Yan-Jian Wang

Cellular microRNA Expression Correlates with Susceptibility of Monocytes/Macrophages to HIV-1 Infection

Although both monocytes and macrophages possess essential requirements for HIV-1 entry, peripheral blood monocytes are infrequently infected with HIV-1 in vivo and in vitro. In contrast, tissue macrophages and monocyte-derived macrophages in vitro are highly susceptible to infection with HIV-1 R5 tropic strains. We investigated intracellular anti-HIV-1 factors that contribute to differential susceptibility of monocytes/macrophages to HIV-1 infection. Freshly isolated monocytes from peripheral blood had significantly higher levels of the anti-HIV-1 microRNAs (miRNA, miRNA-28, miRNA-150, miRNA-223, and miRNA-382) than monocyte-derived macrophages. The suppression of these anti-HIV-1 miRNAs in monocytes facilitates HIV-1 infectivity, whereas increase of the anti-HIV-1 miRNA expression in macrophages inhibited HIV-1 replication. These findings provide compelling and direct evidence at the molecular level to support the notion that intracellular anti-HIV-1 miRNA-mediated innate immunity may have a key role in protecting monocytes/macrophages from HIV-1 infection.

Methamphetamine enhances HIV infection of macrophages.

Epidemiological studies have demonstrated that the use of methamphetamine (meth), a sympathomimetic stimulant, is particularly common among patients infected with HIV. However, there is a lack of direct evidence that meth promotes HIV infection of target cells. This study examined whether meth is able to enhance HIV infection of macrophages, the primary target site for the virus. Meth treatment resulted in a significant and dose-dependent increase of HIV reverse transcriptase activity in human blood monocyte-derived macrophages. Dopamine D1 receptor antagonists (SCH23390 and SKF83566) blocked this meth-mediated increase in the HIV infectivity of macrophages. Investigation of the underlying mechanisms of meth action showed that meth up-regulated the expression of the HIV entry co-receptor CCR5 on macrophages. Additionally, meth inhibited the expression of endogenous interferon-alpha and signal transducer and activator of transcription-1 in macrophages. These findings provide direct in vitro evidence to support the possibility that meth may function as a cofactor in the immunopathogenesis of HIV infection and may lead to the future development of innate immunity-based intervention for meth users with HIV infection.

Dopamine D2L receptor knockout mice display deficits in positive and negative reinforcing properties of morphine and in avoidance learning.

The dopamine D2 receptor (D2) is implicated in drug addiction, learning and memory. Two isoforms of the D2 receptor, termed D2L (long form) and D2S (short form), have been identified. We previously generated mice lacking D2L (D2L-/-), but expressing functional D2S. In this study, we investigated the role of D2L in the positive and negative reinforcing properties of abused drugs and electrical stimuli, using D2L-/- mice as a model system. Mice were trained in three associative learning tasks: conditioned place preference to morphine and cocaine, conditioned place aversion to naloxone-precipitated morphine withdrawal, and active avoidance. D2L-/- mice, like wild type mice, developed a place preference to cocaine. In contrast to wild type mice, D2L-/- mice did not develop a place preference to morphine, nor did they attain a place aversion to morphine withdrawal. D2L-/- mice also failed to acquire avoidance behavior in response to electrical stimuli. There were no significant differences between D2L-/- and wild type mice in mu-opioid receptor density, morphine-induced locomotor stimulation and morphine withdrawal symptoms. These results suggest that D2L may have a greater impact than D2S on the rewarding aspects of morphine, and the aversive properties of morphine withdrawal and electrical stimulus. These findings also suggest that the presence of D2L is critical in the acquisition (learning) and/or retention (memory) of context-stimulus associations in certain situations. On the other hand, D2L is not essential for the rewarding aspects of cocaine and for the development of morphine dependence. Thus, these studies reveal distinct functional roles of D2L and/or D2S in drug addiction and avoidance learning, which may lead to a better understanding of the neurobiological basis underlying these behaviors.

