Suppression of viral rebound by a Rev-dependent lentiviral particle in SIV-infected rhesus macaques.

Persistence of human immunodeficiency virus (HIV) reservoirs prevents viral eradication, and consequently HIV-infected patients require lifetime treatment with antiretroviral therapy (ART) [1-5]. Currently, there are no effective therapeutics to prevent HIV rebound upon ART cessation. Here we describe an HIV/SIV Rev-dependent lentiviral particle that can be administered to inhibit viral rebound [6-9]. Using simian immunodeficiency virus (SIV)-infected rhesus macaques as a model, we demonstrate that the administration of pre-assembled SIV Rev-dependent lentiviral particles into SIVmac239-infected Indian rhesus macaques can lead to reduction of viral rebound upon ART termination. One of the injected animals, KC50, controlled plasma and CNS viremia to an undetectable level most of the time for over two years after ART termination. Surprisingly, detailed molecular and immunological characterization revealed that viremia control was concomitant with the induction of neutralizing antibodies (nAbs) following the administration of the Rev-dependent vectors. This study emphasizes the importance of neutralizing antibodies (nAbs) for viremia control [10-15], and also provides proof of concept that the Rev-dependent vector can be used to target viral reservoirs, including the CNS reservoirs, in vivo. However, future large-scale in vivo studies are needed to understand the potential mechanisms of viremia control induced by the Rev-dependent vector.

Inhibitory receptor CD47 binding to plasma TSP1 suppresses NK-cell IFN-γ production via activating the JAK/STAT3 pathway during HIV infection.

BACKGROUND: Natural killer (NK) cells play an important first-line role against tumour and viral infections and are regulated by inhibitory receptor expression. Among these inhibitory receptors, the expression, function, and mechanism of cluster of differentiation 47 (CD47) on NK cells during human immunodeficiency virus (HIV) infection remain unclear. METHODS: Fresh peripheral blood mononuclear cells (PBMCs) were collected from people living with HIV (PLWH) and HIV negative controls (NC) subjects. Soluble ligand expression levels of CD47 were measured using ELISA. HIV viral proteins or Toll-like receptor 7/8 (TLR7/8) agonist was used to investigate the mechanisms underlying the upregulation of CD47 expression. The effect of CD47 on NK cell activation, proliferation, and function were evaluated by flow cytometry. RNA-seq was used to identify downstream pathways for CD47 and its ligand interactions. A small molecule inhibitor was used to restore the inhibition of NK cell function by CD47 signalling. RESULTS: CD47 expression was highly upregulated on the NK cells from PLWH, which could be due to activation of the Toll-like receptor 7/8 (TLR7/8) pathway. Compared with NC subjects, PLWH subjects exhibited elevated levels of CD47 ligands, thrombospondin-1 (TSP1), and counter ligand signal regulatory protein-α (SIRPα). The TSP1-CD47 axis drives the suppression of interferon gamma (IFN-γ) production and the activation of the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway in NK cells. After treatment with a STAT3 inhibitor, the NK cells from PLWH showed significantly improved IFN-γ production. CONCLUSIONS: The current data indicate that the binding of the inhibitory receptor CD47 to plasma TSP1 suppresses NK cell IFN-γ production by activating the JAK/STAT3 pathway during HIV infection. Our results suggest that CD47 and its related signalling pathways could be targets for improving NK cell function in people living with HIV.

Alterations in levels of cytokine following treatment to predict outcome of sepsis: A meta-analysis.

