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The administration of therapeutics to peripheral nerve tissue is challenging due to the complexities of peripheral neuroanatomy and the limitations imposed by the blood–nerve barrier (BNB). Therefore, there is a pressing need to enhance delivery effectiveness and implement targeted delivery methods. Recently, erythrocyte-derived exosomes (Exos) have gained widespread attention as biocompatible vehicles for therapeutics in clinical applications. However, engineering targeted Exos for the peripheral nervous system (PNS) is still challenging. This study aims to develop a targeted Exo delivery system specifically designed for presynaptic terminals of peripheral nerve tissue. The clostridium neurotoxin, tetanus toxin-C fragment (TTC), was tethered to the surface of red blood cell (RBC)-derived Exos via a facile and efficient bio-orthogonal click chemistry method without a catalyst. Additionally, Cyanine5 (Cy5), a reactive fluorescent tag, was also conjugated to track Exo movement in both in vitro and in vivo models. Subsequently, Neuro-2a, a mouse neuronal cell line, was treated with dye-labeled Exos with/without TTC in vitro, and the results indicated that TTC-Exos exhibited more efficient accumulation along the soma and axonal circumference, compared to their unmodified counterparts. Further investigation, using a mouse model, revealed that within 72 h of intramuscular administration, engineered TTC-Exos were successfully transported into the neuromuscular junction and sciatic nerve tissues. These results indicated that TTC played a crucial role in the Exo delivery system, improving the affinity to peripheral nerves. These promising results underscore the potential of using targeted Exo carriers to deliver therapeutics for treating peripheral neuropathies.

Peripheral nerve regeneration remains a significant clinical challenge due to the unsatisfactory functional recovery and public health burden. Exosomes, especially those derived from mesenchymal stem cells (MSCs), are promising as potential cell-free therapeutics and gene therapy vehicles for promoting neural regeneration. In this study, we reported the differentiation of human adipose derived MSCs (hADMSCs) towards the Schwann cell (SC) phenotype (hADMSC-SCs) and then isolated exosomes from hADMSCs with and without differentiation (i.e., dExo vs uExo). We assessed and compared the effects of uExo and dExo on antioxidative, angiogenic, anti-inflammatory, and axon growth promoting properties by using various peripheral nerve-related cells. Our results demonstrated that hADMSC-SCs secreted more neurotrophic factors and other growth factors, compared to hADMSCs without differentiation. The dExo isolated from hADMSC-SCs protected rat SCs from oxidative stress and enhanced HUVEC migration and angiogenesis. Compared to uExo, dExo also had improved performances in downregulating pro-inflammatory gene expressions and cytokine secretions and promoting axonal growth of sensory neurons differentiated from human induced pluripotent stem cells. Furthermore, microRNA (miRNA) sequencing analysis revealed that exosomes and their parent cells shared some similarities in their miRNA profiles and exosomes displayed a distinct miRNA signature. Many more miRNAs were identified in dExo than in uExo. Several upregulated miRNAs, like miRNA-132-3p and miRNA-199b-5p, were highly related to neuroprotection, anti-inflammation, and angiogenesis. The dExo can effectively modulate various peripheral nerve-related cellular functions and is promising for cell-free biological therapeutics to enhance neural regeneration.

Abstract Coronary artery disease (CAD) is one of the major cardiovascular diseases and represents the leading causes of global mortality. Developing new diagnostic and therapeutic approaches for CAD treatment are critically needed, especially for an early accurate CAD detection and further timely intervention. In this study, we successfully isolated human plasma small extracellular vesicles (sEVs) from four stages of CAD patients, that is, healthy control, stable plaque, non‐ST‐elevation myocardial infarction, and ST‐elevation myocardial infarction. Surface‐enhanced Raman scattering (SERS) measurement in conjunction with five machine learning approaches, including Quadratic Discriminant Analysis, Support Vector Machine (SVM), K‐Nearest Neighbor, Artificial Neural network, were then applied for the classification and prediction of the sEV samples. Among these five approaches, the overall accuracy of SVM shows the best predication results on both early CAD detection (86.4%) and overall prediction (92.3%). SVM also possesses the highest sensitivity (97.69%) and specificity (95.7%). Thus, our study demonstrates a promising strategy for noninvasive, safe, and high accurate diagnosis for CAD early detection.

