Effect of Cocaine on HIV Infection and Inflammasome Gene Expression Profile in HIV Infected Macrophages

We have observed significantly increased HIV infection in HIV infected macrophages in the presence of cocaine that could be due to the downregulation of BST2 restriction factor in these cells. In human inflammasome PCR array, among different involved in inflammasome formation, in HIV infected macrophages in the presence of cocaine, we have observed significant upregulation of NLRP3, AIM2 genes and downstream genes IL-1? and PTGS2. Whereas negative regulatory gene MEFV was upregulated, CD40LG and PYDC1 were significantly downregulated. Among various NOD like receptors, NOD2 was significantly upregulated in both HIV alone and HIV plus cocaine treated cells. In the downstream genes, chemokine (C-C motif) ligand 2 (CCL2), CCL7 and IL-6 were significantly up regulated in HIV plus cocaine treated macrophages. We have also observed significant ROS production (in HIV and/or cocaine treated cells) which is one of the indirect-activators of inflammasomes formation. Further, we have observed early apoptosis in HIV alone and HIV plus cocaine treated macrophages which may be resultant of inflammasome formation and cspase-1 activation. These results indicate that in case of HIV infected macrophages exposed to cocaine, increased ROS production and IL-1? transcription serve as an activators for the formation of NLRP3 and AIM2 mediated inflammasomes that leads to caspase 1 mediated apoptosis

Inflammasome Activation by Methamphetamine Potentiates Lipopolysaccharide Stimulation of IL-1β Production in Microglia

Methamphetamine (Meth) is a psychostimulant drug that is widely abused all around the world. The administration of Meth causes a strong instant euphoria effect, and long-term of abuse is correlative of drug-dependence and neurotoxicity. The neuroimaging studies demonstrated that the long-term abuse of Meth is associated with the reduction of the dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) in the striatum. Neuroinflammation is well-accepted as an important mechanism underlying the Meth-induced neurotoxicity. The over-activated microglia were found both in Meth human abusers and animal models. NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is the most predominant Nod-like receptor (NLR) expressed in microglia and is involved in the pathogenesis of many neurodegenerative diseases. In recent years, multiple lines of evidence suggest that the activation of NLRP3 inflammasome is associated with drug abuse induced innate immune system activation both in central nervous system (CNS) and peripheral nervous system (PNS). We investigated the role of NLRP3 inflammasome in Meth-induced microglial activation. Meth induced the production of mitochondrial ROS and disruption of lysosomal membrane integrity, which served as the second activation signal of NLRP3 inflammasome. The activation of NLRP3 inflammasome led to the cleavage of pro-IL-1β and subsequent release of biologically active IL-1β. By blocking the inflammasome activation, we successfully attenuated the neuronal apoptosis induced by supernatants of Meth-treated microglia. It is well-known that Meth abuse exacerbates HIV-1-associated neurocognitive disorders (HAND). However, the mechanism of how Meth potentiates HAND is not fully understood. Ample evidence indicates that both Meth and HIV-1 cause microglial activation and resultant secretion of proinflammatory molecules leading to neuronal injury and ultimately the development of HAND. Inflammasome is the key signaling platform involved in HIV-1-associated microglia activation and the production of proinflammatory molecules. We studied the synergistic effects of HIV-1 glycoprotein protein 120 (gp120) and Meth in microglial NLRP3 inflammasome activation. Gp120 upregulated the pro-IL-1β and thus, primed the microglia as the first signal. The subsequent stimulation of Meth as the second signal further activates the inflammasome that promotes the processing and release of IL-1β. The overactivated ROS system is potentially relative to gp120- and Meth-induced inflammasome activation

HIV-1 activates oxidative phosphorylation in infected CD4 T cells in a human tonsil explant model

IntroductionHuman immunodeficiency virus type 1 (HIV-1) causes a chronic, incurable infection leading to immune activation and chronic inflammation in people with HIV-1 (PWH), even with virologic suppression on antiretroviral therapy (ART). The role of lymphoid structures as reservoirs for viral latency and immune activation has been implicated in chronic inflammation mechanisms. Still, the specific transcriptomic changes induced by HIV-1 infection in different cell types within lymphoid tissue remain unexplored.MethodsIn this study, we utilized human tonsil explants from healthy human donors and infected them with HIV-1 ex vivo. We performed single-cell RNA sequencing (scRNA-seq) to analyze the cell types represented in the tissue and to investigate the impact of infection on gene expression profiles and inflammatory signaling pathways.ResultsOur analysis revealed that infected CD4+ T cells exhibited upregulation of genes associated with oxidative phosphorylation. Furthermore, macrophages exposed to the virus but uninfected showed increased expression of genes associated with the NLRP3 inflammasome pathway.DiscussionThese findings provide valuable insights into the specific transcriptomic changes induced by HIV-1 infection in different cell types within lymphoid tissue. The activation of oxidative phosphorylation in infected CD4+ T cells and the proinflammatory response in macrophages may contribute to the chronic inflammation observed in PWH despite ART. Understanding these mechanisms is crucial for developing targeted therapeutic strategies to eradicate HIV-1 infection in PWH