BACKGROUND: The mortality rate of patients with sepsis has been increasing in recent years. Alterations of biomarkers levels during treatment are important in evaluating treatment efficacy and predicting outcomes in sepsis. This meta-analysis investigated the relationship between changes in cytokine levels after treatment compared with those on hospital admission, and their relationship with the prognosis of patients with sepsis. METHODS: From conception until August 4, 2021, a complete literature search of the PubMed, Web of Science, and Cochrane Library electronic databases was done. Observational studies where the outcomes of sepsis patients were divided into non-survivors and survivors and which reported cytokine levels at least before treatment in ICU were included in the current study. Standardized mean difference (SMD) with 95% confidence intervals (CI) values from individual studies were pooled using a random-effects model. Quality assessment, subgroup analysis, publication bias, and sensitivity analyses were all carried out. RESULTS: A total of 2570 patients with sepsis from 25 eligible studies were included, and 14 of them measured the cytokine levels before and after treatment in ICU. Among IL-6, TNF-α, IL-1ß and IL-10 levels, those of IL-6 were significantly lower after treatment in ICU than at baseline in patients with sepsis in the survival group (SMD = -0.69, P < 0.0001), but were comparable in the non-survival group (SMD = -0.99, P = 0.0575). Similarly, post-treatment TNF-α levels were significantly lower than those at baseline only in patients with sepsis in the survival group (SMD = -0.44, P < 0.0001), but not in the non-survival group (SMD =-0.17, P = 0.0842). CONCLUSION: This meta-analysis shows that reduced IL-6 and TNF-α levels after sepsis treatment in ICU may be indicators of better prognosis and survival of patients with sepsis.

Mechanical force induces mitophagy-mediated anaerobic oxidation in periodontal ligament stem cells.

BACKGROUND: The preference for glucose oxidative mode has crucial impacts on various physiological activities, including determining stem cell fate. External mechanical factors can play a decisive role in regulating critical metabolic enzymes and pathways of stem cells. Periodontal ligament stem cells (PDLSCs) are momentous effector cells that transform mechanical force into biological signals during the reconstruction of alveolar bone. However, mechanical stimuli-induced alteration of oxidative characteristics in PDLSCs and the underlying mechanisms have not been fully elucidated. METHODS: Herein, we examined the expression of LDH and COX4 by qRT-PCR, western blot, immunohistochemistry and immunofluorescence. We detected metabolites of lactic acid and reactive oxygen species for functional tests. We used tetramethylrhodamine methyl ester (TMRM) staining and a transmission electron microscope to clarify the mitochondrial status. After using western blot and immunofluorescence to clarify the change of DRP1, we further examined MFF, PINK1, and PARKIN by western blot. We used cyclosporin A (CsA) to confirm the regulation of mitophagy and ceased the stretching as a rescue experiment. RESULTS: Herein, we ascertained that mechanical force could increase the level of LDH and decrease the expression of COX4 in PDLSCs. Simultaneously, the yield of reactive oxygen species (ROS) in PDLSC reduced after stretching, while lactate acid augmented significantly. Furthermore, mitochondrial function in PDLSCs was negatively affected by impaired mitochondrial membrane potential (MMP) under mechanical force, and the augment of mitochondrial fission further induced PRKN-dependent mitophagy, which was confirmed by the rescue experiments via blocking mitophagy. As a reversible physiological stimulation, the anaerobic preference of PDLSCs altered by mechanical force could restore after the cessation of force stimulation. CONCLUSIONS: Altogether, our study demonstrates that PDLSCs under mechanical force preferred anaerobic oxidation induced by the affected mitochondrial dynamics, especially mitophagy. Our findings support an association between mechanical stimulation and the oxidative profile of stem cells, which may shed light on the mechanical guidance of stem cell maintenance and commitment, and lay a molecular foundation for periodontal tissue regeneration.

PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is expressed primarily on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits HIV-1 replication, the mechanism of PSGL-1-mediated anti-HIV activity remains to be elucidated. Here we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein or vesicular stomatitis virus G glycoprotein or even lacking a viral glycoprotein is impaired by PSGL-1. Mapping studies show that the extracellular N-terminal domain of PSGL-1 is necessary for its anti-HIV-1 activity, and that the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1-related monomeric E-selectin-binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, down-regulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1-mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses, such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a unique mechanism of action.

Rapid HIV Progression Is Associated with Extensive Ongoing Somatic Hypermutation.

The Ab response to HIV is of great interest, particularly in the context of a protective vaccine and broadly neutralizing Abs, but research is typically geared toward elite controllers because of their ability to successfully control the virus. In this study, we studied the evolution of the Ab repertoire over the first year of HIV infection in people classified as rapid progressors (RP) compared with typical progressors. HIV RPs are an important yet understudied group of HIV patients classified by a rapid decline in CD4 counts and accelerated development of AIDS. We found that the global IgG somatic hypermutation load negatively correlated with disease progression, possibly because of exaggerated isotype switching of unmutated sequences in patients with low CD4 counts. We measured Ab sequence evolution over time using longitudinal samples taken during the early stages of infection and 1 year postinfection. Within clonal lineages spanning both timepoints, visit 2-derived sequences harbored considerably more mutations than their visit 1 relatives. Despite extensive ongoing somatic hypermutation, the initially strong signs of Ag selection pressure observed in visit 1-derived sequences decayed by visit 2. These data suggest that excessive immune activation in RPs leads to a hyperactive B cell response that fails to confer protection.

