Blockade of beta adrenergic receptors protects the blood brain barrier and reduces systemic pathology caused by HIV-1 Nef protein.

Combination antiretroviral therapy (cART) targets viral replication, but early viral protein production by astrocytes may still occur and contribute to the progression of HIV-1 associated neurocognitive disorders and secondary complications seen in patients receiving cART. In prior work with our model, astrocytic HIV-1 Nef expression exhibits neurotoxic effects leading to neurological damage, learning impairment, and immune upregulation that induces inflammation in the lungs and small intestine (SI). In this follow-up study, we focus on the sympathetic nervous system (SNS) as the important branch for peripheral inflammation resulting from astrocytic Nef expression. Male and female Sprague Dawley rats were infused with transfected astrocytes to produce Nef. The rats were divided in four groups: Nef, Nef + propranolol, propranolol and naïve. The beta-adrenergic blocker, propranolol, was administered for 3 consecutive days, starting one day prior to surgery. Two days after the surgery, the rats were sacrificed, and then blood, brain, small intestine (SI), and lung tissues were collected. Levels of IL-1ß were higher in both male and female rats, and treatment with propranolol restored IL-1ß to basal levels. We observed that Nef expression decreased staining of the tight junction protein claudin-5 in brain tissue while animals co-treated with propranolol restored claudin-5 expression. Lungs and SI of rats in the Nef group showed histological signs of damage including larger Peyer's Patches, increased tissue thickness, and infiltration of immune cells; these findings were abrogated by propranolol co-treatment. Results suggest that interruption of the beta adrenergic signaling reduces the peripheral organ inflammation caused after Nef expression in astrocytes of the brain.

Activation of ventral tegmental area neurotensin Receptor-1 neurons promotes weight loss.

Dopamine (DA) neurons in the ventral tegmental area (VTA) modulate physical activity and feeding behaviors that are disrupted in obesity. Yet, the heterogeneity of VTA DA neurons has hindered determination of which ones might be leveraged to support weight loss. We hypothesized that increased activity in the subset of VTA DA neurons expressing neurotensin receptor-1 (NtsR1) might promote weight loss behaviors. To test this, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to activate VTA NtsR1 neurons in normal weight and diet-induced obese mice. Acute activation of VTA NtsR1 neurons (24hr) significantly decreased body weight in normal weight and obese mice by reducing food intake and increasing physical activity. Moreover, daily activation of VTA NtsR1 neurons in obese mice sustained weight loss over 7 days. Activating VTA NtsR1 neurons also suppressed how much mice worked to obtain sucrose rewards, even when there was high motivation to consume. However, VTA NtsR1 neural activation was not reinforcing, nor did it invoke liabilities associated with whole-body NtsR1 agonism such as anxiety, vasodepressor response or hypothermia. Activating VTA NtsR1 neurons therefore promotes dual behaviors that support weight loss without causing adverse effects, and is worth further exploration for managing obesity.

Identification of the predominant human NK cell effector subset mediating ADCC against HIV-infected targets coated with BNAbs or plasma from PLWH.

The potential of immunotherapy strategies utilizing broadly neutralizing antibodies (BNAbs), such as 3BNC117 and 10-1074, to limit viral replication while also facilitating clearance of HIV infected cells has heightened interest in identifying the predominant NK effector subset(s) capable of mediating antibody dependent cellular cytotoxicity (ADCC). Utilizing advanced polychromatic flow cytometry, we identified that CD57 positive NK cells from ART-suppressed in People Living With HIV (PLWH) expressed significantly higher levels of the CD16 FcγR receptor, 2B4 ADCC coreceptor, and HLA-DR activation marker while NKG2C positive NK cells expressed significantly higher levels of the CD2 ADCC coreceptor (p < 0.001, n = 32). Functionally, CD57 positive NK cells from ART-suppressed PLWH with either high or low NKG2C expansion exhibited significantly enhanced degranulation and IFN-γ production against heterologous gp120-coated ADCC targets coated with HIV reference plasma compared to CD57 negative NK cells (p = 0.0029, n = 11). CD57 positive NK cells from control donors lacking NKG2C expansion also exhibited significantly more degranulation and IFN-γ production at every timepoint tested against both heterologous ADCC targets (p = 0.019, n = 9) and HIV-1 infected autologous CD4+ primary T cells coated with BNAbs. Together, our data support CD57 positive and NKG2C positive NK cells as the predominant ADCC effector subsets capable of targeting HIV-infected CD4+ cells in the presence of 3BNC117 and 10-1074 immunotherapy.

Persons who inject drugs (PWID) retain functional NK cells, dendritic cell stimulation, and adaptive immune recall responses despite prolonged opioid use.

