RESUMEN
Dendritic cells (DC) subsets, like Langerhans cells (LC), are immune cells involved in pathogen sensing. They express specific antimicrobial cellular factors that are able to restrict infection and limit further pathogen transmission. Here, we identify the alarmin S100A9 as a novel intracellular antiretroviral factor expressed in human monocyte-derived and skin-derived LC. The intracellular expression of S100A9 is decreased upon LC maturation and inversely correlates with enhanced susceptibility to HIV-1 infection of LC. Furthermore, silencing of S100A9 in primary human LC relieves HIV-1 restriction while ectopic expression of S100A9 in various cell lines promotes intrinsic resistance to both HIV-1 and MLV infection by acting on reverse transcription. Mechanistically, the intracellular expression of S100A9 alters viral capsid uncoating and reverse transcription. S100A9 also shows potent inhibitory effect against HIV-1 and MMLV reverse transcriptase (RTase) activity in vitro in a divalent cation-dependent manner. Our findings uncover an unexpected intracellular function of the human alarmin S100A9 in regulating antiretroviral immunity in Langerhans cells.
Asunto(s)
Alarminas/genética , Calgranulina B/genética , VIH-1/fisiología , Células de Langerhans/virología , Virus de la Leucemia Murina de Moloney/fisiología , Infecciones por Retroviridae/prevención & control , Animales , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Cricetulus , VIH-1/genética , Interacciones Huésped-Patógeno , Humanos , Células de Langerhans/inmunología , Leucemia Experimental/prevención & control , Ratones , Virus de la Leucemia Murina de Moloney/genética , Transcripción Reversa , Factor de Crecimiento Transformador beta/inmunología , Infecciones Tumorales por Virus/prevención & control , Replicación ViralRESUMEN
Recent experiments provide sound arguments in favor of the in vivo expression of the AntiSense Protein (ASP) of HIV-1. This putative protein is encoded on the antisense strand of the provirus genome and entirely overlapped by the env gene with reading frame -2. The existence of ASP was suggested in 1988, but is still controversial, and its function has yet to be determined. We used a large dataset of â¼23,000 HIV-1 and SIV sequences to study the origin, evolution, and conservation of the asp gene. We found that the ASP ORF is specific to group M of HIV-1, which is responsible for the human pandemic. Moreover, the correlation between the presence of asp and the prevalence of HIV-1 groups and M subtypes appeared to be statistically significant. We then looked for evidence of selection pressure acting on asp Using computer simulations, we showed that the conservation of the ASP ORF in the group M could not be due to chance. Standard methods were ineffective in disentangling the two selection pressures imposed by both the Env and ASP proteins-an expected outcome with overlaps in frame -2. We thus developed a method based on careful evolutionary analysis of the presence/absence of stop codons, revealing that ASP does impose significant selection pressure. All of these results support the idea that asp is the 10th gene of HIV-1 group M and indicate a correlation with the spread of the pandemic.
Asunto(s)
Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/genética , Pandemias , Proteínas Virales/genética , Secuencia de Bases , Secuencia Conservada/genética , Evolución Molecular , Genoma Viral , Infecciones por VIH/genética , Filogenia , Sistemas de Lectura/genética , Selección GenéticaRESUMEN
BACKGROUND: CD8+ T cells recognize HIV-1 epitopes translated from a gene's primary reading frame (F1) and any one of its five alternative reading frames (ARFs) in the forward (F2, F3) or reverse (R1-3) directions. The 3' end of HIV-1's proviral coding strand contains a conserved sequence that is directly overlapping but antiparallel to the env gene (ARF R2) and encodes for a putative antisense HIV-1 protein called ASP. ASP expression has been demonstrated in vitro using HIV-transfected cell lines or infected cells. Although antibodies to ASP were previously detected in patient sera, T cell recognition of ASP-derived epitopes has not been evaluated. We therefore investigated the ex vivo and in vitro induction of ASP-specific T