Chemokines and chemokine receptors in allergic rhinitis: from mediators to potential therapeutic targets.

Allergic rhinitis (AR) is an immune-mediated inflammatory condition characterized by immune cell infiltration of the nasal mucosa, with symptoms of rhinorrhea, sneezing, nasal obstruction, and itchiness. Currently, common medication for AR is anti-inflammatory treatment including intranasal steroids, oral, or intranasal anti-histamines, and immunotherapy. These strategies are effective to the majority of patients with AR, but some patients under medication cannot achieve symptom relieve and suffer from bothersome side effects, indicating a demand for novel anti-inflammatory treatment as alternatives. Chemokines, a complex superfamily of small, secreted proteins, were initially recognized for their chemotactic effects on various immune cells. Chemokines constitute both physiological and inflammatory cell positioning systems and mediate cell localization to certain sites via interaction with their receptors, which are expressed on responding cells. Chemokines and their receptors participate in the sensitization, early phase response, and late phase response of AR by promoting inflammatory cell recruitment, differentiation, and allergic mediator release. In this review, we first systemically summarize chemokines and chemokine receptors that are important in AR pathophysiology and then discuss potential strategies targeting chemokines and their receptors for AR therapy.

Online flipped classroom with team-based learning promoted learning activity in a clinical laboratory immunology class: response to the COVID-19 pandemic.

BACKGROUND: Given the rapid development of clinical immunology technologies, students majoring in laboratory medicine should master the technological principles and application of clinical laboratory immunology. However, many are required to take online courses due to COVID-19 restrictions, which highlights the need to revisit teaching strategies. Recently, various medical education courses (such as Biochemistry, Physiology, etc.) have implemented the flipped classroom (FC) and team-based learning (TBL) methods, resulting in more positive teaching evaluations. To promote the students' mastery of the difficult knowledge effectively during the online teaching work, we evaluated the performance of online FC-TBL in a clinical laboratory immunology course. METHODS: Sixty-two third-year students from two classes majoring in Laboratory Medicine were recruited and divided into two groups, including one group with traditional lecture-based learning teaching strategy (LBL group) and the other group with LBL or online FC combined with TBL teaching strategy (FC-TBL group). We selected three chapters to conduct FC-TBL teaching in class. All participants took in-class quizzes and final examinations that targeted the same knowledge points. Finally, all participants completed anonymous questionnaires asking for their perceptions of the respective teaching models. In addition, we conducted a survey of teaching suggestions by a FC-TBL class of students majoring in Laboratory Medicine. RESULTS: The FC-TBL group (vs LBL group) had significantly higher scores on the in-class quizzes and final examinations, and also reported high satisfaction with the FC-TBL model. These findings indicate that FC-TBL is suitable for clinical laboratory immunology, as the participants quickly gained essential knowledge. Specifically, FC-TBL helped to "increase learning motivation," "promote self-directed learning skills," "extend more related knowledge," "enhance problem-solving abilities," "enhance clinical reasoning abilities," and "enhance communication skills." For participants' suggestions, 48.38% (15/31) students held positive attitude to FC-TBL teaching strategy compared to 25.81% (8/31) students who considered FC-TBL teaching strategy still needs continuous improvement, and 25.81% (8/31) students reported that they believed FC-TBL teaching strategy was perfect and no further suggestions. CONCLUSIONS: Online FC-TBL effectively enhanced learning activity among students of a clinical laboratory immunology course. This is particularly useful in the COVID-19 context.

The inhibitory receptor Tim-3 fails to suppress IFN-γ production via the NFAT pathway in NK-cell, unlike that in CD4+ T cells.

