Background: The incidence of cardiovascular disease (CVD) is higher in HIV + patients than it is in the average population, and combination antiretroviral therapy (cART) is a recognized risk factor for CVD. However, the molecular mechanisms that link cART and CVD are currently unknown. Our study explores the role of the activation of p90RSK, a reactive oxygen species (ROS)-sensitive kinase, in engendering senescent phenotype in macrophages and accelerating atherogenesis in patients undergoing cART. Methods: Peripheral whole blood from cART-treated HIV + individuals and non-treated HIV − individuals was treated with H 2 O 2 (200 μM) for 4 minutes, and p90RSK activity in CD14 + monocytes was measured. Plaque formation in the carotids were also analyzed in these individuals. Macrophage senescence was determined by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. The involvement of p90RSK-NRF2 signaling in cART-induced senescence was assessed by p90RSK specific inhibitor (FMK-MEA) or dominant negative p90RSK (DN-p90RSK), and NRF2 activator (NRF2A). Further, the severity of atherosclerosis was determined in myeloid cell-specific wild type and DN-p90RSK transgenic mice. Results: Monocytes from HIV + patients exhibited higher levels of p90RSK activity and were also more sensitive to ROS than monocytes from HIV − individuals. A multiple linear regression analysis involving cART, Reynolds CV risk score, and basal p90RSK activity revealed that cART and basal p90RSK activity were the two significant determinants of plaque formation. Many of the antiretroviral drugs individually activated p90RSK, which simultaneously triggered all components of the macrophage senescent phenotype. cART inhibited antioxidant response element reporter activity via ERK5 S496 phosphorylation. NRF2A reversed the H 2 O 2-induced over-activation of p90RSK in cART-treated macrophages by countering the induction of senescent phenotype. Lastly, the data obtained from our gain-or loss-of-function mice conclusively showed the crucial role of p90RSK in inducing senescent phenotype in macrophages and atherogenesis. Conclusions: cART increased monocyte/macrophage sensitivity to ROS in HIV + individuals by suppressing NRF2-ARE activity via p90RSK-mediated ERK5 S496 phosphorylation, which coordinately elicited senescent phenotypes and pro-inflammatory responses. As such, our report underscores the importance of p90RSK regulation in monocytes/macrophages as a viable biomarker and therapeutic target for preventing CVD, especially in HIV + patients treated with cART.
People living with HIV are at higher risk of atherosclerosis (AS). The pathogenesis of this risk is not fully understood. To assess the regulatory networks involved in AS we sequenced mRNA of the peripheral blood mononuclear cells (PBMCs) and measured cytokine and chemokine levels in the plasma of 13 persons living with HIV and 12 matched HIV-negative persons with and without AS. microRNAs (miRNAs) are known to play a role in HIV infection and may modulate gene regulation to drive AS. Hence, we further assessed miRNA expression in PBMCs of a subset of 12 HIV+ people with and without atherosclerosis. We identified 12 miRNAs differentially expressed between HIV+ AS+ and HIV+ , and validated 5 of those by RT-qPCR. While a few of these miRNAs have been implicated in HIV and atherosclerosis, others are novel. Integrating miRNA measurements with mRNA, we identified 27 target genes including SLC4A7, a critical sodium and bicarbonate transporter, that are potentially dysregulated during atherosclerosis. Additionally, we uncovered that levels of plasma cytokines were associated with transcription factor activity and miRNA expression in PBMCs. For example, BACH2 activity was associated with IL-1β, IL-15, and MIP-1α. IP10 and TNFα levels were associated with miR-124-3p. Finally, integration of all data types into a single network revealed increased importance of miRNAs in network regulation of the HIV+ group in contrast with increased importance of cytokines in the HIV+ AS+ group.