Generation of macrophage models to investigate the effect of host factors restricting HIV and HSV infection

Introduction Macrophages play a central role in the immune response to viral infection. While cellular responses are highly cell-type and pathogen-specific, little is known about innate mechanisms for sensing and restriction of viruses in macrophages and macrophage-like cells. MX2 and SAMHD1 were recently identified as antiviral cellular effectors targeting Human Immunodeficiency Virus Type 1 (HIV-1) and Herpes Simplex Virus Type 1 (HSV-1) in cell lines. Macrophages can be productively infected by HIV-1 and HSV-1 and constitute important cellular reservoirs in vivo, yet the roles of MX2 and SAMHD1 are only partially understood. Aims To fully understand cell type-specific host responses to these viruses, experimental model systems that allow screening approaches, genetic manipulation and physiologically relevant are needed. Here, we pursued the following aims: (i) Establish and validate trans-differentiated BLaER1 cells as a genetically amendable macrophage-like cell model for HIV-1 and HSV-1 infection, (ii) develop a rapid and efficient protocol to knockout genes in monocyte/macrophage primary cells, and (iii) characterize the antiviral potency of MX2 and SAMHD1 in both experimental models. Materials & methods Trans-differentiation and differentiation protocols as well as phenotypic and functional analyses of macrophages were established. Various approaches to study HIV-1 and HSV-1 infection by envelope pseudotyping, use of reporter viruses, virus-like-particles and specific drugs were optimized. Innate responses were monitored by PCR and immunoblotting. Nucleofection-mediated, CRISPR/Cas9-based gene editing in monocytes was developed and knockout efficiencies were validated genetically by immunodetection. Results (i) Trans-differentiated BLaER1 cells adopt a myeloid gene expression profile and display characteristics in response to HIV-1 or HSV-1 infection similar to primary macrophages. (ii) In comparison to siRNA-based approaches, CRISPR/Cas9-edited macrophages are viable, have a stable myeloid marker expression and targeted factors are efficiently depleted. (iii) Knockouts of MX2 or SAMHD1 result in enhanced HIV-1 and HSV-1 infection in trans-differentiated BLaER1 cells and primary macrophages, corroborating and expanding their role as restriction factors against these two human pathogens. (iv) A contamination with squirrel monkey retrovirus (SMRV) is discovered in BLaER1 cells. (v) BLaER1 cells can provide an easily accessible screening platform for cellular factors that are functionally relevant for virus infections. Conclusion Overall, these findings establish critical methodology for different types of experimental studies into virus-macrophage interactions. MX2 and SAMHD1 are broadly acting restriction factors that limit the infection of lentiviruses and herpesviruses in human macrophages

Role of Withaferin A as a Neuroprotectant against Beta Amyloid Induced Toxicity and associated mechanism

Neurological disorders are the biggest concern globally and ageing contributes in worsening the disease scenarios. In AD or AD like diseases, there is abnormal accumulation of extracellular amyloid beta produced due to abnormal processing of the transmembrane amyloid precursor protein, by β and γ-secretases. It spreads in the cortical and limbic regions of the brain leading to neuronal toxicity, impairment in memory and neurological functions. Aβ deposition in the CNS is common in aging HIV patients. Neurotoxic protein Tat, results in increased Aβ in combination with drugs of abuse cocaine. We examined the role of Withaferin A, against Aβ induced neurotoxicity. Our in-vitro dose optimization study demonstrates that lower concentrations (0.5–2 μM) of WA significantly reduce the Aβ40, without inducing cytotoxicity in the APP plasmid transfected SH-SY5Y cells (SHAPP). We demonstrate that Aβ secretion is increased in the presence of Tat (50 ng/ml) and coc (0.1 μM), WA reduces the Tat and coc induced increase in Aβ40. Additionally, we studied the role of WA against NF-kB mediated neuroinflammation, and observed that WA inhibits the expression of NFkB2 and RELA transcription factors, which play a major role in the expression of inflammatory chemokines. Further, to address the issue of minimal drug bioavailability in the CNS, we developed the WA loaded liposomal nanoformulation (WA-LNF) and characterized its size (499+/-50nm), toxicity and drug binding efficacy (28%). Our in-vitro 3D BBB transmigration of WA-LNF demonstrated ~40% transmigration efficiency. Furthermore, it was imperative for us to understand the mechanism of action of WA, therefore we studied the molecular mechanism of interaction of WA with Aβ protein by in-silico molecular dynamics simulations. We demonstrated that WA binds to the middle region of Aβ protein and the amino acid motif involved were FAEDVGS highlighting the mid-region Aβ capture by WA. 3 Hydrogen bonds were formed between WA and the amino acids, ASN17, GLY15 and SER16. This study reports WA as a potent neuroprotectant against amyloid induced neurotoxicity. Our study may have an immense therapeutic potential to target Aβ in the CNS, in the ageing patients and/or PLWH and/or ageing drug abusers