BACKGROUND: T cell immunoglobulin and mucin domain-containing-3 (Tim-3) is a negative regulator expressed on T cells, and is also expressed on natural killer (NK) cells. The function of Tim-3 chiefly restricts IFNγ-production in T cells, however, the impact of Tim-3 on NK cell function has not been clearly elucidated. RESULTS: In this study, we demonstrated down-regulation of Tim-3 expression on NK cells while Tim-3 is upregulated on CD4+ T cells during HIV infection. Functional assays indicated that Tim-3 mediates suppression of CD107a degranulation in NK cells and CD4+ T cells, while it fails to inhibit the production of IFN-γ by NK cells. Analyses of downstream pathways using an antibody to block Tim-3 function demonstrated that Tim-3 can inhibit ERK and NFκB p65 signaling; however, it failed to suppress the NFAT pathway. Further, we found that the NFAT activity in NK cells was much higher than that in CD4+ T cells, indicating that NFAT pathway is important for promotion of IFN-γ production by NK cells. CONCLUSIONS: Thus, our data show that the expression of Tim-3 on NK cells is insufficient to inhibit IFN-γ production. Collectively, our findings demonstrate a potential mechanism of Tim-3 regulation of NK cells and a target for HIV infection immunotherapy.

The accumulation of plasma acylcarnitines are associated with poor immune recovery in HIV-infected individuals.

BACKGROUND: Antiretroviral therapy (ART) can reduce opportunistic infections and mortality rates among individuals infected with human immunodeficiency virus (HIV); however, some HIV-infected individuals exhibit poor immune recovery after ART. Hence, we explored the association between metabolome profiles and immune recovery in HIV-infected individuals following ART. METHODS: An untargeted metabolomics approach was used to analyze plasma samples from 18 HIV-negative individuals and 20 HIV-infected individuals, including 10 immunological non-responders (INR, CD4+ T cell rise < 100 cells/µl) and 10 immunological responders (IR, CD4+ T cell rise > 300 cells/µl) after 2 years of ART. These individuals were followed for the next 6 years and viral loads and CD4+ T cell count were measured regularly. Orthogonal projection on latent structures discriminant analysis (OPLS-DA), ANOVA, correlation, receiver operating characteristic (ROC), and survival analyses were used for selection of discriminant metabolites. RESULTS: Eighteen lipid metabolites were identified which could distinguish among control, INR, and IR groups. Among them, myristoylcarnitine (MC), palmitoylcarnitine (PC), stearoylcarnitine (SC), and oleoylcarnitine (OC) were significantly elevated in INR plasma samples compared with those from the IR and control groups and were negatively associated with CD4+ T cell count. Additionally, ROC analysis using a combination of MC, PC, SC, and OC had high sensitivity and specificity for differentiating INR from IR (AUC = 0.94). Finally, survival analysis for the combination of MC, PC, SC, and OC demonstrated that it could predict CD4+ T cell count in patients undergoing long-term ART. CONCLUSIONS: High levels of lipid metabolites, MC, PC, SC, and OC are associated with poor immune recovery in patients receiving ART and these data provide potential new insights into immune recovery mechanisms.

miR-325-3p Protects Neurons from Oxygen-Glucose Deprivation and Reoxygenation Injury via Inhibition of RIP3.

OBJECTIVE: Recent reports have corroborated that micro-RNAs (miRs) are related to the pathological changes of cerebral ischemia-reperfusion (CIR) induced injury. This work aimed to unearth the role and potential mechanism of miR-325-3p in regulating neuronal survival in CIR injury. METHODS: To conduct this investigation, we established an in vitro model of CIR injury by subjecting neurons to oxygen-glucose deprivation and reoxygenation (OGD/R). Gain and loss of function of miR-325-3p and receptor-interacting serine-threonine kinase 3 (RIP3) in neurons were performed to observe its effect on cell apoptosis and the release of lactate dehydrogenase. The levels of miR-325-3p and RIP3 in neurons were detected by qRT-PCR. Western blot was employed to inspect the levels of caspase3, Bax, and Bcl-2, as well as p38 and JNK phosphorylation. The relationship between miR-325-3p and RIP3 was detected by TargetScan and validated by dual-luciferase reporter assay. RESULTS: Firstly, miR-325-3p expression was obviously downregulated while RIP3 expression was upregulated in neurons following OGD/R treatment. Overexpressed miR-325-3p or downexpressed RIP3 ameliorated OGD/R-induced neuronal injury. Besides, RIP3 was a direct target mRNA of miR-325-3p. Additionally, Western blot revealed the mitogen-activated protein kinase (MAPK) pathway was involved in the regulation of miR-325-3p on OGD/R-induced neuronal injury. Furthermore, miR-325-3p was verified to hinder OGD/R-induced neuronal injury through downregulating RIP3. CONCLUSION: This study demonstrated that miR-325-3p targets RIP3 to inactivate the MAPK pathway, thereby protecting neurons against OGD/R-induced injury.

