DNA damage signaling in Drosophila macrophages modulates systemic cytokine levels in response to oxidative stress.

Environmental factors, infection, or injury can cause oxidative stress in diverse tissues and loss of tissue homeostasis. Effective stress response cascades, conserved from invertebrates to mammals, ensure reestablishment of homeostasis and tissue repair. Hemocytes, the Drosophila blood-like cells, rapidly respond to oxidative stress by immune activation. However, the precise signals how they sense oxidative stress and integrate these signals to modulate and balance the response to oxidative stress in the adult fly are ill-defined. Furthermore, hemocyte diversification was not explored yet on oxidative stress. Here, we employed high-throughput single nuclei RNA-sequencing to explore hemocytes and other cell types, such as fat body, during oxidative stress in the adult fly. We identified distinct cellular responder states in plasmatocytes, the Drosophila macrophages, associated with immune response and metabolic activation upon oxidative stress. We further define oxidative stress-induced DNA damage signaling as a key sensor and a rate-limiting step in immune-activated plasmatocytes controlling JNK-mediated release of the pro-inflammatory cytokine unpaired-3. We subsequently tested the role of this specific immune activated cell stage during oxidative stress and found that inhibition of DNA damage signaling in plasmatocytes, as well as JNK or upd3 overactivation, result in a higher susceptibility to oxidative stress. Our findings uncover that a balanced composition and response of hemocyte subclusters is essential for the survival of adult Drosophila on oxidative stress by regulating systemic cytokine levels and cross-talk to other organs, such as the fat body, to control energy mobilization.

Multiomic spatial landscape of innate immune cells at human central nervous system borders.

The innate immune compartment of the human central nervous system (CNS) is highly diverse and includes several immune-cell populations such as macrophages that are frequent in the brain parenchyma (microglia) and less numerous at the brain interfaces as CNS-associated macrophages (CAMs). Due to their scantiness and particular location, little is known about the presence of temporally and spatially restricted CAM subclasses during development, health and perturbation. Here we combined single-cell RNA sequencing, time-of-flight mass cytometry and single-cell spatial transcriptomics with fate mapping and advanced immunohistochemistry to comprehensively characterize the immune system at human CNS interfaces with over 356,000 analyzed transcriptomes from 102 individuals. We also provide a comprehensive analysis of resident and engrafted myeloid cells in the brains of 15 individuals with peripheral blood stem cell transplantation, revealing compartment-specific engraftment rates across different CNS interfaces. Integrated multiomic and high-resolution spatial transcriptome analysis of anatomically dissected glioblastoma samples shows regionally distinct myeloid cell-type distributions driven by hypoxia. Notably, the glioblastoma-associated hypoxia response was distinct from the physiological hypoxia response in fetal microglia and CAMs. Our results highlight myeloid diversity at the interfaces of the human CNS with the periphery and provide insights into the complexities of the human brain's immune system.

Transcriptome Analysis Identifies Accumulation of Natural Killer Cells with Enhanced Lymphotoxin-ß Expression during Glioblastoma Progression.

Glioblastomas are the most common primary brain tumors. Despite extensive clinical and molecular insights into these tumors, the prognosis remains dismal. While targeted immunotherapies have shown remarkable success across different non-brain tumor entities, they failed to show efficacy in glioblastomas. These failures prompted the field to reassess the idiosyncrasies of the glioblastoma microenvironment. Several high-dimensional single-cell RNA sequencing studies generated remarkable findings about glioblastoma-associated immune cells. To build on the collective strength of these studies, we integrated several murine and human datasets that profiled glioblastoma-associated immune cells at different time points. We integrated these datasets and utilized state-of-the-art algorithms to investigate them in a hypothesis-free, purely exploratory approach. We identified a robust accumulation of a natural killer cell subset that was characterized by a downregulation of activation-associated genes with a concomitant upregulation of apoptosis genes. In both species, we found a robust upregulation of the Lymphotoxin-ß gene, a cytokine from the TNF superfamily and a key factor for the development of adaptive immunity. Further validation analyses uncovered a correlation of lymphotoxin signaling with mesenchymal-like glioblastoma regions in situ and in TCGA and CGGA glioblastoma cohorts. In summary, we identify lymphotoxin signaling as a potential therapeutic target in glioblastoma-associated natural killer cells.