cell responses as a measure of immune recognition and protein expression during HIV-1 infection. RESULTS: A panel of overlapping peptides was initially designed from the full-length ASP sequence to perform a global assessment of T cell responses. Recognition of ASP-derived antigens was evaluated in an IFN-γELISpot assay using PBMCs from HIV-1 seropositive and seronegative individuals. Eight of 25 patients had positive responses to ASP antigens and none of the seronegative donors responded. As a complimentary approach, a second set of antigens was designed using HLA-I binding motifs and affinities. Two ASP-derived peptides with high predicted binding affinities for HLA-A*02 (ASP-YL9) and HLA-B*07 (ASP-TL10) were tested using PBMCs from HIV-1 seropositive and seronegative individuals who expressed the matching HLA-I-restricting allele. We found that HLA-I-restricted ASP peptides were only recognized by CD8+ T cells from patients with the relevant HLA-I and did not induce responses in any of the seronegative donors or patients who do not express the restrictive HLA alleles. Further, ASP-YL9-specific CD8+ T cells had functional profiles that were similar to a previously described HLA-A*02-restricted epitope (Gag-SL9). Specific recognition of ASP-YL9 by CD8+ T cells was also demonstrated by tetramer staining using cells from an HLA-A*02 HIV-infected patient. CONCLUSION: Our results provide the first description of CD8+ T cell-mediated immune responses to ASP in HIV-1-infected patients, demonstrating that ASP is expressed during infection. Our identification of epitopes within ASP has implications for designing HIV vaccines.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Expresión Génica , Antígenos VIH/inmunología , VIH-1/inmunología , VIH-1/fisiología , Proteínas Virales/inmunología , Replicación Viral , Adulto , Anciano , Células Cultivadas , Estudios de Cohortes , Ensayo de Immunospot Ligado a Enzimas , Femenino , Antígenos VIH/biosíntesis , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Proteínas Virales/biosíntesisRESUMEN
To study the transcriptional activity of the HIV-1 LTR, we constructed a vector containing Renilla and Firefly luciferase genes under the control of the LTR (wild-type or mutated version) and oriented in a manner that allowed them to be transcribed in opposite directions. We found that the HIV-1 LTR acted as a bidirectional promoter, which activity was controlled by NF-κB- and Sp1-binding sites in both orientations. We next analyzed with this reporter vector the bidirectional promoter activity of the HTLV-1 LTR and showed that this LTR also possessed a bidirectional transcriptional activity. Interestingly, Sp1-binding elements were also involved in the control of HTLV-1 bidirectional transcription. Moreover, both retroviral trans-activators, Tat and Tax, could preferentially activate sense transcription with no or limited effect on the extent of antisense transcription. We also cloned into this plasmid the MLV LTR and found that the LTR of a simple retrovirus also possessed bidirectional transcriptional activity. This reporter vector represents a powerful tool to analyze the bidirectional transcriptional activity of retrovirus LTRs.
Asunto(s)
Genes Reporteros , Vectores Genéticos , VIH/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Secuencias Repetidas Terminales/genética , Activación Transcripcional/genética , Animales , Clonación Molecular , Productos del Gen tat/genética , Productos del Gen tax/genética , Humanos , Ratones , FN-kappa B/metabolismo , Células 3T3 NIH , Regiones Promotoras Genéticas , TransfecciónRESUMEN
In this study, an antisense luciferase-expressing human immunodeficiency virus type 1 (HIV-1) molecular clone was used to infect primary cells. We found that antisense transcription activity from the 3' long terminal repeat (LTR) was significantly more abundant in monocyte-derived cells than in activated T lymphocytes. Moreover, by analyzing antisense transcription in infected monocyte-derived dendritic cells (MDDCs), we observed that the majority of HIV-1-infected MDDCs with significant antisense transcription activity did not produce Gag. We also confirmed that the negative-strand-encoded antisense protein (ASP) was expressed in monocyte-derived cells.
