Transmission of chimeric HIV by mating in conventional mice: prevention by pre-exposure antiretroviral therapy and reduced susceptibility during estrus.

Heterosexual transmission accounts for the majority of new human immunodeficiency virus (HIV) cases worldwide. The current approach to investigate HIV heterosexual transmission in animals involves application of virus stock to the vaginal surface, a method that does not reproduce the physiological conditions of vaginal intercourse that influence the rate of transmission. We have previously described efficient infection of conventional mice using EcoHIV/NL4-3 and EcoHIV/NDK, chimeric HIV molecular clones constructed to express all HIV structural and regulatory genes except envelope, which is replaced by a rodent-tropic envelope gene. Here we investigated whether EcoHIV/NDK-infected male mice transmit virus to females during coitus, and the sensitivity of this transmission to HIV pre-exposure prophylaxis and the estrus state. Our general approach was to allow mating between EcoHIV/NDK-infected male mice and uninfected females for 1-7 nights. At 1-6 weeks after mating, mice were euthanized and virus burdens were measured by quantitative PCR (qPCR) amplification of HIV RNA or DNA in peritoneal macrophages, inguinal lymph node cells, spleen cells or vas deferens, or by ELISA for antibodies to HIV Gag. We found that 70-100% of female mice mated to EcoHIV/NDK-infected males acquired infection. Pericoital treatment of females with either 2',3'-dideoxcytidine (ddC) or tenofovir largely prevented their EcoHIV/NDK infection by mating (P<0.05 and P<0.003, respectively). In males, T cells were dispensable for virus transmission. The rate of EcoHIV/NDK sexual transmission to females in estrus declined sharply (P=0.003) but their infection by injection was unaffected, indicating that the local environment in the female reproductive tract influences susceptibility to HIV. We conclude that this system of EcoHIV/NDK transmission during mouse mating reproduces key features of heterosexual transmission of HIV in humans and can be used to investigate its biology and control.

Mice chronically infected with chimeric HIV resist peripheral and brain superinfection: a model of protective immunity to HIV.

Infection by some viruses induces immunity to reinfection, providing a means to identify protective epitopes. To investigate resistance to reinfection in an animal model of HIV disease and its control, we employed infection of mice with chimeric HIV, EcoHIV. When immunocompetent mice were infected by intraperitoneal (IP) injection of EcoHIV, they resisted subsequent secondary infection by IP injection, consistent with a systemic antiviral immune response. To investigate the potential role of these responses in restricting neurotropic HIV infection, we established a protocol for efficient EcoHIV expression in the brain following intracranial (IC) inoculation of virus. When mice were inoculated by IP injection and secondarily by IC injection, they also controlled EcoHIV replication in the brain. To investigate their role in EcoHIV antiviral responses, CD8+ T lymphocytes were isolated from spleens of EcoHIV infected and uninfected mice and adoptively transferred to isogenic recipients. Recipients of EcoHIV primed CD8+ cells resisted subsequent EcoHIV infection compared to recipients of cells from uninfected donors. CD8+ spleen cells from EcoHIV-infected mice also mounted modest but significant interferon-γ responses to two HIV Gag peptide pools. These findings suggest EcoHIV-infected mice may serve as a useful system to investigate the induction of anti-HIV protective immunity for eventual translation to human beings.

Inhibition of HIV-1 or bacterial activation of macrophages by products of HIV-1-resistant human cells.

We have recently described the molecular basis of HIV-1 resistance factor (HRF)-mediated anti-viral activity in primary and transformed CD4 T cells. HRF+ cell culture supernatants or partially purified HRF were found to incapacitate the formation of the NF-kappaB/DNA complex, which is indispensable for long terminal promoter-driven transcription of virus genes. In this study, we tested whether HRF might have much broader activity against other organisms whose pathogenesis is linked to NF-kappaB activation. Specifically, we tested the effects of HRF on the NF-kappaB-mediated responses of primary macrophages to HIV-1 or several bacterial antigens. We found that exposure to HRF inhibited HIV-1 expression in macrophages and also induced the production of HRF-like activity by macrophages, which prevented replication of virus in HIV-1-infected peripheral blood lymphocytes cultured in the adjacent compartment. We investigated the mechanism of this inhibition and found that HRF impeded NF-kappaB/DNA binding in macrophages induced by either HIV-1 or lipopolysaccharide from several bacteria species, resulting in impaired tumor necrosis factor-alpha responses to these organisms.