Opioids such as morphine are the most potent and efficacious drugs currently available for pain management. Paradoxically, opioids have also been implicated in inducing neuroinflammation and associated neurocognitive decline. Pericytes, a critical component of the neurovascular unit (NVU), are centrally positioned between endothelial cells and astrocytes, maintaining function of the blood-brain barrier (BBB) nd regulating neuroinflammation by controlling monocyte influx under various pathological conditions. The role of pericytes in morphine-mediated neuroinflammation however, has received less attention, especially in the context of how pericytes crosstalk with other central nervous system (CNS) cells. The current study was undertaken to examine the effect of miRNAs released from morphine-stimulated human primary astrocyte-derived extracellular vesicles (morphine-ADEVs) in mediating pericyte loss at the blood-brain barrier, leading, in turn, to increased influx of peripheral monocytes. Our findings suggest that the heterogeneous nuclear ribonucleoprotein complex A2/B1 (hnRNP A2/B1) plays role in morphine-mediated upregulation and release of miR-23a in ADEVs, and through action of morphine via mu opioid receptor.We further demonstrated that miR-23a in morphine-ADEVs could be taken up by pericytes, resulting in downregulation of PTEN expression, ultimately leading to increased pericyte migration. Furthermore, both overexpression of PTEN and blocking the miR-23a target site at PTEN 3UTR (by transfecting miR-23a-PTEN target protector), attenuated morphine-ADEV-mediated pericyte migration. We also demonstrated that in the microvessels isolated from morphine-administered mice, there were fewer PDGFβR + pericytes co-localizing with CD31+ brain endothelial cells compared with those from saline mice. In line with these findings, we also observed increased loss of pericytes and a concomitantly increased influx of monocytes in the brains of morphine-administered pericyte-labeled NG2-DsRed mice compared with saline mice. In conclusion, our findings indicate morphine-ADEVs mediated loss of pericyte coverage at the brain endothelium, thereby increasing the influx of peripheral monocytes in the central nervous system, leading to neuroinflammation.

Extracellular vesicles (EVs) are small lipid membrane-bound vesicles that can pass the blood–brain barrier. Therefore, EVs could be used for the delivery of therapeutics to the brain. Herein, we investigated the biodistribution of intranasal perfusion of ultrasmall superparamagnetic iron oxide (USPIO)-labeled astrocyte-derived EVs (ADEVs) in mice. We used Western blotting, transmission electron microscopy (TEM), and nanoparticle uptake assay to characterize ADEVs. In addition, intranasal perfusion coupled with magnetic resonance imaging (MRI) was employed to determine the distribution of USPIO-labeled ADEVs in mice. Our results showed the uptake of USPIO by mouse astrocytes and ADEVs. In addition, we confirmed the biodistribution of ADEVs in the brain and other internal organs, including the kidneys, liver, and spleen. Our results suggest that USPIO did not affect mouse astrocyte cell survivability and EV release. Therefore, intranasal delivery of therapeutic loaded EVs could be used for the treatment of various brain disorders.

Morphine administration causes system-level metabolic changes. Here, we show that morphine-tolerant mice exhibited distinct plasma metabolic signatures upon acute and chronic administration. We utilized a mouse model of morphine tolerance by exposing mice to increasing doses of the drug over 4 days. We collected plasma samples from mice undergoing acute or chronic morphine or saline injections and analyzed them using targeted GC–MS-based metabolomics to profile approximately 80 metabolites involved in the central carbon, amino acid, nucleotide, and lipid metabolism. Our findings reveal distinct alterations in plasma metabolite concentrations in response to acute or chronic morphine intake, and these changes were linked to the development of tolerance to morphine’s analgesic effects. We identified several metabolites that had been differentially affected by acute versus chronic morphine use, suggesting that metabolic changes may be mitigated by prolonged exposure to the drug. Morphine-tolerant mice showed a restoration of amino acid and glycolytic metabolites. Additionally, we conducted reconstructed metabolic network analysis on the first 30 VIP-ranked metabolites from the PLSDA of the saline, acute, and morphine-tolerant mice groups, which uncovered four interaction networks involving the amino acid metabolism, the TCA cycle, the glutamine-phenylalanine-tyrosine pathway, and glycolysis. These pathways were responsible for the metabolic differences observed following distinct morphine administration regimens. Overall, this study provides a valuable resource for future investigations into the role of metabolites in morphine-induced analgesia and associated effects following acute or chronic use in mice.