Atherosclerosis (AS)-associated cardiovascular disease is an important cause of mortality in an aging population of people living with HIV (PLWH). This elevated risk has been attributed to viral infection, anti-retroviral therapy, chronic inflammation, and lifestyle factors. However, the rates at which PLWH develop AS vary even after controlling for length of infection, treatment duration, and for lifestyle factors. To investigate the molecular signaling underlying this variation, we sequenced 9368 peripheral blood mononuclear cells (PBMCs) from eight PLWH, four of whom have atherosclerosis (AS+). Additionally, a publicly available dataset of PBMCs from persons before and after HIV infection was used to investigate the effect of acute HIV infection. To characterize dysregulation of pathways rather than just measuring enrichment, we developed the single-cell Boolean Omics Network Invariant Time Analysis (scBONITA) algorithm. scBONITA infers executable dynamic pathway models and performs a perturbation analysis to identify high impact genes. These dynamic models are used for pathway analysis and to map sequenced cells to characteristic signaling states (attractor analysis). scBONITA revealed that lipid signaling regulates cell migration into the vascular endothelium in AS+ PLWH. Pathways implicated included AGE-RAGE and PI3K-AKT signaling in CD8+ T cells, and glucagon and cAMP signaling pathways in monocytes. Attractor analysis with scBONITA facilitated the pathway-based characterization of cellular states in CD8+ T cells and monocytes. In this manner, we identify critical cell-type specific molecular mechanisms underlying HIV-associated atherosclerosis using a novel computational method.
Mesenchymal stem cells (MSCs) are rare progenitor cells present in adult bone marrow that have the capacity to differentiate into a variety of tissue types, including bone, cartilage and fat. The biological activities of MSCs suggest a number of potential clinical applications, where each particular application is related to a specific MSC activity mediated by a different mechanism. Osiris Therapeutics has developed a technology for isolation and expansion of hMSCs from adult bone marrow for clinical use. Data from pre-clinical and clinical studies suggest that the ability of MSCs to migrate to inflammatory sites, modulate immune response, down-regulate inflammation, and accelerate tissue repair in the local environment may have therapeutic effects. Therefore in developing therapeutic applications, the MSCs should be verified to display one or more of above-mentioned functions, calling for the need to develop predictive functional assays. Modulation of the immune response is an apparent in vivo therapeutic property of the MSC necessary for successful Graft versus Host Disease (GVHD) treatment. Based on previous knowledge regarding mechanisms underlying MSC-mediated immunosuppressive effects, several markers for developing an MSC potency assay have been proposed. In the present study a relationship between selected markers and hMSC-mediated immunosuppression was investigated in vitro. Results show that co-culture of hMSCs with anti-CD3/CD28-activated peripheral blood mononuclear cells (hPBMCs) caused inhibition of lymphocyte proliferation. The hMSC effect on lymphocyte proliferation is dose-dependent, causing > 50% inhibition at approximately 1:10–1:25 MSC: T-lymphocyte ratio. Supernatants of parallel co-cultures taken on days 1, 3, and 5 were analyzed for prostaglandin 2 (PGE2), tumor necrosis factor-α (TNF-α), and tryptophan. The results showed increased levels of PGE2, decreased levels of TNF-α and increased depletion of tryptophan related to indoleamine 2,3-dioxygenase (IDO) enzyme activity, associated with increasing number of MSCs in each well. The quantity of PGE2 on day 1 and the level of tryptophan on day 5 in the MSC-PBMC co-culture supernatants correlated to the level of inhibition of proliferation, with the PGE2 range from approximately 11,000 to 22,000 pg/mL and 50% tryptophan depletion resulting in a 50% inhibition of the lymphocyte proliferation point. Further studies demonstrated that the addition of TNF-α to MSCs induced PGE2 secretion at a level which was similar to that detected in the co-culture studies of MSCs-PBMC. Thus, a strong correlation between inducible PGE2 secretion/IDO enzyme activity and the inhibition of lymphocyte proliferation by hMSCs in vitro indicates key molecules responsible for hMSC functional activity related to the immunological responses involved with diseases such as GVHD, solid organ transplantation and autoimmune diseases.
