CRISPR-gene-engineered CYBB knock-out PLB-985 cells, a useful model to study functional impact of X-linked chronic granulomatous disease mutations: application to the G412E X91+-CGD mutation

International audienceAbstract Chronic granulomatous disease (CGD) is a rare primary immune disorder caused by mutations in one of the five subunits of the NADPH oxidase complex expressed in phagocytes. Two-thirds of CGD cases are caused by mutations in CYBB that encodes NOX2 or gp91phox. Some rare X91+-CGD point mutations lead to a loss of function but with a normal expression of the mutated NOX2 protein. It is therefore necessary to ensure that this mutation is indeed responsible for the loss of activity in order to make a safe diagnosis for genetic counselling. We previously used the X-CGD PLB-985 cell model of M.C. Dinauer obtained by homologous recombination in the original PLB-985 human myeloid cell line, in order to study the functional impact of such mutations. Although the PLB-985 cell line was originally described by K.A. Tucker et al. in1987 as a distinct cell line isolated from a patient with acute nonlymphocytic leukemia, it is actually identified as a subclone of the HL-60 cells. In order to use a cellular model that meets the quality standard for the functional study of X91+-CGD mutations in CGD diagnosis, we developed our own model using the CRISPR-Cas9 technology in a certified PLB-985 cell line from DSMZ-German Collection of Microorganisms and Cell Cultures. Thanks to this new X-CGD model, we demonstrated that the G412E mutation in NOX2 found in a X91+-CGD patient prohibits access of the electron donor NADPH to its binding site explaining the absence of superoxide production in his neutrophils

Identification of NOX2 regions for normal biosynthesis of cytochrome b558 in phagocytes highlighting essential residues for p22phox binding.

International audienceCytochrome b558, the redox core of the NADPH oxidase (NOX) complex in phagocytes, is composed of NOX2 and p22phox, the synthesis of which is intimately connected but not fully understood. We reproduced 10 rare X-minus chronic granulomatous disease (CGD) mutations of highly conserved residues in NOX1-NOX4, in X0-CGD PLB-985 cells in order to analyse their impact on the synthesis of cytochrome b558. According to the impact of these mutations on the level of expression of NADPH oxidase 2 (NOX2) and its activity, mutants were categorized into group A (W18C, E309K, K315del and I325F), characterized by a linear relationship between NOX2 expression and NOX activity, and group B (H338Y, P339H, G389A and F656-F570del), showing an absence of NOX activity associated with variable levels of NOX2 expression. These last residues belong to the FAD-binding pocket of NOX2, suggesting that this functional domain also plays a role in the structural integrity of NOX2. Finally, we observed an abnormal accumulation of p65 (65-kDa monomer), the NOX2 precursor and p65-p22phox dissociation in the W18C, E309K, I325F and G389A mutants, pointing out a possible role of the first transmembrane domain (Trp18), and the region between the membrane and the dehydrogenase domain of NOX2 (Glu309, Ile325 and Gly389), in the binding with p22phox

NOX4 is the main NADPH oxidase involved in the early stages of hematopoietic differentiation from human induced pluripotent stem cells

International audienceReactive oxygen species (ROS) produced in hematopoietic stem cells (HSCs) are involved in the balance between quiescence, self-renewal, proliferation and differentiation processes. However the role of NOX enzymes on the early stages of hematopoietic differentiation is poorly investigated. For that, we used induced pluripotent stem cells (iPSCs) derived from X-linked Chronic Granulomatous Disease (X0CGD) patients with deficiency in NOX2, and AR220CGD patients with deficiency in p22phox subunit which decreases NOX1, NOX2, NOX3 and NOX4 activities. CD34+ hematopoietic progenitors were obtained after 7, 10 and 13 days of iPS/OP9 co-culture differentiation system. Neither NOX expression nor activity was found in Wild-type (WT), X0CGD and AR220CGD iPSCs. Although NOX2 and NOX4 mRNA were found in WT, X0CGD and AR220CGD iPSC-derived CD34+ cells at day 10 and 13 of differentiation, NOX4 protein was the only NOX enzyme expressed in these cells. A NADPH oxidase activity was measured in WT and X0CGD iPSC-derived CD34+ cells but not in AR220CGD iPSC-derived CD34+ cells because of the absence of p22phox, which is essential for the NOX4 activity. The absence of NOX4 activity and the poor NOX-independent ROS production in AR220CGD iPSC-derived CD34+ cells favored the CD34+ cells production but lowered their hematopoietic potential compared to WT and X0CGD iPSC-derived CD34+ cells. In addition we found a large production of primitive AR220CGD iPSC-derived progenitors at day 7 compared to the WT and X0CGD cell types. In conclusion NOX4 is the major NOX enzyme involved in the early stages of hematopoietic differentiation from iPSCs and its activity can modulate the production, the hematopoietic potential and the phenotype of iPSC-derived CD34+