HIV-1 infection in the era of combined antiretroviral therapy has been associated with premature aging. Among the various features of HIV-1 associated neurocognitive disorders, astrocyte senescence has been surmised as a potential cause contributing to HIV-1-induced brain aging and neurocognitive impairments. Recently, lncRNAs have also been implicated to play essential roles in the onset of cellular senescence. Herein, using human primary astrocytes (HPAs), we investigated the role of lncRNA TUG1 in HIV-1 Tat-mediated onset of astrocyte senescence. We found that HPAs exposed to HIV-1 Tat resulted in significant upregulation of lncRNA TUG1 expression that was accompanied by elevated expression of p16 and p21, respectively. Additionally, HIV-1 Tat-exposed HPAs demonstrated increased expression of senescence-associated (SA) markers—SA-β-galactosidase (SA-β-gal) activity and SA-heterochromatin foci—cell-cycle arrest, and increased production of reactive oxygen species and proinflammatory cytokines. Intriguingly, gene silencing of lncRNA TUG1 in HPAs also reversed HIV-1 Tat-induced upregulation of p21, p16, SA-β gal activity, cellular activation, and proinflammatory cytokines. Furthermore, increased expression of astrocytic p16 and p21, lncRNA TUG1, and proinflammatory cytokines were observed in the prefrontal cortices of HIV-1 transgenic rats, thereby suggesting the occurrence of senescence activation in vivo. Overall, our data indicate that HIV-1 Tat-induced astrocyte senescence involves the lncRNA TUG1 and could serve as a potential therapeutic target for dampening accelerated aging associated with HIV-1/HIV-1 proteins.

Brain disorders are characterized by the progressive loss of structure and function of the brain as a consequence of progressive degeneration and/or death of nerve cells. Aging is a major risk factor for brain disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and stroke. Various cellular and molecular events have been shown to play a role in the progress of neurodegenerative diseases. Emerging studies suggest that primary cilia could be a key regulator in brain diseases. The primary cilium is a singular cellular organelle expressed on the surface of many cell types, such as astrocytes and neurons in the mature brain. Primary cilia detect extracellular cues, such as Sonic Hedgehog (SHH) protein, and transduce these signals into cells to regulate various signaling pathways. Abnormalities in ciliary length and frequency (ratio of ciliated cells) have been implicated in various human diseases, including brain disorders. This review summarizes current findings and thoughts on the role of primary cilia and ciliary signaling pathways in aging and age-related brain disorders.

Abstract Major depressive disorder (MDD) is the most prevalent psychiatric disorder worldwide and severely limits psychosocial function and quality of life, but no effective medication is currently available. Circular RNAs (circRNAs) have been revealed to participate in the MDD pathological process. Targeted delivery of circRNAs without blood‐brain barrier (BBB) restriction for remission of MDD represents a promising approach for antidepressant therapy. In this study, RVG‐circDYM‐extracellular vesicles (RVG‐circDYM‐EVs) were engineered to target and preferentially transfer circDYM to the brain, and the effect on the pathological process in a chronic unpredictable stress (CUS) mouse model of depression was investigated. The results showed that RVG‐circDYM‐EVs were successfully purified by ultracentrifugation from overexpressed circDYM HEK 293T cells, and the characterization of RVG‐circDYM‐EVs was successfully demonstrated in terms of size, morphology and specific markers. Beyond demonstrating proof‐of‐concept for an RNA drug delivery technology, we observed that systemic administration of RVG‐circDYM‐EVs efficiently delivered circDYM to the brain, and alleviated CUS‐induced depressive‐like behaviours, and we discovered that RVG‐circDYM‐EVs notably inhibited microglial activation, BBB leakiness and peripheral immune cells infiltration, and attenuated astrocyte disfunction induced by CUS. CircDYM can bind mechanistically to the transcription factor TAF1 (TATA‐box binding protein associated factor 1), resulting in the decreased expression of its downstream target genes with consequently suppressed neuroinflammation. Taken together, our findings suggest that extracellular vesicle‐mediated delivery of circDYM is effective for MDD treatment and promising for clinical applications.