Brain endothelial STING1 activation by Plasmodium-sequestered heme promotes cerebral malaria via type I IFN response.

Cerebral malaria (CM) is a life-threatening form of Plasmodium falciparum infection caused by brain inflammation. Brain endothelium dysfunction is a hallmark of CM pathology, which is also associated with the activation of the type I interferon (IFN) inflammatory pathway. The molecular triggers and sensors eliciting brain type I IFN cellular responses during CM remain largely unknown. We herein identified the stimulator of interferon response cGAMP interactor 1 (STING1) as the key innate immune sensor that induces Ifnß1 transcription in the brain of mice infected with Plasmodium berghei ANKA (Pba). This STING1/IFNß-mediated response increases brain CXCL10 governing the extent of brain leukocyte infiltration and blood-brain barrier (BBB) breakdown, and determining CM lethality. The critical role of brain endothelial cells (BECs) in fueling type I IFN-driven brain inflammation was demonstrated in brain endothelial-specific IFNß-reporter and STING1-deficient Pba-infected mice, which were significantly protected from CM lethality. Moreover, extracellular particles (EPs) released from Pba-infected erythrocytes activated the STING1-dependent type I IFN response in BECs, a response requiring intracellular acidification. Fractionation of the EPs enabled us to identify a defined fraction carrying hemoglobin degradation remnants that activates STING1/IFNß in the brain endothelium, a process correlated with heme content. Notably, stimulation of STING1-deficient BECs with heme, docking experiments, and in vitro binding assays unveiled that heme is a putative STING1 ligand. This work shows that heme resultant from the parasite heterotrophic activity operates as an alarmin, triggering brain endothelial inflammatory responses via the STING1/IFNß/CXCL10 axis crucial to CM pathogenesis and lethality.

MyD88 signaling by neurons induces chemokines that recruit protective leukocytes to the virus-infected CNS.

Viral encephalitis initiates a series of immunological events in the brain that can lead to brain damage and death. Astrocytes express IFN-ß in response to neurotropic infection, whereas activated microglia produce proinflammatory cytokines and accumulate at sites of infection. Here, we observed that neurotropic vesicular stomatitis virus (VSV) infection causes recruitment of leukocytes into the central nervous system (CNS), which requires MyD88, an adaptor of Toll-like receptor and interleukin-1 receptor signaling. Infiltrating leukocytes, and in particular CD8+ T cells, protected against lethal VSV infection of the CNS. Reconstitution of MyD88, specifically in neurons, restored chemokine production in the olfactory bulb as well as leukocyte recruitment into the infected CNS and enhanced survival. Comparative analysis of the translatome of neurons and astrocytes verified neurons as the critical source of chemokines, which regulated leukocyte infiltration of the infected brain and affected survival.

The roles of microglia in viral encephalitis: from sensome to therapeutic targeting.

Viral encephalitis is a devastating disease with high mortality, and survivors often suffer from severe neurological complications. Microglia are innate immune cells of the central nervous system (CNS) parenchyma whose turnover is reliant on local proliferation. Microglia express a diverse range of proteins, which allows them to continuously sense the environment and quickly react to changes. Under inflammatory conditions such as CNS viral infection, microglia promote innate and adaptive immune responses to protect the host. However, during viral infection, a dysregulated microglia-T-cell interplay may result in altered phagocytosis of neuronal synapses by microglia that causes neurocognitive impairment. In this review, we summarize the current knowledge on the role of microglia in viral encephalitis, propose questions to be answered in the future and suggest possible therapeutic targets.

Type I Interferon Receptor Signaling in Astrocytes Regulates Hippocampal Synaptic Plasticity and Cognitive Function of the Healthy CNS.