Previous literature suggests that acute opioid use results in the functional impairment of the immune response, thereby decreasing resistance to viral infection. Here, we assessed if innate and adaptive immune responses are compromised ex vivo in persons who inject drugs (PWID) and whether long-term injection drug use may impact host susceptibility to in vitro HIV infection. We measured the frequency, activation state, and functional profile of NK cells, dendritic cells, and CD4+ and CD8+ T cells in low-risk PWID who do not share needles, high-risk needle-sharing PWID, and control donors who did not inject drugs. We also assessed plasma levels of inflammatory markers and CD4+ T cell susceptibility to HIV infection. We observed a significant increase in the amount of sCD14 (P = 0.0023, n = 16) and sCD163 (P = 0.0001, n = 16) in the plasma of PWID compared to controls. Evidence of constitutive activation was noted in PWID as compared to controls with increased CD69 expression in CD56dim NK cells (P = 0.0103, n = 26) and increased CD38 and HLA-DR expression in CD4+ T cells (P = 0.0355, n = 23). However, no innate or adaptive functional differences were detected between PWID and controls, including: NK cell direct or antibody-dependent cellular cytotoxicity poly-functional response, TLR-stimulated dendritic cell/NK crosstalk, CD8+ T cell response to Staphylococcal enterotoxin B or CMV/EBV/FLU peptides, or constitutive or anti-CD3/CD28-stimulated CD4+ T cell infectivity with CCR5-tropic or CXCR4-tropic HIV-1 isolates. Our data indicate that PWID who utilize opioids over as prolonged time frame can retain a functional ex vivo immune response without a measurable increase in CD4+ T cell infectivity suggesting that leukocytes from PWID are not intrinsically more susceptibility to infection with HIV than non-PWID controls.

Lateral hypothalamic area neuropeptides modulate ventral tegmental area dopamine neurons and feeding.

Understanding how the brain coordinates energy status with the motivation to eat is crucial to identify strategies to improve disordered body weight. The ventral tegmental area (VTA), known as the core of the mesolimbic system, is of particular interest in this regard because it controls the motivation to consume palatable, calorie-dense foods and to engage in volitional activity. The VTA is largely composed of dopamine (DA) neurons, but modulating these DA neurons has been alternately linked with promoting and suppressing feeding, suggesting heterogeneity in their function. Subsets of VTA DA neurons have recently been described based on their anatomical distribution, electrophysiological features, connectivity and molecular expression, but to date there are no signatures to categorize how DA neurons control feeding. Assessing the neuropeptide receptors expressed by VTA DA neurons may be useful in this regard, as many neuropeptides mediate anorexic or orexigenic responses. In particular, the lateral hypothalamic area (LHA) releases a wide variety of feeding-modulating neuropeptides to the VTA. Since VTA neurons intercept LHA neuropeptides known to either evoke or suppress feeding, expression of the cognate neuropeptide receptors within the VTA may point to VTA DA neuronal mechanisms to promote or suppress feeding, respectively. Here we review the role of the VTA in energy balance and the LHA neuropeptide signaling systems that act in the VTA, whose receptors might be used to classify how VTA DA neurons contribute to energy balance.

S100A14 Is Increased in Activated NK Cells and Plasma of HIV-Exposed Seronegative People Who Inject Drugs and Promotes Monocyte-NK Crosstalk.

BACKGROUND: HIV-exposed seronegative people who inject drugs (HESN-PWID) have been shown to have increased natural killer (NK) cell and myeloid activation when compared with control donors. METHODS: We investigated potential mechanisms maintaining NK activation by conducting quantitative proteome comparisons of NK cells from HESN-PWID subjects and control donors. Proteins upregulated in NK cells were measured in the plasma of HESN-PWID subjects by ELISA and further investigated for their ability to induce innate immune activation in vitro. RESULTS: The NK cell proteome comparison showed markedly higher levels of interferon-stimulated proteins and S100 proteins, including S100A14. Consistent with these results, we observed significantly higher levels of S100A14 in the plasma of HESN-PWID subjects compared with controls (P = 0.033, n = 25). In vitro, the addition of recombinant S100A14 protein significantly activated NK cells in a peripheral blood mononuclear cell mixture (P = 0.011, n = 9), but not purified NK cells alone. Treatment of purified monocytes with recombinant S100A14 protein induced secretion of TNF-alpha and led to significantly higher NK CD69 activation (P = 0.0156, n = 7) in a co-culture through a TLR4-dependent interaction. CONCLUSIONS: Our study identified S100A14 as a novel protein increased within NK cells and plasma of HESN-PWID subjects with the capacity to sustain NK activation through TLR4-dependent activation of myeloid cells.