Chronic low-grade inflammation remains an essential feature of HIV-1 infection under combined antiretroviral therapy (cART) and contributes to the accelerated cognitive defects and aging in HIV-1 infected populations, indicating cART limitations in suppressing viremia. Interestingly, ~50% of the HIV-1 infected population on cART that develops cognitive defects is complicated by drug abuse, involving the activation of cells in the central nervous system (CNS) and neurotoxin release, altogether leading to neuroinflammation. Neuroinflammation is the hallmark feature of many neurodegenerative disorders, including HIV-1-associated neurocognitive disorders (HAND). Impaired autophagy has been identified as one of the underlying mechanisms of HAND in treated HIV-1-infected people that also abuse drugs. Several lines of evidence suggest that autophagy regulates CNS cells’ responses and maintains cellular hemostasis. The impairment of autophagy is associated with low-grade chronic inflammation and immune senescence, a known characteristic of pathological aging. Therefore, autophagy impairment due to CNS cells, such as neurons, microglia, astrocytes, and pericytes exposure to HIV-1/HIV-1 proteins, cART, and drug abuse could have combined toxicity, resulting in increased neuroinflammation, which ultimately leads to accelerated aging, referred to as neuroinflammaging. In this review, we focus on the potential role of autophagy in the mechanism of neuroinflammaging in the context of HIV-1 and drug abuse.

HIV trans-activator of transcription (Tat), one of the cytotoxic proteins secreted from HIV-infected cells, is also known to facilitate chemokine-mediated transmigration of monocytes into the brain leading, in turn, to neuroinflammation and thereby contributing to the development of HIV-associated neurocognitive disorders (HAND). The mechanism(s) underlying HIV Tat-mediated enhancement of monocyte transmigration, however, remain largely unknown. CXC chemokine receptor 3 (CXCR3) that is expressed by the peripheral monocytes is known to play a role in the monocyte influx and accumulation. In the present study, we demonstrate for the first time that exposure of human monocytes to HIV Tat protein resulted in upregulated expression of CXCR3 leading, in turn, to increased monocyte transmigration across the blood–brain barrier (BBB) both in the in vitro and in vivo model systems. This process involved activation of toll-like receptor 4 (TLR4), with downstream phosphorylation and activation of TANK-binding kinase 1 (TBK1), and subsequent phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3), ultimately leading to enhanced expression of CXCR3 in human monocytes. These findings imply a novel molecular mechanism underlying HIV Tat-mediated increase of monocyte transmigration across the BBB, while also implicating a novel role of CXCR3-dependent monocyte transmigration in HIV Tat-mediated neuroinflammation.

Long noncoding RNAs (lncRNAs) are large RNA transcripts that do not code for proteins but exert their effects in the form of RNA. To date many thousands of lncRNAs have been identified, their molecular functions and mechanisms of action however are largely unknown. The development of high-throughput experimental technologies, such as ChIRP (Chromatin isolation by RNA purification), CHART (Capture Hybridization Analysis of RNA Targets), RAP (RNA antisense purification), RIP (RNA Immunoprecipitation), CLIP (cross-linking and immunoprecipitation) and RNA pull-down, has led to a rapid expansion of lncRNA research and resulted in many publicly-available databases. This review provides an overview of the current methodologies available for discovering and investigating functions of lncRNAs in various human diseases. A comparison and application of these methods are also included. Finally, this paper surveys current databases containing annotations, interactome networks and functions of lncRNAs. The appropriate use of these methods and databases will provide not only high-resolution functional features of lncRNAs, but also enhance our understanding of the underlying mechanisms by which lncRNAs regulate a variety of biological processes.