Hepatitis C virus inhibits intracellular interferon alpha expression in human hepatic cell lines

The chronicity of hepatitis C virus (HCV) infection raises the question of how HCV is able to persist in hepatic cells. We show that human primary hepatocytes and human hepatic cell lines (Huh7 and HepG2) spontaneously produce interferon (IFN)‐α that is inhibited in the HCV replicon cells (Huh.8 and FCA‐1). Silencing IFN‐α gene expression by IFN‐α small interfering RNA (siRNA) in the HCV replicon cells resulted in increased HCV replicon expression. The activation of IFN‐α expression by interferon regulatory factor (IRF‐7) led to the inhibition of HCV replicon expression, whereas the anti–IFN‐α receptor antibody could partially block IRF‐7–mediated HCV replicon inhibition. In addition, the blockade of IFN‐α receptor by anti–IFN‐α receptor antibody on the replicon cells increased HCV replicon expression. Among the HCV nonstructural (NS) proteins tested, NS5A is the most potent inhibitor of IFN‐α expression by the hepatic cells. Investigation of the mechanism of HCV action on IFN‐α showed that IRF‐7–induced IFN‐α promoter activation was inhibited in the HCV replicon cells. Furthermore, IRF‐7 expression was restricted in the HCV replicon cells. In conclusion, we provide direct evidence that HCV undermines the intracellular innate immunity of the target cells, which may account for HCV persistence in hepatic cells. (HEPATOLOGY 2005;42:00–00.) (HEPATOLOGY 2005;42:819–827.)

Activation of Toll-like receptor-3 induces interferon-λ expression in human neuronal cells

We examined the gene expression and regulation of type III human interferon (IFN), IFN-lambda, in human neuronal cells. Human neuronal cells expressed endogenous IFN-lambda1 but not IFN-lambda2/3. Upon the activation of Toll-like receptor (TLR)-3 expressed in the neuronal cells by polyriboinosinic polyribocytidylic acid (PolyI:C), both IFN-lambda1 and IFN-lambda2/3 expression was significantly induced. The activation of TLR-3 also exhibited antiviral activity against pseudotyped human immunodeficiency virus (HIV)-1 infection of the neuronal cells. Human neuronal cells also expressed functional IFN-lambda receptor complex, interleukin-28 receptor alpha subunit (IL-28Ralpha) and IL-10Rbeta, as evidenced by the observations that exogenous IFN-lambda treatment inhibited pseudotyped HIV-1 infection of the neuronal cells and induced the expression of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC)3G/3F, the newly identified anti-HIV-1 cellular factors. These data provide direct and compelling evidence that there is intracellular expression and regulation of IFN-lambda in human neuronal cells, which may have an important role in the innate neuronal protection against viral infections in the CNS.

Natural killer cells suppress full cycle HCV infection of human hepatocytes

Summary.  The role of natural killer (NK) cells in controlling hepatitis C virus (HCV) infection and replication has not been fully delineated. We examined NK cell‐mediated noncytolytic effect on full cycle HCV infection of human hepatocytes. Human hepatocytes (Huh7.5.1 cells) co‐cultured with NK cells or treated with supernatants (SN) from NK cells cultures had significantly lower levels of HCV RNA and protein than control cells. This NK cell‐mediated anti‐HCV activity could be largely abolished by antibody to interferon‐gamma (IFN‐γ). The investigation of the mechanisms for NK cell‐mediated anti‐HCV activity showed that NK SN‐treated hepatocytes expressed higher levels of IFN‐α/β than the control cells. NK SN also enhanced IFN regulatory factor‐3 and 7 expression in the hepatocytes. In addition, NK SN enhanced the expression of signal transducer and activator of transcription 1 and 2, the nuclear factors that are essential for the activation of IFN‐mediated antiviral pathways. These data provide direct evidence at cellular and molecular levels that NK cells have a key role in suppressing HCV infection of and replication in human hepatocytes.

Expression of the endoplasmic reticulum stress response marker, BiP, in the central nervous system of HIV‐positive individuals

The prevalence of HIV‐associated neurocognitive impairment (NCI), which includes HIV‐associated dementia (HAD) and minor cognitive and motor disorder (MCMD), has been increasing. HIV‐infected and/or activated macrophages/microglia in the brain initiate the neurodegeneration seen in HIV‐associated NCI via soluble neurotoxic mediators, including reactive oxygen species, viral proteins and excitotoxins. Neurotoxic factors released by macrophages/microglia injure neurones directly and alter astrocytic homeostatic functions, which can lead to excitotoxicity and oxidative stress‐mediated neuronal injury. Often, cells respond to oxidative stress by initiating the endoplasmic reticulum (ER) stress response. Thus, we hypothesize that ER stress response is activated in HIV‐infected cortex. We used immunofluorescence and immunoblotting to assess expression patterns of the ER stress proteins, BiP and ATF6, in HIV‐positive cortical autopsy tissue. Additionally, we performed immunofluorescence using cell type‐specific markers to examine BiP staining in different cell types, including neurones, astrocytes and macrophages/microglia. We observed a significant increase in BiP expression by both immunoblotting and immunofluorescence in HIV‐positive cortex compared with control tissue. Additionally, phenotypic analysis of immunofluorescence showed cell type‐specific increases in BiP levels in neurones and astrocytes. Further, ATF‐6β, an ER stress response initiator, is up‐regulated in the same patient group, as assessed by immunoblotting. These results suggest that ER stress response is activated in HIV‐infected cortex. Moreover, data presented here indicate for the first time that numbers of macrophages/microglia increase in brains of MCMD patients, as has been observed in HAD.

