Mitochondrial damage-associated molecular patterns from fractures suppress pulmonary immune responses via formyl peptide receptors 1 and 2

Abstract

Background: No known biologic mechanisms link tissue injury with pneumonia (PNA). Neutrophils (PMNs) are innate immune cells that clear bacteria from the lung by migration toward chemoattractants and killing bacteria in neutrophil extracellular traps (NETs). We predicted that tissue injury would suppress PMN antimicrobial function in the lung. We have also shown that mitochondria-derived damage-associated molecular pattern molecules from the bone can alter PMN phenotype and so hypothesized that formyl peptides (FPs) from fractures predispose to PNA by suppressing PMN activity in the lung.

Methods: Animal studies involved the following. (1) Rats were divided into three groups (10 per condition) as follows: (a) saline injection in the thigh (b) Staphylococcus aureus (SA, 3 × 10) injected intratracheally, or (c) pseudofracture (PsFx; bone supernatant injected in the thigh) plus intratracheally injected SA. (2) Rats were divided into four groups as follows: (a) control, (b) pulmonary contusion (PC), (c) PsFx, and (d) PC + PsFx. Bronchoalveolar lavage was performed 16 hours later. Clinical studies involved the following. (3) Human bone supernatant was assayed for its FP-receptor (FPR) stimulation. (4) Trauma patients' PMN (n = 32; mean ± SE Injury Severity Score [ISS], 27 ± 10) were assayed for chemotaxis (CTX) or treated with Phorbol 12-myristate 13-acetate (PMA, Phorbol ester) and analyzed for NET formation.

Results: In the animal studies, (1) SA was rapidly cleared by the uninjured mice and PsFx markedly suppressed lung bacterial clearance (p < 0.01). (2a) PC induces PMN traffic to the lung, but PsFx decreases PC-induced PMN traffic (p < 0.01). (2b) SA increased bronchoalveolar lavage PMN, and PsFx decreased that influx (p < 0.01). In the clinical studies, (3) bone supernatant activates PMN both via FPR-1 and FPR-2. (4) Trauma decreases PMN CTX to multiple chemokines. Circulating PMNs show NETs spontaneously after trauma, but maximal NET formation is markedly attenuated.

Conclusion: Fractures may decrease lung bacterial clearance because FP suppresses PMN CTX to other chemoattractants via FPR-1/2. Trauma activates NETosis but suppresses maximal NETosis. Fractures decrease lung bacterial clearance by multiple mechanisms. PNA after fractures may reflect damage-associated molecular pattern-mediated suppression of PMN antimicrobial function in the lung.

Figure 1

Bacterial counts in bronchoalveolar lavage fluids (BALF). Almost no bacteria were detected in saline controls. S. aureus (SA) injected i.t. were cleared overnight. BALF bacterial counts after SA plus pseudo-fracture (SA+PsFx) were more than five times the counts seen with SA alone (p=0.003).

Figure 2

PMN in BALF. A: BALF PMN after pulmonary contusion (PC). PC induced a 16-fold increase in PMN count compared to controls (p<0.001). PsFx per se did not induce a rise in BALF PMN but when combined with PC (PC+PsFx) PMN number was significantly less than PC alone (p=0.002). B: BALF PMN after bacterial inoculation: S. aureus increased airway PMN and PsFx decreased airway PMN accumulation in response to bacterial inoculation (p<0.01).

Figure 3

Ca²⁺ flux responses to bone supernatants depend on FPR-1 and FPR-2. Ca²⁺ flux is stimulated in human PMN at t=30 seconds by bone supernatants. There was a significant reduction in responses seen in cells pre-incubated with antibodies to FPR-1 (p = 0.027) or anti FPR-2 (p = 0.007). Incubation with both antibodies blocks better but inhibition is still incomplete. This may be due to the presence of other FPRs or FPR heterodimerization.

Figure 4

FP suppress CTX to LTB₄: LTB₄ stimulates CTX of human PMN at both high and low doses (1 nM and 10 nM). Both effects are markedly reduced by fMLP pre-treatment.

Figure 5

CTX in trauma patients (ISS 27±10[SE]) and matched volunteers (n=32/group). PMN CTX to IL-8, GRO-α and fMLP were all significantly suppressed by injury (p<0.01). Suppression was most significant on Day 3.

Figure 6

NETosis. NETosis is not seen in unstimulated volunteer PMN. Unstimulated PMN in trauma patients consistently show modest NET formation. PMA induces maximal activation of NETs in volunteers but PMN from trauma patients treated with PMA remain largely unchanged when compared to unstimulated cells.