Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation

Abstract

Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SCGB1A1 gene is triplicated in equids and gives rise to two distinct proteins. In this study we produced the endogenously expressed forms of SCGBs (SCGB 1A1 and 1A1A) as recombinant proteins, and analyzed their effects on reactive oxygen species production, phagocytosis, chemotaxis and neutrophil extracellular trap (NET) formation ex vivo. We further evaluated whether NETs are present in vivo in control and inflamed lungs. Our data show that SCGB 1A1A but not SCGB 1A1 increase neutrophil oxidative burst and phagocytosis; and that both proteins markedly reduce neutrophil chemotaxis. SCGB 1A1A reduced chemotaxis significantly more than SCGB 1A1. NET formation was significantly reduced in a time- and concentration-dependent manner by SCGB 1A1 and 1A1A. SCGB mRNA in bronchial biopsies, and protein concentration in bronchoalveolar lavage fluid, was lower in horses with RAO. NETs were present in bronchoalveolar lavage fluid from horses with exacerbated RAO, but not in fluid from horses with RAO in remission or in challenged healthy horses. These findings indicate that SCGB 1A1 and 1A1A have overlapping and diverging functions. Considering disparities in the relative abundance of SCGB 1A1 and 1A1A in airway secretions of animals with RAO suggests that these functional differences may contribute to the pathogenesis of RAO and other neutrophilic inflammatory lung diseases.

Characterization of Circulating Low-Density Neutrophils Intrinsic Properties in Healthy and Asthmatic Horses

“…The increased circulating LDNs we observed in asthmatic horses indicates that they possibly contribute to the disease expression, by enhanced NET production. The presence of NETs in the lungs of asthmatic horses but not in controls has been reported 42 .…”

Club Cell Protein 16 (CC16) Augmentation: A Potential Disease-modifying Approach for Chronic Obstructive Pulmonary Disease (COPD)

“…For example, plasmid-mediated over-expression of CC16 in human bronchial epithelial cells reduced IL-1β-induced release of IL-8 ( Figure 3 ), suppressed the proliferation of human lung cancer cells in vitro and increased their rates of apoptosis 151;152 , and reduced NFκB activation in the cells 113 . Treating cells involved in the pathogenesis of COPD (e.g., fibroblasts 129 , PMNs 153 , and macrophages 118 ) with recombinant CC16 also inhibited their migration to chemoattractants in vitro ( Figure 3 ).…”

Extracellular Traps: An Ancient Weapon of Multiple Kingdoms

“…Equine NET formation was analysed with respect to the role of SCGB1 [132], a protein described as being associated with reduced neutrophil migration and decreased ROS production during recurrent airway obstruction [133,134]. Horse neutrophils released NETs in a presumably ROS-independent Biology 2020, 9, 34 9 of 23 manner, which were inhibited by the addition of SCGB proteins [132].…”

“…Equine NET formation was analysed with respect to the role of SCGB1 [132], a protein described as being associated with reduced neutrophil migration and decreased ROS production during recurrent airway obstruction [133,134]. Horse neutrophils released NETs in a presumably ROS-independent Biology 2020, 9, 34 9 of 23 manner, which were inhibited by the addition of SCGB proteins [132]. Co-incubation of equine PMNs with various pathogens causing endometriosis in mares (S. equi subspecies zooepidermicus, E. coli, and S. capitis) resulted in NET release from equine PMNs and all pathogens were found entrapped within the meshwork [2].…”