Circulating mitochondrial DAMPs cause inflammatory responses to injury

Abstract

Injury causes a systemic inflammatory response syndrome (SIRS) that is clinically much like sepsis. Microbial pathogen-associated molecular patterns (PAMPs) activate innate immunocytes through pattern recognition receptors. Similarly, cellular injury can release endogenous 'damage'-associated molecular patterns (DAMPs) that activate innate immunity. Mitochondria are evolutionary endosymbionts that were derived from bacteria and so might bear bacterial molecular motifs. Here we show that injury releases mitochondrial DAMPs (MTDs) into the circulation with functionally important immune consequences. MTDs include formyl peptides and mitochondrial DNA. These activate human polymorphonuclear neutrophils (PMNs) through formyl peptide receptor-1 and Toll-like receptor (TLR) 9, respectively. MTDs promote PMN Ca(2+) flux and phosphorylation of mitogen-activated protein (MAP) kinases, thus leading to PMN migration and degranulation in vitro and in vivo. Circulating MTDs can elicit neutrophil-mediated organ injury. Cellular disruption by trauma releases mitochondrial DAMPs with evolutionarily conserved similarities to bacterial PAMPs into the circulation. These signal through innate immune pathways identical to those activated in sepsis to create a sepsis-like state. The release of such mitochondrial 'enemies within' by cellular injury is a key link between trauma, inflammation and SIRS.

FIGURE 1. PMN [Ca²⁺]_i responses to MTD

Rhabdomyosarcoma-derived125125 MTD (1.2μg/ml protein) induces Ca²⁺ store depletion (a) and Ca²⁺ influx (a, b) in human PMN. (*p=0.01, t-test). c, PMN serially exposed to MTD and GRO-α (CXCL1) exhibit heterologous desensitization. d, PMN stimulated with GRO-α or MTD show equal store release by ionomycin. e, PMN show homologous desensitization of [Ca²⁺]_i responses to MTD. f, Systemic injection of MTD increases rat PMN responses to PAF. Traces with error bars are from ≥3 experiments, other traces are exemplary. Traces may be displaced temporally for clarity.

FIGURE 2. MTD activate PMN

Human PMN exposed to human MTD (muscle) were immunoblotted for phosphorylated and total (control) p38 (a) or p44/42 MAPK (b). MMP-8 was immunoblotted in supernatants (c,d are from same gel). αFPR1 denotes anti-FPR1. e–f, MTD elicits PMN IL-8 synthesis: */** denote p<0.05 versus control. *** denotes p<0.05 (ANOVA/Tukey) versus control or MTD (n=3). PMN chemotaxis to fMLF and MTD was analyzed by video-microscopy (g,h, Videos 1,2). CsH or anti-FPR1 drastically inhibit chemotaxis (i,j, Videos 3,4). MTD injection into the mouse peritoneum (k) causes rapid, CsH-inhibitable neutrophil influx (n=6, *p<0.05, ANOVA/Dunn) compared with saline or 10nM W-peptide controls.

FIGURE 3. mtDNA activates PMN via CpG/TLR9 interactions

a, Incubation of PMN (10⁶) with 1μg/ml mtDNA activates p38 MAPK (n=3, *p<0.05 vs unstimulated cells). b, mtDNA-induced activation of p38 MAPK was inhibited by pre-treatment with the inhibitory ODN TTAGGG. Inhibition was overcome at higher mtDNA concentration. c, PMN were co-incubated in 1nM fMLF plus mtDNA at clinical concentrations (1–10μg/ml, see Supplementary Fig. 1d). Neither CpG-DNA nor mtDNA caused IL-8 release alone, but each caused significant release along with low dose fMLF. (n=3, *p< 0.05 compared with unstimulated control) (all tests ANOVA/Dunn).

FIGURE 4. MTD cause systemic inflammation and organ injury in vivo

Rats given intravenous MTD equivalent to mitochondria from a 5% liver injury show marked evidence of lung injury by H&E histology (a–b) and 4-HNE stain for oxidant injury (c–d). MTD increased pulmonary albumin permeability (e), lung wet/dry weight (f), accumulation of IL-6 in lung (g) and PMN infiltration into the airways (h). Early (3h) appearance of TNF-α (i) and late (6h) appearance of IL-6 (j) were noted in lung lavage fluid. Whole lung (k) and liver (l) MMP-8 confirmed increased PMN infiltration. (all studies n≥3, *p<0.05, ANOVA/post-hoc).