Prolyl-Isomerase Pin1 Controls Key fMLP-Induced Neutrophil Functions

International audienceNeutrophils are key cells of the innate immune and inflammatory responses. They are the first blood cells to migrate to the infection site where they release high amounts of reactive oxygen species (ROS) and several peptides and enzymes required for microbial killing. However, excessive neutrophil activation can induce tissue injury participating in inflammation, thus the characterization of the enzymes involved in neutrophil activation could help to identify new pharmacological targets to treat inflammation. The prolyl-isomerase Pin1 is a ubiquitous enzyme involved in several functions, however, its role in neutrophil functions is less known. In this study, we show that the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP or fMLF), a G-protein coupled receptor (GPCR) agonist-induced Pin1 activation in human neutrophils. PiB and juglone, two Pin1 inhibitors inhibited Pin1 activity in neutrophils and consequently inhibited fMLP-induced chemotaxis and -degranulation of azurophil and specific granules as measured by myeloperoxidase and neutrophil gelatinase-associated lipocalin (NGAL) release respectively. We also showed that PiB inhibited TNFα + fMLP-induced superoxide production, confirming the effect of juglone. These data show that inhibitors of Pin1 impaired key pro-inflammatory neutrophil functions elicited by GPCR activation and suggest that Pin1 could control neutrophil inflammatory functions

Eugenol prevents fMLF-induced superoxide anion production in human neutrophils by inhibiting ERK1/2 signaling pathway and p47phox phosphorylation

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Activation of the phagocyte NADPH oxidase/NOX2 and myeloperoxidase in the mouse brain during pilocarpine-induced temporal lobe epilepsy and inhibition by ketamine

International audienceExcessive reactive oxygen species (ROS) production can induce tissue injury involved in a variety of neurodegenerative disorders such as neurodegeneration observed in pilocarpine-induced temporal lobe epilepsy. Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist has beneficial effects in pilocarpine-induced temporal lobe epilepsy when administered within minutes of seizure to avoid the harmful neurological lesions induced by pilocarpine. However, the enzymes involved in ROS productions and the effect of ketamine on this process remain less documented. Here we show that during pilocarpine-induced epilepsy in mice, the expression of the phagocyte NADPH oxidase NOX2 subunits (NOX2/gp91 phox , p22 phox , and p47 phox) and the expression of myeloperoxidase (MPO) were dramatically increased in mice brain treated with pilocarpine. Interestingly, treatment of mice with ketamine before or after pilocarpine administration decreased this process mainly when injected before pilocarpine. Finally, our results showed that pilocarpine induced p47 phox phosphorylation and H 2 O 2 production in mice brain and ketamine was able to inhibit these processes. Our results show that pilocarpine-induced NOX2 activation to produce ROS in mice brain and that administration of ketamine before or after the induction of temporal lobe epilepsy by pilocarpine inhibited this activation in mice brain. These results suggest a key role of the phagocyte NADPH oxidase NOX2 and MPO in epilepsy and identify a novel effect of ketamine

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The data used to produce the figure 6 are presented in this tabl

Using long-term experimental evolution to uncover the patterns and determinants of molecular evolution of an Escherichia coli natural isolate in the streptomycin-treated mouse gut.

International audienceAlthough microbial ecology of the gut is now a major focus of interest, little is known about the molecular determinants of microbial adaptation in the gut. Experimental evolution coupled with whole-genome sequencing can provide insights of the adaptive process. In vitro experiments have revealed some conserved patterns: intermediate convergence, and epistatic interactions between beneficial mutations and mutations in global regulators. To test the relevance of these patterns and to identify the selective pressures acting in vivo, we have performed a long-term adaptation of an E. coli natural isolate, the streptomycin-resistant strain 536, in the digestive tract of streptomycin-treated mice. After a year of evolution, a clone from 15 replicates was sequenced. Consistently with in vitro observations, the identified mutations revealed a strong pattern of convergence at the mutation, gene, operon and functional levels. Yet, the rate of molecular evolution was lower than in in vitro, and no mutations in global regulators were recovered. More specific targets were observed: the dgo operon, involved in the galactonate pathway that improved growth on D-galactonate, and rluD and gidB, implicated in the maturation of the ribosomes, which mutations improved growth only in the presence of streptomycin. As in vitro, the nonrandom associations of mutations within the same pathways suggested a role of epistasis in shaping the adaptive landscape. Overall, we show that 'evolve and sequence' approach coupled with an analysis of convergence, when applied to a natural isolate, can be used to study adaptation in vivo and uncover the specific selective pressures of that environment