Asunto(s)
VIH-1/genética , Monocitos/virología , ARN sin Sentido/genética , Transcripción Genética , Células Cultivadas , Genes Virales , Duplicado del Terminal Largo de VIH , HumanosRESUMEN
A determinant of human T-lymphotropic virus-1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) development is the HTLV-1-infected cell burden. Viral proteins Tax and HBZ, encoded by the sense and antisense strands of the pX region, respectively, play key roles in HTLV-1 persistence. Tax drives CD4(+)-T cell clonal expansion and is the immunodominant viral antigen recognized by the immune response. Valproate (2-n-propylpentanoic acid, VPA), a histone deacetylase inhibitor, was thought to trigger Tax expression, thereby exposing the latent HTLV-1 reservoir to immune destruction. We evaluated the impact of VPA on Tax, Gag, and HBZ expressions in cultured lymphocytes from HTLV-1 asymptomatic carriers and HAM/TSP patients. Approximately one-fifth of provirus-positive CD4(+) T cells spontaneously became Tax-positive, but this fraction rose to two-thirds of Tax-positive-infected cells when cultured with VPA. Valproate enhanced Gag-p19 release. Tax- and Gag-mRNA levels peaked spontaneously, before declining concomitantly to HBZ-mRNA increase. VPA enhanced and prolonged Tax-mRNA expression, whereas it blocked HBZ expression. Our findings suggest that, in addition to modulating Tax expression, another mechanism involving HBZ repression might determine the outcome of VPA treatment on HTLV-1-infected-cell proliferation and survival.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/biosíntesis , Regulación Viral de la Expresión Génica/efectos de los fármacos , Productos del Gen tax/biosíntesis , Virus Linfotrópico T Tipo 1 Humano/fisiología , Ácido Valproico/farmacología , Proteínas Virales/biosíntesis , Elementos sin Sentido (Genética)/efectos de los fármacos , Apoptosis/efectos de los fármacos , Enfermedades Asintomáticas , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Células Cultivadas/efectos de los fármacos , Células Cultivadas/virología , Genes gag , Genes pX , Histona Acetiltransferasas/antagonistas & inhibidores , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/virología , Paraparesia Espástica Tropical , Provirus/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Viral/biosíntesis , ARN Viral/genética , Proteínas de los Retroviridae , Proteínas Virales/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/biosíntesisRESUMEN
BACKGROUND: Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. RESULTS: We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. CONCLUSION: These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.
Asunto(s)
Membrana Celular/virología , Regulación Viral de la Expresión Génica , Infecciones por VIH/virología , VIH-1/genética , ARN sin Sentido/genética , ARN Viral/genética , Linfocitos T/virología , Proteínas Virales/genética , Línea Celular , Membrana Celular/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Humanos , Mutación , Transporte de Proteínas , ARN sin Sentido/metabolismo , ARN Viral/metabolismo , Linfocitos T/metabolismo , Proteínas Virales/metabolismoRESUMEN
It was first predicted in 1988 that there may be an Open Reading Frame (ORF) on the negative strand of the Human Immunodeficiency Virus type 1 (HIV-1) genome that could encode a protein named AntiSense Protein (ASP). In spite of some controversy, reports began to emerge some years later describing the detection of HIV-1 antisense transcripts, the presence of ASP in transfected and infected cells, and the existence of an immune response targeting ASP. Recently, it was established that the asp gene is exclusively conserved within the pandemic group M of HIV-1. In this review, we summarize the latest findings on HIV-1 antisense transcripts and ASP, and we discuss their potential functions in HIV-1 infection together with the role played by antisense transcripts and ASPs in some other viruses. Finally, we suggest pathways raised by the study of antisense transcripts and ASPs that may warrant exploration in the future.
RESUMEN
The existence of an antisense Open Reading Frame (ORF) that encodes a putative AntiSense Protein (ASP) on the proviral genome of Human Immunodeficiency Virus type 1 (HIV-1) was a source of debate for 30 years. During the last years, some progresses have been made to characterize the cellular immune response against ASP in HIV-1 seropositive patients. However, no tools were available for the detection of antibodies to ASP in the plasma of HIV-1-infected patients during the natural course of the infection. The aim of our study was to develop a Luciferase Immuno-Precipitation System (LIPS) to monitor the quantitative detection of ASP-specific antibodies in the plasma of HIV-1-infected patients [antiretroviral therapy (ART) naive-patients, patients under ART and HIV-1 controllers], patients who discontinued antiretroviral drugs (ARV). We further used this approach to delineate the epitopes of ASP targeted by antibodies. Antibodies directed against ASP were detected in 3 out of 19 patients who discontinued ARV (15%) and in 1 out of 10 ART-naive patients (10%), but were neither detected in HIV-1 infected patients under ART nor in HIV-1 controllers. Individual variations in levels of ASP-specific antibodies were detected overtime. Both the conserved prolin-rich motif and the core 60-189 region of ASP were found to be essential for antibody recognition in the four patients tested positive for anti-ASP antibodies, who were all untreated at the time of sampling. Moreover, for two of these patients, increased levels of ASP-specific antibodies were observed concomitantly to viremia declines. Overall, our method may represent a useful tool to detect a humoral response to ASP in HIV-1-infected patients, which allowed us to confirm the expression of ASP during the course of HIV-1 infection. Further studies will be needed to fully characterize the humoral response to ASP in HIV-1-infected patients.