Transcriptional insights into the CD8+ T cell response in mono-HIV and HCV infection.

BACKGROUND: Disease progression in the absence of therapy varies significantly in mono-HIV and HCV infected individuals. Virus-specific CD8+ T cells play an important role in restricting lentiviral replication and determining the rate of disease progression during HIV and HCV mono- and co-infection. Thus, understanding the similarities in the characteristics of CD8+ T cells in mono-HIV and HCV infection at the transcriptomic level contributes to the development of antiviral therapy. In this study, a meta-analysis of CD8+ T cell gene expression profiles derived from mono-HIV and HCV infected individuals at different stages of disease progression, was conducted to understand the common changes experienced by CD8+ T cells. METHODS: Five microarray datasets, reporting CD8+ T cell mRNA expression of the mono-HIV and HCV infected patients, were retrieved from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were identified via integrative meta-analysis of expression data (INMEX) program. Network analysis methods were used to assess protein-protein interaction (PPI) networks, Gene Ontology (GO) terms and pathway enrichment for DEGs. MirDIP and miRDB online prediction tools were used to predict potential microRNAs (miRNAs) targeting hub genes. RESULTS: First, we identified 625 and 154 DEGs in the CD8+ T cells originating from mono-HIV and HCV chronic progressor patients, respectively, compared to healthy individuals. Among them, interferon-stimulated genes (ISGs) including ISG15, IFIT3, ILI44L, CXCL8, FPR1 and TLR2, were upregulated after mono-HIV and HCV infection. Pathway enrichment analysis of DEGs showed that the "cytokine-cytokine receptor interaction" and "NF-kappa B" signaling pathways were upregulated after mono-HIV and HCV infection. In addition, we identified 92 and 50 DEGs in the CD8+ T cells of HIV non-progressor and HCV resolver patients, respectively, compared with corresponding chronic progressors. We observed attenuated mitosis and reduced ISG expression in HIV non-progressors and HCV resolvers compared with the corresponding chronic progressors. Finally, we identified miRNA-143-3p, predicted to target both IFIT3 in HIV and STAT5A in HCV infection. CONCLUSIONS: We identified DEGs and transcriptional patterns in mono-HIV and HCV infected individuals at different stages of disease progression and identified miRNA-143-3p with potential to intervene disease progression, which provides a new strategy for developing targeted therapies.

CD4+CD38+ central memory T cells contribute to HIV persistence in HIV-infected individuals on long-term ART.

BACKGROUND: Despite the effective antiretroviral treatment (ART) of HIV-infected individuals, HIV persists in a small pool. Central memory CD4+ T cells (Tcm) make a major contribution to HIV persistence. We found that unlike HLA-DR, CD38 is highly expressed on the Tcm of HIV-infected subjects receiving ART for > 5 years. It has been reported that the half-life of total and episomal HIV DNA in the CD4+CD38+ T cell subset, exhibits lower decay rates at 12 weeks of ART. Whether CD38 contributes to HIV latency in HIV-infected individuals receiving long-term ART is yet to be addressed. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of HIV-infected subjects receiving suppressive ART. The immunophenotyping, proliferation and apoptosis of CD4+ T cell subpopulations were detected by flow cytometry, and the level of CD38 mRNA and total HIV DNA were measured using real-time PCR and digital droplet PCR, respectively. A negative binomial regression model was used to determine the correlation between CD4+CD38+ Tcm and total HIV DNA in CD4+ T cells. RESULTS: CD38 was highly expressed on CD4+ Tcm cells from HIV infected individuals on long-term ART. Comparing with HLA-DR-Tcm and CD4+HLA-DR+ T cells, CD4+CD38+ Tcm cells displayed lower levels of activation (CD25 and CD69) and higher levels of CD127 expression. The proportion of CD38+ Tcm, but not CD38- Tcm cells can predict the total HIV DNA in the CD4+ T cells and the CD38+ Tcm subset harbored higher total HIV DNA copy numbers than the CD38- Tcm subset. After transfected with CD38 si-RNA in CD4+ T cells, the proliferation of CD4+ T cells was inhibited. CONCLUSION: The current date indicates that CD4+CD38+ Tcm cells contribute to HIV persistence in HIV-infected individuals on long-term ART. Our study provides a potential target to resolve HIV persistence.