Inebriated Immunity: Alcohol Affects Innate Immune Signaling in the Gut-Liver-Brain Axis

Alcohol is a commonly consumed beverage, a drug of abuse and an important molecule affecting nearly every organ-system in the body. This project seeks to investigate the interplay between alcohol’s effects on critical organ-systems making up gut-liver-brain axis. Alcohol initially interacts with the gastrointestinal tract. Our research describes the alterations seen in intestinal microbiota following alcohol consumption in an acute-on-chronic model of alcoholic hepatitis and indicates that reducing intestinal bacteria using antibiotics protects from alcohol-induced intestinal cytokine expression, alcoholic liver disease and from inflammation in the brain. Alcohol-induced liver injury can occur due to direct hepatocyte metabolic dysregulation and from leakage of bacterial products from the intestine that initiates an immune response. Here, we will highlight the importance of this immune response, focusing on the role of infiltrating immune cells in human patients with alcoholic hepatitis and alcoholic cirrhosis. Using a small molecule inhibitor of CCR2/CCR5 chemokine receptor signaling in mice, we can protect the liver from damage and alcohol-induced inflammation. In the brain, we observe that chronic alcohol leads to the infiltration of macrophages in a region-specific manner. CCR2/CCR5 inhibition reduced macrophage infiltration, alcohol-induced inflammation and microglial changes. We also report that chronic alcohol shifts excitatory/inhibitory synapses in the hippocampus, possibly through complement-mediated remodeling. Finally, we show that anti-inflammasome inhibitors altered behavior by reducing alcohol consumption in female mice. Together, these data advance our understanding of the gut-liver-brain axis in alcoholism and suggest novel avenues of therapeutic intervention to inhibit organ pathology associated with alcohol consumption and reduce drinking

Role of Astrocyte-Derived Extracellular Vesicles in Neuroinflammation Mediated by Drug Abuse

Neuronal damage and neuroinflammation is a hallmark feature of HIV-associated neurological disorders (HANDs). Opioids abuse accelerates the incidence and progression of HAND; however, the mechanisms underlying the potentiation of neuropathogenesis by these drugs remain elusive. Extracellular vesicles (EVs) are essential conduits in HIV and drug abuse-mediated synaptodendritic injury and neuroinflammation. Findings from our group have demonstrated that astrocyte-derived EV (ADEV)-miRNA-29b mediates HIV Tat and morphine-induced neuronal injury, thus underscoring the importance of such interactions in NeuroHIV. Besides, HIV Tat and morphine-mediated synaptodendritic injury via ADEVs, we are also interested in whether ADEVs contributes to neuroinflammation. Microglia are critical players in neuroinflammation. Morphine could regulate microglial function; However, the role of ADEVs in morphine-mediated dysregulation of microglia remains elusive. Additionally, drugs of abuse such as opioids can result in a breach of the blood-brain barrier (BBB), ultimately leading to enhanced monocyte transmigration and ensuing neuroinflammation. Recently studies provide compelling evidence that pericyte loss on the microvessels results in increased extravasation of peripheral immune cells. Mechanism(s) by which pericytes contribute to morphine-mediated neuroinflammation, however, remains less understood. The overarching goal of this thesis is to explore another undefined role of the morphine-mediated release of EV-miRNAs in regulating microglial and pericyte function(s), which, in turn, leading to neuroinflammation. We found that 1) morphine stimulated ADEVs can be taken up by microglial cells, leading, in turn, to impaired microglial phagocytosis via the TLR7-NF-kB -lincRNA-Cox2 axis and intranasal delivery of lincRNA-Cox2 siRNA restored microglial phagocytic activity of morphine-administered mice; 2) exposure of astrocytes in culture to morphine resulted in increased expression and secretion of miR-138 in the ADEVs which in turn, were taken up by the microglia, resulting in microglial activation, via binding to endosomal TLR7; 3) exposure of astrocytes to morphine resulted in induction and release the of miR-23a in the ADEVs, which, taken up by pericytes, leading to loss of pericyte thus lead to influx of monocyte (Figure 1). Our findings could have clinical ramifications in the future for developing EV-loaded RNA-based therapeutics that aimed at managing neuroinflammation-associated cognitive disorders in the context of chronic morphine abuse and HIV-opiate comorbidities