TNFα counteracts interleukin-10 anti-inflammatory pathway through the NOX2-Lyn-SHP-1 axis in human monocytes

TNFα-mediated signaling pathways play a pivotal role in the pathogenesis of inflammatory diseases such as rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) by promoting phagocyte inflammatory functions, notably cytokine release and reactive oxygen species (ROS) production by NOX2. In contrast, interleukin-10 (IL-10), a powerful anti-inflammatory cytokine, potently shuts down phagocyte activation, making IL-10 an attractive therapeutic candidate. However, IL-10 therapy has shown limited efficacy in patients with inflammatory diseases. Here, we report that TNFα blocks IL-10 anti-inflammatory pathways in human monocytes, thereby prolonging inflammation. TNFα decreased IL-10-induced phosphorylation of STAT3 and consequently IL-10-induced expression of the major anti-inflammatory factor, SOCS3. Decreased STAT3 phosphorylation was due to a SHP1/2 phosphatase, as NSC-87877, a SHP1/2 inhibitor, restored STAT3 phosphorylation and prevented the TNFα-induced inhibition of IL-10 signaling. TNFα activated only SHP1 in human monocytes and this activation was NOX2-dependent, as diphenyleneiodonium, a NOX2 inhibitor, suppressed SHP1 activation and STAT3 dephosphorylation triggered by TNFα. ROS-induced activation of SHP1 was mediated by the redox-sensitive kinase, Lyn, as its inhibition impeded TNFα-induced SHP1 activation and STAT3 dephosphorylation. Furthermore, H2O2 recapitulated TNFα-inhibitory activity on IL-10 signaling. Finally, NSC-87877 dampened collagen antibody-induced arthritis (CAIA) in mice. These results reveal that TNFα disrupts IL-10 signaling by inducing STAT3 dephosphorylation through a NOX2-ROS-Lyn-SHP1 axis in human monocytes and that inhibition of SHP1/2 in vivo protects against CAIA. These new findings might explain the poor efficacy of IL-10 therapy in patients with inflammatory diseases and suggest that anti-TNFα agents and SHP1/2 inhibitors could improve the therapeutic use of IL-10

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Quantification of E. coli 536 in the feces in each mouse along the experimen

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The data used to produce the box plots of the figure 5 are presented in this tabl

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The results of flux cytometry are presented in this tabl

Impaired p47phox phosphorylation in neutrophils from p67phox-deficient chronic granulomatous disease patients Short title: p67phox regulates p47phox phosphorylation

International audienceAbstract Superoxide production by the phagocyte reduced NAD phosphate (NADPH) oxidase is essential for innate immunity as shown in chronic granulomatous disease (CGD), an immunodeficiency disease resulting from mutations in 1 of its genes. The NADPH oxidase is composed of 2 membrane proteins (gp91phox/NOX2 and p22phox) and 4 cytosolic proteins (p47phox, p67phox, p40phox, and Rac1/2). The phosphorylation of p47phox is required for NADPH oxidase activation in cells. As p47phox and p67phox can form a tight complex in cells, we hypothesized that p67phox could regulate p47phox phosphorylation. To investigate this hypothesis, we used phospho-specific antibodies against 5 major p47phox-phosphorylated sites (Ser304, Ser315, Ser320, Ser328, and Ser345) and neutrophils from healthy donors and from p67phox−/− CGD patients. Results showed that formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate induced a time- and a concentration-dependent phosphorylation of p47phox on Ser304, Ser315, Ser320, and Ser328 in healthy human neutrophils. Interestingly, in neutrophils and Epstein-Barr virus-transformed B lymphocytes from p67phox−/− CGD patients, phosphorylation of p47phox on serine residues was dramatically reduced. In COSphox cells, the presence of p67phox led to increased phosphorylation of p47phox. In vitro studies showed that recombinant p47phox was phosphorylated on Ser304, Ser315, Ser320, and Ser328 by different PKC isoforms and the addition of recombinant p67phox alone or in combination with p40phox potentiated this process. Thus, p67phox and p40phox are required for optimal p47phox phosphorylation on Ser304, Ser315, Ser320, and Ser328 in intact cells. Therefore, p67phox and p40phox are novel regulators of p47phox-phosphorylation