Impairment of microglial functions, such as phagocytosis and/or dysregulation of immune responses, has been implicated as an underlying factor involved in the pathogenesis of various neurodegenerative disorders. Our previous studies have demonstrated that long intergenic noncoding RNA (lincRNA)-Cox2 expression is influenced by nuclear factor κB (NF-κB) signaling and serves as a coactivator of transcriptional factors to regulate the expression of a vast array of immune-related genes in microglia. Extracellular vesicles (EVs) have been recognized as primary facilitators of cell-to-cell communication and cellular regulation. Herein, we show that EVs derived from astrocytes exposed to morphine can be taken up by microglial endosomes, leading, in turn, to activation of Toll-like receptor 7 (TLR7) with a subsequent upregulation of lincRNA-Cox2 expression, ultimately resulting in impaired microglial phagocytosis. This was further validated in vivo, wherein inhibition of microglial phagocytic activity was also observed in brain slices isolated from morphine-administrated mice compared with control mice. Additionally, we also showed that intranasal delivery of EVs containing lincRNA-Cox2 siRNA (small interfering RNA) was able to restore microglial phagocytic activity in mice administered morphine. These findings have ramifications for the development of EV-loaded RNA-based therapeutics for the treatment of various disorders involving functional impairment of microglia. Keywords: phagocytosis, drug abuse, morphine, extracellular vesicle, exosome, lincRNA, microglia, CNS

MicroRNA-124 (miR-124), a brain-enriched microRNA, is known to regulate microglial quiescence. Psychostimulants such as cocaine have been shown to activate microglia by downregulating miR-124, leading, in turn, to neuroinflammation. We thus rationalized that restoring the levels of miR-124 could function as a potential therapeutic approach for cocaine-mediated neuroinflammation. Delivering miRNA based drugs in the brain that are effective and less invasive, however, remains a major challenge in the field. Herein we engineered extracellular vesicles (EVs) and loaded them with miR-124 for delivery in the brain. Approach involved co-transfection of mouse dendritic cells with Dicer siRNA and RVG-Lamp2b plasmid to deplete endogenous miRNAs and for targeting the CNS, respectively. Mouse primary microglia (mPm) were treated with purified engineered EVs loaded with either Cy5-miR-124 or Cy5-scrambled miRNA oligos in the presence or absence of cocaine followed by assessing EV uptake and microglial activation. In vivo studies involved pretreating mice intranasally with engineered EVs followed by cocaine injection (20 mg/kg, i.p.). mPm exposed to EV-miR-124 exhibited reduced expression of miR-124 targets – TLR4 and STAT3 as well as ERK-1/2 and Iba1. In cocaine administered mice, EV-Cy5-miR-124 delivered intranasally were detected in the CNS and significantly reduced the expression of inflammatory markers TLR4, MYD88, STAT3 and NF-kB p65 while also downregulating the microglial activation marker, Iba1. Collectively, these findings suggest that engineered EVs can deliver miR-124 into the CNS, thereby alleviating cocaine-mediated microglial activation. Manipulating EV miRNAs can thus be envisioned as an efficient means for delivery of RNA-based therapeutics to target organs.

Increased life expectancy of patients diagnosed with HIV in the current era of antiretroviral therapy is unfortunately accompanied with the prevalence of HIV-associated neurocognitive disorders (HANDs) and risk of comorbidities such as Alzheimer-like pathology. HIV-1 transactivator of transcription (Tat) protein has been shown to induce the production of toxic neuronal amyloid protein and also enhance neurotoxicity. The contribution of astrocytes in Tat-mediated amyloidosis remains an enigma. We report here, in simian immunodeficiency virus (SIV)+ rhesus macaques and patients diagnosed with HIV, brain region-specific up-regulation of amyloid precursor protein (APP) and Aβ (40 and 42) in astrocytes. In addition, we find increased expression of β-site cleaving enzyme (BACE1), APP, and Aβ in human primary astrocytes (HPAs) exposed to Tat. Mechanisms involved up-regulation of hypoxia-inducible factor (HIF-1α), its translocation and binding to the long noncoding RNA (lncRNA) BACE1-antisense transcript (BACE1-AS), resulting, in turn, in the formation of the BACE1-AS/BACE1 RNA complex, subsequently leading to increased BACE1 protein, and activity and generation of Aβ-42. Gene silencing approaches confirmed the regulatory role of HIF-1α in BACE1-AS/BACE1 in Tat-mediated amyloidosis. This is the first report implicating the role of the HIF-1α/lncRNABACE1-AS/BACE1 axis in Tat-mediated induction of astrocytic amyloidosis, which could be targeted as adjunctive therapies for HAND-associated Alzheimer-like comorbidity.

