-infected TEC transmitted the virus to a CD4+ T cell line and to CD4+ T cells from healthy donors, during in vitro cellular co-cultures. Altogether, our data point to the possibility that the human thymic epithelial cells play a role in the establishment and progression of HTLV-1 infection, functioning as a reservoir and transmitting the virus to maturing CD4+ T lymphocytes, which in turn will cause disease in the periphery.

Protective effect of gedunin on TLR-mediated inflammation by modulation of inflammasome activation and cytokine production: Evidence of a multitarget compound.

Activation of toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) triggers an innate immune response, via cytokine production and inflammasome activation. Herein, we have investigated the modulatory effect of the natural limonoid gedunin on TLR activation in vitro and in vivo. Intraperitoneal (i.p.) pre- and post-treatments of C57BL/6 mouse with gedunin impaired the influx of mononuclear cells, eosinophils and neutrophils, as well as the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and nitric oxide (NO), triggered by lipopolysaccharide (LPS) in mouse pleura. Accordingly, in vitro post-treatment of immortalized murine macrophages with gedunin also impaired LPS-induced production of such mediators. Gedunin diminished LPS-induced expression of the nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) on pleural leukocytes in vivo and in immortalized macrophages in vitro. In line with this, gedunin inhibited LPS-induced caspase-1 activation and the production of IL-1ß in vivo and in vitro. In addition, gedunin treatment triggered the generation of the anti-inflammatory factors IL-10 and heme oxigenase-1 (HO-1) at resting conditions or upon stimulation. We also demonstrate that gedunin effect is not restricted to TLR4-mediated response, since this compound diminished TNF-α, IL-6, NO, NLRP3 and IL-1ß, as well as enhanced IL-10 and HO-1, by macrophages stimulated with the TLR2 and TLR3 agonists, palmitoyl-3-Cys-Ser-(Lys)4 (PAM3) and polyriboinosinic:polyribocytidylic acid (POLY I:C), in vitro. In silico modeling studies revealed that gedunin efficiently docked into caspase-1, TLR2, TLR3 and to the myeloid differentiation protein-2 (MD-2) component of TLR4. Overall, our data demonstrate that gedunin modulates TLR4, TLR3 and TLR2-mediated responses and reveal new molecular targets for this compound.

HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR.

HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling.

Inhibition of ecto-ATPase activities impairs HIV-1 infection of macrophages.

Nucleotides and nucleosides are secreted into extracellular media at different concentrations as a consequence of different physiologic and pathological conditions. Ecto-nucleotidases, enzymes present on the surface of most cells, hydrolyze these extracellular nucleotides and reduce the concentration of them, thus affecting the activation of different nucleotide and nucleoside receptors. Also, ecto-nucleotidases are present in a number of microorganisms and play important roles in host-pathogen interactions. Here, we characterized the ecto-ATPase activities present on the surface of HIV-1 particle and human macrophages as well. We found that the kinetic properties of HIV-1 and macrophage ecto-ATPases are similar, suggesting that the enzyme is the same. This ecto-ATPase activity was increased in macrophages infected in vitro with HIV-1. Using three different non-related ecto-ATPase inhibitors-POM-1, ARL67156 and BG0-we showed that the inhibition of these macrophage and viral ecto-ATPase activities impairs HIV-1 infection. In addition, we also found that elevated extracellular concentrations of ATP inhibit HIV-1 production by infected macrophages.

IL-27 enhances Leishmania amazonensis infection via ds-RNA dependent kinase (PKR) and IL-10 signaling.

The protozoan parasite Leishmania infects and replicates in macrophages, causing a spectrum of diseases in the human host, varying from cutaneous to visceral clinical forms. It is known that cytokines modulate the immunological response against Leishmania and are relevant for infection resolution. Here, we report that Interleukin (IL)-27 increases Leishmania amazonensis replication in human and murine macrophages and that the blockage of the IL-10 receptor on the surface of infected cells abolished the IL-27-mediated enhancement of Leishmania growth. IL-27 induced the activation/phosphorylation of protein kinase R (PKR) in macrophages, and PKR blockage or PKR gene deletion abrogated the enhancement of the parasite growth driven by IL-27, as well as the L. amazonensis-induced macrophage production of IL-27. We also observed that L. amazonensis-induced expression of IL-27 depends on type I interferon signaling and the engagement of Toll-like receptor 2. Treatment of Leishmania-infected mice with IL-27 increased lesion size and parasite loads in the footpad and lymph nodes of infected animals, indicating that this cytokine exerts a local and a systemic effect on parasite growth and propagation. In conclusion, we show that IL-27 is a L. amazonensis-enhancing factor and that the PKR/IFN1 axis and IL-10 are critical mediators of this IL-27 induced effect.

