TLR9 activation induces normal neutrophil responses in a child with IRAK-4 deficiency: involvement of the direct PI3K pathway.

International audiencePolymorphonuclear neutrophils (PMN) play a key role in innate immunity. Their activation and survival are tightly regulated by microbial products via pattern recognition receptors such as TLRs, which mediate recruitment of the IL-1R-associated kinase (IRAK) complex. We describe a new inherited IRAK-4 deficiency in a child with recurrent pyogenic bacterial infections. Analysis of the IRAK4 gene showed compound heterozygosity with two mutations: a missense mutation in the death domain of the protein (pArg12Cys) associated in cis-with a predicted benign variant (pArg391His); and a splice site mutation in intron 7 that led to the skipping of exon 7. A nontruncated IRAK-4 protein was detected by Western blotting. The patient's functional deficiency of IRAK-4 protein was confirmed by the absence of IRAK-1 phosphorylation after stimulation with all TLR agonists tested. The patient's PMNs showed strongly impaired responses (L-selectin and CD11b expression, oxidative burst, cytokine production, cell survival) to TLR agonists which engage TLR1/2, TLR2/6, TLR4, and TLR7/8; in contrast, the patient's PMN responses to CpG-DNA (TLR9) were normal, except for cytokine production. The surprisingly normal effect of CpG-DNA on PMN functions and apoptosis disappeared after pretreatment with PI3K inhibitors. Together, these results suggest the existence of an IRAK-4-independent TLR9-induced transduction pathway leading to PI3K activation. This alternative pathway may play a key role in PMN control of infections by microorganisms other than pyogenic bacteria in inherited IRAK-4 deficiency

European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies

Peptide-Based Inhibitors Of The Phagocyte Nadph Oxidase

International audiencePhagocytes such as neutrophils, monocytes and macrophages play an essential role in host defenses against pathogens. To kill these pathogens, phagocytes produce and release large quantities of antimicrobial molecules such as reactive oxygen species (ROS), microbicidal peptides, and proteases. The enzyme responsible for ROS generation is called NADPH oxidase, or respiratory burst oxidase, and is composed of six proteins: gp91phox, p22phox, p47phox, p67phox, p40phox and Rac1/2. The vital importance of this enzyme in host defenses is illustrated by a genetic disorder called chronic granulomatous disease (CGD), in which the phagocyte NADPH oxidase is dysfunctional, leading to life-threatening recurrent bacterial and fungal infections. However, excessive NADPH oxidase activation and ROS overproduction can damage surrounding tissues and participate in exaggerated inflammatory processes. As ROS production is believed to be involved in several inflammatory diseases, specific phagocyte NADPH oxidase inhibitors might have therapeutic value. In this commentary, we summarize the structure and activation of the phagocyte NADPH oxidase, and describe pharmacological inhibitors of this enzyme, with particular emphasis on peptide-based inhibitors derived from gp91phox, p22phox and p47phox

Starting‐NOX2‐Up: Rac unrolls p67 phox

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Live or Die: PD-L1 delays neutrophil apoptosis

International audienceIn this issue of Blood, Wang et al. uncover a key role for programmed death ligand 1 (PD-L1), which delays neutrophil apoptosis at the inflammatory site through activation of the PI3K-AKT survival pathway 1. Polymorphonuclear neutrophils (PMN) are the most abundant circulating leukocytes, constituting 60 to 70% of circulating white blood cells. PMN are terminally differentiated and have a short life span, but are essential for innate immunity and host defense against microbes 2. They are the first cells to migrate out of the circulation and to be massively recruited at the infection site where they recognize microbes via different receptors expressed at their cell surface, inducing engulfment of the microbe into a phagosome 3,4. Killing of microbes by PMN occurs through the release into the phagosome of highly toxic agents such as reactive oxygen species (ROS) and granule contents including myeloperoxidase, glucosidases, proteases and anti-bacterial peptides. Microbes can also be trapped and killed by neutrophil extracellular traps (NETs) 5. Many processes such as apoptosis, NETosis, autophagy, pyroptosis, necroptosis and necrosis can induce the death of neutrophils 5 , upon which they are phagocytized and eliminated by local macrophages through a process called efferocytosis, resulting in the cleaning up of the infection site. Thus, PMN are critical anti-inflammatory components of the innate immune system as their physiological role i

Immunochemical identification and translocation of protein kinase C zeta in human neutrophils

AbstractWestern blots of human polymorphonuclear leukocyte (PMN) extracts were immunostained with antibodies specific for various protein kinase C (PKC) isoforms. Two bands corresponding to PKC type ξ with apparent molecular masses of 81 kDa and 76 kDa were identified in the cytosolic fraction of resting cells, in addition to PKC types α and β. PKCξ was apparently abundant, like PKCβ, whereas PKCδ, -ε, and -γ were not detectable. Following short stimulation (5 min) of PMN with phorbol-12-myristate-13-acetate (1 μ/ml), physical translocation of PKCξ from the cytosol to the plasma membrane fraction occurred, although this isoform does not bind phorbol esters. These data show that, in addition to the two calcium-dependent isoenzymes α and β, human PMN express a calcium-independent isoenzyme ξ which translocates in stimulated cells, suggesting a role in the regulation of antibacterial activities

Oleuropein and hydroxytyrosol inhibit the N-formyl-methionyl-leucyl-phenylalanine-induced neutrophil degranulation and chemotaxis via AKT, p38, and ERK1/2 MAP-Kinase inhibition

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