Effects of Coal Fly Ash Particulate Matter on the Antimicrobial Activity of Airway Surface Liquid

Abstract

Background: Sustained exposure to ambient particulate matter (PM) is a global cause of mortality. Coal fly ash (CFA) is a byproduct of coal combustion and is a source of anthropogenic PM with worldwide health relevance. The airway epithelia are lined with fluid called airway surface liquid (ASL), which contains antimicrobial proteins and peptides (AMPs). Cationic AMPs bind negatively charged bacteria to exert their antimicrobial activity. PM arriving in the airways could potentially interact with AMPs in the ASL to affect their antimicrobial activity.

Objectives: We hypothesized that PM can interact with ASL AMPs to impair their antimicrobial activity.

Methods: We exposed pig and human airway explants, pig and human ASL, and the human cationic AMPs β-defensin-3, LL-37, and lysozyme to CFA or control. Thereafter, we assessed the antimicrobial activity of exposed airway samples using both bioluminescence and standard colony-forming unit assays. We investigated PM-AMP electrostatic interaction by attenuated total reflection Fourier-transform infrared spectroscopy and measuring the zeta potential. We also studied the adsorption of AMPs on PM.

Results: We found increased bacterial survival in CFA-exposed airway explants, ASL, and AMPs. In addition, we report that PM with a negative surface charge can adsorb cationic AMPs and form negative particle-protein complexes.

Conclusion: We propose that when CFA arrives at the airway, it rapidly adsorbs AMPs and creates negative complexes, thereby decreasing the functional amount of AMPs capable of killing pathogens. These results provide a novel translational insight into an early mechanism for how ambient PM increases the susceptibility of the airways to bacterial infection. https://doi.org/10.1289/EHP876.

Figure 1.

Coal fly ash (CFA) impairs ASL antimicrobial activity. (A) Effects of CFA exposure on pig nasal epithelium explant antimicrobial activity assessed by luminescence. Explants exposed to increasing concentrations of CFA (0.3, 3, and $30{\mathrm{\mu }\mathrm{g}/\mathrm{cm}}^2$) had increased live bacteria in relative light units (RLU) (46.62%, 66.32%, and 112.9%, respectively) compared with control at 2 min after bacterial challenge ($\text{mean}\text{inoculum}=2214.8\text{RLU}$; $n=5$). (B) Effecst of CFA exposure on pig nasal epithelium explant antimicrobial activity assessed by standard colony-forming unit (CFU) counting. Explants exposed to CFA ($3{\mathrm{\mu }\mathrm{g}/\mathrm{cm}}^2$) that were harvested after 20 min of bacterial challenge had higher CFUs than the control ($\text{mean}\text{inoculum}=5\times {10}^6\text{CFU}$; $n=7$). (C) Effects of CFA exposure on human airway bronchial epithelium explant (6 mm) antimicrobial activity assessed by luminescence. Explants exposed to $3{\mathrm{\mu }\mathrm{g}/\mathrm{cm}}^2$ CFA had an increased number of live bacteria in RLU after bacterial challenge ($\text{mean}\text{inoculum}=2598.6\text{RLU}$; $n=3$). (D, E, F) Human β-defensin-3 (HBD-3) and LL-37 antimicrobial activity assessed by luminescence plotted as a function of time. CFAs 2689, 2690 and 2691 impaired (E) HBD-3 ($n=3$) and (F) LL-37 ($n=3$) antimicrobial activity in vitro compared with control at 6 min [A, E, and F were compared with control by analysis of variance (ANOVA). B, C, and D pig ASL ex vivo, and in vitro, were compared to control by paired Student’s t-test. D, human ex vivo samples were compared with each other by multiple comparisons ANOVA. * $p<0.05$; ** $p<0.001$; *** $p<0.0001$].

Figure 2.