2´,3´-Dialdehyde of ATP, ADP, and adenosine inhibit HIV-1 reverse transcriptase and HIV-1 replication.

The 2´3´-dialdehyde of ATP or oxidized ATP (oATP) is a compound known for specifically making covalent bonds with the nucleotide-binding site of several ATP-binding enzymes and receptors. We investigated the effects of oATP and other oxidized purines on HIV-1 infection and we found that this compound inhibits HIV-1 and SIV infection by blocking early steps of virus replication. oATP, oxidized ADP (oADP), and oxidized Adenosine (oADO) impact the natural activity of endogenous reverse transcriptase enzyme (RT) in cell free virus particles and are able to inhibit viral replication in different cell types when added to the cell cultures either before or after infection. We used UFLC-UV to show that both oADO and oATP can be detected in the cell after being added in the extracellular medium. oATP also suppresses RT activity and replication of the HIV-1 resistant variants M184V and T215Y. We conclude that oATP, oADP and oADO display anti HIV-1 activity that is at in least in part due to inhibitory activity on HIV-1 RT.

HIV-1 and its gp120 inhibits the influenza A(H1N1)pdm09 life cycle in an IFITM3-dependent fashion.

HIV-1-infected patients co-infected with A(H1N1)pdm09 surprisingly presented benign clinical outcome. The knowledge that HIV-1 changes the host homeostatic equilibrium, which may favor the patient resistance to some co-pathogens, prompted us to investigate whether HIV-1 infection could influence A(H1N1)pdm09 life cycle in vitro. We show here that exposure of A(H1N1)pdm09-infected epithelial cells to HIV-1 viral particles or its gp120 enhanced by 25% the IFITM3 content, resulting in a decrease in influenza replication. This event was dependent on toll-like receptor 2 and 4. Moreover, knockdown of IFITM3 prevented HIV-1 ability to inhibit A(H1N1)pdm09 replication. HIV-1 infection also increased IFITM3 levels in human primary macrophages by almost 100%. Consequently, the arrival of influenza ribonucleoproteins (RNPs) to nucleus of macrophages was inhibited, as evaluated by different approaches. Reduction of influenza RNPs entry into the nucleus tolled A(H1N1)pdm09 life cycle in macrophages earlier than usual, limiting influenza's ability to induce TNF-α. As judged by analysis of the influenza hemagglutin (HA) gene from in vitro experiments and from samples of HIV-1/A(H1N1)pdm09 co-infected individuals, the HIV-1-induced reduction of influenza replication resulted in delayed viral evolution. Our results may provide insights on the mechanisms that may have attenuated the clinical course of Influenza in HIV-1/A(H1N1)pdm09 co-infected patients during the recent influenza form 2009/2010.

The effects of neurotrophins and the neuropeptides VIP and PACAP on HIV-1 infection: histories with opposite ends.

The nerve growth factor (NGF) and other neurotrophins, and the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are largely present in human tissue and can exert modulatory activities on nervous, endocrine and immune system functions. NGF, VIP and PACAP receptors are expressed systemically in organisms, and thus these mediators exhibit pleiotropic natures. The human immunodeficiency virus type 1 (HIV-1), the causal agent of the acquired immunodeficiency syndrome (AIDS), infects immune cells, and its replication is modulated by a number of endogenous factors that interact with HIV-1-infected cells. NGF, VIP and PACAP can also affect HIV-1 virus particle production upon binding to their receptors on the membranes of infected cells, which triggers cell signaling pathways that modify the HIV-1 replicative cycle. These molecules exert opposite effects on HIV-1 replication, as NGF and other neurotrophins enhance and VIP and PACAP reduce viral production in HIV-1-infected human primary macrophages. The understanding of AIDS pathogenesis should consider the mechanisms by which the replication of HIV-1, a pathogen that causes chronic morbidity, is influenced by neurotrophins, VIP and PACAP, i.e. molecules that exert a broad spectrum of physiological activities on the neuroimmunoendocrine axis. In this review, we will present the main effects of these two groups of mediators on the HIV-1 replicative cycle, as well as the mechanisms that underlie their abilities to modulate HIV-1 production in infected immune cells, and discuss the possible repercussion of the cross talk between NGF and both neuropeptides on the pathogenesis of HIV-1 infection.