RASAL3 Is a Putative RasGAP Modulating Inflammatory Response by Neutrophils

Abstract

As first responder cells in host defense, neutrophils must be carefully regulated to prevent collateral tissue injury. However, the intracellular events that titrate the neutrophil’s response to inflammatory stimuli remain poorly understood. As a molecular switch, Ras activity is tightly regulated by Ras GTPase activating proteins (RasGAP) to maintain cellular active-inactive states. Here, we show that RASAL3, a RasGAP, is highly expressed in neutrophils and that its expression is upregulated by exogenous stimuli in neutrophils. RASAL3 deficiency triggers augmented neutrophil responses and enhanced immune activation in acute inflammatory conditions. Consequently, mice lacking RASAL3 (RASAL3-KO) demonstrate accelerated mortality in a septic shock model via induction of severe organ damage and hyperinflammatory response. The excessive neutrophilic hyperinflammation and increased mortality were recapitulated in a mouse model of sickle cell disease, which we found to have low neutrophil RASAL3 expression upon LPS activation. Thus, RASAL3 functions as a RasGAP that negatively regulates the cellular activity of neutrophils to modulate the inflammatory response. These results demonstrate that RASAL3 could serve as a therapeutic target to regulate excessive inflammation in sepsis and many inflammatory disease states.

Creatine supplementation enhances immunological function of neutrophils by increasing cellular adenosine triphosphate

“…1G, 1H ). Neutrophil extracellular traps (NETs) formation is another dynamic antibacterial response in neutrophils [ 28 ]. In the NETosis induced by PMA in this study, creatine treatment enhanced the formation of NET structures, which could be observed under fluorescence microscope ( Fig.…”

“…Finally, the samples were stained with 7-AAD and analyzed on the same day without fixation. The NETosis assay was performed by a previously reported method with minor modification [ 28 , 29 ]. Briefly, peripheral blood was treated with 1× red blood cell (RBC) lysis buffer at room temperature (RT) for 10 min, washed and fixed with 1% paraformaldehyde (PFA) at RT for 10 min, and subjected to staining for surface markers as well as nuclear and DNA staining.…”

“…BM neutrophils were isolated from naive or treated (saline or creatine supplementation for 7 days) C57BL/6 J WT mice by following a protocol described in a previous report [ 28 ]. Briefly, tibias and femurs were extracted from the mice, and then BM cells were flushed out by using a 10 mL syringe with a 27G needle and cell culture medium (RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin).…”

Direct activation of the Ras-Akt network mediates polarity and organizes protrusions in human neutrophil migration

“…We studied a RasGAP, RASAL3, which was recently characterized for its immunomodulatory functions in neutrophils. It is possible that these effects on immune response were due to inhibition of migration, but this was not assessed 95 . For our experiment, we transiently recruited RASAL3 to the leading front by shining light periodically near the existing protrusions, as shown by the dashed white box (Figure 3G, Video S8).…”

Lymphocyte antigen 6 complex locus G6D downregulation is a novel parameter for functional impairment of neutrophils in aged mice

“…Neutrophils are frequently studied due to their central role in the innate immune system, specifically their involvement in antibacterial immunity (5). Under bacterial invasion, neutrophils function to eliminate the bacteria through several activities, such as phagocytosis, neutrophil extracellular trap (NET) formation, and reactive oxygen species (ROS) production (5)(6)(7). Thus, it is expected that the impairment of neutrophil functioning will result in the collapse of the primary defense against pathogen colonization.…”