Project description:IL-10 is produced by macrophages in diverse immune settings and is critical in limiting immune-mediated pathology. In helminth infections macrophages are an important source of IL-10, however the molecular mechanism underpinning production of IL-10 by these cells is poorly characterized. Here, bone marrow derived macrophages exposed to Excretory/Secretory (E/S) products released by Schistosoma mansoni cercariae rapidly produce IL-10 as a result of MyD88-mediated activation of MEK/ERK/RSK and p38. The phosphorylation of these kinases was triggered by TLR2 and TLR4 and converged on activation of the transcription factor CREB. Uptake of cercarial E/S products by phagocytosis was critical for the production of IL-10 and the activation of MAPKs and CREB, which indicates that interactions between E/S products with TLR2 and TLR4, may be occurring in endosomes. Following phosphorylation, CREB is recruited to a novel regulatory element in the Il10 promoter and is also responsible for regulating a network of genes involved in metabolic processes, such as glycolysis, the tricarboxylic acid cycle and oxidative phosphorylation. Moreover, skin resident tissue macrophages, which encounter S. mansoni E/S products during infection, are the first monocytes to produce IL-10 in vivo early after infection with S. mansoni cercariae. The early and rapid release of IL-10 by these cells has the potential to condition the dermal microenvironment encountered by immune cells recruited to this infection site. To conclude, we propose a mechanism by which CREB regulates the production of IL-10 by macrophages in the skin, but also has a major effect on their metabolic state. CREB binding sites 3 pooled samples of stimulated bone marrow macrophages
Project description:Pattern recognition receptors (PRRs) at the plasma membrane promote plant immunity through the detection of conserved microbe-associated molecular patterns (MAMPs). In plants, the PRR for bacterial flagellin (flg22) is encoded by the receptor kinase FLS2. One of the earliest MAMP responses is the rapid and transient increase of cytosolic calcium (Ca2+) ions, which is required for many of the well-described downstream responses, e.g. generation of reactive oxygen species (ROS) and the transcriptional activation of defence-associated genes. Despite its relevance, the molecular components regulating the Ca2+ burst remain largely unknown. Here, we show that the plasma membrane P2B-type Ca2+ ATPase ACA8 forms a dynamic complex with the PRR FLS2. ACA8 and its closest homologue ACA10 are required for immunity against virulent bacteria. Mutant aca8 aca10 plants are reduced in the flg22-induced Ca2+ burst, show reduced ROS production and exhibit altered transcriptional reprogramming. In particular, flg22-induced gene expression is elevated downstream of signalling mitogen-activated protein (MAP) kinases, but reduced downstream of the calcium-dependent protein (CDP) kinase cascade. These results demonstrate that the fine regulation of Ca2+ fluxes in the cytosol is critical for the coordination of the downstream MAMP responses and provide for the first time a link between the FLS2 receptor complex and signalling kinases via the secondary messenger Ca2+. ACA8 also interacted with the BRI1 and CLV1 receptor kinases, which correlated with the developmental phenotypes of aca8 aca10 mutants suggesting a broader role for Ca2+ ATPases in receptor-mediated signalling. We used Affymetrix Arabidopsis Tiling 1.0R Array to compare global transcript levels in 7 days-old sterile grown seedlings. Steady-state mRNA levels in total RNA samples of 7 days old sterile seedlings
Project description:Pathogenic Th17 cells play an important role in many autoimmune and inflammatory diseases. Their function is dependent on signaling through the T cell receptor (TCR) and cytokines that activate the transcription factor signal transducer and activator of transcription 3 (STAT3). TCR engagement activates stromal interaction molecule 1 (STIM1) and calcium (Ca2+) influx through the Ca2+ release-activated Ca2+ (CRAC) channel. We here show that deletion of STIM1 and Ca2+ influx in T cells expressing a hyperactive form of STAT3 (STAT3C) attenuates pathogenic Th17 cell function and multiorgan inflammation associated with STAT3C expression. Deletion of STIM1 in pathogenic Th17 cells impairs the expression of nuclear encoded mitochondrial electron transport chain genes and oxidative phosphorylation (OXPHOS) but enhances reactive oxygen species (ROS) production. Deletion of STIM1 or inhibition of OXPHOS is associated with impaired Th17 cell function and a non-pathogenic Th17 gene expression signature. Our findings establish STIM1 and Ca2+ signals as a critical regulator of OXPHOS and oxidative stress in pathogenic Th17 cells and multiorgan inflammation.