NOX4 is the main NADPH oxidase involved in the early stages of hematopoietic differentiation from human induced pluripotent stem cells

International audienceReactive oxygen species (ROS) produced in hematopoietic stem cells (HSCs) are involved in the balance between quiescence, self-renewal, proliferation and differentiation processes. However the role of NOX enzymes on the early stages of hematopoietic differentiation is poorly investigated. For that, we used induced pluripotent stem cells (iPSCs) derived from X-linked Chronic Granulomatous Disease (X0CGD) patients with deficiency in NOX2, and AR220CGD patients with deficiency in p22phox subunit which decreases NOX1, NOX2, NOX3 and NOX4 activities. CD34+ hematopoietic progenitors were obtained after 7, 10 and 13 days of iPS/OP9 co-culture differentiation system. Neither NOX expression nor activity was found in Wild-type (WT), X0CGD and AR220CGD iPSCs. Although NOX2 and NOX4 mRNA were found in WT, X0CGD and AR220CGD iPSC-derived CD34+ cells at day 10 and 13 of differentiation, NOX4 protein was the only NOX enzyme expressed in these cells. A NADPH oxidase activity was measured in WT and X0CGD iPSC-derived CD34+ cells but not in AR220CGD iPSC-derived CD34+ cells because of the absence of p22phox, which is essential for the NOX4 activity. The absence of NOX4 activity and the poor NOX-independent ROS production in AR220CGD iPSC-derived CD34+ cells favored the CD34+ cells production but lowered their hematopoietic potential compared to WT and X0CGD iPSC-derived CD34+ cells. In addition we found a large production of primitive AR220CGD iPSC-derived progenitors at day 7 compared to the WT and X0CGD cell types. In conclusion NOX4 is the major NOX enzyme involved in the early stages of hematopoietic differentiation from iPSCs and its activity can modulate the production, the hematopoietic potential and the phenotype of iPSC-derived CD34+

Differential impact of glucose levels and advanced glycation end-products on tubular cell viability and pro-inflammatory/profibrotic functions

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Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

The X+-linked chronic granulomatous disease (X+-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X+-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X+-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2

Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase.

International audienceFlavocytochrome b(558) (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22(phox) and gp91(phox). The latter subunit, also known as Nox2, has a cytosolic C-terminal "dehydrogenase domain" containing FAD/NADPH-binding sites. The N-terminal half of Nox2 contains six predicted transmembrane α-helices coordinating two hemes. We studied the role of the second transmembrane α-helix, which contains a "hot spot" for mutations found in rare X(+) and X(-) chronic granulomatous disease. By site-directed mutagenesis and transfection in X-CGD PLB-985 cells, we examined the functional and structural impact of seven missense mutations affecting five residues. P56L and C59F mutations drastically influence the level of Nox2 expression indicating that these residues are important for the structural stability of Nox2. A53D, R54G, R54M, and R54S mutations do not affect spectral properties of oxidized/reduced cytb, oxidase complex assembly, FAD binding, nor iodonitrotetrazolium (INT) reductase (diaphorase) activity but inhibit superoxide production. This suggests that Ala-53 and Arg-54 are essential in control of electron transfer from FAD. Surprisingly, the A57E mutation partially inhibits FAD binding, diaphorase activity, and oxidase assembly and affects the affinity of immunopurified A57E cytochrome b(558) for p67(phox). By competition experiments, we demonstrated that the second transmembrane helix impacts on the function of the first intracytosolic B-loop in the control of diaphorase activity of Nox2. Finally, by comparing INT reductase activity of immunopurified mutated and wild type cytb under aerobiosis versus anaerobiosis, we showed that INT reduction reflects the electron transfer from NADPH to FAD only in the absence of superoxide production

Characterization of superoxide overproduction by the D-Loop(Nox4)-Nox2 cytochrome b(558) in phagocytes-Differential sensitivity to calcium and phosphorylation events.