RESUMEN
Dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to infection with the intracellular bacteria Brucella. Infection with living Brucella prevents infected human DCs from engaging in maturation processes, thus impairing their capacity to present antigens to naïve T cells and to secrete IL-12. Recently, we have established that several attenuated mutants of Brucella (rough, omp25, bvrR) are unable to control DCs maturation and thus effectively stimulate naïve T cells, which could be the origin of the protective immunity elicited by these mutants in vivo. In this study, we investigate the interactions of a VirB-defective Brucella mutant with human DCs to determine whether its attenuation could be attributed to the induction of an adaptive immune response. We show here that in contrast to previously studied strains and similar to wild-type strains, this virB mutant was unable to trigger significant DC maturation. Together with recently published data describing infection with virB mutants in vivo, these results suggest that Brucella T4SS VirB is not involved in the control of DC maturation and does not interfere with the establishment of a T-helper type 1 adaptive immune response.
Asunto(s)
Brucella suis/inmunología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Proteínas de Transporte de Membrana/inmunología , Factores de Virulencia/inmunología , Brucella suis/genética , Proliferación Celular , Eliminación de Gen , Humanos , Proteínas de Transporte de Membrana/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factores de Virulencia/genéticaRESUMEN
Most HIV-1 Tat is unconventionally secreted by infected cells following Tat interaction with phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane. Extracellular Tat is endocytosed by uninfected cells before escaping from endosomes to reach the cytosol and bind PI(4,5)P2. It is not clear whether and how incoming Tat concentrates in uninfected cells. Here we show that, in uninfected cells, the S-acyl transferase DHHC-20 together with the prolylisomerases cyclophilin A (CypA) and FKBP12 palmitoylate Tat on Cys31 thereby increasing Tat affinity for PI(4,5)P2. In infected cells, CypA is bound by HIV-1 Gag, resulting in its encapsidation and CypA depletion from cells. Because of the lack of this essential cofactor, Tat is not palmitoylated in infected cells but strongly secreted. Hence, Tat palmitoylation specifically takes place in uninfected cells. Moreover, palmitoylation is required for Tat to accumulate at the plasma membrane and affect PI(4,5)P2-dependent membrane traffic such as phagocytosis and neurosecretion.
Asunto(s)
Membrana Celular/metabolismo , Ciclofilina A/metabolismo , VIH-1/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Aciltransferasas/metabolismo , Animales , Animales Recién Nacidos , Membrana Celular/virología , Ciclofilina A/genética , Células HEK293 , VIH-1/fisiología , Humanos , Células Jurkat , Lipoilación , Ratones , Ratones Endogámicos C57BL , Células PC12 , Fosfatidilinositol 4,5-Difosfato/metabolismo , Unión Proteica , Células RAW 264.7 , RatasRESUMEN
Brucella is a facultative intracellular pathogen of various mammals and the etiological agent of brucellosis. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to Brucella infection. Furthermore, Brucella prevented the infected DCs from engaging in maturation processes and impaired their capacity to present antigen to naive T cells and to secrete interleukin-12 (IL-12). The lipopolysaccharide (LPS) phenotype is largely associated with the virulence of Brucella. Depending on whether they express the O-side chain of LPS or not, the bacteria display a smooth or rough phenotype. Rough Brucella mutants are attenuated and induce a potent protective T-cell-dependent immune response. Due to the essential role of DCs in the initiation of T-cell-dependent adaptive immune responses, it seemed pertinent to study the interaction between rough Brucella strains and human DCs. In the present paper, we report that, in contrast to smooth bacteria, infection of DCs with rough mutants of Brucella suis or Brucella abortus leads to both phenotypic and functional maturation of infected cells. Rough mutant-infected DCs then acquire the capacity to produce IL-12 and to stimulate naive CD4+ T lymphocytes. Experiments with rough and smooth purified LPS of Brucella supported the hypothesis of an indirect involvement of the O-side chain. These results provide new data concerning the role of LPS in Brucella virulence strategy and illuminate phenomena contributing to immune protection conferred by rough vaccine strains.
