Mitochondrial N-formyl peptides cause airway contraction and lung neutrophil infiltration via formyl peptide receptor activation

Abstract

Respiratory failure is a common characteristic of systemic inflammatory response syndrome (SIRS) and sepsis. Trauma and severe blood loss cause the release of endogenous molecules known as damage-associated molecular patterns (DAMPs). Mitochondrial N-formyl peptides (F-MITs) are DAMPs that share similarities with bacterial N-formylated peptides, and are potent immune system activators. Recently, we observed that hemorrhagic shock-induced increases in plasma levels of F-MITs associated with lung damage, and that antagonism of formyl peptide receptors (FPR) ameliorated hemorrhagic shock-induced lung injury in rats. Corroborating these data, in the present study, it was observed that F-MITs expression is higher in plasma samples from trauma patients with SIRS or sepsis when compared to control trauma group. Therefore, to better understand the role of F-MITs in the regulation of lung and airway function, we studied the hypothesis that F-MITs lead to airway contraction and lung inflammation. We observed that F-MITs induced concentration-dependent contraction in trachea, bronchi and bronchioles. However, pre-treatment with mast cells degranulator or FPR antagonist decreased this response. Finally, intratracheal challenge with F-MITs increased neutrophil elastase expression in lung and inducible nitric oxide synthase and cell division control protein 42 expression in all airway segments. These data suggest that F-MITs could be a putative target to treat respiratory failure in trauma patients.

Keywords: Airway and lung inflammation; Mitochondrial N-formyl peptides; Trauma.

Fig. 1.

Representative blots and densitometric analyses from protein expression for mitochondrial NADH dehydrogenase 6 (ND6) (F-MITs) in plasma from trauma patients. Control (no evidence of bacteria in blood cultures and no signs of SIRS); SIRS (no evidence of bacteria in blood cultures) and SEPSIS (evidence of bacteria in blood cultures). Mean ± SEM, n = 3–5. One-way ANOVA *vs. control, p < 0.05.

Fig. 2.

Immunofluorescence: (A) top image represents neutrophil elastase (confocal microscopy 40 X) in lung from Wistar rats that were intratracheally challenged with an equal volume of F-MITs (0.02 mg/rat) or non-formylated peptide control (vehicle, VEH) (B). Immunohistochemistry (optical microscopy 10 X) confirms the presence of neutrophil elastase in lung. (C) and (D) demonstrate representative blots and densitometric analyses from protein expression for neutrophil elastase and iNOS of lungs, trachea, bronchus and bronchiole from animals that were intratracheally challenged with F-MITs or VEH. (E) single and double-staining for neutrophils in lung tissue using flow cytometry. Cells positively stained with LY6G and neutrophil elastase were considered neutrophils, and LY6G and CD18 were considered activated neutrophil. Mean ± SEM, n = 3–5. T-test *vs. vehicle (VEH), p < 0.05.

Fig. 3.

Typical trace for concentration–response curve to F-MITs (1–30 μM) in trachea, bronchus and bronchiole from naive Wistar rats (A). The rings were previously precontracted with acetylcholine (1 μM). Graphs represent concentration–response curve to F-MITs (1–30 μM) in the absence or presence of WRW4 (FPR-2 antagonist) (B), cyclosporin H (CsH, FPR-1 antagonist) (C) and C48/80 compound (mast cell degranulator) (D). Mean ± SEM, n = 4–8. Two-way ANOVA: *vs. F-MITs, p < 0.05.

Fig. 4.

Representative blots and densitometric analyses from protein expression for CDC42 of lungs, trachea, bronchus and bronchiole from Wistar rats that were intratracheally challenged with an equal volume of F-MITs (0.02 mg/rat) or non-formylated peptide (VEH). Mean ± SEM, n = 4–5. t-test *vs. vehicle (VEH), p < 0.05.

Fig. 5.

Scheme shows that trauma induces the release of mitochondrial N-formyl peptides leading to formyl peptide receptor (FPR) activation, airway contractility and lung injury.