Dimethyl Fumarate Prevents HIV-Induced Lysosomal Dysfunction and Cathepsin B Release from Macrophages.

HIV-associated neurocognitive disorders (HAND) are prevalent despite combined antiretroviral therapy, affecting nearly half of HIV-infected patients worldwide. During HIV infection of macrophages secretion of the lysosomal protein, cathepsin B, is increased. Secreted cathepsin B has been shown to induce neurotoxicity. Oxidative stress is increased in HIV-infected patients, while antioxidants are decreased in monocytes from patients with HIV-associated dementia (HAD). Dimethyl fumarate (DMF), an antioxidant, has been reported to decrease HIV replication and neurotoxicity mediated by HIV-infected macrophages. Thus, we hypothesized that DMF will decrease cathepsin B release from HIV-infected macrophages by preventing oxidative stress and enhancing lysosomal function. Monocyte-derived macrophages (MDM) were isolated from healthy donors, inoculated with HIV-1ADA, and treated with DMF following virus removal. After 12 days post-infection, HIV-1 p24 and total cathepsin B levels were measured from HIV-infected MDM supernatants using ELISA; intracellular reactive oxygen and nitrogen species (ROS/RNS) were measured from MDM lysates, and functional lysosomes were assessed using a pH-dependent lysosomal dye. Neurons were incubated with serum-free conditioned media from DMF-treated MDM and neurotoxicity was determined using TUNEL assay. Results indicate that DMF reduced HIV-1 replication and cathepsin B secretion from HIV-infected macrophages in a dose-dependent manner. Also, DMF decreased intracellular ROS/RNS levels, and prevented HIV-induced lysosomal dysfunction and neuronal apoptosis. In conclusion, the improvement in lysosomal function with DMF treatment may represent the possible mechanism to reduce HIV-1 replication and cathepsin B secretion. DMF represents a potential therapeutic strategy against HAND.

Macrophage secretome from women with HIV-associated neurocognitive disorders.

PURPOSE: Thirty to 50% of HIV patients develop HIV-associated neurocognitive disorders (HANDs) despite combined antiretroviral therapy. HIV-1-infected macrophages release viral and cellular proteins that induce neuronal degeneration and death. We hypothesize that changes in the macrophage secretome of HIV-1 seropositive patients with HAND may dissect proteins related to neurotoxicity. EXPERIMENTAL DESIGN: Monocyte-derived macrophages (MDMs) were isolated from the peripheral blood of 12 HIV+ and four HIV- women characterized for neurocognitive function. Serum-free MDM supernatants were collected for protein isolation and quantification with iTRAQ® labeling. Protein identification was performed using a LTQ Orbitrap Velos mass spectrometer and validated in MDM supernatants and in plasma using ELISA. RESULTS: Three proteins were different between normal cognition (NC) and asymptomatic neurocognitive disorders (ANI), six between NC and HIV-associated dementia (HAD), and six between NC and HAD. Among these, S100A9 was decreased in plasma from patients with ANI, and metalloproteinase 9 was decreased in the plasma of all HIV+ patients regardless of cognitive status, and was significantly reduced in supernatant of MDM isolated from patients with ANI. CONCLUSIONS AND CLINICAL RELEVANCE: S100A9 and metalloproteinase 9 have been associated with inflammation and cognitive impairment, and therefore represent potential targets for HAND treatment.

HIV gp120 sequence variability associated with HAND in Hispanic Women.

OBJECTIVE: HIV-1 variants with different tropisms are associated with various neuropathologies. This study intends to determine if this correlation is determined by unique viral env sequences. We hypothesize that HIV-1 envelope gene sequence changes are associated with cognition status. METHODS: Viral RNA was extracted from peripheral blood mononuclear cells (PBMCs) co-cultures derived from HIV-1 infected Hispanic women that had been characterized for HIV associated neurocognitive disorders (HAND). RESULTS: Analyses of the C2V4 region of HIV gp120 demonstrated that increased sequence diversity correlates with cognition status as sequences derived from subjects with normal cognition exhibited less diversity than sequences derived from subjects with cognitive impairment. In addition, differences in V3 and V4 loop charges were also noted as well as differences in the N-glycosylation of the V4 region. CONCLUSIONS: Our data suggest that the genetic signature within the C2V4 region may contribute to the pathogenesis of HAND. HIV env sequence characteristics for the isolates grouped in milder forms of HAND can provide insightful information of prognostic value to assess neurocognitive status in HIV+ subjects, particularly during the era of highly prevalent milder forms of HAND.

Macrophage derived cystatin B/cathepsin B in HIV replication and neuropathogenesis.