Human immunodeficiency virus type 1 efficiently binds to human fetal astrocytes and induces neuroinflammatory responses independent of infection.

BACKGROUND: HIV-1 infects human astrocytes in vitro and in vivo but the frequency of infected cells is low and its biological significance is unknown. In studies in vitro, recombinant gp120 alone can induce profound effects on astrocyte biology, suggesting that HIV-1 interaction with astrocytes and its functional consequences extend beyond the limited levels of infection in these cells. Here we determined the relative efficiencies of HIV-1 binding and infection in human fetal astrocytes (HFA), mainly at the single cell level, using HIV-1 tagged with green fluorescence protein (GFP)-Vpr fusion proteins, termed HIV-GFP, to detect virus binding and HIV-1 expressing Rev and NefGFP fusion proteins to detect productive infection. RESULTS: Essentially all HFA in a population bound HIV-GFP specifically and independently of CCR5 and CXCR4. The dynamics of this binding at 37 degrees C resembled binding of an HIV fusion mutant to CD4-positive cells, indicating that most of HIV-GFP arrested infection of HFA at the stage of virus-cell fusion. Despite extensive binding, only about 1% of HFA were detectably infected by HIV-RevGFP or HIV-NefGFP, but this proportion increased to the majority of HFA when the viruses were pseudotyped with vesicular stomatitis virus envelope glycoprotein G, confirming that HFA impose a restriction upon HIV-1 entry. Exposure of HFA to HIV-1 through its native proteins rapidly induced synthesis of interleukin-6 and interleukin-8 with increased mRNA detected within 3 h and increased protein detected within 18 h of exposure. CONCLUSION: Our results indicate that HIV-1 binding to human astrocytes, although extensive, is not generally followed by virus entry and replication. Astrocytes respond to HIV-1 binding by rapidly increased cytokine production suggesting a role of this virus-brain cell interaction in HIV-1 neuropathogenesis.

Gene expression profiling of human primary astrocytes exposed to manganese chloride indicates selective effects on several functions of the cells.

Exposure of adult humans to manganese (Mn) has long been known to cause neurotoxicity. Recent evidence also suggests that exposure of children to Mn is associated with developmental neurotoxicity. Astrocytes are critical for the proper functioning of the nervous system, and they play active roles in neurogenesis, synaptogenesis and synaptic neurotransmission. In this report, to help elucidate the molecular events underlying Mn neurotoxicity, we systematically identified the molecular targets of Mn in primary human astrocytes at a genome-wide level, by using microarray gene expression profiling and computational data analysis algorithms. We found that Mn altered the expression of diverse genes ranging from those encoding cytokines and transporters to signal transducers and transcriptional regulators. Particularly, 28 genes encoding proinflammatory chemokines, cytokines and related functions were up-regulated, whereas 15 genes encoding functions involved in DNA replication and repair and cell cycle checkpoint control were down-regulated. Consistent with the increased expression of proinflammatory factors, analysis of common regulators revealed that 16 targets known to be positively affected by the interferon-gamma signaling pathway were up-regulated by Mn(2+). In addition, 68 genes were found to be similarly up- or down-regulated by both Mn(2+) and hypoxia. These results from genomic analysis are further supported by data from real-time RT-PCR, Western blotting, flow cytometric and toxicological analyses. Together, these analyses show that Mn(2+) selectively affects cell cycle progression, the expression of hypoxia-responsive genes, and the expression of proinflammatory factors in primary human astrocytes. These results provide important insights into the molecular mechanisms underlying Mn neurotoxicity.

The common insecticides cyfluthrin and chlorpyrifos alter the expression of a subset of genes with diverse functions in primary human astrocytes.