Asunto(s)
Brucella abortus/inmunología , Brucella suis/inmunología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Humanos , Interleucina-12/inmunología , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Antígenos O/química , Antígenos O/inmunología , Linfocitos T/inmunología , Células TH1/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Most macrophages remain uninfected in HIV-1-infected patients. Nevertheless, the phagocytic capacity of phagocytes from these patients is impaired, favouring the multiplication of opportunistic pathogens. The basis for this phagocytic defect is not known. HIV-1 Tat protein is efficiently secreted by infected cells. Secreted Tat can enter uninfected cells and reach their cytosol. Here we found that extracellular Tat, at the subnanomolar concentration present in the sera of HIV-1-infected patients, inhibits the phagocytosis of Mycobacterium avium or opsonized Toxoplasma gondii by human primary macrophages. This inhibition results from a defect in mannose- and Fcγ-receptor-mediated phagocytosis, respectively. Inhibition relies on the interaction of Tat with phosphatidylinositol (4,5)bisphosphate that interferes with the recruitment of Cdc42 to the phagocytic cup, thereby preventing Cdc42 activation and pseudopod elongation. Tat also inhibits FcγR-mediated phagocytosis in neutrophils and monocytes. This study provides a molecular basis for the phagocytic defects observed in uninfected phagocytes following HIV-1 infection.
Asunto(s)
VIH-1/fisiología , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacología , Efecto Espectador , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Lectinas Tipo C/antagonistas & inhibidores , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Macrófagos/ultraestructura , Receptor de Manosa , Lectinas de Unión a Manosa/antagonistas & inhibidores , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Monocitos/metabolismo , Monocitos/ultraestructura , Mycobacterium avium/crecimiento & desarrollo , Neutrófilos/metabolismo , Neutrófilos/ultraestructura , Fosfatidilinositol 4,5-Difosfato/antagonistas & inhibidores , Fosfatidilinositol 4,5-Difosfato/metabolismo , Cultivo Primario de Células , Transporte de Proteínas/efectos de los fármacos , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Seudópodos/ultraestructura , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/genética , Receptores de IgG/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Transducción de Señal , Toxoplasma/crecimiento & desarrollo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/biosíntesisRESUMEN
Pathogens have developed different strategies to survive and multiply within their host. Among them is the ability to control phagocyte apoptosis while another is to affect the expression of cytokines which is necessary for a normal protective function of the immune response. To establish themselves and cause chronic disease in humans and animals, Brucella spp. invade and proliferate within monocytic phagocytes. We have established that in humans, Brucella suis impairs the apoptosis of monocytes and macrophages, thus preventing its host cell elimination. In mice, which are not naturally colonized by the bacteria, Brucella infection results in Type1 (Th1) cellular immune response which promotes a clearance of the bacterial organism. The development of this response is under the control of major cytokines like TNF-alpha, IFN-gamma and IL-12 produced at the onset of infection. We have observed that in humans, B. suis-infected macrophages which produce IL-1, IL-6, IL-10 and several chemokines including IL-8, do not secrete TNF-alpha. By constructing null mutants, we demonstrated that this inhibition involves the outer membrane protein Omp25 of Brucella, however the mechanism regulating the inhibition has not yet been clearly defined. It is likely that the Omp25-induced effect on TNF-alpha production assists bacterial evasion of antimicrobial defences at different levels. Firstly, by preventing the autocrine activation of macrophages thus inhibiting innate immunity and secondly by impairing the production of IL-12 and the development of a Th1 type specific immunity. In addition to the central role of the macrophage in Brucella infection, others cells of the innate immune response are recruited and influenced by the interactions between bacteria and host. For instance, human Vgamma9Vdelta2 T-cells play an important role in the early response to infection with intracellular pathogens. Evidence has been presented that their number dramatically increased in the peripheral blood of patients with acute brucellosis. We have shown that human Vgamma9Vdelta2 T-cells can be specifically activated by non-peptidic low molecular weight compound(s) from B. suis lysate or by soluble factors produced by B. suis-infected macrophages. Under these conditions, they produce TNF-alpha and IFN-gamma and reduce the bacterial multiplication inside infected autologous macrophages. This impairment of B. suis multiplication is due to both soluble factors released from activated gammadeltaT-cells (including TNF-alpha and IFN-gamma) and to a contact-dependent cytotoxicity directed against the infected cells. The interactions between the bacteria and these cells can counteract the intramacrophagic development of the bacteria and finally influence the further development of the host defense. We hypothesize that the chronicity or the elimination of the infection will depend on the balance between contradictory effects induced by the bacteria which favor either the host or the pathogen. Moreover, the interrelationship between the different cells must be taken into account in the analysis of the virulence of the bacteria and in the development of in vitro models of human macrophage infection.