Type I interferon receptor (IFNAR) signaling is a hallmark of viral control and host protection. Here, we show that, in the hippocampus of healthy IFNAR-deficient mice, synapse number and synaptic plasticity, as well as spatial learning, are impaired. This is also the case for IFN-ß-deficient animals. Moreover, antibody-mediated IFNAR blocking acutely interferes with neuronal plasticity, whereas a low-dose application of IFN-ß has a positive effect on dendritic spine structure. Interfering with IFNAR signaling in different cell types shows a role for cognitive function and synaptic plasticity specifically mediated by astrocytes. Intriguingly, levels of the astrocytic glutamate-aspartate transporter (GLAST) are reduced significantly upon IFN-ß treatment and increase following inhibition of IFNAR signaling. These results indicate that, besides the prominent role for host defense, IFNAR is important for synaptic plasticity as well as cognitive function. Astrocytes are at the center stage of this so-far-unknown signaling cascade.

Selective reconstitution of IFN­Î³ gene function in Ncr1+ NK cells is sufficient to control systemic vaccinia virus infection.

IFN-γ is an enigmatic cytokine that shows direct anti-viral effects, confers upregulation of MHC-II and other components relevant for antigen presentation, and that adjusts the composition and balance of complex cytokine responses. It is produced during immune responses by innate as well as adaptive immune cells and can critically affect the course and outcome of infectious diseases, autoimmunity, and cancer. To selectively analyze the function of innate immune cell-derived IFN-γ, we generated conditional IFN-γOFF mice, in which endogenous IFN-γ expression is disrupted by a loxP flanked gene trap cassette inserted into the first intron of the IFN-γ gene. IFN-γOFF mice were intercrossed with Ncr1-Cre or CD4-Cre mice that express Cre mainly in NK cells (IFN-γNcr1-ON mice) or T cells (IFN-γCD4-ON mice), respectively. Rosa26RFP reporter mice intercrossed with Ncr1-Cre mice showed selective RFP expression in more than 80% of the NK cells, while upon intercrossing with CD4-Cre mice abundant RFP expression was detected in T cells, but also to a minor extent in other immune cell subsets. Previous studies showed that IFN-γ expression is needed to promote survival of vaccinia virus (VACV) infection. Interestingly, during VACV infection of wild type and IFN-γCD4-ON mice two waves of serum IFN-γ were induced that peaked on day 1 and day 3/4 after infection. Similarly, VACV infected IFN-γNcr1-ON mice mounted two waves of IFN-γ responses, of which the first one was moderately and the second one profoundly reduced when compared with WT mice. Furthermore, IFN-γNcr1-ON as well as IFN-γCD4-ON mice survived VACV infection, whereas IFN-γOFF mice did not. As expected, ex vivo analysis of splenocytes derived from VACV infected IFN-γNcr1-ON mice showed IFN-γ expression in NK cells, but not T cells, whereas IFN-γOFF mice showed IFN-γ expression neither in NK cells nor T cells. VACV infected IFN-γNcr1-ON mice mounted normal cytokine responses, restored neutrophil accumulation, and showed normal myeloid cell distribution in blood and spleen. Additionally, in these mice normal MHC-II expression was detected on peripheral macrophages, whereas IFN-γOFF mice did not show MHC-II expression on such cells. In conclusion, upon VACV infection Ncr1 positive cells including NK cells mount two waves of early IFN-γ responses that are sufficient to promote the induction of protective anti-viral immunity.

The olfactory epithelium as a port of entry in neonatal neurolisteriosis.

Bacterial infections of the central nervous system (CNS) remain a major cause of mortality in the neonatal population. Commonly used parenteral infection models, however, do not reflect the early course of the disease leaving this critical step of the pathogenesis largely unexplored. Here, we analyzed nasal exposure of 1-day-old newborn mice to Listeria monocytogenes (Lm). We found that nasal, but not intragastric administration, led to early CNS infection in neonate mice. In particular, upon bacterial invasion of the olfactory epithelium, Lm subsequently spread along the sensory neurons entering the brain tissue at the cribriform plate and causing a significant influx of monocytes and neutrophils. CNS infection required listeriolysin for penetration of the olfactory epithelium and ActA, a mediator of intracellular mobility, for translocation into the brain tissue. Taken together, we propose an alternative port of entry and route of infection for neonatal neurolisteriosis and present a novel infection model to mimic the clinical features of late-onset disease in human neonates.

