Localization and functionality of the inflammasome in neutrophils.

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1ß and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1ß protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1ß release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1ß, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1ß and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.

Expression and regulation of interferon-related development regulator-1 in cystic fibrosis neutrophils.

A genome-wide association study identified interferon-related development regulator-1 (IFRD1), a protein expressed by neutrophils, as a key modifier gene in cystic fibrosis (CF) lung disease. Here, we investigated the expression and regulation of IFRD1 in CF neutrophils. IFRD1 expression was quantified in peripheral blood and airway neutrophils from patients with CF, patients with non-CF lung disease, and healthy control subjects. The regulation of IFRD1 expression was analyzed using isolated neutrophils and ex vivo stimulation assays with CF airway fluids. IFRD1 single-nucleotide polymorphisms (SNPs) were analyzed in a CF cohort (n = 572) and correlated with longitudinal lung function and IFRD1 expression. Patients with CF expressed higher protein levels of IFRD1 in peripheral blood neutrophils compared with healthy or non-CF disease control subjects. Within patients with CF, IFRD1 protein expression levels in neutrophils were lower in airway fluids compared with peripheral blood. High IFRD1 expression was positively associated with the production of reactive oxygen species (ROS) in CF neutrophils. In vitro regulation studies showed that CF airway fluid and the CF-characteristic chemokines CXCL8 and CXCL2 down-regulated IFRD1 expression in neutrophils, an effect that was mediated through CXCR2. Genetic analyses showed that three IFRD1 SNPs were associated with longitudinal declines in lung function, and modulated IFRD1 expression. These studies demonstrate that IFRD1 expression is systemically up-regulated in human CF neutrophils, is linked to the production of ROS, and is modulated by chemokines in CF airway fluids, depending on the IFRD1 genotype. Understanding the regulation of IFRD1 may pave the way for novel therapeutic approaches to target neutrophilic inflammation in CF.