Decreased CD4+CD8low T cells in early HIV infection are associated with rapid disease progression.

BACKGROUND: HIV rapid progressors (RPs) present with a rapid decline of CD4+ T cells within a few years of infection. Determining the underlying mechanisms throughout this decline is important to identify prognostic biomarkers and intervention strategies. Determining the numbers of CD4+ and CD8+ T cells is essential for monitoring the immune status of HIV infected patients. There are additional kinds of cell subtypes in T cells, but their relationship to the rapid progression of HIV disease is not well defined. METHODS: Nineteen RPs and twenty-one chronic progressors (CPs) were enrolled in this study. Based on the intensity of CD4 and CD8 expression, different T cell subtypes were identified, including CD4+CD8+T cells, CD4-CD8- T cells, CD4+CD8low T cells and CD4-CD8low T cells. Alterations in these T cell subtypes in early HIV infection (within 120 days of infection) between RPs and CPs were measured, and the relationships between these subtypes and HIV disease progression were investigated. In addition, expression of IFN-γ in T cell subtypes after PMA stimulation was analyzed by flow cytometry. RESULTS: We found that during early HIV infection, CD4+CD8low T cells both significantly decreased in numbers and percentages in RPs compared to CPs. Furthermore, baseline CD4+CD8low T cells positively correlated not only with baseline CD4+T cells but also with CD4+T cells 12 months after infection. Moreover, survival analysis indicated that low levels of baseline CD4+CD8low T cells significantly accelerated the decline in CD4+ T cells as well as increased viral loads. CD4+CD8low T cells secreted significantly more IFN-γ after PMA stimulation compared to CD4+CD8-T cells and CD4-CD8+T cells, which may be beneficial for the prevention of disease progression. CONCLUSIONS: Our results identified that in early stage HIV-1 infection, a subtype of T cells, CD4+CD8low, are associated with subsequent disease progression.

Elevated CD54 Expression Renders CD4+ T Cells Susceptible to Natural Killer Cell-Mediated Killing.

BACKGROUND: Natural killer (NK) cells are an important type of effector cell in the innate immune response, and also have a role in regulation of the adaptive immune response. Several studies have indicated that NK cells may influence CD4+ T cells during HIV infection. METHODS: In total, 51 HIV-infected individuals and 15 healthy controls participated in this study. We performed the flow cytometry assays and real-time PCR for the phenotypic analysis and the functional assays of NK cell-mediated deletion of CD4+ T cells, phosphorylation of nuclear factor-κB (NF-κB/p65) and the intervention of metformin. RESULTS: Here we detected high CD54 expression on CD4+ T cells in HIV-infected individuals, and demonstrate that upregulated CD54 is associated with disease progression in individuals infected with HIV. We also show that CD54 expression leads to the deletion of CD4+ T cells by NK cells in vitro, and that this is modulated by NF-κB/p65 signaling. Further, we demonstrate that metformin can suppress CD54 expression on CD4+ T cells by inhibiting NF-κB/p65 phosphorylation. CONCLUSIONS: Our data suggest that further studies to evaluate the potential role of metformin as adjunctive therapy to reconstitute immune function in HIV-infected individuals are warranted.

Reduced eIF3d accelerates HIV disease progression by attenuating CD8+ T cell function.

