IFN-gamma mediates enhancement of HIV replication in astrocytes by inducing an antagonist of the beta-catenin pathway (DKK1) in a STAT 3-dependent manner

Typically, IFN-γ is an antiviral cytokine that inhibits the replication of many viruses, including HIV. However, in the CNS, IFN-γ induces HIV-productive replication in astrocytes. Although astrocytes in vitro are refractory to HIV replication, recent in vivo evidence demonstrated that astrocytes are infected by HIV, and their degree of infection is correlated with proximity to activated macrophages/microglia. The ability of IFN-γ to induce HIV replication in astrocytes suggests that the environmental milieu is critical in regulating the permissiveness of astrocytes to HIV infection. We evaluated the mechanism by which IFN-γ relieves restricted HIV replication in astrocytes. We demonstrate that although astrocytes have robust endogenous β-catenin signaling, a pathway that is a potent inhibitor of HIV replication, IFN-γ diminished β-catenin signaling in astrocytes by 40%, as evaluated by both active β-catenin protein expression and β-catenin-mediated T cell factor/lymphoid enhancer reporter (TOPflash) activity. Further, IFN-γ-mediated inhibition of β-catenin signaling was dependent on its ability to induce an antagonist of the β-catenin signaling pathway, Dickkopf-related protein 1, in a STAT 3-dependent manner. Inhibition of STAT3 and Dickkopf-related protein 1 abrogated the ability of IFN-γ to enhance HIV replication in astrocytes. These data demonstrated that IFN-γ induces HIV replication in astrocytes by antagonizing the β-catenin pathway. To our knowledge, this is the first report to point to an intricate cross-talk between IFN-γ signaling and β-catenin signaling that may have biologic and virologic effects on HIV outcome in the CNS, as well as on broader processes where the two pathways interface.     

Protein vaccines induce uncommitted IL-2-secreting human and mouse CD4 T cells, whereas infections induce more IFN-gamma-secreting cells

Mouse and human CD4 T cells primed during an immune response may differentiate into effector phenotypes such as Th1 (secreting IFN-gamma) or Th2 (secreting IL-4) that mediate effective immunity against different classes of pathogen. However, primed CD4 T cells can also remain uncommitted, secreting IL-2 and chemokines, but not IFN-gamma or IL-4. We now show that human CD4 T cells primed by protein vaccines mostly secreted IL-2, but not IFN-gamma, whereas in the same individuals most CD4 T cells initially primed by infection with live pathogens secreted IFN-gamma. We further demonstrate that many tetanus-specific IL-2+IFN-gamma- cells are uncommitted and that a single IL-2+IFN-gamma- cell can differentiate into Th1 or Th2 phenotypes following in vitro stimulation under appropriate polarizing conditions. In contrast, influenza-specific IL-2+IFN-gamma- CD4 cells maintained a Th1-like phenotype even under Th2-polarizing conditions. Similarly, adoptively transferred OTII transgenic mouse T cells secreted mainly IL-2 after priming with OVA in alum, but were biased toward IFN-gamma secretion when primed with the same OVA peptide presented as a pathogen Ag during live infection. Thus, protein subunit vaccines may prime a unique subset of differentiated, but uncommitted CD4 T cells that lack some of the functional properties of committed effectors induced by infection. This has implications for the design of more effective vaccines against pathogens requiring strong CD4 effector T cell responses.     

IL-12 and type-I IFN synergize for IFN-gamma production by CD4 T cells, whereas neither are required for IFN-gamma production by CD8 T cells after Listeria monocytogenes infection

Differentiation of Ag-specific T cells into IFN-gamma producers is essential for protective immunity to intracellular pathogens. In addition to stimulation through the TCR and costimulatory molecules, IFN-gamma production is thought to require other inflammatory cytokines. Two such inflammatory cytokines are IL-12 and type I IFN (IFN-I); both can play a role in priming naive T cells to produce IFN-gamma in vitro. However, their role in priming Ag-specific T cells for IFN-gamma production during experimental infection in vivo is less clear. In this study, we examine the requirements for IL-12 and IFN-I, either individually or in combination, for priming Ag-specific T cell IFN-gamma production after Listeria monocytogenes (Lm) infection. Surprisingly, neither individual nor combined defects in IL-12 or IFN-I signaling altered IFN-gamma production by Ag-specific CD8 T cells after Lm infection. In contrast, individual defects in either IL-12 or IFN-I signaling conferred partial ( approximately 50%) reductions, whereas combined deficiency in both IL-12 and IFN-I signaling conferred more dramatic (75-95%) reductions in IFN-gamma production by Ag-specific CD4 T cells. The additive effects of IL-12 and IFN-I signaling on IFN-gamma production by CD4 T cells were further demonstrated by adoptive transfer of transgenic IFN-IR(+/+) and IFN-IR(-/-) CD4 T cells into normal and IL-12-deficient mice, and infection with rLm. These results demonstrate an important dichotomy between the signals required for priming IFN-gamma production by CD4 and CD8 T cells in response to bacterial infection.     