Review and Meta-Analyses of TAAR1 Expression in the Immune System and Cancers

Since its discovery in 2001, the major focus of TAAR1 research has been on its role in monoaminergic regulation, drug-induced reward and psychiatric conditions. More recently, TAAR1 expression and functionality in immune system regulation and immune cell activation has become a topic of emerging interest. Here, we review the immunologically-relevant TAAR1 literature and incorporate open-source expression and cancer survival data meta-analyses. We provide strong evidence for TAAR1 expression in the immune system and cancers revealed through NCBI GEO datamining and discuss its regulation in a spectrum of immune cell types as well as in numerous cancers. We discuss connections and logical directions for further study of TAAR1 in immunological function, and its potential role as a mediator or modulator of immune dysregulation, immunological effects of psychostimulant drugs of abuse, and cancer progression

Novel therapeutic strategies for inflammatory cardiomyopathy: from bench to bedside

Die entzündliche Kardiomyopathie ist eine heterogene Erkrankung. Die häufigste Ursache ist eine Virusinfektion, wobei Parvovirus B19 (B19V) der bedeutendste Erreger ist. In dieser Arbeit wurden potenzielle Therapie für die speziellen klinischen Verläufe und deren Phänotypen untersucht. In vitro konnte gezeigt werden, dass Telbivudin in B19V-infizierten/B19V-non-structural protein-1-stimulierten humanen mikrovaskulären Endothelzellen (HMEC-1) endothelial-protektiv wirkt. In einem klinischen Versuch wurden dann 4 Patienten, bei denen eine aktive Transkription des B19V nachgewiesen wurde, für 6 Monate mit Telbivudin behandelt. Alle Patienten verbesserten sich. Ein anderes klinisches Szenario stellt die schwere Entzündung des Myokards dar, welche gewöhnlich mit einer inaktiven/persistierenden Infektion des B19V verbunden ist. Eine Behandlung mit Immunsuppressiva ist hier umstritten, da eine Reaktivierung des Virus befürchtet wird. Um diesen Aspekt weiter zu untersuchen, würde eine Therapie mit Prednisolon in Kombination mit Azathioprin bei 51 B19V-positiven und 17 B19V-negativen Patienten angewandt. Beide Gruppen profitierten in ähnlichem Maße von der Kombinationstherapie, wobei sich die Virusmenge nicht signifikant veränderte. Bei B19V-negativen Patienten konnte über die Persistenz von CD20+ B-Lymphozyten in den EMBs die Untergruppe der „Steroide non-responder“ klassifiziert werden. Im weiteren Verlauf wurden 6 Patienten mit Rituximab, einem monoklonalen Antikörper, der spezifisch gegen CD20+ B-Lymphozyten gerichtet ist, behandelt. Hiervon zeigten 5 Patienten eine ausgezeichnete klinische Verbesserung. Ein Patient mit Myokarditis-induzierten kardiogenen Schock zeigt, dass die Entlastung des linken Ventrikels mittels eines Mikroaxialpumpensystems zu einer rapiden Abnahme der Entzündungszellen führt. Zusammenfassend liefert diese Arbeit Belege für die Wirksamkeit und die Notwendigkeit einer phänotypbasierten Behandlung bei der entzündlichen Kardiomyopathie.Inflammatory cardiomyopathy is a heterogenous disease. Viral etiologies are the most common, with parvovirus B19 (B19V) being the most prominent culprit. Currently, no specific treatment for inflammatory cardiomyopathy exists. In this study, tailored treatment strategies were investigated as potential therapies for specific clinical scenarios. The antiviral drug telbivudine was investigated in the setting of EMB-proven B19V-associated inflammatory cardiomyopathy. In cell culture, telbivudine exhibited endothelial-protective effects on B19V-infected/B19V-non-structural protein-1-stimulated human microvascular endothelial cells (HMEC-1). Clinically, four B19V-positive patients improved following six-month telbivudine regimen in a single-patient use approach. The results were translated to the “PreTOPIC” clinical study, for further evaluation in a randomized placebo-controlled setting. In a different clinical scenario, severe myocardial inflammation is usually associated with inactive/persistent B19V. Here, the use of immunosuppression is controversial, fearing viral flare-up. We investigated combined prednisolone/azathioprine therapy in 51 B19V-positive and 17 B19V negative patients in a single-center observational study. Both groups gained similar benefit, while viral loads did not significantly vary. Among virus-negative phenotypes, EMB-proven CD20+ B lymphocyte persistence characterized a subgroup of steroid non-responders. In this cohort, six patients were treated with rituximab, a monoclonal antibody selectively targeting CD20+ B lymphocytes. Five patients showed outstanding clinical improvement parallel to CD20+ B lymphocyte depletion. Lastly, in a single case of myocarditis-induced cardiogenic shock, mechanical left ventricular unloading via axial flow pump proved to exert disease-modifying effects. In conclusion, this thesis provides evidence for the efficacy and need for phenotype-based inflammatory cardiomyopathy treatment