BACKGROUND: In human immunodeficiency virus (HIV) infection, 10-15% of individuals exhibit a rapid decline in CD4+ T cells and become rapid progressors (RPs). Overall, understanding the factors affecting rapid disease progression in early HIV infection (EHI) can aid in treatment initiation. Recent studies show that eIF3s, classic scaffold proteins during the translation initiation process, can directly promote or inhibit the translation of mRNA, therefore participating in the regulation of cell function. However, to our knowledge, it has not been addressed whether eIF3s are involved in the diverse prognosis of HIV infection. METHODS: Expression of eIF3s in primary cells from early or chronic HIV-infected patients was detected by real-time PCR. To investigate the potential mechanisms of eIF3d in the regulation of CD8+ T cell function, complete transcriptomes of eIF3d-inhibited Jurkat T cells were sequenced by RNA sequencing (RNA-Seq). Additionally, to examine the effect of eIF3d on CD8+ T cell function, eIF3d expression was inhibited alone or in combination with SOCS-7 knockdown by siRNA in isolated CD8+ T cells. CD8+ T cell proliferation, IFN-r secretion and apoptosis were detected by flow cytometry. Moreover, the effect of eIF3d on HIV replication was evaluated in Jurkat cells, peripheral blood mononuclear cells (PBMCs) and CD4+ T cells with eIF3d knockdown using a pNL4-3 pseudotyped virus. RESULTS: At approximately 100 days of infection, only eIF3d was markedly decreased in RPs compared with chronic progressors (CPs). Expression of eIF3d correlated significantly with disease progression in EHI. Based on in vitro analyses, reduced eIF3d expression led to decreased proliferation and IFN-γ secretion and increased apoptosis in CD8+ T cells. Inhibited expression of eIF3d caused enhanced expression of SOCS-7, and inhibiting SOCS-7 expression by siRNA rescued the attenuated CD8+ T cell function caused by eIF3d. Finally, when eIF3d was inhibited in Jurkat cells, PBMCs and CD4+ T cells, pNL4-3-VSV-G virus replication was enhanced. CONCLUSIONS: The current data highlight the importance of eIF3d in HIV infection by inhibiting CD8+ T cell function and promoting viral replication. Our study provides potential targets for improved immune intervention.

MicroRNA-206 Downregulates Connexin43 in Cardiomyocytes to Induce Cardiac Arrhythmias in a Transgenic Mouse Model.

BACKGROUND: MicroRNAs (miRNAs) are critical modulators of various physiological and pathological processes, but their role in cardiac arrhythmias remains yet to be completely understood. Connexin43 (Cx43) is an important cardiac gap junction protein and a potential target of miR-206, and downregulation of Cx43 induces ventricular tachyarrhythmias. METHODS: We investigated the effects of miR-206 overexpression on the adult mouse heart and in cardiac arrhythmias. Luciferase activity assay was employed to validate Cx43 as a direct target of miR-206. Expression of Cx43 was measured in cardiac muscle cell line HL-1 securely expressing miR-206. An inducible miR-206 overexpression mouse model was established to evaluate the in vivo effect of miR-206 on Cx43 expression and cardiac rhythm. RESULTS: MiR-206 directly recognised 3'-untranslated region of Cx43 mRNA to inhibit its expression in HL-1 cells. Induction of miR-206 in the adult mouse heart suppressed Cx43 expression, particularly in the atria and ventricle. Importantly, miR-206 overexpression also induced abnormal heart-rate and PR interval, and shortened life-span in the experimental mice. CONCLUSIONS: In cardiomyocytes, miR-206 is a upstream regulator of Cx43, and its overexpression downregulates Cx43 to induce abnormal heart-rate and PR interval.

Alteration of CCR6+CD95+CD4+ naïve T cells in HIV-1 infected patients: Implication for clinical practice.

The profound deficiency of Th17 cells contributes to HIV disease progression. The mechanisms of their perturbation remain unclear. Recently, CCR6+CD95+CD4+ naïve T cells (CCR6+CD95+CD4+ TNA), identified as pre-committed Th17 precursors, were recognized as a subpopulation of CD4+ T cells with stem cell properties. Following phenotypical identification, we evaluated their level in patients during chronic HIV infection and following antiretroviral therapy (ART) using flow cytometry. The levels of CCR6+CD95+CD4+ TNA were decreased during chronic HIV infection and correlated with CD4+ T cell counts. Immunological responders harbored higher frequency of CCR6+CD95+CD4+ TNA, which was associated with CD4/CD8 T cell ratio. Immunological non-responders with lower frequency of CCR6+CD95+CD4+ TNA failed to exhibit a correlation between CCR6+CD95+CD4+ TNA and CCR6+CD95+CD4+ TCM, and displayed elevated ratio of CCR6+CD95+CD4+ TCM/TNA. The number of CCR6+CD95+CD4+ TNA was increased following early ART. These findings shed light on the importance of targeting pre-committed Th17 precursors that enhance immune reconstitution.