CD56+ T cells inhibit hepatitis C virus replication in human hepatocytes

CD56+ T cells are abundant in liver and play an important role in defense against viral infections. However, the role of CD56+ T cells in control of hepatitis C virus (HCV) infection remains to be determined. We investigated the noncytolytic anti‐HCV activity of primary CD56+ T cells in human hepatocytes. When HCV Japanese fulminant hepatitis‐1 (JFH‐1)–infected hepatocytes were co‐cultured with CD56+ T cells or incubated in media conditioned with CD56+ T cell culture supernatants (SN), HCV infectivity and replication were significantly inhibited. The antibodies to interferon (IFN)‐γ or IFN‐γ receptor could largely block CD56+ T cell–mediated anti‐HCV activity. Investigation of mechanism(s) responsible for CD56+ T cell–mediated noncytolytic anti‐HCV activity showed that CD56+ T SN activated the multiple elements of janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and enhanced the expression of IFN regulatory factors (IRFs) 1, 3, 7, 8, and 9, resulting in the induction of endogenous IFN‐α/β expression in hepatocytes. Moreover, CD56+ T SN treatment inhibited the expression of HCV‐supportive micro RNA (miRNA)‐122 and enhanced the levels of anti‐HCV miRNA‐196a in human hepatocytes. Conclusion: These findings provide direct in vitro evidence at cellular and molecular levels that CD56+ T cells may have an essential role in innate immune cell–mediated defense against HCV infection. (HEPATOLOGY 2009.)

Expression and regulation of antiviral protein APOBEC3G in human neuronal cells

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) has recently been identified as a potent antiviral protein. Here, we examined the expression and regulation of APOBEC3G in human brain tissues and the cells of central nervous system (CNS). Similar to the immune cells, human brain tissue and the CNS cells expressed APOBEC3G at both mRNA and protein levels. The expression of APOBEC3G could be up-regulated in human neuronal cells (NT2-N) and astrocytes (U87-MG) by interferons (IFN-alpha, beta and gamma), interleukin-1 (IL-1), and tumor necrosis factor. Other cytokines (IL-4, IL-6 and transforming growth factor beta1) and CC-chemokines (CCL3, 4 and 5), however, had little impact on the expression of APOBEC3G. In addition, pseudotyped HIV-1 infection and cytokine/chemokine-enriched supernatants from lipopolysaccharide-stimulated macrophage cultures induced APOBEC3G expression in NT2-N cells. APOBEC3G expressed in the neuronal cells and astrocytes was biologically functional, as the suppression of APOBEC3G expression by the specific siRNA led to increase of pseudotyped HIV-1 replication in these cells. These findings provide direct and compelling evidence that there is intracellular expression and regulation of functional APOBEC3G in the neuronal cells, which may be one of innate defense mechanisms involved in the neuronal protection in the CNS.

Morphine inhibits CD8+ T cell‐mediated, noncytolytic, anti‐HIV activity in latently infected immune cells

Opiates have profound effects on the function of human immune cells and are a possible cofactor in the immunopathogenesis of human immunodeficiency virus (HIV) disease. We investigated the impact of morphine on CD8+ T cell‐mediated, noncytotoxic, anti‐HIV activity in latently infected human immune cells. Morphine inhibited the noncytotoxic, anti‐HIV activity of CD8+ T cells in HIV latently infected cells (U1 and J1.1). Naltrexone abrogated the morphine‐mediated, inhibitory effect on the noncytotoxic, anti‐HIV activity of CD8+ T cells. Interferon‐γ (IFN‐γ), a potent antiviral cytokine produced by CD8+ T cells, was partially responsible for CD8+ T cell‐mediated, noncytotoxic, anti‐HIV activity. The anti‐HIV activity of IFN‐γ was also compromised by morphine treatment. Further, morphine attenuated CD8+ T cell‐mediated suppression of the HIV long‐terminal repeat promoter activation. Morphine also inhibited CD8+ T cell‐induced expression of the signal transducer and activator of transcription‐1, an important transcriptional factor in the IFN signaling pathway. These data provide additional evidence to support the notion that opioids play a role in impairing the anti‐HIV function of the immune system.