Background: In people living with HIV (PLWH) on combination antiretroviral therapy (cART), persistent systemic inflammation is a driving force for the progression of comorbidities, such as cardiovascular and cerebrovascular diseases. In this context, monocyte- and macrophage-related inflammation rather than T cell activation is a major cause of chronic inflammation. However, the underlying mechanism of how monocytes cause persistent systemic inflammation in PLWH is elusive. Methods and Results: In vitro, we demonstrated that lipopolysaccharides (LPS) or tumor necrosis factor alpha (TNFα), induced a robust increase of Delta-like ligand 4 (Dll4) mRNA and protein expression in human monocytes and Dll4 secretion (extracellular Dll4, exDll4) from monocytes. Enhanced membrane-bound Dll4 (mDll4) expression in monocytes triggered Notch1 activation to promote pro-inflammatory factors expression. Dll4 silencing and inhibition of Nocth1 activation diminished the LPS or TNFα -induced inflammation. exDll4 releases in response to cytokines occurred in monocytes but not endothelial cells or T cells. In clinical specimens, we found that PLWH, both male and female, on cART, showed a significant increase in mDll4 expression, activation of Dll4-Notch1 signaling, and inflammatory markers in monocytes. Although there was no sex effect on mDII4 in PLWH, plasma exDll4 was significantly elevated in males but not females compared to HIV uninfected individuals. Furthermore, exDll4 plasma levels paralleled with monocytes mDll4 in male PLWH. Circulating exDll4 was also positively associated with pro-inflammatory monocytes phenotype and negatively associated with classic monocytes phenotype in male PLWH. Conclusion: Pro-inflammatory stimuli increase Dll4 expression and Dll4-Notch1 signaling activation in monocytes and enhance monocyte proinflammatory phenotype, contributing to persistent systemic inflammation in male and female PLWH. Therefore, monocyte mDll4 could be a potential biomarker and therapeutic target of systemic inflammation. Plasma exDll4 may also play an additional role in systemic inflammation but primarily in men.
Increased cardiovascular (CV) events in HIV patients and cancer survivors and the fact that combination antiretroviral therapy (cART) and chemotherapy induce similar unwanted CV effects are becoming evident, but the reason for this is unclear. In this study, we examined the role of p90RSK in the CV toxicity of cART and anti-cancer drugs. The level of p90RSK activation by H2O2 was higher in peripheral monocytes from cART-treated patients than in those from untreated patients. Multiple linear regression analysis involving HIV+/-, Reynolds CV risk score, and basal p90RSK activation revealed HIV+/- and basal p90RSK activation to be strong determinants for plaque formation when adjusted for other independent variables. In murine macrophages most of the cART and several chemotherapy agents activated p90RSK and reduced antioxidant molecule expression and telomere (TL) lengths. p90RSK-mediated ERK5 S496 phosphorylation inhibited transcriptional activity of ERK5 and NRF2, decreasing efferocytosis, antioxidant production and TL lengths. NRF2 activator normalized cART-induced sensitization of p90RSK to H2O2. Lastly, we generated myeloid cells-specific wild type (WT) and dominant negative (DN) p90RSK transgenic mice, and ERK5 S496A knock-in mice, crossed with Ldlr-/- mice or treated with a single injection of adeno-associated virus vector (AAV) encoding a gain-of-function mutant of PCSK9 and fed a high fat diet. Our animal studies showed the crucial role of p90RSK-mediated ERK5 S496 phosphorylation in suppressing effecrocytosis and antioxidant production, and up-regulating senescence and inflammation-related molecules expression, leading to atherogenesis. Our results taken together show that p90RSK is activated by anti-HIV and anti-cancer drugs, and this activation sensitizes the monocyte/macrophages to the secondary oxidative insult by reducing NRF2 transcriptional activity and TL length. The p90RSK activation also reduces macrophage efferocytosis and antioxidant capacity, and increases inflammation and senescence via up-regulating ERK5 S496 phosphorylation, thereby accelerating atherosclerosis. Monocyte/macrophage p90RSK-ERK5 S496 phosphorylation could be a good target to prevent drugs-induced CV diseases.
