Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome

Abstract Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40phox by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40phox and p67phox at the phagosomal membrane. Pharmacologic inhibition of PI(3)P synthesis at the phagosome decreased the ROS production both in differentiated PLB-985 cells and human neutrophils. It also releases p67phox, the key cytosolic subunit of the oxidase, and p40phox from the phagosome. The knockdown of the PI(3)P phosphatase MTM1 or Rubicon or both increases the level of PI(3)P at the phagosome. That increase enhances ROS production inside the phagosome and triggers an extended accumulation of p67phox at the phagosome. Furthermore, the overexpression of MTM1 at the phagosomal membrane induces the disappearance of PI(3)P from the phagosome and prevents sustained ROS production. In conclusion, PI(3)P, indeed, regulates ROS production by maintaining p40phox and p67phox at the phagosomal membrane.</jats:p

Distinct timing of neutrophil spreading and stiffening during phagocytosis

International audiencePhagocytic cells form the first line of defense in an organism, engulfing microbial pathogens. Phagocytosis involves cell mechanical changes that are not yet well understood. Understanding these mechanical modifications promises to shed light on the immune processes that trigger pathological complications. Previous studies showed that phagocytes undergo a sequence of spreading events around their target followed by an increase in cell tension. Seemingly in contradiction, other studies observed an increase in cell tension concomitant with membrane expansion. Even though phagocytes are viscoelastic, few studies have quantified viscous changes during phagocytosis. It is also unclear whether cell lines behave mechanically similarly to primary neutrophils. We addressed the question of simultaneous versus sequential spreading and mechanical changes during phagocytosis by using immunoglobulin-G-coated 8-and 20-mm-diameter beads as targets. We used a micropipettebased single-cell rheometer to monitor viscoelastic properties during phagocytosis by both neutrophil-like PLB cells and primary human neutrophils. We show that the faster expansion of PLB cells on larger beads is a geometrical effect reflecting a constant advancing speed of the phagocytic cup. Cells become stiffer on 20-than on 8-mm beads, and the relative timing of spreading and stiffening of PLB cells depends on target size: on larger beads, stiffening starts before maximal spreading area is reached but ends after reaching maximal area. On smaller beads, the stiffness begins to increase after cells have engulfed the bead. Similar to PLB cells, primary cells become stiffer on larger beads but start spreading and stiffen faster, and the stiffening begins before the end of spreading on both bead sizes. Our results show that mechanical changes in phagocytes are not a direct consequence of cell spreading and that models of phagocytosis should be amended to account for causes of cell stiffening other than membrane expansion

Radiation-induced reactive oxygen species partially assemble neutrophil NADPH oxidase

International audienceNeutrophils are key cells from the innate immune system that destroy invading bacteria or viruses, thanks mainly to the non-mitochondrial reactive oxygen species (ROS) generated by the enzyme NADPH oxidase. Our aim was to study the response of neutrophils to situations of oxidative stress with emphasis on the impact on the NADPH oxidase complex. To mimic oxidative stress, we used gamma irradiation that generated ROS (OH•, O2•- and H2O2) in a quantitative controlled manner. We showed that, although irradiation induces shorter half-lives of neutrophil (reduced by at least a factor of 2), it triggers a pre-activation of surviving neutrophils. This is detectable by the production of a small but significant amount of superoxide anions, proportional to the dose (about 3 times that of sham). Investigations at the molecular level showed that this ROS increase was generated by the NADPH oxidase enzyme after neutrophils irradiation. The NADPH oxidase complex undergoes an incomplete assembly which includes p47phox and p67phox but excludes the G-protein Rac. Importantly, this irradiation-induced pre-activation is capable of considerably improving neutrophil reactivity. Indeed, we have observed that this leads to an increase in the production of ROS and the capacity of phagocytosis, leading to the conclusion that radiation induced ROS clearly behave as neutrophil primers

Class I phosphoinositide 3-kinases control sustained NADPH oxidase activation in adherent neutrophils

Place: Oxford Publisher: Pergamon-Elsevier Science Ltd WOS:000551658300034Phagocytes, especially neutrophils, can produce reactive oxygen species (ROS), through the activation of the NADPH oxidase (NOX2). Although this enzyme is crucial for host-pathogen defense, ROS production by neutrophils can be harmful in several pathologies such as cardiovascular diseases or chronic pulmonary diseases. The ROS production by NOX2 involves the assembly of the cytosolic subunits (p67(phox), p47(phox), and p40(phox)) and Rac with the membrane subunits (gp91(phox) and p22(phox)). Many studies are devoted to the activation of NOX2. However, the mechanisms that cause NADPH oxidase deactivation and thus terminate ROS production are not well known. Here we investigated the ability of class I phosphoinositide 3-kinases (PI3Ks) to sustain NADPH oxidase activation. The NADPH oxidase activation was triggered by seeding neutrophil-like PLB-985 cells, or human neutrophils on immobilized fibrinogen. Adhesion of the neutrophils, mediated by beta 2 integrins, induced activation of the NADPH oxidase and translocation of the cytosolic subunits at the plasma membrane. Inhibition of class I PI3Ks, and especially PI3K beta, terminated ROS production. This deactivation of NOX2 is due to the release of the cytosolic subunits, p67(phox) and p47(phox) from the plasma membrane. Overexpression of an active form of Rac 1 did not prevent the drop of ROS production upon inhibition of class I PI3Ks. Moreover, the phosphorylation of p47(phox) at S328, a potential target of kinases activated by the PI3K pathway, was unchanged. Our results indicate that the experimental downregulation of class I PI3K products triggers the plasma membrane NADPH oxidase deactivation. Release of p47(phox) from the plasma membrane may involve its PX domains that bind PI3K products

