A type I IFN-dependent DNA damage response regulates the genetic program and inflammasome activation in macrophages

Macrophages produce genotoxic agents, such as reactive oxygen and nitrogen species, that kill invading pathogens. Here we show that these agents activate the DNA damage response (DDR) kinases ATM and DNA-PKcs through the generation of double stranded breaks (DSBs) in murine macrophage genomic DNA. In contrast to other cell types, initiation of this DDR depends on signaling from the type I interferon receptor. Once activated, ATM and DNA-PKcs regulate a genetic program with diverse immune functions and promote inflammasome activation and the production of IL-1β and IL-18. Indeed, following infection with Listeria monocytogenes, DNA-PKcs-deficient murine macrophages produce reduced levels of IL-18 and are unable to optimally stimulate IFN-γ production by NK cells. Thus, genomic DNA DSBs act as signaling intermediates in murine macrophages, regulating innate immune responses through the initiation of a type I IFN-dependent DDR.</jats:p

MECANISMES DE REGULATION DE L'EXPRESSION DE LA PHOSPHOLIPASE A2 SECRETEE DE GROUPE IIA PAR LES ACIDES GRAS ET LEURS METABOLITES DANS LES MACROPHAGES ALVEOLAIRES (DOCTORAT MICROBIOLOGIE)

CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Autophagy and Inflammasome activation triggered by LPS-negative <i>Ehrlichia</i> is dependent on both mTOR activation and MyD88 signaling

Abstract Lipopolysaccharide (LPS) positive pathogens trigger autophagy and inflammasome activation. Autophagy is an innate anti-microbial host defense that maintains tissue homeostasis and is regulated by the nutrient-sensing mTORC1 complex. However it is unknown how the LPS-negative, Gram negative Ehrlichia elicits the same response. Monocytic Ehrlichia triggers Type I IFN-dependent NLRP3 inflammasome activation, which detrimentally effects host response to Ehrlichia. In this study, we examined the regulatory mechanisms of autophagy and inflammasome activation. With toll like receptor (TLR)-adaptor molecule MYD88 deficient mice, we reveal a crucial role for MYD88 in mediation of Ehrlichia-dependent inflammasome activation and hepatic inflammation. This role is demonstrated by decreased Caspase 1 and IL-1β levels in infected liver tissues, and decreased inflammatory cytokines (TNF-α and IL-6) when compared to wild type. Ehrlichia-induced inflammasome activation in wild type mice correlated with inhibition of autophagy. Ehrlichia-induced inhibition of autophagy in infected macrophages was dependent on TLR9, TLR2, and their adaptor MYD88. Inhibition was enhanced by IFN-β stimulation. We also demonstrate that mTORC1 autophagy inhibition acts downstream of MYD88. These data suggest that LPS-negative Ehrlichia inhibit autophagy and activate the inflammasome through mTORC1 activation and MYD88 signaling, contributing to Ehrlichia-induced inflammatory disregulation and liver injury.</jats:p

Ionic Strength- and Temperature-Induced K(Ca) Shifts in the Uncoating Reaction of Rotavirus Strains RF and SA11: Correlation with Membrane Permeabilization

The hydrodynamic diameters of native rotavirus particles, bovine RF and simian SA11 strains, were determined by quasielastic light scattering. By using this method and agarose gel electrophoresis, the Ca(2+) dissociation constant, K(Ca), governing the transition from triple-layer particles (TLPs) to double-layer particles (DLPs), was shown to increase, at constant pH, as the temperature and/or the ionic strength of the incubation medium increased. We report the novel observation that, under physiological conditions, K(Ca) values for both RF and SA11 rotaviruses were well above the intracytoplasmic Ca(2+) concentrations of various cells, which may explain why TLP uncoating takes place within vesicles (possibly endosomes) during the entry process. A correlation between TLP uncoating and cell membrane permeabilization was found, as shown by the release of carboxyfluorescein (CF) from CF-loaded intestinal brush-border membrane vesicles. Conditions stabilizing the virion in the TLP form inhibited CF release, whereas conditions favoring the TLP-to-DLP transformation activated this process. We conclude that membrane permeabilization must be preceded by the loss of the outer-capsid proteins from trypsinized TLP and that physiological ionic strength is required for permeabilization to take place. Finally, the paper develops an alternative explanation for the mechanism of rotavirus entry, compatible with the Ca(2+)-dependent endocytic pathway. We propose that there must be an iterative process involving tight coupling in time between the lowering of endosomal Ca(2+) concentration, virion decapsidation, and membrane permeabilization, which would cause the transcriptionally active DLPs to enter the cytoplasm of cells

Effect of surfactant on pulmonary expression of type IIA PLA 2 in an animal model of acute lung injury

International audienceWe previously showed that the seminatural surfactant Curosurf inhibits the in vitro synthesis of secretory type IIA phospholipase A(2) (sPLA(2)-IIA) in alveolar macrophages (AM). These cells are the main source of sPLA(2)-IIA in a guinea pig model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Here, we investigate the effect of Curosurf on the pulmonary synthesis of sPLA(2)-IIA in this ALI model. Our results showed that intratracheal administration of LPS (330 microg/kg) induced an increase in pulmonary expression of sPLA(2)-IIA, which was inhibited when animals received Curosurf (16 mg/guinea pig) 30 min or 8 h after LPS instillation. When AM were isolated from LPS-treated animals and cultured in conditioned medium, they expressed higher levels of sPLA(2)-IIA than AM from saline-treated animals. This ex vivo sPLA(2)-IIA expression was significantly reduced when guinea pigs received Curosurf 30 min after LPS instillation. Finally, we examined the effect of Curosurf on pulmonary inflammation measured 8 or 24 h after LPS administration. Curosurf instillation 30 min or 8 h after LPS reversed the increase in tumor necrosis factor-alpha expression, polymorphonuclear cell extravasation, and protein concentration in bronchoalveolar lavage fluids. Curosurf also decreased the bronchial reactivity induced by LPS. We conclude that Curosurf inhibits the pulmonary expression of sPLA(2)-IIA and exhibits palliative anti-inflammatory effects in an animal model of ALI

Arachidonic acid differentially affects basal and lipopolysaccharide-induced sPLA2-IIA expression in alveolar macrophages through NF-kappa B and PPAR-gamma -dependent pathways

ABSTRACT Secretory type IIA phospholipase A 2 (sPLA 2 -IIA) is a critical enzyme involved in inflammatory diseases. We have previously identified alveolar macrophages (AMs) as the major pulmonary source of lipopolysaccharide (LPS)-induced sPLA 2 -IIA expression in a guinea pig model of acute lung injury (ALI). Here, we examined the role of arachidonic acid (AA) in the regulation of basal and LPS-induced sPLA 2 -IIA expression in AMs. We showed that both AA and its nonmetabolizable analog, 5,8,11,14-eicosatetraynoic acid (ETYA), inhibited sPLA 2 -IIA synthesis in unstimulated AMs. However, only AA inhibited sPLA 2 -IIA expression in LPS-stimulated cells, suggesting that this effect requires metabolic conversion of AA. Indeed, cyclooxygenase inhibitors abolished this down-regulation. Prostaglandins PGE 2 , PGA 2 , and 15d-PGJ 2 also inhibited the LPSinduced sPLA 2 -IIA expression. Nuclear factor-B (NF-B) was found to regulate sPLA 2 -IIA expression in AMs. Both AA and ETYA inhibited basal activation of NF-B but had no effect on LPS-induced NF-B translocation, suggesting that suppression of sPLA 2 -IIA synthesis by AA in LPS-stimulated cells occurs via a NF-B-independent pathway. 15-Deoxy-⌬ 12,14 -PGJ 2 and ciglitazone, which are, respectively, natural and synthetic ligands for peroxisome proliferator-activated receptor-␥ (PPAR-␥), inhibited LPS-induced sPLA 2 -IIA synthesis, whereas PPAR-␣ ligands were ineffective. Moreover, electrophoretic mobility shift assay showed PPAR activation by AA and PPAR-␥ ligands in LPS-stimulated AMs. Our results suggest that the down-regulation of basal sPLA 2 -IIA expression is unrelated to the metabolic conversion of AA but is dependent on the impairment of NF-B activation. In contrast, the inhibition of LPS-stimulated sPLA 2 -IIA expression is mediated by cyclooxygenase-derived metabolites of AA and involves a PPAR-␥-dependent pathway. These findings provide new insights for the treatment of ALI

