Heroin, Temperature, HIV-1 Infection And Innate Immunity

Part I Exosomal miRNA and HIV-1 InfectionHuman immunodeficiency virus (HIV-1) infection is the cause of acquired immunodeficiency syndrome (AIDS). HIV-1 infection impairs host immune system, resulting in the depletion of CD4+T cells. Patients with AIDS often have opportunistic infections caused by other viruses, bacteria and fungi, which contributes to the increased risk for cancer, neurological disorders and other conditions, even facing life-threatening. Injecting use of heroin is known to have a major role in HIV-1 transmission, as a result of collective use of needle. Many of heroin users are infected with HIV-1. Exosomes are small (30-100 nm) membrane vesicles which are produced by most of the cell types, making them abundant in a variety of body fluids, including plasma. Exosomes are important in cell-to-cell communication. MicroRNAs (miRNAs) are a class of small (-22 nucleotides) noncoding RNA that were found in plants and animals and regulate gene expression at the posttranscriptional level. MiRNAs are reported to be related to host cell innate immunity. MiRNAs and exosomes both play a role in human immunodeficiency virus disease. It is recently revealed that exosomes contain functional microRNAs (miRNAs) which can be delivered into recipient cells, exerting biological functions. However, the relationship between exosomal miRNAs and HIV-1 infection remains unclear.Objective:In the present study, we investigated the impact of HIV-1 infection and/or heroin use on the expression of miRNAs derived from plasma exosomes. We examined plasma exosomal levels of miRNAs in healthy donors (N=32), heroin users with (N=19) or without (N=25) HIV-1 infection, and individuals with HIV-1 infection but without heroin use (N=37).Methods:Experiment was divided into two parts. The first part is to investigate the in vivo exosomal miRNAs expression in four groups by real time PCR and array. The second part is to examine the TNF-a expression of macrophages on exosomes from HIV-1 infected cells by immunofluorescence, real time PCR and ELISA in vitro.Results:We found that six miRNAs (16, let-7a,146a,29c,21,126) were dramatically increased in heroin users with HIV-1 infection. However, the long-term (>5 years) methadone treatment of heroin users restore the levels of these miRNAs. We have also observed an increase of plasma miRNAs (150,146a,29b, let-7a) in subjects with advanced disease (CD4 cell count<350 cells/mm3 or viral load above detection level). In addition, miRNA150 and miRNA29b were reduced in no therapy to ART individuals while let-7a was enhanced. Furthermore, we analyzed expression of 84 plasma exosomal miRNAs by array and 56 miRNA expressions were changed by HIV-1 infection and/or heroin use. Finally, we detect the immune response of macrophages on exosomes from HIV-1 infected cells and find that exosomes from HIV-1 infected cells (J1.1) promote TNF-? expression both in mRNA and protein levels. Exosomal miRNA 16 related to inflammation was increased in J1.1 exosome and could be delivered into macrophage.Conclusions:These data provide evidence that HIV-1 infection and/or heroin use dysregulate the plasma exosomal miRNAs expression, which might contribute to HIV-1-mediated dysregulation of host immune system and viral infection/persistence in the host cells.Part II Effect of Temperature on Innate ImmunitySimian immunodeficiency virus (SIV) infection of rhesus macaques has been widely used as a well-established nonhuman primate (NHP) model for HIV-1/AIDS research. The physiological body temperature of rhesus macaques is higher (about 38.5?) than that of humans (37?). At present, there are no report about the impact of rhesus macaques normal body temperature (38.5?) on cell cultures and SIV infections.Objective:To investigated the impact of in vitro culture temperatures (37? vs 38.5?) on SIV infection of peripheral blood mononuclear cells (PBMCs) and macrophages of rhesus macaques and optimize the in vitro cell culture model of rhesus macaques.Methods:The impact of temperature on rhesus macaque cell (PBMCs and macrophages) viability was first evaluated by MTT assay. Then, higher levels of SIV or SHIV infection were observed in PBMCs and macrophages cultured at 38.5? compared to those infected at 37? by real time PCR. Next, the mechanisms which promote viral replication were studied by real time PCR and flow cytometry.Results:We observed that 38.5? had little effect on cell viability as compared to 37?. CD4 and CCR5 are important to virus entry. Little difference in CD4 and CCR5 expression on PBMCs was observed between 37? and 38.5? culture conditions. However, higher level of CD4 expression was observed on macrophages. CD4+T cells activation and monocyte differentiation have been implicated in HIV-l disease progression. Further investigations demonstrated that cells cultured at 38.5? expressed higher levels of activation marker (CD69) on CD4+T cells while another activation marker (CD25) and monocyte differentiation marker (CD 16) were not changed. In addition, the PBMCs cultured at 38.5? showed reduced expression of intracellular IFN regulatory factor3 (IRF3) and IFN-stimulated gene 54 (ISG54), which are significant to antiviral state. MiRNAs play important role in host and virus interaction. Four SIV restriction miRNAs (29a,29b,9,146a) have been found decreased in macrophages at 38.5?.Conclusions:These findings suggest that physiological body temperature (38.5?) enhanced SIV replication in PBMCs possibly through increasing CD4 T cell activation and suppressing IRF3 and ISG54, while promoting SIV replication in macrophage by means of elevating entry receptor and inhibiting anti-SIV miRNAs expression which should be taken into the consideration for in vitro studies of primary immune cells from Chinese rhesus macaques.