Membrane Dynamics and Organization of the Phagocyte NADPH Oxidase in PLB-985 Cells

Publisher: FrontiersNeutrophils are the first cells recruited at the site of infections, where they phagocytose the pathogens. Inside the phagosome, pathogens are killed by proteolytic enzymes that are delivered to the phagosome following granule fusion, and by reactive oxygen species (ROS) produced by the NADPH oxidase. The NADPH oxidase complex comprises membrane proteins (NOX2 and p22phox), cytoplasmic subunits (p67phox, p47phox, p40phox) and the small GTPase Rac. These subunits assemble at the phagosomal membrane upon phagocytosis. In resting neutrophils the catalytic subunit NOX2 is mainly present at the plasma membrane and in the specific granules. We show here that NOX2 is also present in early and recycling endosomes in human neutrophils and in the neutrophil-like cell line PLB-985 expressing GFP-NOX2. In the latter cells, an increase in NOX2 at the phagosomal membrane was detected by live-imaging after phagosome closure, probably due to fusion of endosomes with the phagosome. Using super-resolution microscopy in PLB-985 WT cells, we observed that NOX2 forms discrete clusters in the plasma membrane. The number of clusters increased during frustrated phagocytosis. In PLB-985NCF1GT cells that lack p47phox and do not assemble a functional NADPH oxidase, the number of clusters remained stable during phagocytosis. Our data suggest a role for p47phox and possibly ROS production in NOX2 recruitment at the phagosome

Dual function of MIPS1 as a metabolic enzyme and transcriptional regulator.

International audienceBecause regulation of its activity is instrumental either to support cell proliferation and growth or to promote cell death, the universal myo-inositol phosphate synthase (MIPS), responsible for myo-inositol biosynthesis, is a critical enzyme of primary metabolism. Surprisingly, we found this enzyme to be imported in the nucleus and to interact with the histone methyltransferases ATXR5 and ATXR6, raising the question of whether MIPS1 has a function in transcriptional regulation. Here, we demonstrate that MIPS1 binds directly to its promoter to stimulate its own expression by locally inhibiting the spreading of ATXR5/6-dependent heterochromatin marks coming from a transposable element. Furthermore, on activation of pathogen response, MIPS1 expression is reduced epigenetically, providing evidence for a complex regulatory mechanism acting at the transcriptional level. Thus, in plants, MIPS1 appears to have evolved as a protein that connects cellular metabolism, pathogen response and chromatin remodeling

Multiple functions of Kip-related protein5 connect endoreduplication and cell elongation.

International audienceDespite considerable progress in our knowledge regarding the cell cycle inhibitor of the Kip-related protein (KRP) family in plants, less is known about the coordination of endoreduplication and cell differentiation. In animals, the role of cyclin-dependent kinase (CDK) inhibitors as multifunctional factors coordinating cell cycle regulation and cell differentiation is well documented and involves not only the inhibition of CDK/cyclin complexes but also other mechanisms, among them the regulation of transcription. Interestingly, several plant KRPs have a punctuated distribution in the nucleus, suggesting that they are associated with heterochromatin. Here, one of these chromatin-bound KRPs, KRP5, has been studied in Arabidopsis (Arabidopsis thaliana). KRP5 is expressed in endoreduplicating cells, and loss of KRP5 function decreases endoreduplication, indicating that KRP5 is a positive regulator of endoreduplication. This regulation relies on several mechanisms: in addition to its role in cyclin/CDK kinase inhibition previously described, chromatin immunoprecipitation sequencing data combined with transcript quantification provide evidence that KRP5 regulates the transcription of genes involved in cell wall organization. Furthermore, KRP5 overexpression increases chromocenter decondensation and endoreduplication in the Arabidopsis trithorax-related protein5 (atxr5) atxr6 double mutant, which is deficient for the deposition of heterochromatin marks. Hence, KRP5 could bind chromatin to coordinately control endoreduplication and chromatin structure and allow the expression of genes required for cell elongation

Chloroplast dysfunction causes multiple defects in cell cycle progression in the Arabidopsis crumpled leaf mutant

The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants