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    The explosive duo PCNA-p47phox controls neutrophil’s fate: a promising target for inflammatory disease treatment

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    A study directed by Véronique Witko-Sarsat

    The team directed by Véronique Witko-Sarsat publishes in Journal of Experimental Medicine the discovery of a promising target of anti-inflammatory treatments.

    The neutrophil is a key cell in the defense against the bacteria it kills through the generation of very powerful mediators such as the reactive oxygen species produced in large quantities by NADPH oxidase (NOX2) during what has been called the "respiratory burst". If the activation of neutrophils and its removal from the inflammatory site are not controlled, the inflammation persists and the neutrophil becomes a dangerous and deleterious actor for the tissues.
    The work of Véronique Witko-Sarsat's team has shown that the cytoplasmic proliferating cell nuclear antigen (PCNA) is associated with the p47phox protein thereby controlling the NADPH oxidase activity and the survival of neutrophils.
    From a therapeutic point of view, the targeting of the PCNA-p47phox interaction in the neutrophils, allows to have a powerful anti-inflammatory effect in a mouse model of colitis, promoting the resolution of inflammation and decreasing oxidative stress.

    Neutrophils account for 70% of circulating leukocytes and are major players in innate immunity. Historically, neutrophils were studied mainly for their function of phagocytosis and destruction of bacteria. It is now known that neutrophils are endowed with immunomodulatory capacity and that they are involved in many non-infectious pathologies, in particular in cancer, autoimmune, cardiovascular and allergic diseases.

    Neutrophils are the first cells that migrate to the inflammatory site to destroy bacteria by means of powerful molecules that are reactive oxygen species generated in huge quantities by the activation of NADPH oxidase which has been called "respiratory burst". Their mission accomplished, neutrophils must be eliminated by macrophages. Neutrophil apoptosis, a programmed cell death that prevents the release of neutrophilic toxins, is tightly controlled to limit the destruction of nearby tissues. The phagocytosis of apoptotic neutrophils by macrophages allows these macrophages to synthesize many anti-inflammatory mediators. If these steps are deregulated, chronic inflammation sets in.

    Mechanisms for coordinating neutrophil activation and apoptosis, essential for the resolution of inflammation, are poorly understood. The work of Véronique Witko-Sarsat's team has shown that proliferating cell nuclear antigen (PCNA) is an essential key in this coordination.

    PCNA, named the "Maestro of Replication" is an ancient protein whose ring-shaped trimeric structure is highly conserved in evolution. In eukaryotic cells, PCNA was well-known as an exclusively nuclear "scaffolding" protein with the function of assembling other proteins to orchestrate DNA replication. About ten years ago, Véronique Witko-Sarsat's team described the unexpected presence of PCNA in the neutrophil cytoplasm, where it promotes its survival by inhibiting apoptosis via procaspase sequestration (1).

    In the recent work of the team, proteomic analysis of the PCNA interactome in the cytosol of neutrophils made it possible to identify numerous partner proteins including the components of NADPH oxidase. Structural, molecular and functional analysis shed light on the specific association of PCNA with p47phox. Molecular modeling techniques allowed to identify at the molecular level with atomic precision the respective sites of interaction between PCNA and P47phox. Meticulous dissection at the cellular level allowed to demonstrate that PCNA participates in the assembly of NADPH oxidase and in the production of ROS. Finally, in a model of colitis in mice in which neutrophils play a major role in tissue lesions, the inhibition of the PCNA-p47phox interaction by a small molecule, T2AA, was shown to decrease very significantly the presence of neutrophils and the inflammatory lesions.

    This work therefore has obvious clinical implications since it provides the proof of concept that targeting PCNA in neutrophils has a powerful anti-inflammatory therapeutic effect, favoring resolution of inflammation and reduction of oxidative stress.

    This study has been carried out in collaboration with the team of Jamel El-Benna (Bichat Hospital, Paris), the team of Philippe Frachet (Institute of Structural Biology, Grenoble) and the platforms of the Institut Cochin (Imaging of the living animals, histology). 

     

    Figure legend

    In neutrophils, p47phox protein which is a cytosolic component of NADPH oxidase is associated with PCNA (left panel). The murine model of TNBS (Tetra-Nitro-Benzene Sulfonic acid)-induced colitis is characterized by an acute inflammation dominated by neutrophils whic generate huge amounts of ROS. T2AA by interfering with the PCNA-p47phox interaction has a potent anti-oxidant and anti-inflammatory effect characterized by an accelerated healing of intestinal epithelium (Right panel).

     

    Reference

    Ohayon D, De Chiara A, Dang PM, Thieblemont N, Chatfield S, Marzaioli V, Burgener SS, Mocek J, Candalh C, Pintard C, Tacnet-Delorme P, Renault G, Lagoutte I, Favier M, Walker F, Hurtado-Nedelec M, Desplancq D, Weiss E, Benarafa C, Housset D, Marie JC, Frachet P, El-Benna J, Witko-Sarsat V. Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils. J Exp Med. 2019 Sep 6. pii: jem.20180371.

     

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    (1) Witko-Sarsat V, Mocek J, Bouayad D, Tamassia N, Ribeil JA, Candalh C, Davezac N, Reuter N, Mouthon L, Hermine O, Pederzoli-Ribeil M, Cassatella MA. Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival. J Exp Med. 2010 Nov 22;207(12):2631-45.