Polymorphonuclear neutrophils are the most numerous circulating leukocytes and play an important role in innate immunity and inflammation. During phagocytosis, neutrophils are activated to produce high quantities of superoxide anion which generates other reactive oxygen species (ROS) such hydrogen peroxide, hydroxyl radical and hypochlorous acid. The enzyme responsible for superoxide anion production by neutrophils is called the phagocyte NADPH oxidase. This enzyme complex is composed of two membrane-bound proteins, p22phox and gp91phox/NOX2 and four cytosolic proteins, p47phox, p67phox, p40phox and Rac2. In resting cells, these proteins are distributed between the cytosol and membranes of granules and the plasma membrane. Upon activation, p47phox, p67phox, p40phox, p22phox and gp91phox are phosphorylated, Rac2 is activated and the system assembles into an active complex. ROS production is required for host defense, as illustrated by the genetic immunodeficiency disease called chronic granulomatous disease (CGD) in which phagocytes do not produce ROS and affected patients have more fatal infections than healthy individuals. CGD is a rare disease found in 1 of 200,000 to 250,000 individuals. It is caused by a gene mutation in one of the NADPH oxidase components, the most frequent CGD form being caused by mutations in the gp91phox/CYBB gene (65%), followed by mutation in the p47phox/NCF1 gene (20%), the p22phox/CYBA gene (less than 5%), the p67phox/NCF2 gene (less than 5%), the p40phox/NCF4 and Rac2/NCF3 genes (both forms less than 5%). In addition to host defense, ROS production can participate to tissue injury and excessive inflammation if not well controlled. Thus, activation of the phagocyte NADPH oxidase must be tightly regulated to ensure that ROS are produced only when and where required. One mechanism by which the NADPH oxidase is regulated is the phosphorylation of p47phox. However the pathways which regulate p47phox phosphorylation are not completely known.
In the study published in Blood*, we used phospho-specific antibodies against five major p47phox-phosphorylated sites (Ser304, Ser315, Ser320, Ser328 and Ser345) developed by our team and found that in neutrophils isolated from p67phox-deficient CGD patients (p67phox-/-) phosphorylation of p47phox on serine residues was dramatically reduced. This result was confirmed in Epstein-Barr virus (EBV)-transformed B- lymphocytes from p67phox-/- CGD patients and In COSphox cells transfected with all the NADPH oxidase components except p67phox. 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, the use of neutrophils from p67phox-/- CGD patients uncovers that 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.
*Belambri SA, Marzailoli V, Hurtado-Nedelec M, Pintard C, Liang S, Liu Y, Boussetta T, Gougerot-Pocidalo MA, Ye RD, Dang PM, El Benna J. Impaired p47phox phosphorylation in neutrophils from p67phox-deficient chronic granulomatous disease patients. Blood. 2022 Feb 2:blood.2021011134. doi: 10.1182/blood.2021011134. Epub ahead of print. PMID: 35108370.