RESUMEN
To identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latent HIV-1 infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome, and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. A select set of candidate genes was functionally validated using short hairpin RNA (shRNA)-mediated depletion in latent HIV-1 infected J-Lat A2 and 11.1 T cell lines. We confirmed ADK, CHD9, CMSS1, EVI2B, EXOSC8, FAM19A, GRIK5, IRF2BP2, NF1, and USP15 as novel host factors involved in the maintenance of HIV-1 latency. Chromatin immunoprecipitation assays indicated that CHD9, a chromodomain helicase DNA-binding protein, maintains HIV-1 latency via direct association with the HIV-1 5' long terminal repeat (LTR), and its depletion results in increased histone acetylation at the HIV-1 promoter, concomitant with HIV-1 latency reversal. FDA-approved inhibitors 5-iodotubercidin, trametinib, and topiramate, targeting ADK, NF1, and GRIK5, respectively, were characterized for their latency reversal potential. While 5-iodotubercidin exhibited significant cytotoxicity in both J-Lat and primary CD4+ T cells, trametinib reversed latency in J-Lat cells but not in latent HIV-1 infected primary CD4+ T cells. Importantly, topiramate reversed latency in cell line models, in latently infected primary CD4+ T cells, and crucially in CD4+ T cells from three people living with HIV-1 (PLWH) under suppressive antiretroviral therapy, without inducing T cell activation or significant toxicity. Thus, using an adaptation of a haploid forward genetic screen, we identified novel and druggable host factors contributing to HIV-1 latency. IMPORTANCE A reservoir of latent HIV-1 infected cells persists in the presence of combination antiretroviral therapy (cART), representing a major obstacle for viral eradication. Reactivation of the latent HIV-1 provirus is part of curative strategies which aim to promote clearance of the infected cells. Using a two-color haploid screen, we identified 69 candidate genes as latency-maintaining host factors and functionally validated a subset of 10 of those in additional T-cell-based cell line models of HIV-1 latency. We further demonstrated that CHD9 is associated with HIV-1's promoter, the 5' LTR, while this association is lost upon reactivation. Additionally, we characterized the latency reversal potential of FDA compounds targeting ADK, NF1, and GRIK5 and identify the GRIK5 inhibitor topiramate as a viable latency reversal agent with clinical potential.
Asunto(s)
Infecciones por VIH/genética , VIH-1/fisiología , Haploidia , Latencia del Virus , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , Interacciones Huésped-Patógeno , Humanos , Receptores de Ácido Kaínico/genética , Receptores de Ácido Kaínico/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismo , Activación ViralRESUMEN
Public policy requires public support, which in turn implies a need to enable the public not just to understand policy but also to be engaged in its development. Where complex science and technology issues are involved in policy making, this takes time, so it is important to identify emerging issues of this type and prepare engagement plans. In our horizon scanning exercise, we used a modified Delphi technique. A wide group of people with interests in the science and policy interface (drawn from policy makers, policy adviser, practitioners, the private sector and academics) elicited a long list of emergent policy issues in which science and technology would feature strongly and which would also necessitate public engagement as policies are developed. This was then refined to a short list of top priorities for policy makers. Thirty issues were identified within broad areas of business and technology; energy and environment; government, politics and education; health, healthcare, population and aging; information, communication, infrastructure and transport; and public safety and national security.
Asunto(s)
Técnica Delphi , Formulación de Políticas , Política Pública/tendencias , Ciencia/tendencias , Tecnología/tendencias , Cambio Climático , Conservación de los Recursos Naturales , Toma de Decisiones , Atención a la Salud , Demografía , Ambiente , Gobierno , Humanos , Invenciones , Esperanza de Vida , Política , Dinámica Poblacional , Sector Privado , Asignación de RecursosRESUMEN
The presence of an anti-bacterial T cell response and evidence of bacterial products in inflamed joints of reactive arthritis patients suggests an antigen transportation role in this disease for macrophages and dendritic cells. We have investigated the functional properties and in vivo migration of macrophages and DC after infection with Salmonella enterica serovar Typhimurium (S. typhimurium). BM-derived macrophages and DC displayed enhanced expression of costimulatory molecules (CD40 and CD86) and increased production of pro-inflammatory cytokines (TNF-alpha, IL-6 and IL-12p40) and nitric oxide after infection. Upon adoptive transfer into mice, infected DC migrated to lymphoid tissues and induced an anti-Salmonella T cell response, whereas infected macrophages did not. Infection of DC with S. typhimurium was associated with strong up-regulation of the chemokine receptor CCR7 and acquisition of responsiveness to chemokines acting through this receptor. Moreover, S. typhimurium-infected CCR7-deficient DC were unable to migrate to lymph nodes after adoptive transfer, although they did reach the spleen. Our data demonstrate distinct roles for macrophages and DC as antigen transporters after S. typhimurium infection and a dependence on CCR7 for migration of DC to lymph nodes after bacterial infection.
