Scripps Research scientists led a team that reports that it has uncovered key details of an immune-cell process that frequently underlies excessive inflammation in the body. The findings could lead to new ways of preventing and/or treating inflammation-related conditions such as sepsis, arthritis, and coronary artery disease, according to the researchers.

The study “Differential dysregulation of granule subsets in WASH-deficient neutrophil leukocytes resulting in inflammation” appears in Nature Communications and shows that a multi-protein “molecular machine” called WASH has an important role in restraining excessive inflammatory activity by neutrophils.

“Dysregulated secretion in neutrophil leukocytes associates with human inflammatory disease. The exocytosis response to triggering stimuli is sequential; gelatinase granules modulate the initiation of the innate immune response, followed by the release of pro-inflammatory azurophilic granules, requiring stronger stimulation. Exocytosis requires actin depolymerization which is actively counteracted under non-stimulatory conditions,” write the investigators.

“Here we show that the actin nucleator, WASH, is necessary to maintain azurophilic granules in their refractory state by granule actin entrapment and interference with the Rab27a-JFC1 exocytic machinery. On the contrary, gelatinase granules of WASH-deficient neutrophil leukocytes are characterized by decreased Rac1, shortened granule-associated actin comets and impaired exocytosis. Rac1 activation restores exocytosis of these granules.

“ In vivo, WASH deficiency induces exacerbated azurophilic granule exocytosis, inflammation, and decreased survival. WASH deficiency thus differentially impacts neutrophil granule subtypes, impairing exocytosis of granules that mediate the initiation of the neutrophil innate response while exacerbating pro-inflammatory granule secretion.”

Scripps Research scientists found a protein in neutrophils that could be a target against harmful inflammation. Stochastic Optical Reconstruction super-resolution Microscopy (STORM, right image) shows that in resting neutrophils, the WASH protein complex (green) forms a molecular clustered complex with microfilaments called F-actin (red) and with granule cargo (blue) near the plasma membrane (left image). In the absence of WASH, the actin nucleator Arp2/3 (right, pink) has reduced activity, facilitating toxic granule (right, green) secretion, leading to systemic inflammation. [Scripps Research]
“Our findings point to the possibility of future treatments that target this WASH-regulated pathway to inhibit neutrophil-driven inflammation while preserving most of neutrophils’ anti-microbial effectiveness,” says study senior author Sergio Catz, PhD, professor in the Department of Molecular Medicine at Scripps Research.

Neutrophils are workhorses of the mammalian immune system, comprising about two-thirds of the white blood cells that circulate through our bloodstreams. They combat invading microbes by engulfing and digesting them, and by releasing a variety of antimicrobial molecules via exocytosis.

Many of the antimicrobial molecules that neutrophils release via exocytosis are potent enough to harm healthy cells. There is evidence that excessive and/or chronic release of these molecules at least partly underlies serious medical conditions and types of tissue injury, including sepsis, arthritis, smoke inhalation injury to lungs, inflammatory bowel disease, some cancers, and the artery-thickening atherosclerosis that leads to heart attacks and strokes.

In experiments, neutrophils without WASH released excessive amounts of azurophilic granules. Mice with these neutrophils had blood levels of toxic azurophilic molecules that are normally found in cases of harmful systemic inflammation. The mortality rate of such mice when experiencing an experimental sepsis-like condition was more than triple that of normal mice.

“WASH seems to be an important molecular switch that controls neutrophils’ responses to infection and inflammation by regulating the release of these two kinds of antimicrobial cargoes,” Catz says. “When WASH is dysfunctional, the result is likely to be excessive and chronic inflammation.”

“In this study, using [advanced] cell biology approaches, we reveled how neutrophils control their timely response through sequential exocytosis, and have identified a molecular system that acts as the gatekeeper of this process,” Catz adds.

Catz and his colleagues are continuing to study WASH and other molecules involved in neutrophil exocytosis, with the goal of finding candidate drug molecules that can damp excessive azurophilic granule exocytosis—to treat inflammatory conditions—without impairing neutrophils’ functions as immune first responders.

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