RESUMEN
Streptococcus pneumoniae pneumolysin (PLY) is a virulence factor that causes toxic effects contributing to pneumococcal pneumonia. To date, deriving a PLY candidate vaccine with the appropriate detoxification and immune profile has been challenging. A pneumolysin protein that is appropriately detoxified and that retains its immunogenicity is a desirable vaccine candidate. In this study, we assessed the protective efficacy of our novel PlyD1 detoxified PLY variant and investigated its underlying mechanism of protection. Results have shown that PlyD1 immunization protected mice against lethal intranasal (i.n.) challenge with pneumococci and lung injury mediated by PLY challenge. Protection was associated with PlyD1-specific IgG titers and in vitro neutralization titers. Pretreatment of PLY with PlyD1-specific rat polyclonal antiserum prior to i.n. delivery of toxin reduced PLY-mediated lung lesions, interleukin-6 (IL-6) production, and neutrophil infiltration into lungs, indicating that protection from lung lesions induced by PLY is antibody mediated. Preincubation of PLY with a neutralizing monoclonal PLY antibody also specifically reduced the cytotoxic effects of PLY after i.n. inoculation in comparison to nonneutralizing monoclonal antibodies. These results indicate that the induction of neutralizing antibodies against PLY can contribute to protection against bacterial pneumonia by preventing the development of PLY-induced lung lesions and inflammation. Our detoxified PlyD1 antigen elicits such PLY neutralizing antibodies, thus serving as a candidate vaccine antigen for the prevention of pneumococcal pneumonia.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Anticuerpos Neutralizantes/sangre , Vacunas Bacterianas , Lesión Pulmonar/prevención & control , Neumonía Neumocócica/prevención & control , Estreptolisinas/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Líquido del Lavado Bronquioalveolar , Femenino , Lesión Pulmonar/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Streptococcus pneumoniae/metabolismo , Estreptolisinas/químicaRESUMEN
Hematopoietic development is controlled by combinatorial interactions between E-protein transcription factors and other lineage regulators that operate in the context of gene-regulatory networks. The E-proteins HEB and E2A are critical for T cell and B cell development, but the mechanisms by which their activities are directed to different genes in each lineage are unclear. We found that a short form of HEB, HEBAlt, acts downstream of Delta-like (DL)-Notch signaling to promote T cell development. In this paper, we show that forced expression of HEBAlt in mouse hematopoietic progenitors inhibited B cell development, but it allowed them to adopt a myeloid fate. HEBAlt interfered with the activity of E2A homodimers and with the expression of the transcription factor Pax5, both of which are critical for B cell development. However, when combined with DL-Notch signaling, HEBAlt enhanced the generation of T cell progenitors at the expense of myeloid cells. The longer form of HEB, HEBCan, also inhibited E47 activity and Pax5 expression, but it did not collaborate with DL-Notch signaling to suppress myeloid potential. Therefore, HEBAlt can suppress B cell or myeloid potential in a context-specific manner, which suggests a role for this factor in maintaining T lineage priming prior to commitment.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Linaje de la Célula/genética , Regulación de la Expresión Génica/inmunología , Células Madre Hematopoyéticas/citología , Linfopoyesis/genética , Linfocitos T/citología , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Expresión Génica , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Linfopoyesis/inmunología , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/inmunología , Factor de Transcripción PAX5/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/inmunología , Factores de Transcripción TCF/metabolismo , Proteína 1 Similar al Factor de Transcripción 7RESUMEN
Hematopoiesis is controlled by the interplay between transcription factors and environmental signals. One of the primary determinants of the T-lineage choice is Delta-like (DL)-Notch signaling, which promotes T-cell development and inhibits B-cell development. We have found that the transcription factor HEBAlt is up-regulated in early hematopoietic precursors in response to DL-Notch signaling and that it can promote early T-cell development. Here, we identified a population of lineage-negative Sca-1â»c-kit(+) (LK) cells in the mouse fetal liver that rapidly gave rise to myeloid cells and B cells but exhibited very little T-cell potential. However, forced expression of HEBAlt in these precursors restored their ability to develop into T cells. We also showed that Ikaros and Notch1 are up-regulated in response to HEBAlt over-expression and that activated Notch1 enhances the ability of LK cells to enter the T-cell lineage. Furthermore, the myeloid transcription factor C/EBPα is down-regulated in response to HEBAlt. We therefore propose that HEBAlt plays a role in the network that enforces the T-lineage fate and limits myeloid fate during hematopoiesis.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Hematopoyesis/inmunología , Linfocitos T/citología , Animales , Antígenos Ly , Diferenciación Celular , Linaje de la Célula/inmunología , Feto/inmunología , Factor de Transcripción Ikaros/inmunología , Factor de Transcripción Ikaros/metabolismo , Hígado/embriología , Hígado/inmunología , Proteínas de la Membrana/deficiencia , Ratones , Células Progenitoras Mieloides/inmunología , Células Precursoras de Linfocitos T/inmunología , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Receptor Notch1/metabolismo , Regulación hacia ArribaRESUMEN
The basic helix-loop-helix (bHLH) transcription factors HEB and E2A are critical mediators of gene regulation during lymphocyte development. We have cloned a new transcription factor, called HEBAlt, from a pro-T cell cDNA library. HEBAlt is generated by alternative transcriptional initiation and splicing from the HEB gene locus, which also encodes the previously characterized E box protein HEBCan. HEBAlt contains a unique N-terminal coding exon (the Alt domain) that replaces the first transactivation domain of HEBCan. Downstream of the Alt domain, HEBAlt is identical to HEBCan, including the DNA binding domain. HEBAlt is induced in early thymocyte precursors and down-regulated permanently at the double negative to double positive (DP) transition, whereas HEBCan mRNA expression peaks at the DP stage of thymocyte development. HEBAlt mRNA is up-regulated synergistically by a combination of HEBCan activity and Delta-Notch signaling. Retroviral transduction of HEBAlt or HEBCan into hemopoietic stem cells followed by OP9-DL1 coculture revealed that HEBAlt-transduced precursors generated more early T lineage precursors and more DP pre-T cells than control transduced cells. By contrast, HEBCan-transduced cells that maintained high level expression of the HEBCan transgene were inhibited in expansion and progression through T cell development. HEB(-/-) fetal liver precursors transduced with HEBAlt were rescued from delayed T cell specification, but HEBCan-transduced HEB(-/-) precursors were not. Therefore, HEBAlt and HEBCan are functionally distinct transcription factors, and HEBAlt is specifically required for the efficient generation of early T cell precursors.