Arachidonic Acid Differentially Affects Basal and Lipopolysaccharide-Induced sPLA 2 -IIA Expression in Alveolar Macrophages through NF-κB and PPAR-γ–Dependent Pathways

International audienceSecretory type IIA phospholipase A(2) (sPLA(2)-IIA) is a critical enzyme involved in inflammatory diseases. We have previously identified alveolar macrophages (AMs) as the major pulmonary source of lipopolysaccharide (LPS)-induced sPLA(2)-IIA expression in a guinea pig model of acute lung injury (ALI). Here, we examined the role of arachidonic acid (AA) in the regulation of basal and LPS-induced sPLA(2)-IIA expression in AMs. We showed that both AA and its nonmetabolizable analog, 5,8,11,14-eicosatetraynoic acid (ETYA), inhibited sPLA(2)-IIA synthesis in unstimulated AMs. However, only AA inhibited sPLA(2)-IIA expression in LPS-stimulated cells, suggesting that this effect requires metabolic conversion of AA. Indeed, cyclooxygenase inhibitors abolished this down-regulation. Prostaglandins PGE(2), PGA(2), and 15d-PGJ(2) also inhibited the LPS-induced sPLA(2)-IIA expression. Nuclear factor-kappaB (NF-kappaB) was found to regulate sPLA(2)-IIA expression in AMs. Both AA and ETYA inhibited basal activation of NF-kappaB but had no effect on LPS-induced NF-kappaB translocation, suggesting that suppression of sPLA(2)-IIA synthesis by AA in LPS-stimulated cells occurs via a NF-kappaB-independent pathway. 15-Deoxy-Delta(12,14)-PGJ(2) and ciglitazone, which are, respectively, natural and synthetic ligands for peroxisome proliferator-activated receptor-gamma (PPAR-gamma), inhibited LPS-induced sPLA(2)-IIA synthesis, whereas PPAR-alpha ligands were ineffective. Moreover, electrophoretic mobility shift assay showed PPAR activation by AA and PPAR-gamma ligands in LPS-stimulated AMs. Our results suggest that the down-regulation of basal sPLA(2)-IIA expression is unrelated to the metabolic conversion of AA but is dependent on the impairment of NF-kappaB activation. In contrast, the inhibition of LPS-stimulated sPLA(2)-IIA expression is mediated by cyclooxygenase-derived metabolites of AA and involves a PPAR-gamma-dependent pathway. These findings provide new insights for the treatment of ALI

c-Jun NH(2)-Terminal Kinase-Mediated Signaling Is Essential for Pseudomonas aeruginosa ExoS-Induced Apoptosis

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa mainly affects immunocompromised individuals as well as patients with cystic fibrosis. In a previous study, we showed that ExoS of P. aeruginosa, when injected into host cells through a type III secretion apparatus, functions as an effector molecule to trigger apoptosis in various tissue culture cells. Here, we show that injection of the ExoS into HeLa cells activates c-Jun NH(2)-terminal kinase (JNK) phosphorylation while shutting down ERK1/2 and p38 phosphorylation. Inhibiting JNK activation by expression of a dominant negative JNK1 or with a specific JNK inhibitor abolishes ExoS-triggered apoptosis, demonstrating the requirement for JNK-mediated signaling. Following JNK phosphorylation, cytochrome c is released into the cytosol, leading to the activation of caspase 9 and eventually caspase 3. Although c-Jun phosphorylation is also observed as a result of JNK activation, ongoing host protein synthesis is not essential for the apoptotic induction, suggesting that c-Jun- or other AP-1-driven activation of gene expression is dispensable in this process. Therefore, ExoS has opposing effects on different cellular pathways that regulate apoptosis: it shuts down host cell survival signal pathways by inhibiting ERK1/2 and p38 activation, and it activates proapoptotic pathways through activation of JNK1/2 leading ultimately to cytochrome c release and activation of caspases. These results highlight the modulation of host cell signaling by the type III secretion system during interaction between P. aeruginosa and host cells