Mononuclear phagocytes including monocytes and macrophages, are important defense components of innate immunity, but can be detrimental in HIV-1 infection by serving as the principal reservoirs of virus in brain and triggering a strong immune response. These viral reservoirs represent a challenge to HIV-1 eradication since they continue producing virus in tissue despite antiretroviral therapy. HIV-1 associated neurocognitive disorders (HAND) involve alterations to the blood-brain barrier and migration of activated HIV-1 infected monocytes to the brain with subsequent induced immune activation response. Our group recently showed that HIV replication in monocyte-derived macrophages is associated with increased cystatin B. This cysteine protease inhibitor also inhibits the interferon-induced antiviral response by decreasing levels of tyrosine phosphorylated STAT-1. These recent discoveries reveal novel mechanisms of HIV persistence that could be targeted by new therapeutic approaches to eliminate HIV in macrophage reservoirs. However, cystatin B has been also associated with neuroprotection. Cystatin B is an inhibitor of the cysteine protease cathepsin B, a potent neurotoxin. During HIV-1 infection cystatin B and cathepsin B are upregulated in macrophages. Reduction in cystatin/cathepsin interactions in infected macrophages leads to increased cathepsin B secretion and activity which contributes to neuronal apoptosis. Increased intracellular expression of both proteins was recently found in monocytes from Hispanic women with HAND. These findings provide new evidence for the role of cathepsin /cystatin system in the neuropathogenesis induced by HIV-infected macrophages. We summarize recent research on cystatin B and one of its substrates, cathepsin B, in HIV replication in macrophages and neuropathogenesis.

Inhibition of interferon response by cystatin B: implication in HIV replication of macrophage reservoirs.

Cystatin B and signal transducer and activator of transcription-1 (STAT-1) phosphorylation have recently been shown to increase human immunodeficiency virus-1 (HIV-1) replication in monocyte-derived macrophages (MDM), but the molecular pathways by which they do are unknown. We hypothesized that cystatin B inhibits the interferon (IFN) response and regulates STAT-1 phosphorylation by interacting with additional proteins. To test if cystatin B inhibits the IFN-ß response, we performed luciferase reporter gene assays in Vero cells, which are IFN deficient. Interferon-stimulated response element (ISRE)-driven expression of firefly luciferase was significantly inhibited in Vero cells transfected with a cystatin B expression vector compared to cells transfected with an empty vector. To determine whether cystatin B interacts with other key players regulating STAT-1 phosphorylation and HIV-1 replication, cystatin B was immunoprecipitated from HIV-1-infected MDM. The protein complex was analyzed by liquid chromatography tandem mass spectrometry. Protein interactions with cystatin B were verified by Western blots and immunofluorescence with confocal imaging. Our findings confirmed that cystatin B interacts with pyruvate kinase M2 isoform, a protein previously associated cocaine enhancement of HIV-1 replication, and major vault protein (MVP), an IFN-responsive protein that interferes with JAK/STAT signals. Western blot studies confirmed the interaction with pyruvate kinase M2 isoform and MVP. Immunofluorescence studies of HIV-1-infected MDM showed that upregulated MVP colocalized with STAT-1. To our knowledge, the current study is the first to demonstrate the coexpression of cystatin B, STAT-1, MVP, and pyruvate kinase M2 isoform with HIV-1 replication in MDM and thus suggests novel targets for HIV-1 restriction in macrophages, the principal reservoirs for HIV-1 in the central nervous system.

Proteomic analysis of HIV-infected macrophages.

Mononuclear phagocytes (monocytes, macrophages, and microglia) play an important role in innate immunity against pathogens including HIV. These cells are also important viral reservoirs in the central nervous system and secrete inflammatory mediators and toxins that affect the tissue environment and function of surrounding cells. In the era of antiretroviral therapy, there are fewer of these inflammatory mediators. Proteomic approaches including surface enhancement laser desorption ionization, one- and two-dimensional difference in gel electrophoresis, and liquid chromatography tandem mass spectrometry have been used to uncover the proteins produced by in vitro HIV-infected monocytes, macrophages, and microglia. These approaches have advanced the understanding of novel mechanisms for HIV replication and neuronal damage. They have also been used in tissue macrophages that restrict HIV replication to understand the mechanisms of restriction for future therapies. In this review, we summarize the proteomic studies on HIV-infected mononuclear phagocytes and discuss other recent proteomic approaches that are starting to be applied to this field. As proteomic instruments and methods evolve to become more sensitive and quantitative, future studies are likely to identify more proteins that can be targeted for diagnosis or therapy and to uncover novel disease mechanisms.