RESUMEN
IL-33 is an IL-1-related cytokine that can act as an alarmin when released from necrotic cells. Once released, it can target various immune cells including mast cells, innate lymphoid cells and T cells to elicit a Th2-like immune response. We show here that bone marrow-derived mast cells produce IL-13, IL-6, TNF, GM-CSF, CCL3 and CCL4 in response to IL-33 stimulation. Inhibition of the p38 MAPK, or inhibition or knockout of its downstream kinases MK2 and MK3, blocked the production of these cytokines in response to IL-33. The mechanism downstream of MK2/3 was cytokine specific; however, MK2 and MK3 were able to regulate TNF and GM-CSF mRNA stability. Previous studies in macrophages have shown that MK2 regulates mRNA stability via phosphorylation of the RNA-binding protein TTP (Zfp36). The regulation of cytokine production in mast cells was, however, independent of TTP. MK2/3 were able to phosphorylate the TTP-related protein Brf1 (Zfp36 l1) in IL-33-stimulated mast cells, suggesting a mechanism by which MK2/3 might control mRNA stability in these cells. In line with its ability to regulate in vitro IL-33-stimulated cytokine production, double knockout of MK2 and 3 in mice prevented neutrophil recruitment following intraperitoneal injection of IL-33.
Asunto(s)
Interleucina-33/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Infiltración Neutrófila/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Células Cultivadas , Citocinas/biosíntesis , Interleucina-33/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMEN
Phosphoinositide-dependent kinase-1 (PDK1) controls the activation of a subset of AGC kinases. Using a conditional knockout of PDK1 in haematopoietic cells, we demonstrate that PDK1 is essential for B cell development. B-cell progenitors lacking PDK1 arrested at the transition of pro-B to pre-B cells, due to a cell autonomous defect. Loss of PDK1 decreased the expression of the IgH chain in pro-B cells due to impaired recombination of the IgH distal variable segments, a process coordinated by the transcription factor Pax5. The expression of Pax5 in pre-B cells was decreased in PDK1 knockouts, which correlated with reduced expression of the Pax5 target genes IRF4, IRF8 and Aiolos. As a result, Ccnd3 is upregulated in PDK1 knockout pre-B cells and they have an impaired ability to undergo cell-cycle arrest, a necessary event for Ig light chain rearrangement. Instead, these cells underwent apoptosis that correlated with diminished expression of the pro-survival gene Bcl2A1. Reintroduction of both Pax5 and Bcl2A1 together into PDK1 knockout pro-B cells restored their ability to differentiate in vitro into mature B cells.
Asunto(s)
Linfocitos B/metabolismo , Puntos de Control del Ciclo Celular/fisiología , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinación V(D)J/fisiología , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Linfocitos B/citología , Ciclina D3/genética , Ciclina D3/metabolismo , Técnicas de Silenciamiento del Gen , Factor de Transcripción Ikaros , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Ratones , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
The polarization of macrophages into a regulatory-like phenotype and the production of IL-10 plays an important role in the resolution of inflammation. We show in this study that PGE(2), in combination with LPS, is able to promote an anti-inflammatory phenotype in macrophages characterized by high expression of IL-10 and the regulatory markers SPHK1 and LIGHT via a protein kinase A-dependent pathway. Both TLR agonists and PGE(2) promote the phosphorylation of the transcription factor CREB on Ser(133). However, although CREB regulates IL-10 transcription, the mutation of Ser(133) to Ala in the endogenous CREB gene did not prevent the ability of PGE(2) to promote IL-10 transcription. Instead, we demonstrate that protein kinase A regulates the phosphorylation of salt-inducible kinase 2 on Ser(343), inhibiting its ability to phosphorylate CREB-regulated transcription coactivator 3 in cells. This in turn allows CREB-regulated transcription coactivator 3 to translocate to the nucleus where it serves as a coactivator with the transcription factor CREB to induce IL-10 transcription. In line with this, we find that either genetic or pharmacological inhibition of salt-inducible kinases mimics the effect of PGE(2) on IL-10 production.
