Li Song

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.

A critical function of toll-like receptor-3 in the induction of anti-human immunodeficiency virus activities in macrophages

Toll‐like receptor‐3 (TLR‐3) recognizes double‐stranded RNA and induces multiple intracellular events responsible for innate anti‐viral immunity against a number of viral infections. Activation of TLR‐3 inhibits human immunodeficiency virus (HIV) replication, but the mechanism(s) underlying the action of TLR‐3 activation on HIV are largely unknown. Here we demonstrate that treatment of monocyte‐derived macrophages with poly I:C, a synthetic ligand for TLR‐3, significantly inhibited HIV infection and replication. Investigation of the mechanisms showed that TLR‐3 activation resulted in the induction of type I interferon inducible antiviral factors, including APOBEC3G and tetherin, the newly identified anti‐HIV cellular proteins. In addition, poly I:C‐treated macrophages expressed increased levels of CC chemokines, the ligands for CCR5. Furthermore, TLR‐3 activation in macrophages induced the expression of cellular microRNAs (miRNA‐28, ‐125b, ‐150, ‐223 and ‐382), the newly identified intracellular HIV restriction factors. These findings indicate that TLR‐3‐mediated induction of multiple anti‐HIV factors should be beneficial for the treatment of HIV disease where innate immune responses are compromised by the virus.

Morphine enhances HIV infection of neonatal macrophages.

Perinatal transmission of HIV accounts for almost all new HIV infections in children. There is an increased risk of perinatal transmission of HIV with maternal illicit substance abuse. Little is known about neonatal immune system alteration and subsequent susceptibility to HIV infection after morphine exposure. We investigated the effects of morphine on HIV infection of neonatal monocyte-derived macrophages (MDM). Morphine significantly enhanced HIV infection of neonatal MDM. Morphine-induced HIV replication in neonatal MDM was completely suppressed by naltrexone, the opioid receptor antagonist. Morphine significantly up-regulated CCR5 receptor expression and inhibited the endogenous production of macrophage inflammatory protein-1β in neonatal MDM. Thus, morphine, most likely through alteration of β-chemokines and CCR5 receptor expression, enhances the susceptibility of neonatal MDM to HIV infection, and may have a cofactor role in perinatal HIV transmission and infection.

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.

Alcohol Potentiates HIV‐1 Infection of Human Blood Mononuclear Phagocytes

BACKGROUND Acute and chronic alcohol abuse impairs various functions of the immune system and thus has been implicated as a cofactor in HIV infection. The mechanisms by which alcohol affects the function of human immune cells that are the targets for HIV are unknown. METHODS Human blood monocyte-derived macrophages (MDM) were incubated with or without alcohol (10-40 mM) for 24 hr and then infected with HIV for 24 hr. Culture supernatants were harvested for HIV reverse transcription assay. HIV entry receptor (CCR5, CD4, and CXCR4) expression was determined by reverse transcription-polymerase chain reaction and flow cytometry assays. Beta-chemokines were analyzed using enzyme-linked immunosorbent assay. Different HIV strains (Bal, SF-162, 89.6, and UG024) were used for infection experiments. In addition, ADA (macrophage-tropic strain) and murine leukemia virus envelope-pseudotyped HIV infection was carried out. RESULTS Although alcohol had little effect on HIV T-lymphocyte-tropic strain infection, it significantly enhanced HIV R5 strain infection in MDM. The enhancing effect of alcohol on the HIV R5 strain was further evidenced by the observation that the R5 (ADA) strain envelope-pseudotyped HIV infection is markedly increased by alcohol, whereas murine leukemia virus envelope-pseudotyped HIV infection was not affected. Alcohol significantly up-regulated CCR5 receptor expression and inhibited the endogenous production of beta-chemokines by MDM. CONCLUSION Alcohol, through the down-regulation of beta-chemokine production and the up-regulation of CCR5 receptor expression, enhances HIV R5 strain infection of MDM and may have an important role as a cofactor in the progression of HIV disease.

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.)

Alpha-defensins inhibit HIV infection of macrophages through upregulation of CC-chemokines