Type I Interferon Receptor Signaling of Neurons and Astrocytes Regulates Microglia Activation during Viral Encephalitis.

In sterile neuroinflammation, a pathological role is proposed for microglia, whereas in viral encephalitis, their function is not entirely clear. Many viruses exploit the odorant system and enter the CNS via the olfactory bulb (OB). Upon intranasal vesicular stomatitis virus instillation, we show an accumulation of activated microglia and monocytes in the OB. Depletion of microglia during encephalitis results in enhanced virus spread and increased lethality. Activation, proliferation, and accumulation of microglia are regulated by type I IFN receptor signaling of neurons and astrocytes, but not of microglia. Morphological analysis of myeloid cells shows that type I IFN receptor signaling of neurons has a stronger impact on the activation of myeloid cells than of astrocytes. Thus, in the infected CNS, the cross talk among neurons, astrocytes, and microglia is critical for full microglia activation and protection from lethal encephalitis.

Chemokine receptors CCR2 and CX3CR1 regulate viral encephalitis-induced hippocampal damage but not seizures.

Viral encephalitis is a major risk factor for the development of seizures, epilepsy, and hippocampal damage with associated cognitive impairment, markedly reducing quality of life in survivors. The mechanisms underlying seizures and hippocampal neurodegeneration developing during and after viral encephalitis are only incompletely understood, hampering the development of preventive treatments. Recent findings suggest that brain invasion of blood-born monocytes may be critically involved in both seizures and brain damage in response to encephalitis, whereas the relative role of microglia, the brain's resident immune cells, in these processes is not clear. CCR2 and CX3CR1 are two chemokine receptors that regulate the responses of myeloid cells, such as monocytes and microglia, during inflammation. We used Ccr2-KO and Cx3cr1-KO mice to understand the role of these receptors in viral encephalitis-associated seizures and neurodegeneration, using the Theiler's virus model of encephalitis in C57BL/6 mice. Our results show that CCR2 as well as CX3CR1 plays a key role in the accumulation of myeloid cells in the CNS and activation of hippocampal myeloid cells upon infection. Furthermore, by using Cx3cr1-creER+/-tdTomatoSt/Wt reporter mice, we show that, with regard to CD45 and CD11b expression, some microglia become indistinguishable from monocytes during CNS infection. Interestingly, the lack of CCR2 or CX3CR1 receptors was associated with almost complete prevention of hippocampal damage but did not prevent seizure development after viral CNS infection. These data are compatible with the hypothesis that CNS inflammatory mechanism(s) other than the infiltrating myeloid cells trigger the development of seizures during viral encephalitis.

Microglia have a protective role in viral encephalitis-induced seizure development and hippocampal damage.

In the central nervous system (CNS), innate immune surveillance is mainly coordinated by microglia. These CNS resident myeloid cells are assumed to help orchestrate the immune response against infections of the brain. However, their specific role in this process and their interactions with CNS infiltrating immune cells, such as blood-borne monocytes and T cells are only incompletely understood. The recent development of PLX5622, a specific inhibitor of colony-stimulating factor 1 receptor that depletes microglia, allows studying the role of microglia in conditions of brain injury such as viral encephalitis, the most common form of brain infection. Here we used this inhibitor in a model of viral infection-induced epilepsy, in which C57BL/6 mice are infected by a picornavirus (Theiler's murine encephalomyelitis virus) and display seizures and hippocampal damage. Our results show that microglia are required early after infection to limit virus distribution and persistence, most likely by modulating T cell activation. Microglia depletion accelerated the occurrence of seizures, exacerbated hippocampal damage, and led to neurodegeneration in the spinal cord, which is normally not observed in this mouse strain. This study enhances our understanding of the role of microglia in viral encephalitis and adds to the concept of microglia-T cell crosstalk.