Despite the efficacy of combination antiretroviral therapy (ART) in controlling human immunodeficiency virus (HIV-1) replication, cytotoxic viral proteins such as HIV-1 transactivator of transcription (Tat) persist in tissues such as the brain. Although HIV-1 does not infect neuronal cells, it is susceptible to viral Tat protein-mediated toxicity, leading to neuroinflammation that underlies HIV-associated neurocognitive disorders (HAND). Given the role of extracellular vesicles (EVs) in both cellular homoeostasis and under pathological conditions, we sought to investigate the alterations in the quantity of neuronal-derived EVs in the brain – as defined by the presence of cell adhesion molecule L1 (L1CAM) and to evaluate the presence of L1CAM+ EVs in the peripheral circulation of HIV-1 transgenic (HIV-1 Tg) rats. The primary goal of this study was to investigate the effect of long-term exposure of HIV-1 viral proteins on the release of neuronal EVs in the brain and their transfer in the systemic compartment. Brain and serum EVs were isolated from both wild type and HIV-1 Tg rats using differential ultracentrifugation with further purification using the Optiprep gradient method. The subpopulation of neuronal EVs was further enriched using immunoprecipitation. The current findings demonstrated increased presence of L1CAM+ neuronal-derived EVs both in the brain and serum of HIV-1 Tg rats.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Almost all types of cells release extracellular vesicles (EVs) into the extracellular space. EVs such as exosomes and microvesicles are membrane-bound vesicles ranging in size from 30 to 1000 nm in diameter. Under normal conditions, EVs mediate cell to cell as well as inter-organ communication via the shuttling of their cargoes which include RNA, DNA and proteins. Under pathological conditions, however, the number, size and content of EVs are found to be altered and have been shown to play crucial roles in disease progression. Emerging studies have demonstrated that EVs are involved in many aspects of viral infection-mediated neurodegenerative diseases. In the current review, we will describe the interactions between EV biogenesis and the release of virus particles while also reviewing the role of EVs in various viral infections, such as HIV-1, HTLV, Zika, CMV, EBV, Hepatitis B and C, JCV, and HSV-1. We will also discuss the potential uses of EVs and their cargoes as biomarkers and therapeutic vehicles for viral infections.

Extracellular vesicles (EVs) are globular, membrane bound nanovesicles (30–100 nm range) that are shed both during normal cellular functioning and under pathological conditions by most cell types. In recent years, there has been significant interest in the study of these vesicles as conduits for the delivery of information between cells from both analogous and disparate tissues. Their ability to carry specialised cargo including signalling mediators, proteins, messenger RNA and miRNAs characterises these vesicles as primary facilitators of cell-to-cell communication and regulation. EVs have also been demonstrated to play important roles in the field of cancer biology and metastasis. However, significant knowledge gaps exist in the role these vesicles play in the context of HIV infection and drug abuse. To foster discussion in this area a satellite symposium on “HIV, NeuroAIDS, Drug Abuse & EVs”, was held in conjunction with the annual meeting of the International Society for Extracellular Vesicles (ISEV) in Bethesda, in April 2015. Experts in HIV and drug abuse fields were invited to share their findings on the role of EVs in HIV-1 infection and drug addiction. Additional discussion included current areas of research in EV biology in HIV infection and drug abuse.

Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.

