Class I Phosphoinositide-3-Kinases and Src Kinases Play a Nonredundant Role in Regulation of Adhesion-Independent and -Dependent Neutrophil Reactive Oxygen Species Generation.

Chemoattractant-induced reactive oxygen species (ROS) generation by adherent neutrophils occurs in two phases: the first is very rapid and transient, and the second one is delayed and lasts up to 30-40 min. We examined the role of phosphoinositide 3-kinases (PI3Ks) and Src-family kinases (SFKs) in these responses using human neutrophils treated with inhibitory compounds or murine neutrophils deficient of PI3K\u3b3 or Hck, Fgr, and Lyn. Our studies show that PI3K\u3b3 is indispensable for the early, fMLF-induced ROS generation and AKT and ERK phosphorylation, but is dispensable for the late response to fMLF. Additionally, the response to TNF, an agonist triggering only the delayed phase of ROS generation, was also unaffected in PI3K\u3b3-deficient neutrophils. In contrast, inhibition of SFKs by a selective inhibitor in human, or SFK deficiency in murine, neutrophils resulted in the inhibition of both the early and late phase of ROS generation, without affecting the early phase of AKT phosphorylation, but inhibiting the late one. Selective inhibitors of PI3K\u3b1 and PI3K\u3b4 markedly reduced both the early and late response to fMLF and TNF in human neutrophils. These findings suggest that class IA PI3Ks may be activated by PI3K\u3b3 via Ras in the early phase of the response and by SFKs in the late phase. The evidence that inhibition of SFKs in human, or SFK deficiency in murine, neutrophils results in suppression of Vav phosphorylation at all time points of the response to fMLF or TNF suggests that SFKs are indispensable for Vav phosphorylation

The RacGAP ArhGAP15 is a master negative regulator of neutrophil functions

In phagocytes, GTPases of the Rac family control crucial antimicrobial functions. The RacGAP ArhGAP15 negatively modulates Rac activity in leukocytes, but its in vivo role in innate immunity remains largely unknown. Here we show that neutrophils and macrophages derived from mice lacking ArhGAP15 presented higher Rac activity but distinct phenotypes. In macrophages, the loss of ArhGAP15 induced increased cellular elongation and membrane protrusions but did not modify chemotactic responses. Conversely, the lack of ArhGAP15 in neutrophils affected critical Rac-dependent antimicrobial functions, specifically causing enhanced chemotactic responses, straighter directional migration, amplified reactive oxygen species production, increased phagocytosis, and improved bacterial killing. In vivo, in a model of severe abdominal sepsis, these effects contributed to increase neutrophil recruitment to the site of infection, thereby limiting bacterial growth, controlling infection spread, reducing systemic inflammation, and ultimately improving survival in ArhGAP15-null mice. Altogether, these results demonstrate the relevance of ArhGAP15 in the selective regulation of multiple neutrophil functions, suggesting that ArhGAP15 targeting might be beneficial in specific pathologic settings like severe sepsis

The RacGAP ArhGAP15 is a master negative regulator of neutrophil functions.

In phagocytes, GTPases of the Rac family control crucial antimicrobial functions. The RacGAP ArhGAP15 negatively modulates Rac activity in leukocytes, but its in vivo role in innate immunity remains largely unknown. Here we show that neutrophils and macrophages derived from mice lacking ArhGAP15 presented higher Rac activity but distinct phenotypes. In macrophages, the loss of ArhGAP15 induced increased cellular elongation and membrane protrusions but did not modify chemotactic responses. Conversely, the lack of ArhGAP15 in neutrophils affected critical Rac-dependent antimicrobial functions, specifically causing enhanced chemotactic responses, straighter directional migration, amplified reactive oxygen species production, increased phagocytosis, and improved bacterial killing. In vivo, in a model of severe abdominal sepsis, these effects contributed to increase neutrophil recruitment to the site of infection, thereby limiting bacterial growth, controlling infection spread, reducing systemic inflammation, and ultimately improving survival in ArhGAP15-null mice. Altogether, these results demonstrate the relevance of ArhGAP15 in the selective regulation of multiple neutrophil functions, suggesting that ArhGAP15 targeting might be beneficial in specific pathologic settings like severe sepsis

Brief report

Neutrophil adhesion and extravasation into tissue at sites of injury or infection depend on binding of integrin lymphocyte function-associated antigen-1 (LFA-1) to ICAM-1 expressed on activated endothelial cells. The activation-dependent conformational change of LFA-1 to the high affinity conformation (H+) requires kindlin-3 binding to the beta2-integrin cytoplasmic domain. Here we show that genetic deletion of the known kindlin-interactor integrin linked kinase (ILK) impaired neutrophil adhesion and extravasation in the cremaster muscle and in a clinically relevant model of renal-ischemia-reperfusion injury. Utilizing in vitro microfluidic adhesion chambers and conformation specific antibodies, we show that knockdown of ILK in HL-60 cells reduces the conformational change of beta2-integrins to the H+ conformation. Mechanistically, we found that ILK is required for PKC membrane targeting and for chemokine-induced upregulation of its kinase activity. Moreover, PKC-alpha deficiency also results in impaired leukocyte adhesion in bone marrow chimeric mice. Mass spectrometric and western blot analyses revealed a stimulation- and ILK-dependent phosphorylation of kindlin-3 upon activation. In sum our data indicate an important role of ILK in kindlin-3-dependent conformational activation of LFA-1. Copyright © 2020 American Society of Hematology