Type I interferon receptor signaling delays Kupffer cell replenishment during acute fulminant viral hepatitis.

BACKGROUND & AIM: Virus-induced fulminant hepatitis is a major cause of acute liver failure. During acute viral hepatitis the impact of type I interferon (IFN-I) on myeloid cells, including liver-resident Kupffer cells (KC), is only partially understood. Herein, we dissected the impact of locally induced IFN-I responses on myeloid cell function and hepatocytes during acute liver inflammation. METHODS: Two different DNA-encoded viruses, vaccinia virus (VACV) and murine cytomegalovirus (MCMV), were studied. In vivo imaging was applied to visualize local IFN-ß induction and IFN-I receptor (IFNAR) triggering in VACV-infected reporter mice. Furthermore, mice with a cell type-selective IFNAR ablation were analyzed to dissect the role of IFNAR signaling in myeloid cells and hepatocytes. Experiments with Cx3cr1+/gfp mice revealed the origin of reconstituted KC. Finally, mixed bone marrow chimeric mice were studied to specifically analyze the effect of IFNAR triggering on liver infiltrating monocytes. RESULTS: VACV infection induced local IFN-ß responses, which lead to IFNAR signaling primarily within the liver. IFNAR triggering was needed to control the infection and prevent fulminant hepatitis. The severity of liver inflammation was independent of IFNAR triggering of hepatocytes, whereas IFNAR triggering of myeloid cells protected from excessive inflammation. Upon VACV or MCMV infection KC disappeared, whereas infiltrating monocytes differentiated to KC afterwards. During IFNAR triggering such replenished monocyte-derived KC comprised more IFNAR-deficient than -competent cells in mixed bone marrow chimeric mice, whereas after the decline of IFNAR triggering both subsets showed an even distribution. CONCLUSION: Upon VACV infection IFNAR triggering of myeloid cells, but not of hepatocytes, critically modulates acute viral hepatitis. During infection with DNA-encoded viruses IFNAR triggering of liver-infiltrating blood monocytes delays the development of monocyte-derived KC, pointing towards new therapeutic strategies for acute viral hepatitis. LAY SUMMARY: Viral infection can cause fulminant hepatitis, which in turn is a major cause of acute liver failure. Herein, we aimed to study the role of type 1 interferon responses in acute viral hepatitis. We identified that during infection with DNA-encoded viruses, type 1 interferon receptor triggering of blood monocytes delays the development of monocyte-derived Kupffer cells. This points to new therapeutic strategies for acute viral hepatitis.

Macrophage depletion by liposome-encapsulated clodronate suppresses seizures but not hippocampal damage after acute viral encephalitis.

Viral encephalitis is a major risk factor for the development of seizures and epilepsy, but the underlying mechanisms are only poorly understood. Mouse models such as viral encephalitis induced by intracerebral infection with Theiler's virus in C57BL/6 (B6) mice allow advancing our understanding of the immunological and virological aspects of infection-induced seizures and their treatment. Previous studies using the Theiler's virus model in B6 mice have indicated that brain-infiltrating inflammatory macrophages and the cytokines released by these cells are key to the development of acute seizures and hippocampal damage in this model. However, approaches used to prevent or reduce macrophage infiltration were not specific, so contribution of other mechanisms could not be excluded. In the present study, we used a more selective and widely used approach for macrophage depletion, i.e., systemic administration of clodronate liposomes, to study the contribution of macrophage infiltration to development of seizures and hippocampal damage. By this approach, almost complete depletion of monocytic cells was achieved in spleen and blood of Theiler's virus infected B6 mice, which was associated with a 70% decrease in the number of brain infiltrating macrophages as assessed by flow cytometry. Significantly less clodronate liposome-treated mice exhibited seizures than liposome controls (P<0.01), but the development of hippocampal damage was not prevented or reduced. Clodronate liposome treatment did not reduce the increased Iba1 and Mac3 labeling in the hippocampus of infected mice, indicating that activated microglia may contribute to hippocampal damage. The unexpected mismatch between occurrence of seizures and hippocampal damage is thought-provoking and suggests that the mechanisms involved in degeneration of specific populations of hippocampal neurons in encephalitis-induced epilepsy are more complex than previously thought.