Asunto(s)
Células Dendríticas/inmunología , Macrófagos/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Animales , Antígeno B7-2/metabolismo , Antígenos CD40/metabolismo , Movimiento Celular , Citocinas/metabolismo , Células Dendríticas/microbiología , Células Dendríticas/trasplante , Activación de Macrófagos , Macrófagos/microbiología , Ratones , Ratones Transgénicos , Mutación , Receptores CCR7 , Receptores de Quimiocina/genética , Linfocitos T/inmunología , Regulación hacia ArribaRESUMEN
HLA-B27 transgenic rats and strains of HLA-B27-transgenic beta(2)-microglobulin (beta(2)m)-deficient mice develop a multisystem inflammatory disease affecting the joints, skin, and bowel with strong similarity to human spondyloarthritis. We show that HLA-B27 transgenic mice and rats express HC10-reactive, beta(2)m-free HLA-B27 homodimers (B27(2)) and multimers, both intracellularly and at the cell surface of leukocytes, including rat dendritic cells. Fluorescent-labeled tetrameric complexes of HLA-B27 homodimers (B27(2) tetramers) bind to populations of lymphocytes, monocytes, and dendritic cells. The murine (and probably rat) paired Ig-like receptors (PIRs) are ligands for B27(2). Thus, B27(2) tetramers stain RBL cells transfected with murine activating PIR-A4 and inhibitory PIR-B receptors. Murine PIR-A and -B can be immunoprecipitated from the RAW264.7 macrophage cell line, and murine PIR-A can be immunoprecipitated from the J774.A1 line using B27(2). B27(2) tetramer staining corresponds to the distribution of PIR expression on lymphoid and myeloid cells and on murine macrophage cell lines. B27(2) can induce TNF-alpha release from the J774.A1 macrophage cell line. The binding of B27(2) to PIR is inhibited by HC10, an mAb that ameliorates arthritis in HLA-B27(+) beta(2)m(-/-) mice. The expression and PIR recognition of B27(2) could explain the pathogenesis of rodent spondyloarthritis.
Asunto(s)
Modelos Animales de Enfermedad , Antígeno HLA-B27/inmunología , Receptores Inmunológicos/inmunología , Espondiloartritis/inmunología , Animales , Animales Modificados Genéticamente , Anticuerpos Monoclonales/inmunología , Biopolímeros , Biotinilación , Línea Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Dimerización , Antígeno HLA-B27/química , Antígeno HLA-B27/genética , Antígeno HLA-B27/metabolismo , Ligandos , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Pruebas de Precipitina , Unión Proteica , Ratas , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Espondiloartritis/genética , Espondilitis Anquilosante/genética , Espondilitis Anquilosante/inmunología , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genéticaRESUMEN
The strong association of HLA B27 with spondyloarthropathies contrasts strikingly with most autoimmune diseases, which are HLA class II associated and thought to be mediated by CD4+ T lymphocytes. By introducing a human-derived HLA B27-restricted TCR into HLA B27 transgenic mice, we have obtained a functional TCR transgenic model, GRb, dependent on HLA B27 for response. Surprisingly, HLA B27 supported CD4+ as well as CD8+ T cell responses in vivo and in vitro. Further, HLA B27-restricted CD4+ T cells were capable of differentiation into a range of Th1 and Th2 T cell subsets with normal patterns of cytokine expression. The transgenic T cells were also able to enhance clearance of recombinant vaccinia virus containing influenza nucleoprotein in vivo. This is the first description of a human HLA class I-restricted TCR transgenic line. The existence of CD4+ MHC class I-restricted T cells has significant implications for immune regulation in autoimmunity and, in particular, in HLA B27-associated arthritis. We believe that this model provides a novel system for the study of unusual T cell behavior in vivo.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno HLA-B27/genética , Proteínas de Unión al ARN , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Citocinas/biosíntesis , Femenino , Antígeno HLA-B27/fisiología , Humanos , Virus de la Influenza A/inmunología , Interferón gamma/biosíntesis , Depleción Linfocítica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de la Nucleocápside , Nucleoproteínas/inmunología , Nucleoproteínas/farmacología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Células TH1/citología , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/virología , Células Th2/citología , Células Th2/inmunología , Células Th2/metabolismo , Células Th2/virología , Transgenes/inmunología , Proteínas del Núcleo Viral/inmunología , Proteínas del Núcleo Viral/farmacologíaRESUMEN
The human major histocompatibility complex (MHC) class I allele HLA-B27 is strongly associated with seronegative spondyloarthropathies including ankylosing spondylitis and reactive arthritis. Although of unknown aetiology, one hypothesis suggests that a cytotoxic T cell (CTL) response against a self-antigen at sites of inflammation, such as entheses or joints may be involved. The chondrocyte is one of the major specialized cell types found both in articular cartilage and cartilaginous entheses and therefore is a possible source of such an antigen. CTL recognition of these cells is a potential mechanism for inflammation and cartilage damage, both through direct lysis of chondrocytes and the secretion of pro-inflammatory cytokines such as tumour necrosis factor and interferon-gamma (IFN-gamma). We test the feasibility of this hypothesis by examining the ability of chondrocytes to present antigen to CTL in vitro. Chondrocytes isolated from the ribcages of mice did not constitutively express detectable levels of MHC class I by fluorescence-activated cell sorting analysis. In addition, they were resistant to lysis by alloreactive and influenza A virus nucleoprotein (NP)-specific CTL. However, treatment of chondrocytes with IFN-gamma up-regulated MHC class I expression and rendered the cells susceptible to lysis by CTL. Similarly, IFN-gamma-treated chondrocytes infected with influenza A virus were recognized by NP-specific CTL, though with variable efficiency. Thus, we suggest that under certain circumstances CTL-mediated lysis of chondrocytes is potentially a potent mechanism for cartilage damage in vivo, but that low levels of MHC class I on healthy chondrocytes protects from immune recognition in health.