title: Role of neprilysin in airway inflammation induced by diesel exhaust emissions.
abstract: In this study, we examined the role of neprilysin (NEP), a key membrane-bound endopeptidase, in the inflammatory response induced by diesel exhaust emissions (DEE) in the airways through a number of approaches: in vitro, animal, and controlled human exposure. Our specific aims were (1) to examine the role of NEP in inflammatory injury induced by diesel exhaust particles (DEP) using Nep-intact (wild-type) and Nep-null mice; (2) to examine which components of DEP are associated with NEP downregulation in vitro; (3) to determine the molecular impact of DEP exposure and decreased NEP expression on airway epithelial cells' gene expression in vitro, using a combination of RNA interference (RNAi) and microarray approaches; and (4) to evaluate the effects on NEP activity of human exposure to DEE. We report four main results: First, we found that exposure of normal mice to DEP consisting of standard reference material (SRM) 2975 via intratracheal installation can downregulate NEP expression in a concentration-dependent manner. The changes were accompanied by increases in the number of macrophages and epithelial cells, as well as proinflammatory cytokines, examined in bronchoalveolar lavage (BAL) fluid and cells. Nep-null mice displayed increased and/or additional inflammatory responses when compared with wild-type mice, especially in response to exposure to the higher dose of DEP that we used. These in vivo findings suggest that loss of NEP in mice could cause increased susceptibility to injury or exacerbate inflammatory responses after DEP exposure via release of specific cytokines from the lungs. Second, we found evidence, using in vitro studies, that downregulation of NEP by DEP in cultured human epithelial BEAS-2B cells was mostly attributable to DEP-adsorbed organic compounds, whereas the carbonaceous core and transition metal components of DEP had little or no effect on NEP messenger RNA (mRNA) expression. This NEP downregulation was not a specific response to DEP or its contents because the change also occurred after exposure to urban dust (SRM 1649a), which differs in physical and chemical composition from DEP. Third, we also collected the transcriptome profiles of the concentration-effects of SRM 2975 in cultured BEAS-2B cells through a 2 X 3 factorial design. DEP exposure upregulated 151 genes and downregulated 59 genes. Cells with decreased NEP expression (accomplished by transfecting an NEP-specific small interfering RNA [siRNA]) substantially altered the expression of genes (upregulating 17 and downregulating 14) associated with DNA/protein binding, calcium channel activities, and the cascade of intracellular signaling by cytokines. Data generated from the combined RNAi and microarray approaches revealed that there is a complex molecular cascade mediated by NEP in different subcellular compartments, possibly influencing the inflammatory response. Fourth, in a controlled human exposure study, we observed significant increases in soluble NEP in sputum after acute exposure to DEE, with an average net increase of 31%. We speculate that the change in NEP activity in sputum, if confirmed in larger epidemiologic investigations at ambient exposure levels to DEE, may provide a useful endpoint and promote insight into the mechanism of DEE-induced airway alterations.
authors.affiliation: Department of Pediatrics and Steele Children's Research Center, University of Arizona Health Science Center, Tucson, AZ, USA. shengjun@email.arizona.edu
authors.forename: Simon S
authors.initials: SS
authors.lastname: Wong
authors.forename: Nina N
authors.initials: NN
authors.lastname: Sun
authors.forename: Cynthia D
authors.initials: CD
authors.lastname: Fastje
authors.forename: Mark L
authors.initials: ML
authors.lastname: Witten
authors.forename: R Clark
authors.initials: RC
authors.lastname: Lantz
authors.forename: Bao
authors.initials: B
authors.lastname: Lu
authors.forename: Duane L
authors.initials: DL
authors.lastname: Sherrill
authors.forename: Craig J
authors.initials: CJ
authors.lastname: Gerard
authors.forename: Jefferey L
authors.initials: JL
authors.lastname: Burgess
mesh_terms: D000328:Adult; D000818:Animals; D001991:Bronchitis / Q000139:chemically induced* / Q000201:enzymology*; D015536:Down-Regulation; D004847:Epithelial Cells / Q000201:enzymology; D005260:Female; D015870:Gene Expression; D006801:Humans; D007249:Inflammation; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D015260:Neprilysin / Q000235:genetics / Q000378:metabolism*; D010316:Particle Size; D013183:Sputum / Q000201:enzymology; D001335:Vehicle Emissions / Q000506:poisoning*; D055815:Young Adult
chemical_list: D001335:Vehicle Emissions; D015260:Neprilysin