Systemic Virus Infections Differentially Modulate Cell Cycle State and Functionality of Long-Term Hematopoietic Stem Cells In Vivo.

Quiescent long-term hematopoietic stem cells (LT-HSCs) are efficiently activated by type I interferon (IFN-I). However, this effect remains poorly investigated in the context of IFN-I-inducing virus infections. Here we report that both vesicular stomatitis virus (VSV) and murine cytomegalovirus (MCMV) infection induce LT-HSC activation that substantially differs from the effects triggered upon injection of synthetic IFN-I-inducing agents. In both infections, inflammatory responses had to exceed local thresholds within the bone marrow to confer LT-HSC cell cycle entry, and IFN-I receptor triggering was not critical for this activation. After resolution of acute MCMV infection, LT-HSCs returned to phenotypic quiescence. However, non-acute MCMV infection induced a sustained inflammatory milieu within the bone marrow that was associated with long-lasting impairment of LT-HSC function. In conclusion, our results show that systemic virus infections fundamentally affect LT-HSCs and that also non-acute inflammatory stimuli in bone marrow donors can affect the reconstitution potential of bone marrow transplants.

The antiviral drug ganciclovir does not inhibit microglial proliferation and activation.

Ganciclovir is effective in the treatment of human infections with viruses of the Herpesviridae family. Beside antiviral properties, recently ganciclovir was described to inhibit microglial proliferation and disease severity of experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis. Microglial activation and proliferation are main characteristics of neuroinflammatory CNS diseases and inhibition of microglial functions might be beneficial in autoimmune diseases, or detrimental in infectious diseases. The objective of this study was to determine potential inhibitory effects of ganciclovir in three different murine animal models of CNS neuroinflammation in which microglia play an important role: Theiler´s murine encephalomyelitis, the cuprizone model of de- and remyelination, and the vesicular stomatitis virus encephalitis model. In addition, in vitro experiments with microglial cultures were performed to test the hypothesis that ganciclovir inhibits microglial proliferation. In all three animal models, neither microglial proliferation or recruitment nor disease activity was changed by ganciclovir. In vitro experiments confirmed that microglial proliferation was not affected by ganciclovir. In conclusion, our results show that the antiviral drug ganciclovir does not inhibit microglial activation and proliferation in the murine CNS.

Upon intranasal vesicular stomatitis virus infection, astrocytes in the olfactory bulb are important interferon Beta producers that protect from lethal encephalitis.