Cardiac involvement is a well-documented complication of human immunodeficiency virus-1 (HIV-1) infection. Previous studies have demonstrated increased adhesion of monocytes to human vascular endothelial cells in HIV-infected individuals. HIV Tat protein, which is the transactivator of transcription (Tat), plays a key role in activating endothelial cells. In the present study, we demonstrated that exposure of HUVECs to HIV Tat protein resulted in induced expression of cell adhesion molecules specifically ICAM-1, leading to increased adhesion of monocytes to the endothelium. This effect of Tat was mediated through activation of mitogen-activated protein kinases and downstream transcription factor NF-κB. Increased expression of ICAM-1 was regulated by microRNA (miRNA) miR-221 and to some extent by miR-222, both of which are known to target ICAM-1. Functional inhibition of the respective miRNAs with anti-miR oligonucleotides resulted in induction of ICAM-1 protein in HUVECs. Furthermore, Tat-stimulated regulation of ICAM-1 via miR-221/-222 involved the NF-kB-dependent pathway. Functional implication and specificity of up-regulated ICAM-1 was confirmed using the ICAM-1 neutralizing antibody in the in vitro cell adhesion assays. These findings were further confirmed in vivo using the HIV transgenic (Tg) rats. These animals not only demonstrated increased expression of ICAM-1 mRNA, with a concomitant reduction in the expression of miR-221 in the aorta and heart, but also had increased expression of the ICAM-1 protein that was predominantly in the endothelial cell layer. Taken together, these findings implicate that Tat-mediated induction of ICAM-1 expression plays a critical role in monocyte adhesion observed in HIV-1-associated cardiomyopathies.

The signaling pathways associated with the Toll-like receptors (TLRs) and nuclear factor-kappaB (NF-κB) are essential to pro-inflammatory cytokine and chemokine expression, as well as initiating innate epithelial immune responses. The TLR/NF-κB signaling pathways must be stringently controlled through an intricate network of positive and negative regulatory elements. MicroRNAs (miRNAs) are non-coding small RNAs that regulate the stability and/or translation of protein-coding mRNAs. Herein we report that miR-16 promotes NF-κB-regulated transactivation of the IL-8 gene by suppression of the silencing mediator for retinoid and thyroid hormone receptor (SMRT). LPS stimulation activated miR-16 gene transcription in human monocytes (U937) and biliary epithelial cells (H69) through MAPK-dependent mechanisms. Transfection of cells with the miR-16 precursor promoted LPS-induced production of IL-8, IL-6, and IL-1α, without a significant effect on their RNA stability. Instead, an increase in NF-κB-regulated transactivation of the IL-8 gene was confirmed in cells following transfection of miR-16 precursor. Importantly, miR-16 targeted the 3'-untranslated region of SMRT and caused translational suppression of SMRT. LPS decreased SMRT expression via upregulation of miR-16. Moreover, functional manipulation of SMRT altered NF-κB-regulated transactivation of LPS-induced IL-8 expression. These data suggest that miR-16 targets SMRT and modulates NF-κB-regulated transactivation of the IL-8 gene.

Cryptosporidium parvum is a protozoan parasite that infects the gastrointestinal epithelium and causes diarrheal disease worldwide. Innate epithelial immune responses are key mediators of the host's defense to C. parvum. MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level and are involved in regulation of both innate and adaptive immune responses. Using an in vitro model of human cryptosporidiosis, we analyzed C. parvum-induced miRNA expression in biliary epithelial cells (i.e., cholangiocytes). Our results demonstrated differential alterations in the mature miRNA expression profile in cholangiocytes following C. parvum infection or lipopolysaccharide stimulation. Database analysis of C. parvum-upregulated miRNAs revealed potential NF-kappaB binding sites in the promoter elements of a subset of miRNA genes. We demonstrated that mir-125b-1, mir-21, mir-30b, and mir-23b-27b-24-1 cluster genes were transactivated through promoter binding of the NF-kappaB p65 subunit following C. parvum infection. In contrast, C. parvum transactivated mir-30c and mir-16 genes in cholangiocytes in a p65-independent manner. Importantly, functional inhibition of selected p65-dependent miRNAs in cholangiocytes increased C. parvum burden. Thus, we have identified a panel of miRNAs regulated through promoter binding of the NF-kappaB p65 subunit in human cholangiocytes in response to C. parvum infection, a process that may be relevant to the regulation of epithelial anti-microbial defense in general.