Project description:Reactive oxygen species (ROS) are important messengers in eukaryotic organisms. Extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase (RLK)-dependent signaling networks. This ROS production is tightly controlled by multiple mechanisms including phosphorylation by different kinases. Here we show that the cysteine-rich RLK CRK2 exists in a preformed complex with the NADPH oxidase RBOHD at the plasma membrane. Functional CRK2 is required for the full pathogen-induced ROS burst and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae. We identified phosphorylation sites in the C-terminal region of RBOHD and mutations of the phosphorylation sites modulate ROS production in response to biotic stimuli. Our work demonstrates that CRK2 occupies a central role in controlling ROS production and highlights that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region is an ancient mechanism which is conserved between animals and plants.
Project description:IL-10 is produced by macrophages in diverse immune settings and is critical in limiting immune-mediated pathology. In helminth infections macrophages are an important source of IL-10, however the molecular mechanism underpinning production of IL-10 by these cells is poorly characterized. Here, bone marrow derived macrophages exposed to Excretory/Secretory (E/S) products released by Schistosoma mansoni cercariae rapidly produce IL-10 as a result of MyD88-mediated activation of MEK/ERK/RSK and p38. The phosphorylation of these kinases was triggered by TLR2 and TLR4 and converged on activation of the transcription factor CREB. Uptake of cercarial E/S products by phagocytosis was critical for the production of IL-10 and the activation of MAPKs and CREB, which indicates that interactions between E/S products with TLR2 and TLR4, may be occurring in endosomes. Following phosphorylation, CREB is recruited to a novel regulatory element in the Il10 promoter and is also responsible for regulating a network of genes involved in metabolic processes, such as glycolysis, the tricarboxylic acid cycle and oxidative phosphorylation. Moreover, skin resident tissue macrophages, which encounter S. mansoni E/S products during infection, are the first monocytes to produce IL-10 in vivo early after infection with S. mansoni cercariae. The early and rapid release of IL-10 by these cells has the potential to condition the dermal microenvironment encountered by immune cells recruited to this infection site. To conclude, we propose a mechanism by which CREB regulates the production of IL-10 by macrophages in the skin, but also has a major effect on their metabolic state.
Project description:Pattern recognition receptors (PRRs) at the plasma membrane promote plant immunity through the detection of conserved microbe-associated molecular patterns (MAMPs). In plants, the PRR for bacterial flagellin (flg22) is encoded by the receptor kinase FLS2. One of the earliest MAMP responses is the rapid and transient increase of cytosolic calcium (Ca2+) ions, which is required for many of the well-described downstream responses, e.g. generation of reactive oxygen species (ROS) and the transcriptional activation of defence-associated genes. Despite its relevance, the molecular components regulating the Ca2+ burst remain largely unknown. Here, we show that the plasma membrane P2B-type Ca2+ ATPase ACA8 forms a dynamic complex with the PRR FLS2. ACA8 and its closest homologue ACA10 are required for immunity against virulent bacteria. Mutant aca8 aca10 plants are reduced in the flg22-induced Ca2+ burst, show reduced ROS production and exhibit altered transcriptional reprogramming. In particular, flg22-induced gene expression is elevated downstream of signalling mitogen-activated protein (MAP) kinases, but reduced downstream of the calcium-dependent protein (CDP) kinase cascade. These results demonstrate that the fine regulation of Ca2+ fluxes in the cytosol is critical for the coordination of the downstream MAMP responses and provide for the first time a link between the FLS2 receptor complex and signalling kinases via the secondary messenger Ca2+. ACA8 also interacted with the BRI1 and CLV1 receptor kinases, which correlated with the developmental phenotypes of aca8 aca10 mutants suggesting a broader role for Ca2+ ATPases in receptor-mediated signalling. We used Affymetrix Arabidopsis Tiling 1.0R Array to compare global transcript levels in 7 days-old sterile grown seedlings.