Background: Atherosclerosis (AS)-associated cardiovascular disease is an important cause of mortality in an aging population of people living with HIV (PLWH). This elevated risk of atherosclerosis has been attributed to viral infection, prolonged usage of anti-retroviral therapy, and subsequent chronic inflammation. Methods: To investigate dysregulated immune signaling in PLWH with and without AS, we sequenced 9368 peripheral blood mononuclear cells (PBMCs) from 8 PLWH, 4 of whom also had atherosclerosis (AS+). To develop executable models of signaling pathways that drive cellular states in HIV-associated atherosclerosis, we developed the single-cell Boolean Omics Network Invariant Time Analysis (scBONITA) algorithm. ScBONITA (a) uses single-cell RNA sequencing data to infer Boolean rules for topologically characterized networks, (b) prioritizes genes based on their impact on signaling, (c) performs pathway analysis, and (d) maps sequenced cells to characteristic signaling states. We used scBONITA to identify dysregulated pathways in different cell-types from AS+ PLWH and AS- PLWH. To compare our findings with pathways associated with HIV infection, we used scBONITA to analyze a publicly available dataset of PBMCs from subjects before and after HIV infection. Additionally, the executable Boolean models characterized by scBONITA were used to analyze observed cellular states corresponding to the steady states of signaling pathways Results: We identified an increased subpopulation of CD8+ T cells and a decreased subpopulation of monocytes in AS+ PLWH. Dynamic modeling of signaling pathways and pathway analysis with scBONITA provided a new perspective on the mechanisms of HIV-associated atherosclerosis. Lipid metabolism and cell migration pathways are induced by AS rather than by HIV infection. These pathways included AGE-RAGE and PI3K-AKT signaling in CD8+ T cells, and glucagon and cAMP signaling pathways in monocytes. Further analysis of other cell subpopulations suggests that the highly interconnected PI3K-AKT signaling pathway drives cell migratory state in response to dyslipidemia. scBONITA attractor analysis mapped cells to pathway-specific signaling states that correspond to distinct cellular states. Conclusions: Dynamic network modeling and pathway analysis with scBONITA indicates that dysregulated lipid signaling regulates cell migration into the vascular endothelium in AS+ PLWH. Attractor analysis with scBONITA facilitated pathway-based characterization of cellular states that are not apparent in gene expression analyses.
Despite antiretroviral treatment (cART), people living with HIV (PLWH) are more susceptible to neurocognitive impairment (NCI), probably due to synergistic/additive contribution of traditional cerebrovascular risk factors. Specifically, altered blood brain barrier (BBB) and transmigration of inflammatory monocytes are risk factors for developing cerebral small vessel disease (CSVD). In order to investigate if inflammatory monocytes exacerbate CSVD and cognitive impairment, 110 PLWH on cART and 110 age-, sex- and Reynolds cardiovascular risk score-matched uninfected individuals were enrolled. Neuropsychological testing, brain magnetic resonance imaging and whole blood analyses to measure platelet-monocyte interaction and monocyte, endothelial activation were performed. Results demonstrated that PLWH exhibited increased levels of platelet-monocyte complexes (PMCs) and higher expression of activation molecules on PMCs. PLWH with CSVD had the poorest cognitive performance and the highest circulating levels of non-classical monocytes which exhibited significant inverse correlation with each other. Furthermore, markers of monocyte and endothelium activation were significantly positively correlated indicating BBB impairment. Our results confirm that interaction with platelets activates and drives monocytes towards an inflammatory phenotype in PLWH. In particular, elevated levels of non-classical monocytes may represent a common pathway to neuroinflammation, CSVD and subsequent cognitive impairment, warranting further longitudinal studies to evaluate responsiveness of this potential biomarker.
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