IL-10+ NK and TGF-ß+ NK cells play negative regulatory roles in HIV infection.

BACKGROUND: Natural killer (NK) cells play cytotoxic roles by targeting tumor cells or virus infected cells, they also play regulatory roles by secreting cytokines and chemokines. Transforming growth factor (TGF)-ß and interleukin (IL)-10 are important immunosuppressive cytokines potentially related to the immune dysregulation that occurs in the infection of human immunodeficiency virus (HIV). NK cells are an important source of TGF-ß and a main early producer of IL-10 in response to viral infection. Here, we evaluated the percentages of IL-10+ and TGF-ß+ NK cells in HIV-infected patients relative to healthy controls (HCs). METHODS: Study participants (n = 63) included 31 antiretroviral treatment (ART)-naïve HIV-infected patients, 17 ART-treated HIV-infected patients, and 15 HIV-negative HCs. Expression of IL-10 or TGF-ß in NK cells was examined by flow cytometry, and the influences of recombinant IL-10 (rIL-10) or recombinant TGF-ß (rTGF-ß) on NK cell function were investigated in vitro. RESULTS: Compared with HCs, ART-naïve HIV-infected patients had increased percentages of IL-10+ (2.0% vs. 0.4%, p = 0.015) and TGF-ß+ (4.5% vs. 2.1%, p = 0.022) NK cells, and ART-treated patients also had a higher percentage of IL-10+ NK cells (2.5% vs. 0.4%, p = 0.002). The percentages of IL-10+ and TGF-ß+ NK cells were positively correlated (r = 0.388; p = 0.010). The results of in vitro experiments demonstrated that rIL-10 and rTGF-ß inhibited NK cell CD107a expression (p = 0.037 and p = 0.024, respectively), IFN-γ secretion (p = 0.006, p = 0.016, respectively), and granzyme B release after stimulation (p = 0.014, p = 0.040, respectively). CONCLUSIONS: Our data suggest that the percentages of IL-10+ or TGF-ß+ NK cells are increased in HIV-infected patients, and that rIL-10 and/or rTGF-ß can inhibit NK cell functions in vitro, providing a potential therapeutic target for strategies aimed at combating HIV infection.

Transcriptomic meta-analysis identifies gene expression characteristics in various samples of HIV-infected patients with nonprogressive disease.

BACKGROUND: A small proportion of HIV-infected patients remain clinically and/or immunologically stable for years, including elite controllers (ECs) who have undetectable viremia (<50 copies/ml) and long-term nonprogressors (LTNPs) who maintain normal CD4+ T cell counts for prolonged periods (>10 years). However, the mechanism of nonprogression needs to be further resolved. In this study, a transcriptome meta-analysis was performed on nonprogressor and progressor microarray data to identify differential transcriptome pathways and potential biomarkers. METHODS: Using the INMEX (integrative meta-analysis of expression data) program, we performed the meta-analysis to identify consistently differentially expressed genes (DEGs) in nonprogressors and further performed functional interpretation (gene ontology analysis and pathway analysis) of the DEGs identified in the meta-analysis. Five microarray datasets (81 cases and 98 controls in total), including whole blood, CD4+ and CD8+ T cells, were collected for meta-analysis. RESULTS: We determined that nonprogressors have reduced expression of important interferon-stimulated genes (ISGs), CD38, lymphocyte activation gene 3 (LAG-3) in whole blood, CD4+ and CD8+ T cells. Gene ontology (GO) analysis showed a significant enrichment in DEGs that function in the type I interferon signaling pathway. Upregulated pathways, including the PI3K-Akt signaling pathway in whole blood, cytokine-cytokine receptor interaction in CD4+ T cells and the MAPK signaling pathway in CD8+ T cells, were identified in nonprogressors compared with progressors. In each metabolic functional category, the number of downregulated DEGs was more than the upregulated DEGs, and almost all genes were downregulated DEGs in the oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle in the three types of samples. CONCLUSIONS: Our transcriptomic meta-analysis provides a comprehensive evaluation of the gene expression profiles in major blood types of nonprogressors, providing new insights in the understanding of HIV pathogenesis and developing strategies to delay HIV disease progression.