Coal fly ash (CFA) adsorbs lysozyme and inhibits its antimicrobial activity. (A) Effect of CFA exposure on lysozyme antimicrobial activity assessed by luminescence. CFA 2691 inhibited lysozyme antimicrobial activity in a concentration-dependent manner ($n=3$). (B) CFA bulk adsorption assay of lysozyme. CFA, coincubated with lysozyme, decreased the percentage of available free protein in a concentration-dependent manner ($n=3$). (C) CFA desorption assay. We recovered more lysozyme from the particles as we increased the amount of CFA in micrograms $(n=3)$. (D) Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) spectra of lysozyme on CFA. A lysozyme solution was flowed over the surface of a thin layer of CFA. Every 5 min, the surface was washed with water and spectra of the lysozyme amide groups were obtained using ATR-FTIR. Each line on the graph represents the spectra of lysozyme on the CFA particles every 5 min. The lysozyme absorbance on CFA increased as a function of time. (E) Integrated lysozyme absorbance ($\mathrm{1,460}-\mathrm{1,800}{\mathrm{cm}}^{-1}$) plotted as a function of time. Lysozyme adsorption onto CFA particles increased as a function of time. [A, B, and C were compared with the control by multiple comparison analysis of variance (ANOVA). * $p<0.05$, ** $p<0.001$, *** $p<0.0001$].

Figure 3.

Coal fly ash (CFA) creates a negative particle-protein complex. (A) Zeta potential measurements of CFA 2691, human β-defensin-3 (HBD-3), and HBD-3–CFA suspensions. CFA 2691 had a negative zeta potential, HBD-3 had a positive zeta potential. When they were coincubated, $\text{HBD}-3+\text{CFA}2691$ formed a less negative complex compared with CFA alone ($n=6$). (B–F) Zeta potential measurements of several ambient particulate matters (PMs). (B) CFA 2691, (C) CFA 2690, (D) CFA 2689, (E) urban PM, and (F) volcanic ash all had a negative surface charge and formed negatively charged complexes when coincubated with increasing concentrations of lysozyme ($n=3$).

Figure 4.

Ethylene glycol tetraacetic acid (EGTA) prevents the adsorption and the antimicrobial activity impaired by coal fly ash (CFA). (A) Effects of CFA exposure on the antimicrobial activity of human explants ($\text{mean}\text{inoculum}=2598.6\text{relative}\text{light}\text{units}\left(\text{RLU}\right)$; $n=6$), (B) pig explants ($\text{mean}\text{inoculum}=5\times {10}^6\text{CFU}$, $n=6$), (C) pig in vitro airway surface liquid (ASL) and antimicrobial proteins and peptides (AMPs) [human β-defensin-3 (HBD-3) and lysozyme]. CFA treated with EGTA prevented the impairment of antimicrobial activity by CFA. (D) Effects of increasing concentrations of EGTA on the zeta potential of CFA. Treatment of CFA with EGTA increased its surface charge in a concentration-dependent manner. (E) Lysozyme desorption assay for CFA treated with EGTA. CFA treated with EGTA bound less lysozyme per microgram $(n=3)$. (F) Integrated absorbance of lysozyme ($\mathrm{1,480}{\mathrm{cm}}^{-1}$ to $\mathrm{1,800}{\mathrm{cm}}^{-1}$) on CFA pretreated with EGTA, plotted as a function of time. CFA treated with EGTA showed decreased lysozyme uptake as a function of time (A, B, C, and E were compared with CFA 2691 by paired Student’s t-test. * $p<0.05$).

Figure 5.

Proposed mechanism of particulate matter (PM) impairment of airway surface liquid (ASL) antimicrobial activity. (A) Airborne bacteria are killed by the ASL cationic antimicrobial proteins and peptides (AMPs). (B) Inhaled PM can adsorb cationic AMPs, creating a negatively charged particle–protein complex. (C) The airway, with a decreased concentration of functional AMPs, has an increased probability of bacterial survival.