UNLABELLED: Previously we found that following intranasal (i.n.) infection with neurotropic vesicular stomatitis virus (VSV) type I interferon receptor (IFNAR) triggering of neuroectodermal cells was critically required to constrain intracerebral virus spread. To address whether locally active IFN-ß was induced proximally, we studied spatiotemporal conditions of VSV-mediated IFN-ß induction. To this end, we performed infection studies with IFN-ß reporter mice. One day after intravenous (i.v.) VSV infection, luciferase induction was detected in lymph nodes. Upon i.n. infection, luciferase induction was discovered at similar sites with delayed kinetics, whereas on days 3 and 4 postinfection enhanced luciferase expression additionally was detected in the foreheads of reporter mice. A detailed analysis of cell type-specific IFN-ß reporter mice revealed that within the olfactory bulb IFN-ß was expressed by neuroectodermal cells, primarily by astrocytes and to a lesser extent by neurons. Importantly, locally induced type I IFN triggered distal parts of the brain as indicated by the analysis of ISRE-eGFP mice which after i.n. VSV infection showed enhanced green fluorescent protein (eGFP) expression throughout the brain. Compared to wild-type mice, IFN-ß(-/-) mice showed increased mortality to i.n. VSV infection, whereas upon i.v. infection no such differences were detected highlighting the biological significance of intracerebrally expressed IFN-ß. In conclusion, upon i.n. VSV instillation, IFN-ß responses mounted by astrocytes within the olfactory bulb critically contribute to the antiviral defense by stimulating distal IFN-ß-negative brain areas and thus arresting virus spread. IMPORTANCE: The central nervous system has long been considered an immune privileged site. More recently, it became evident that specialized immune mechanisms are active within the brain to control pathogens. Previously, we showed that virus, which entered the brain via the olfactory route, was arrested within the olfactory bulb by a type I IFN-dependent mechanism. Since peripheral type I IFN would not readily cross the blood-brain barrier and within the brain thus far no abundant type I IFN responses have been detected, here we addressed from where locally active IFN originated from. We found that upon intranasal VSV instillation, primarily astrocytes, and to a lesser extent neurons, were stimulated within the olfactory bulb to mount IFN-ß responses that also activated and protected distal brain areas. Our results are surprising because in other infection models astrocytes have not yet been identified as major type I IFN producers.

HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

BACKGROUND: HIV-1 Tat protein is known to be associated with neuroinflammation, a condition that develops in almost half of patients infected with HIV-1. HIV-1 Tat can alter glial neuroprotective functions, leading to neurotoxicity within the CNS. HIV-1 Tat is known to be secreted from productively infected cells and can affect neighboring uninfected cells by modulating cellular gene expression in a bystander fashion. METHODS: We were interested to study whether exogenous exposure to HIV-1 Tat-C protein perturbs the microRNA (miRNA) expression profile of human microglial cells, leading to altered protein expression. We used protein expression and purification, miRNA overexpression, miRNA knockdown, transfection, site-directed mutagenesis, real-time PCR, luciferase assay and western blotting techniques to perform our study. RESULTS: HIV-1 Tat-C treatment of human microglial cells resulted in a dose-dependent increase in miR-32 expression. We found that tumor necrosis factor-receptor-associated factor 3 TRAF3) is a direct target for miR-32, and overexpression of miR-32 in CHME3 cells decreased TRAF3 both at the mRNA and the protein level. Recovery of TRAF3 protein expression after transfection of anti-miR-32 and the results of the luciferase reporter assay provided direct evidence of TRAF3 regulation by miR-32. We found that the regulation of interferon regulatory factor 3 (IRF3) and IRF7 is controlled by cellular levels of TRAF3 protein in microglial cells, as after overexpression of miR-32 and application of anti-miR-32, expression levels of IRF3 and IRF7 were inversely regulated by expression levels of TRAF3. Thus, our results suggest a novel miRNA mediated mechanism for regulation of TRAF3 in human microglial cells exposed to HIV-1 Tat C protein. These results may help to elucidate the detrimental neuroinflammatory consequences of HIV-1 Tat C protein in bystander fashion. CONCLUSION: HIV-1 Tat protein can modulate TRAF3 expression through miRNA mediated pathway and can change the downstream expression of IRF3 and IRF7. This study demonstrates a novel mechanism of HIV-1 Tat C protein-mediated perturbation of miRNA, resulting in dysregulation of cellular TRAF3.

HIV vaccine: hopes and hurdles.

The AIDS vaccine development effort has already been facing various scientific and economic challenges. The fundamental challenge resides at the level of understanding the basic biology of HIV-1 infection and an effective antiviral immune response. There is a need to design immunogens that can elicit cross-clade neutralizing antibodies (NAbs) along with effective T-cell responses against a wide variety of primary HIV isolates. We must exploit the capabilities of the vaccine-elicited cytotoxic T cells and the NAb responses in controlling HIV-1 replication. A coordinated approach is required to understand the intricacies involved in the basic immune responses against HIV infection as well as the cross-clade effectiveness of an AIDS vaccine.