Given the widespread use of insecticides in the environment, it is important to perform studies evaluating their potential effects on humans. Organophosphate insecticides, such as chlorpyrifos, are being phased out; however, the use of pyrethroids in household pest control is increasing. While chlorpyrifos is relatively well studied, much less is known about the potential neurotoxicity of cyfluthrin and other pyrethroids. To gain insights into the neurotoxicity of cyfluthrin, we compared and evaluated the toxicity profiles of chlorpyrifos and cyfluthrin in primary human fetal astrocytes. We found that at the same concentrations, cyfluthrin exerts as great as, or greater toxic effects on the growth, survival, and proper functioning of human astrocytes. By using microarray gene expression profiling, we systematically identified and compared the potential molecular targets of chlorpyrifos and cyfluthrin, at a genome-wide scale. We found that chlorpyrifos and cyfluthrin affect a similar number of transcripts. These targets include molecular chaperones, signal transducers, transcriptional regulators, transporters, and those involved in behavior and development. Further computational and biochemical analyses show that cyfluthrin and chlorpyrifos upregulate certain targets of the interferon-gamma and insulin-signaling pathways and that they increase the protein levels of activated extracellular signal-regulated kinase 1/2, a key component of insulin signaling; interleukin 6, a key inflammatory mediator; and glial fibrillary acidic protein, a marker of inflammatory astrocyte activation. These results suggest that inflammatory activation of astrocytes might be an important mechanism underlying neurotoxicity of both chlorpyrifos and cyfluthrin.

Neuropathologic and neuroinflammatory activities of HIV-1-infected human astrocytes in murine brain.

The balance between astrocyte and microglia neuroprotection and neurotoxicity defines the tempo of neuronal dysfunction during HIV-1-associated dementia (HAD). Astrocytes maintain brain homeostasis and respond actively to brain damage by providing functional and nutritive neuronal support. In HAD, low-level, continuous infection of astrocytes occurs, but the functional consequences of this infection are poorly understood. To this end, human fetal astrocytes (HFA) and monocyte-derived macrophages (MDM) were infected with HIV-1DJV and HIV-1NL4-3 (neurotropic and lymphotropic strains respectively) and a pseudotyped Vesicular Stomatitis Virus (VSV/HIV-1NL4-3) prior to intracranial injection into the basal ganglia of severe combined immunodeficient mice. Neuropathological and immunohistochemical comparisons for inflammatory and neurotoxic activities were performed amongst the infected cell types at 7 or 14 days. HIV-1-infected MDM induced significant increases in Mac-1, glial fibrillary acidic protein, ionized calcium-binding adapter molecule 1, and proinflammatory cytokine RNA and/or protein expression when compared with HSV/HIV-1- and HIV-1-infected HFA and sham-operated mice. Levels of neuron-specific nuclear protein, microtubule-associated protein 2, and neurofilament antigens were reduced significantly in the brain regions injected with human MDM infected with HIV-1DJV or VSV/HIV-1. We conclude that HIV-1 infection of astrocytes leads to limited neurodegeneration, underscoring the early and active role of macrophage-driven neurotoxicity in disease.

A mouse model for study of systemic HIV-1 infection, antiviral immune responses, and neuroinvasiveness.

We created a model of HIV-1 infection of conventional mice for investigation of viral replication, control, and pathogenesis. To target HIV-1 to mice, the coding region of gp120 in HIV-1/NL4-3 was replaced with that of gp80 from ecotropic murine leukemia virus, a retrovirus that infects only rodents. The resulting chimeric virus construct, EcoHIV, productively infected murine lymphocytes, but not human lymphocytes, in culture. Adult, immunocompetent mice were readily susceptible to infection by a single inoculation of EcoHIV as shown by detection of virus in splenic lymphocytes, peritoneal macrophages, and the brain. The virus produced in animals was infectious, as shown by passage in culture, and immunogenic, as shown by induction of antibodies to HIV-1 Gag and Tat. A second chimeric virus based on clade D HIV-1/NDK was also highly infectious in mice; it was detected in both spleen and brain 3 wk after tail vein inoculation, and it induced expression of infection response genes, MCP-1, STAT1, IL-1beta, and complement component C3, in brain tissue as determined by quantitative real-time PCR. EcoHIV infection of mice forms a useful model of HIV-1 infection of human beings for convenient and safe investigation of HIV-1 therapy, vaccines, and potentially pathogenesis.