International audienceNADPH oxidase is a crucial element of phagocytes involved in microbicidal mechanisms. It becomes active when membrane-bound cytochrome b(558), the redox core, is assembled with cytosolic p47(phox), p67(phox), p40(phox), and rac proteins to produce superoxide, the precursor for generation of toxic reactive oxygen species. In a previous study, we demonstrated that the potential second intracellular loop of Nox2 was essential to maintaining NADPH oxidase activity by controlling electron transfer from FAD to O(2). Moreover, replacement of this loop by the Nox4-D-loop (D-loop(Nox4)-Nox2) in PLB-985 cells induced superoxide overproduction. In the present investigation, we demonstrated that both soluble and particulate stimuli were able to induce this superoxide overproduction. Superoxide overproduction was also observed after phosphatidic acid activation in a purified cell-free-system assay. The highest oxidase activity was obtained after ionomycin and fMLF stimulation. In addition, enhanced sensitivity to Ca(2+) influx was shown by thapsigargin, EDTA, or BTP2 treatment before fMLF activation. Mutated cytochrome b(558) was less dependent on phosphorylation triggered by ERK1/2 during fMLF or PMA stimulation and by PI3K during OpZ stimulation. The superoxide overproduction of the D-loop(Nox4)-Nox2 mutant may come from a change of responsiveness to intracellular Ca(2+) level and to phosphorylation events during oxidase activation. Finally the D-loop(Nox4)-Nox2-PLB-985 cells were more effective against an attenuated strain of Pseudomonas aeruginosa compared to WT-Nox2 cells. The killing mechanism was biphasic, an early step of ROS production that was directly bactericidal, and a second oxidase-independent step related to the amount of ROS produced in the first step

MC1R expression in HaCaT keratinocytes inhibits UVA-induced ROS production via NADPH oxidase- and cAMP-dependent mechanisms.

International audienceUltraviolet A (UVA) radiations are responsible for deleterious effects, mainly due to reactive oxygen species (ROS) production. Alpha-melanocyte stimulating hormone (α-MSH) binds to melanocortin-1 receptor (MC1R) in melanocytes to stimulate pigmentation and modulate cutaneous inflammatory responses. MC1R may be induced in keratinocytes after UV exposure. To investigate the effect of MC1R signaling on UVA-induced ROS (UVA-ROS) production, we generated HaCaT cells that stably express human MC1R (HaCaT-MC1R) or the Arg151Cys (R(151)C) non-functional variant (HaCaT-R(151)C). We then assessed ROS production immediately after UVA exposure and found that: (1) UVA-ROS production was strongly reduced in HaCaT-MC1R but not in HaCaT-R(151)C cells compared to parental HaCaT cells; (2) this inhibitory effect was further amplified by incubation of HaCaT-MC1R cells with α-MSH before UVA exposure; (3) protein kinase A (PKA)-dependent NoxA1 phosphorylation was increased in HaCaT-MC1R compared to HaCaT and HaCaT-R(151)C cells. Inhibition of PKA in HaCaT-MC1R cells resulted in a marked increase of ROS production after UVA irradiation; (4) the ability of HaCaT-MC1R cells to produce UVA-ROS was restored by inhibiting epidermal growth factor receptor (EGFR) or extracellular signal-regulated kinases (ERK) activity before UVA exposure. Our findings suggest that constitutive activity of MC1R in keratinocytes may reduce UVA-induced oxidative stress via EGFR and cAMP-dependent mechanisms

Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation

NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47 and p67 to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the V of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486