Asunto(s)
Brucella/inmunología , Brucelosis/inmunología , Inmunidad Innata , Animales , Apoptosis , Brucella/crecimiento & desarrollo , Brucella/patogenicidad , Brucella suis/inmunología , Humanos , Macrófagos/inmunología , Macrófagos/patología , Ratones , Monocitos/inmunología , Linfocitos T/inmunología , VirulenciaRESUMEN
BACKGROUND: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological inflammatory disease associated with a predominant infiltration of CD4+ T lymphocytes, which are the main subset of HTLV-1-infected cells. It has been demonstrated that in cell line the viral Tax protein transcriptionnally regulate expression of osteopontin, an inflammatory cytokine associated with Th17-related pathologies. OBJECTIVES: The aim of the study was to explore osteopontin expression in HTLV-1 asymptomatic carriers and in HAM/TSP patients and consequences on IL17 expression. STUDY DESIGN: We quantified Tax, osteopontin, RORγ, IL17 and IL22 mRNA expressions in cells from 10 HAM/TSP patients, 6 asymptomatic HTLV-1 carriers (ASY) and 4 HTLV-1-negative healthy donors during ex vivo culture. RESULTS: We observed that the expression of osteopontin was higher in HAM/TSP patients and correlated with Tax expression levels. Positive regulation of RORγ, IL17 and IL22 were also observed during cell culture. CONCLUSIONS: Our results propose a new mechanism which could contribute to HAM/TSP pathogenesis.
Asunto(s)
Portador Sano/patología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Interleucina-17/inmunología , Osteopontina/biosíntesis , Paraparesia Espástica Tropical/patología , Anciano , Enfermedades Asintomáticas , Linfocitos T CD4-Positivos/inmunología , Portador Sano/inmunología , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Productos del Gen tax/biosíntesis , Humanos , Masculino , Persona de Mediana Edad , Paraparesia Espástica Tropical/inmunologíaRESUMEN
To develop intracellularly within phagocytes and cause chronic infection, Brucella must overcome different steps of the host immune responses. IFNgamma is a key mediator of the innate and adaptive responses produced during Brucella infection. Therefore, Brucella would control host defenses by impairing macrophage responses to IFNgamma. We first showed that in infected human macrophages (VD3-differentiated THP-1 cells) Brucella escaped the microbicidal environment generated by IFNgamma. We then analyzed the IFNgamma-mediated signaling in Brucella-infected cells. We observed no decrease in STAT1 tyrosine or serine phosphorylation, or in dimerization of phosphorylated STAT1 (P-STAT1) and P-STAT1 translocation to the nucleus or in P-STAT1 binding to GAS, a minimal IFNgamma-response DNA sequence. In contrast, immuno-precipitation experiments indicated that the IFNgamma-mediated association of P-STAT1 with CBP/P300 transactivators was markedly reduced in infected macrophages, demonstrating that P-STAT1 was unable to normally recruit these transactivators. The host cell cAMP pathway triggered by Brucella could be responsible for this defect, CBP/P300 mobilization by phosphorylated CREB (P-CREB) disrupting the IFNgamma-induced STAT1-CBP/P300 association, required for a normal response of macrophages to IFNgamma. In any case, the inhibition of an essential protein-protein interaction probably lead to a deteriorated response to IFNgamma and thus participated in the pathogen's establishment within its host.