Microarray analysis of changes in cellular gene expression induced by productive infection of primary human astrocytes: implications for HAD.

The role of astrocytes in HIV-1 associated dementia (HAD) is not well understood. HIV-1 binds efficiently to astrocytes but infects only a small fraction of the cells in vitro and in vivo. To gain insight into the biology of HIV-1-expressing astrocytes, we productively infected human fetal astrocytes with pseudotyped HIV-1 and employed Affymetrix oligonucleotide microarrays to determine global changes in cellular gene expression at the peak of virus production. With a twofold change as a cutoff, HIV-1 increased transcription of 266 genes in astrocytes and suppressed expression of 468. The functions of highly expressed genes included interferon-mediated antiviral responses (OAS1, IFIT1), intercellular contacts (SH3, glia-derived nexin), cell homing/adhesion (matrix metalloproteinases), and cell-cell signaling (neuropilin 1 and 2). Surprisingly, genes involved in innate immune responses of astrocytes were largely unaffected. The single most significant effect of HIV-1, however, was down-modulation of at least 55 genes involved in control of cell cycle, DNA replication, and cell proliferation, which were overrepresented in these categories with probability scores of 10(-10)-10(-26). Our data suggest that HIV-1 expression in astrocytes profoundly alters host cell biology, with potential consequences for the physiological function of astrocytes during HIV-1 infection in the brain.

Productive infection of primary murine astrocytes, lymphocytes, and macrophages by human immunodeficiency virus type 1 in culture.

A mouse model of human immunodeficiency virus type 1 (HIV-1) infection would be extremely valuable for evaluation of therapies and vaccines; however, multiple blocks to productive infection of NIH 3T3 and other mouse cell lines have been reported. The authors investigated the replication of HIV-1 in primary mouse astrocytes, lymphocytes, and macrophages in culture by infection with intact HIV-1 pseudotyped with the vesicular stomatitis virus G envelope glycoprotein (VSV-G) or with the envelope glycoprotein of amphotropic murine leukemia virus. Astrocytes, lymphocytes, and macrophages were susceptible to productive infection as variously assayed by detection of p24 and Tat proteins, viral protease-mediated processing of Gag, appropriately spliced viral RNA, and infectious progeny virus. As expected, NIH 3T3 cells were not susceptible to productive infection by VSV/NL4. Susceptibility mapped neither to the Fv locus nor to a possible polymorphism in cyclin T1. This study indicates that there are no intrinsic intracellular barriers to HIV-1 replication in primary mouse cells when virus entry is efficient.

The macrophage response to HIV-1: Intracellular control of X4 virus replication accompanied by activation of chemokine and cytokine synthesis.

During human immunodeficiency virus (HIV)-1 infection, T lymphocytes and macrophages play dual roles. They are the primary targets for virus replication, but they are also primary effector cells in acquired and innate immunity, respectively. The authors are now investigating how these roles come together in the response of human monocyte-derived macrophages (MDM) to certain HIV-1. The authors and others have previously shown that MDM permit entry of some X4 virus strains, but control viral replication intracellularly. In the present study, viral DNA synthesis, entry into the nucleus, and transcription to RNA were all observed in X4 virus-infected MDM. MDM arrested HIV-1 replication prior to expression of mature capsid antigen p24 and production of cell-free infectious viral particles. Cell-associated transmissible HIV-1 was detected by cocultivation of infected MDM and susceptible T lymphocytes. A second protective response of MDM to specific R5 as well as X4 HIV-1 was identified in rapid and extensive secretion of tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, and RANTES. These findings support the view that MDM act aggressively to control HIV-1 replication: X4 strains by severely limiting the progeny virus production and R5 strains by producing beta-chemokines competent to block virus entry into target cells. Optimizing these innate immune responses offers another means to control HIV-1 infection in the human host.