The possible involvement of α-defensins in CD8 T-cell-mediated anti-HIV activities has been the subject of recent investigations [1–3]. HIV host defence mechanisms are partly mediated by CD8 T-cell non-cytotoxic antiviral responses [4]. Walker et al. [5] first demonstrated that this anti-HIV activity involves a soluble factor(s) designated as CD8 cell antiviral factor (CAF) whose identity remains unknown [4]. Zhang et al. [1] proposed that α-defensins are produced by CD8 T cells and contribute to CAF-mediated anti-HIV activities. In contrast, the recent studies by Mackewicz et al. [2] and Chang et al. [3] demonstrated that the α-defensins are not produced by CD8 T cells but unexpectedly were found to be expressed by monocytes [2]. As CAF-mediated anti-HIV activity is also observed for macrophages [6,7] and monocytes express α-defensins [2], we investigated the capacity of α-defensins to suppress HIV infection of macrophages. The addition of α-defensins to peripheral blood monocyte-derived macrophage cultures markedly suppressed HIV Bal replication (Fig. 1a) [8,9]. In order to determine the mechanism(s) responsible for α-defensin-mediated HIV inhibition in macrophages, we investigated whether α-defensins regulate the expression of CC-chemokines. CC-chemokines [macrophage inflammatory protein (MIP)-1α, MIP-1β and Rantes] inhibit infection by competing with HIV M-tropic strains for the CCR5 receptor on macrophages [10,11]. Our experiments demonstrated that α-defensins dramatically enhance expression (as much as a 25-fold increase) of MIP-1α and MIP-1β messenger RNA in macrophages (Fig. 1b) [12]. This increased CC-chemokine gene expression by α-defensins was further confirmed by the demonstration of increased production (as much as a 57-fold increase) of MIP-1α and MIP-1β proteins in α-defensin-treated macrophage cultures (Fig. 1c). In addition, the antibodies to CC-chemokines completely abrogated α-defensin-mediated HIV inhibition in macrophages (Fig. 1d). Our data, therefore, indicate that the α-defensin-mediated inhibition of HIV infection of macrophages is mediated through the upregulation of CC-chemokines. This pathway is distinct from the anti-HIV activity of CAF in macrophages, because CC-chemokines are not responsible for the ability of CAF to suppress HIV infection of these cells [6,7]. Fig. 1 Effect of α-defensins on HIV infection and β-chemokine expression in macrophages The biological interaction of defensins with chemokines and chemokine receptors has been documented. Defensins functionally overlap with chemokines in microbicidal activity [13]. The treatment of dendritic cells with β-defensin-2 upregulated the expression of CC-chemokines (MIP-1α and MIP-1β) and down-regulated CCR5 expression [14]. By utilizing chemokine receptors on immune cells, defensins may contribute to the regulation of host adaptive immunity against microbial invasion [15]. Taken together, our data provide evidence that α-defensins could play a role in host defence against HIV infection of macrophages. The biological interaction of α-defensins with CC-chemokines may constitute a unique mechanism of innate immunity against HIV disease.

Centrifugal enhancement of human immunodeficiency virus type 1 infection and human cytomegalovirus gene expression in human primary monocyte/macrophages in vitro

In an effort to facilitate the efficiency of human immunodeficiency virus type 1 (HIV‐1) and/or human cytomegalovirus (HCMV) infection in primary monocyte/macrophages in vitro, the effect of low‐speed centrifugation was studied. The infectivity of Aree strains (Bai, Ada‐M, and IIIB) of HIV‐1 tested was significantly enhanced by centrifugal inoculation at a force of 1500g for 60 min. Reverse transcriptase activity and HIV‐1 p24 antigen in primary monocyte/macrophages infected by a centrifugal inoculation technique were detectable 3‐7 days earlier and were more than 10‐fold greater in magnitude (at an early stage of the infection) than those of control cells infected by the conventional inoculation technique. Examination of the cells by indirect immunofluorescence revealed higher expression of HIV‐1 p24 protein in the monocyte/macrophages infected by the centrifugal inoculation technique. These differences were directly related to centrifugal inoculation and were evident up to 3 weeks after infection. Enhancement was not observed when centrifugation was carried out before or after HIV‐1 infection. Centrifugal inoculation of HCMV also enhanced its immediate‐early and early gene expression up to 36‐ to 50‐fold, although neither late nuclear antigens and glycoproteins of HCMV nor infectious virus was detected in HCMV‐infected monocyte/ macrophage cultures. These results show that centrifugal inoculation is a useful technique for improving the efficiency of HCMV and HIV‐1 infection in vitro.

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.

Interleukin-1β stimulates macrophage inflammatory protein-1α and -1β expression in human neuronal cells (NT2-N)

Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2‐N) produce macrophage inflammatory protein‐1α and ‐1β (MIP‐1α and MIP‐1β), which could be enhanced by interleukin (IL)‐1β at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte‐derived macrophage (MDM) cultures induced MIP‐1β mRNA expression in NT2‐N cells. Anti‐IL‐1β antibody removed most, but not all, of the MDM culture supernatant‐induced MIP‐1β mRNA expression in NT2‐N cells, suggesting that IL‐1β in the MDM culture supernatants is a major factor in the induction of MIP‐1β expression. Investigation of the mechanism(s) responsible for IL‐1β‐induced MIP‐1α and ‐1β expression demonstrated that IL‐1β activated nuclear factor kappa B (NF‐κB) promoter‐directed luciferase activity in NT2‐N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF‐κB, not only blocked IL‐1β‐induced activation of the NF‐κB promoter but also decreased IL‐1β‐induced MIP‐1α and ‐1β expression in NT2‐N cells. These data suggest that NF‐κB is at least partially involved in the IL‐1β‐mediated action on MIP‐1α and ‐1β in NT2‐N cells. IL‐1β‐mediated up‐regulation of β‐chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.