Interleukin-15 modulates interferon-gamma and beta-chemokine production in patients with HIV infection: implications for immune-based therapy

Interleukin (IL)-15 is a cytokine that has lymphocyte stimulatory activity similar to that of IL-2, and plays important immunoregulatory functions during HIV disease. To evaluate the role of IL-15 in HIV infection the following patients were studied: 18 antiretroviral-naive patients with advanced disease; 19 patients with continuous viral suppression and immunological response after 48-120 weeks of highly active antiretroviral therapy (HAART); and 12 patients with evidence of virological and immunological HAART treatment failure. Nineteen healthy blood donors were included as controls. The production of IL-15 by human peripheral blood monocytes stimulated with lipopolysaccharide and Mycobacterium avium complex, the priming effect of IL-15 on IFN-gamma production from purified CD4(+) and CD8(+) T cells, and the ability of IL-15 to stimulate the beta-chemokine release from purified CD4(+) and CD8(+) T cells were analyzed. In the present work IL-15 production by human peripheral blood monocytes was significantly increased in HIV-infected patients with long-term virological and immunological response to HAART. IL-15 enhanced the in vitro priming of CD4(+) and CD8(+) T cells for IFN-gamma production, also in patients receiving HAART. Finally, IL-15 had positive effects on RANTES, MIP-1alpha, and MIP-1beta release by CD4(+) and CD8(+) T cells. In conclusion IL-15 could affect the immune response of HIV-infected patients by augmenting and/or modulating IFN-gamma production and beta-chemokine release. These data about functional properties of IL-15 could provide new implications for immune-based therapies in HIV infection.     

Expression of simian immunodeficiency virus (SIV) nef in astrocytes during acute and terminal infection and requirement of nef for optimal replication of neurovirulent SIV in vitro

As the most numerous cells in the brain, astrocytes play a critical role in maintaining central nervous system homeostasis, and therefore, infection of astrocytes by human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) in vivo could have important consequences for the development of HIV encephalitis. In this study, we establish that astrocytes are infected in macaques during acute SIV infection (10 days postinoculation) and during terminal infection when there is evidence of SIV-induced encephalitis. Additionally, with primary adult rhesus macaque astrocytes in vitro, we demonstrate that the macrophage-tropic, neurovirulent viruses SIV/17E-Br and SIV/17E-Fr replicate efficiently in astrocytes, while the lymphocyte-tropic, nonneurovirulent virus SIV(mac)239 open-nef does not establish productive infection. Furthermore, aminoxypentane-RANTES abolishes virus replication, suggesting that these SIV strains utilize the chemokine receptor CCR5 for entry into astrocytes. Importantly, we show that SIV Nef is required for optimal replication in primary rhesus macaque astrocytes and that normalizing input virus by particle number rather than by infectivity reveals a disparity between the ability of a Nef-deficient virus and a virus encoding a nonmyristoylated form of Nef to replicate in these central nervous system cells. Since the myristoylated form of Nef has been implicated in functions such as CD4 and major histocompatibility complex I downregulation, kinase association, and enhancement of virion infectivity, these data suggest that an as yet unidentified function of Nef may exist to facilitate SIV replication in astrocytes that may have important implications for in vivo pathogenesis.     

HIV-1 does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo

The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs for IL-1beta, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants. Although IL-1beta, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result in characteristic mRNA profiles for IL-1beta, -2, -4, -6, -8, -10, -12, -15, IFN-gamma, TNF-alpha, TGF-beta, and beta-chemokines in tissue blocks or HLAC. The increased expression of IL-1beta, -6, and -8 in tissue blocks may approximate clinical situations with heightened immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV uses during acute infection.     

Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta

Astrocytes have the capacity to secrete or respond to a variety of cytokines including IL-1, IL-6, IL-3, and TNF-alpha. In this study, we have examined the capacity of astrocytes to secrete TNF-alpha in response to a variety of biologic stimuli, particularly cytokines such as IL-1 and IFN-gamma, which are known to be present in the central nervous system during neurologic diseases associated with inflammation. Rat astrocytes do not constitutively produce TNF-alpha, but have the ability to secrete TNF-alpha in response to LPS, and can be primed by IFN-gamma to respond to a suboptimal dose of LPS. IFN-gamma and IL-1 beta alone do not induce TNF-alpha production, however, the combined treatment of IFN-gamma and IL-1 beta results in a striking synergistic effect on astrocyte TNF-alpha production. Astrocyte TNF-alpha protein production induced by a combined treatment of either IFN-gamma/LPS or IFN-gamma/IL-1 beta occurs in a dose- and time-dependent manner, and appears to require a "priming signal" initiated by IFN-gamma, which then renders the astrocyte responsive to either a suboptimal dose of LPS or IL-1 beta. Astrocyte TNF-alpha production by IFN-gamma/LPS stimulation can be inhibited by the addition of anti-rat IFN-gamma antibody, whereas IFN-gamma/IL-1-induced TNF-alpha production is inhibited by antibody to either IFN-gamma or IL-1 beta. Polyclonal antisera reactive with mouse macrophage-derived TNF-alpha neutralized the cytotoxicity of IFN-gamma/LPS and IFN-gamma/IL-1 beta-induced astrocyte TNF-alpha, demonstrating similarities between these two sources of TNF-alpha. We propose that astrocyte-produced TNF-alpha may have a pivotal role in augmenting intracerebral immune responses and inflammatory demyelination due to its diverse functional effects on glial cells such as oligodendrocytes and astrocytes themselves.     

Infection of human pericytes by HIV‐1 disrupts the integrity of the blood–brain barrier

Human immunodeficiency virus type 1 (HIV‐1) infection of the central nervous system (CNS) affects cross‐talk between the individual cell types of the neurovascular unit, which then contributes to disruption of the blood–brain barrier (BBB) and the development of neurological dysfunctions. Although the toxicity of HIV‐1 on neurons, astrocytes and brain endothelial cells has been widely studied, there are no reports addressing the influence of HIV‐1 on pericytes. Therefore, the purpose of this study was to evaluate whether or not pericytes can be infected with HIV‐1 and how such an infection affects the barrier function of brain endothelial cells. Our results indicate that human brain pericytes express the major HIV‐1 receptor CD4 and co‐receptors CXCR4 and CCR5. We also determined that HIV‐1 can replicate, although at a low level, in human brain pericytes as detected by HIV‐1 p24 ELISA. Pericytes were susceptible to infection with both the X4‐tropic NL4‐3 and R5‐tropic JR‐CSF HIV‐1 strains. Moreover, HIV‐1 infection of pericytes resulted in compromised integrity of an in vitro model of the BBB. These findings indicate that human brain pericytes can be infected with HIV‐1 and suggest that infected pericytes are involved in the progression of HIV‐1‐induced CNS damage.     

Chimeric interferon-gamma receptors demonstrate that an accessory factor required for activity interacts with the extracellular domain.

We determined the species specificity and function of structural domains of the interferon-gamma receptor (IFN-gamma R) by construction of human/murine chimeric IFN-gamma R cDNA clones and their expression in various cells. We demonstrate that we can reconstitute a biologically active IFN-gamma R in eukaryotic cells with chimeric receptors as long as the extracellular domain and an accessory factor are from the same species. These results indicate that the extracellular domain of the receptor interacts directly or indirectly with the species-specific accessory factor.     

Human innate Mycobacterium tuberculosis-reactive alphabetaTCR+ thymocytes

The control of Mycobacterium tuberculosis (Mtb) infection is heavily dependent on the adaptive Th1 cellular immune response. Paradoxically, optimal priming of the Th1 response requires activation of priming dendritic cells with Th1 cytokine IFN-gamma. At present, the innate cellular mechanisms required for the generation of an optimal Th1 T cell response remain poorly characterized. We hypothesized that innate Mtb-reactive T cells provide an early source of IFN-gamma to fully activate Mtb-exposed dendritic cells. Here, we report the identification of a novel population of Mtb-reactive CD4(-) alphabetaTCR(+) innate thymocytes. These cells are present at high frequencies, respond to Mtb-infected cells by producing IFN-gamma directly ex vivo, and display characteristics of effector memory T cells. This novel innate population of Mtb-reactive T cells will drive further investigation into the role of these cells in the containment of Mtb following infectious exposure. Furthermore, this is the first demonstration of a human innate pathogen-specific alphabetaTCR(+) T cell and is likely to inspire further investigation into innate T cells recognizing other important human pathogens.     