Asunto(s)
Brucella/patogenicidad , Proteína de Unión a CREB/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Interferón gamma/inmunología , Macrófagos/microbiología , Factor de Transcripción STAT1/metabolismo , Línea Celular , Humanos , Interferón gamma/metabolismo , Interferón gamma/farmacología , Macrófagos/inmunología , Macrófagos/metabolismo , Receptores de IgG/metabolismoRESUMEN
Brucella is a facultative intracellular pathogen and the etiological agent of brucellosis. In some cases, human brucellosis results in a persistent infection that may reactivate years after the initial exposure. The mechanisms by which the parasite evades clearance by the immune response to chronically infect its host are unknown. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to Brucella infection and are a preferential niche for the development of the bacteria. Here, we report that in contrast to several intracellular bacteria, Brucella prevented the infected DCs from engaging in their maturation process and impaired their capacities to present antigen to naïve T cells and to secrete interleukin-12. Moreover, Brucella-infected DCs failed to release tumor necrosis factor alpha (TNF-alpha), a defect involving the bacterial protein Omp25. Exogenous TNF-alpha addition to Brucella-infected DCs restored cell maturation and allowed them to present antigens. Two avirulent mutants of B. suis, B. suis bvrR and B. suis omp25 mutants, which do not express the Omp25 protein, triggered TNF-alpha production upon DC invasion. Cells infected with these mutants subsequently matured and acquired the ability to present antigens, two properties which were dramatically impaired by addition of anti-TNF-alpha antibodies. In light of these data, we propose a model in which virulent Brucella alters the maturation and functions of DCs through Omp25-dependent control of TNF-alpha production. This model defines a specific evasion strategy of the bacteria by which they can escape the immune response to chronically infect their host.
Asunto(s)
Presentación de Antígeno , Brucella suis/inmunología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Regulación de la Expresión Génica , Factor de Necrosis Tumoral alfa/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas Bacterianas/genética , Proliferación Celular , Células Cultivadas , Eliminación de Gen , Humanos , Interleucina-12/antagonistas & inhibidores , Interleucina-12/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Bacteria from the Brucella genus are able to survive and proliferate within macrophages. Because they are phylogenetically closely related to macrophages, myeloid dendritic cells (DCs) constitute potential targets for Brucella bacteria. Here we report that DCs display a great susceptibility to Brucella infection. Therefore, DCs might serve as a reservoir and be important for the development of Brucella bacteria within their host.
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Brucella abortus/patogenicidad , Brucella melitensis/patogenicidad , Brucella suis/patogenicidad , Células Dendríticas/microbiología , Brucelosis/inmunología , Brucelosis/microbiología , Células Dendríticas/inmunología , Células Dendríticas/ultraestructura , Humanos , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/ultraestructuraRESUMEN
By comparing smooth wild-type Brucella spp. to their rough mutants, we show that the LPS O chain restricted the activation of the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) pathways, thus preventing the synthesis of immune mediators that regulate host defense. We conclude that the MAPKs are a target for immune intervention by virulent smooth Brucella.
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Brucella/crecimiento & desarrollo , Brucella/patogenicidad , Activación Enzimática , Regulación de la Expresión Génica , Macrófagos/microbiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Brucella/clasificación , Brucelosis/microbiología , Línea Celular , Lipopolisacáridos/farmacología , Activación de Macrófagos , Ratones , Ratones Endogámicos BALB C , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fenotipo , Virulencia , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Brucella spp. are stealthy bacteria that enter host cells without major perturbation. The molecular mechanism involved is still poorly understood, although numerous studies have been published on this subject. Recently, it was reported that Brucella abortus utilizes cellular prion protein (PrP(C)) to enter the cells and to reach its replicative niche. The molecular mechanisms involved were not clearly defined, prompting us to analyze this process using blocking antibodies against PrP(C). However, the behavior of Brucella during cellular infection under these conditions was not modified. In a next step, the behavior of Brucella in macrophages lacking the prion gene and the infection of mice knocked out for the prion gene were studied. We observed no difference from results obtained with the wild-type control. Although some contacts between PrP(C) and Brucella were observed on the surface of the cells by using confocal microscopy, we could not show that Brucella specifically bound recombinant PrP(C). Therefore, we concluded from our results that prion protein (PrP(C)) was not involved in Brucella infection.