Muramic acid, endotoxin, 3-hydroxy fatty acids, and ergosterol content explain monocyte and epithelial cell inflammatory responses to agricultural dusts

Abstract

In agricultural and other environments, inhalation of airborne microorganisms is linked to respiratory disease development. Bacterial endotoxins, peptidoglycans, and fungi are potential causative agents, but relative microbial characterization and inflammatory comparisons amongst agricultural dusts are not well described. The aim of this study was to determine the distribution of microbial endotoxin, 3-hydroxy fatty acids (3-OHFA), muramic acid, and ergosterol and evaluate inflammatory responses in human monocytes and bronchial epithelial cells with various dust samples. Settled surface dust was obtained from five environments: swine facility, dairy barn, grain elevator, domestic home (no pets), and domestic home with dog. Endotoxin concentration was determined by recombinant factor C (rFC). 3-OHFA, muramic acid, and ergosterol were measured using gas chromatography-mass spectrometry. Dust-induced inflammatory cytokine secretion in human monocytes and bronchial epithelial cells was evaluated. Endotoxin-independent dust-induced inflammatory responses were evaluated. Endotoxin and 3-OHFA levels were highest in agricultural dusts. Muramic acid, endotoxin, 3-OHFA, and ergosterol were detected in dusts samples. Muramic acid was highest in animal farming dusts. Ergosterol was most significant in grain elevator dust. Agricultural dusts induced monocyte tumor necrosis factor (TNF) alpha, interleukin (IL)-6, IL-8, and epithelial cell IL-6 and IL-8 secretion. Monocyte and epithelial IL-6 and IL-8 secretion was not dependent on endotoxin. House dust(s) induced monocyte TNFalpha, IL-6, and IL-8 secretion. Swine facility dust generally produced elevated responses compared to other dusts. Agricultural dusts are complex with significant microbial component contribution. Large animal farming dust(s)-induced inflammation is not entirely dependent on endotoxin. Addition of muramic acid to endotoxin in large animal farming environment monitoring is warranted.

Figure 1

(A-B). Endotoxin’s 3-hydroxy fatty acid (3-OHFA) analysis of various agricultural and domestic house dusts after Tween extraction. (A) Total 3-OHFA normalized to the total amount of dust (pmol/mg). (B) The percent of the total 3-OHFAs for each carbon chain length reported for each individual dust extract. Mean results ± SEM are presented (N=3). *Statistically different than other dust extracts (p<0.001).

Figure 2

(A-B). Endotoxin’s 3-hydroxy fatty acid (3-OHFA) analysis of various agricultural and domestic house dusts with HBSS extraction, which was utilized in cell culture studies at a 1% concentration. (A) Total 3-OHFA normalized to the total amount of dust in a 1% concentration (pmol/mL). (B) The percent of the total 3-OHFAs for each carbon chain length reported for each individual dust extract. Mean results ± SEM are presented (N=3). *Statistically different from all other dust extracts (p<0.001).

Figure 3

(A-B). Muramic acid analysis of the various agricultural and domestic house dust extracts. (A) Muramic acid concentration normalized to the total amount of dust extracted in Tween (ng/mg). (B) Muramic acid concentration normalized to total volume in the 1% HBSS dust extract (ng/mL). Mean results ± SEM are presented (N=3). *Statistically different from all other dust extracts (p<0.001). #Statistically different from all other dust extracts (p<0.05).

Figure 4

(A-B). Ergosterol analysis of the various agricultural and domestic house dust samples. (A) Ergosterol concentration normalized to the total amount of dust extracted in Tween (ng/mg). (B) Ergosterol concentration normalized to total volume in the 1% HBSS dust extract (ng/mL). Mean results ± SEM are presented (N=3). *Statistically different from all other dust extracts (p<0.001). #Statistically different from all other dust extracts (p<0.05

Figure 5

(A-C). Secretion of TNFα (A), IL-6 (B), and CXCL8/IL-8 (C) in human peripheral blood monocytes stimulated with complete (untreated) and polymyxin B-treated (endotoxin-depleted) various agricultural and domestic house 1% HBSS dust extracts for 5 hours. *Significantly different from respective media control (p<0.05). #Significantly different from matched endotoxin-depleted dust extract (polymyxin B treated vs. untreated dust extract; p<0.05). N=3 separate experiments. Mean results are presented per 1 × 10⁶ cells ± SD. ^aStatistically different from all other endotoxin-depleted dust extracts. ^bStatistically different from endotoxin-depleted domestic house dust extracts.

Figure 6

(A-B). Secretion of IL-6 (A) and CXCL8/IL-8 (B) in human bronchial epithelial cells (BEAS-2B) stimulated with complete (untreated) and polymyxin B-treated (endotoxin-depleted) various agricultural and domestic house 1% HBSS dust extracts for 24 hours. *Significantly different from respective media control (p<0.05). N=3 separate experiments. Mean results ± SD are presented. ^aStatistically different as compared to grain elevator and domestic house dust extracts. ^bStatistically different as compared to all other endotoxin-depleted dust extracts. ^cStatistically different as compared to endotoxin-depleted grain elevator and domestic house dust extracts.