人星形细胞HIV—1膜蛋白gp120新受体的免疫学鉴定

自1994年首次报导HIV－1外膜蛋白gp120与人胎儿星形细胞膜蛋白质位点（推测分子量为260kD,命名为PAG）结合以来[1],这项工作持续集中于研究该蛋白的功能与作用,本研究藉助杂交瘤技术建立了一系列不鼠抗人星形细胞PAG）的单克隆抗体,这些抗体成功的抑制了gp120与PAG的结合,抑制可达50％以上,并几乎完全阻断了gp120介导的由摄入所星表细胞风钙离子的升高,通过ELISA可证实抗体与星形细胞的特异反应,蛋白印迹和免疫沉淀试验结果表明PAG作为实体的存在,试验表明PAG对于gp120与星形细胞的结合,对于gp120个导的星形细胞摄入所致钙离子的升高均有决定性作用,许多方向报导和研究表明gp120可与多种细胞结合而导致HIV－1感染,由此推论PAG是HIV－1gp120在人星形细胞上的新受体,同时也可能对HIV－1脑病的治疗开辟一条崭新的途径,PAG是否为HIV－1感染人星形细胞的受体,仍有等进一步实验证明。     

The antiviral response to gamma interferon

A role for alpha/beta interferon (IFN-alpha/beta) in the IFN-gamma antiviral response has long been suggested. Accordingly, possible roles for autocrine or double-stranded-RNA (dsRNA)-induced IFN-alpha/beta in the IFN-gamma response were investigated. Use was made of wild-type and a variety of mutant human fibrosarcoma cell lines, including mutant U5A cells, which lack a functional IFN-alpha/beta receptor and hence an IFN-alpha/beta response. IFN-gamma did not induce detectable levels of IFN-alpha/beta in any of the cell lines, nor was the IFN-gamma response per se dependent on autocrine IFN-alpha/beta. On the other hand, a number of responses to dsRNA [poly(I). poly(C)] and encephalomyocarditis virus were greatly enhanced by IFN-gamma pretreatment (priming) of wild-type cells or of mutant cells lacking an IFN-alpha/beta response; these include the primary induction of dsRNA-inducible mRNAs, including IFN-beta mRNA, and, to a lesser extent, the dsRNA-mediated activation of the p38 mitogen-activated protein (MAP) kinase(s). IFN-gamma priming of mRNA induction by dsRNA is dependent on JAK1 and shows biphasic kinetics, with an initial rapid (<30-min) response being followed by a more substantial effect on overnight incubation. The IFN-gamma-primed dsRNA responses appear to be subject to modulation through the p38, phosphatidylinositol 3-kinase, and ERK1/ERK2 MAP kinase pathways. It can be concluded that despite efficient priming of IFN-beta production, the IFN-alpha/beta pathways play no significant role in the primary IFN-gamma antiviral response in these cell-virus systems. The observed IFN-gamma priming of dsRNA responses, on the other hand, will likely play a significant role in combating virus infection in vivo.     

Interferon induces astrocyte maturation causing an increase in cholinergic properties of cultured human spinal cord cells

It has been well established that interferon-gamma (IFN-gamma) can modify the immune status of cells in the central nervous system (CNS) by inducing major histocompatibility antigens. Furthermore, it has been shown that endogenous IFN can be produced in the brain following viral infection and a form of IFN-alpha/beta can be produced by astrocytes in culture. Here we show that IFN can induce astrocyte maturation and alter neurotransmitter properties in cultured CNS neurons at a given developmental stage. IFN causes a dose-dependent increase in choline acetyltransferase activity and glial fibrillary acidic protein (GFAP) immunoreactivity in cultures of human embryonic spinal cord neurons. The GABAergic activity and the Thy1 immunoreactivity remain unchanged. IFN-gamma does not act directly on the neurons but via the nonneuronal cells, probably the astrocytes, which in turn stimulate the cholinergic traits. These studies could be important for demonstrating an action of the immune system on glial cell maturation and on the neurotransmitter phenotype expression in CNS neurons.