Inducible lung epithelial resistance requires multisource reactive oxygen species generation to protect against bacterial infections

Abstract

Pneumonia remains a global health threat, in part due to expanding categories of susceptible individuals and increasing prevalence of antibiotic resistant pathogens. However, therapeutic stimulation of the lungs' mucosal defenses by inhaled exposure to a synergistic combination of Toll-like receptor (TLR) agonists known as Pam2-ODN promotes mouse survival of pneumonia caused by a wide array of pathogens. This inducible resistance to pneumonia relies on intact lung epithelial TLR signaling, and inducible protection against viral pathogens has recently been shown to require increased production of epithelial reactive oxygen species (ROS) from multiple epithelial ROS generators. To determine whether similar mechanisms contribute to inducible antibacterial responses, the current work investigates the role of ROS in therapeutically-stimulated protection against Pseudomonas aerugnosa challenges. Inhaled Pam2-ODN treatment one day before infection prevented hemorrhagic lung cytotoxicity and mouse death in a manner that correlated with reduction in bacterial burden. The bacterial killing effect of Pam2-ODN was recapitulated in isolated mouse and human lung epithelial cells, and the protection correlated with inducible epithelial generation of ROS. Scavenging or targeted blockade of ROS production from either dual oxidase or mitochondrial sources resulted in near complete loss of Pam2-ODN-induced bacterial killing, whereas deficiency of induced antimicrobial peptides had little effect. These findings support a central role for multisource epithelial ROS in inducible resistance against a bacterial pathogen and provide mechanistic insights into means to protect vulnerable patients against lethal infections.

Fig 1. Pam2-ODN protects against bacterial pneumonia.

(A) Survival of wildtype mice treated with Pam2-ODN or PBS (sham) by aerosol 24 h before challenge with P. aeruginosa. (B) Pathogen burden of mice in A immediately after challenge, as assessed by serial dilution culture of lung homogenates (left panel) or BAL fluid (right panel). (C) Gross appearance of mouse lungs 24 h after P. aeruginosa challenge following treatment with Pam2-ODN or sham. (D) Hematoxylin-eosin stained histology of lungs in C. Scale bar = 400 μm left panels, 100 μm right panels. Each panels is representative of at least three independent experiments. N = 8 mice/group for survival, N = 4 mice/group for pathogen burden. * p < 0.0002 vs. PBS-treated; ** p < 0.002 vs PBS-treated.

Fig 2. Pam2-ODN induces antibacterial responses in isolated lung epithelial cells.

HBEC3kt (A) or MLE15 (B) cells were treated for 4 h with PBS or the indicated doses of Pam2-ODN, then challenged with P. aeruginosa. Shown are culture bacterial burdens 4 h after challenge. HBEC3kt (C, E) or MLE15 (D, F) cells were treated with PBS or Pam2-ODN (middle dose used in A and B, 2.23 uM Pam2 and 0.56 uM ODN) for the indicated interval relative to challenge with P. aeruginosa. Shown are culture bacterial burdens 4 h after challenge. HBEC3kt (G) or MLE15 (H) cells were treated for 4 h with the indicated treatments, then challenged with P. aeruginosa. Shown are culture bacterial burdens 4 h after challenge. HBEC3kt (I) or MLE15 (J) cells were treated with PBS or Pam2-ODN for 6 h prior to P. aeruginosa challenge. Cell survival determined by Trypan blue exclusion is shown at the indicated time points. Each panels is representative of at least three independent experiments. * p < 0.05 vs. PBS-treated; ** p < 0.005 vs. PBS-treated; † p < 0.05 vs. either single ligand treatment.

Fig 3. Pam2-ODN induces antibacterial ROS from isolated lung epithelial cells.

HBEC3kt (A) or MLE15 (B) cells were exposed to CO-H₂DCFDA, treated with the indicated doses of Pam2-ODN, then fluorescence intensity was measured every 5 min. HBEC3kt (C) or MLE15 (D) cells were pretreated with PEG-HCC or PBS, exposed to CO-H₂DCFDA, then treated with the indicated dose of Pam2-ODN. Shown are fluorescence intensity 100 min after treatment. HBEC3kt (E) or MLE15 (F) cells were pretreated with PEG-HCC or PBS, treated for 4 h with PBS or Pam2-ODN (Pam2 2.23 uM, ODN 0.56 uM), then challenged with P. aeruginosa. Shown are culture bacterial burdens 4 h after challenge. (G) HBEC3kt cells were stably transfected with scrambled (control) shRNA or shRNA targeting DUOX1 or DUOX2, then treated with PBS or Pam2-ODN for 6 h prior to P. aeruginosa challenge. Shown are culture bacterial burdens 4 h after challenge. Each panels is representative of at least three independent experiments. * p < 0.005 vs no Pam2-ODN treatment; † p < 0.005 vs no PEG-HCC, same Pam2-ODN; †† p < 0.02 vs scrambled shRNA + Pan2-ODN; ‡ p < 0.003 vs DUOX1 knockdown + Pam2-ODN.

Fig 4. Mitochondrial ROS are required for Pam2-ODN-induced antibacterial epithelial responses.

HBEC3kt (A) or MLE15 (B) cells were pretreated with MitoTEMPO or PBS, treated for 4 h with PBS or Pam2-ODN (Pam2 2.23 uM, ODN 0.56 uM), then challenged with P. aeruginosa. Shown are culture bacterial burdens 4 h after challenge. (C) HBEC3kt cells were pretreated with FCCP-TTFA or PBS, exposed to MitoSOX, then treated with PBS or Pam2-ODN at the indicated doses. Shown are culture fluorescence intensities at 100 min after treatment. HBEC3kt (D) or MLE15 (E) cells were pretreated with FCCP-TTFA or PBS, treated for 4 h with PBS or Pam2-ODN (Pam2 2.23 uM, ODN 0.56 uM), then challenged with P. aeruginosa. Shown are culture bacterial burdens 4 h after challenge. Each panels is representative of at least three independent experiments. N = 4–5 samples/condition for all experiments. * p < 0.01 vs. PBS-treated without inhibitor/scavenger; † p < 0.02 vs. Pam2-ODN-treated without inhibitor/scavenger.