Trauma is a leading cause of premature death 5
. Injury causes activation of neutrophils (PMN), organ failure, susceptibility to infection and the Systemic Inflammatory Response Syndrome (SIRS) 1,6
. Bacterial translocation from ischemic gut to circulation was long thought to cause SIRS 7
. This was disproven 8
although shock may cause gut inflammation 9
. Crushes and burns however, cause SIRS without shock. Thus the molecular signals linking injury to inflammation remain unclear.
During infection, innate immunity is activated by pathogen-associated molecular patterns (PAMPs) expressed on invading microorganisms. Pattern recognition receptors (PRR) recognize PAMPs 2
. Bacterial proteins are N
, so formyl peptides (FP) activate chemoattractant FP-receptors (FPR). Toll-like receptors (TLR) respond to many PAMPs, like bacterial DNA that stimulates TLR9. Since mitochondria evolved from saprophytic bacteria to endosymbionts to organelles, the mitochondrial genome (mtDNA) contains CpG DNA repeats and also codes for formylated peptides 4,11
. Mechanical trauma disrupts cells, so we hypothesized injury might release mitochondrial “damage” associated molecular patterns (DAMPs) 3
into the circulation, activating immunity and initiating SIRS.
To prove trauma releases mitochondrial DAMPs (MTD) into the circulation we measured plasma mtDNA in 15 major trauma patients (Injury Severity Score [ISS 12
] >25). Sampling was prior to resuscitation. Patients had no open wounds or gastrointestinal injuries (Details: Supplementary Table 1
). Trauma patient mtDNA was markedly elevated (Supplementary Figs. 1a–c
) compared to volunteers (Supplementary Table 2
). mtDNA in trauma plasma was 2.7±0.94[SE] μg/ml where volunteer levels were thousands of fold lower (Supplementary Fig. 1d
). mtDNA was further elevated 24hrs post-injury (Supplementary Fig. 1e
). Ultra-centrifugates of femur reaming specimens obtained during fracture repair contained even higher titers of mtDNA. Thus MTD are mobilized by external or operative injury and enter the circulation. Bacterial 16s-RNA was absent from all specimens (Supplementary Fig. 1f
Mitochondrial formyl peptides (FP) can attract PMN 13
and activate related cell lines 14
. The synthetic peptide fMLF simulates bacterial challenge. But the role of endogenous formyl peptides in trauma, PMN activation and SIRS is unstudied. FPs signal via the G-protein coupled receptors (GPCR); FPR1 and FPRL-1, with high and low affinities respectively. PMN activation via GPCR causes increased intracellular calcium ([Ca2+
, heterologous and homologous GPCR desensitization 16
and activates MAP Kinases 17
. MTD from human myocytes induced human PMN [Ca2+
fluxes equal to 1nM fMLF (). MTD from human liver, muscle and fracture hematoma (Supplementary Fig. 2a
) or from rat muscle or liver produced similar PMN Ca2+
depletion. Whole and fragmented mitochondria had similar potency (Supplementary Fig. 2b
). Thus release of MTD from many cell types activates immunity.
Blocking antibodies to FPR1 abolished Ca2+
depletion (), and Ca2+
entry () responses to MTD. Cyclosporin H (CsH) inhibits FPR118
and abolishes Ca2+
flux to MTD (Supplementary Fig. 3a
). Isotype control (FPRL-1, MMP-2) antibodies have no effects (Supplementary Fig. 3b
). Apyrase-treated and untreated MTD act identical whereas apyrase abolishes [Ca2+
response to ATP (Supplementary Fig. 3c
). ATP was undetectable on random assays of MTD (n=3).
Activating FPR1 desensitizes chemokine receptors, predisposing to infection after trauma 16
. Human PMN treated with MTD became insensitive to GRO-α (CXCL1, ). PMN stimulated by GRO-α, MTD or buffer () show identical Ca2+
release by ionomycin. Since Ca2+
stores are equal, suppression by MTD reflects CXCR2 desensitization by FPR1. PMN also show homologous desensitization when re-challenged with MTD () or fMLF (Supplementary Fig. 4
). Others have shown that PMN MAP kinases are phosphorylated and activated by injury 17
. Skeletal muscle MTD caused phosphorylation of PMN p38 and p44/42 MAP kinases () with p38 being activated at lower concentrations. Thus muscular injury can liberate mitochondrial DAMPs that activate multiple inflammatory signal pathways.
Mitochondrial DAMPs activate PMN signaling, so next we studied whether they elicit an inflammatory PMN phenotype. Matrix metalloproteinase (MMP)-8 is a neutrophil-specific collagenase 19
that aids in PMN tissue penetration and recruitment. Interleukin (IL)-8 causes PMN chemotaxis and activation, and such PMN activation also induces secondary IL-8 release. MTD caused MMP-8 release from human PMN (). Inhibition by CsH or anti-FPR1 demonstrates FPR1-dependence (). Human PMN synthesized and released IL-8 in response to MTD (/f) more rapidly than to LPS. This “bell-shaped” response curve () may reflect FPR1 suppression by high concentrations of MTD (see ). In longer incubation studies, LPS was more potent ().
PMN use lytic enzymes like MMPs to migrate into bystander organs. We assessed the effects of MTD on PMN migration. Under video-microscopy PMN migrated toward MTD from clinical femur fractures (, supplementary videos 1–4
). Speed and directionality of migration were inhibited by CsH 18
(, Video 3
) or by antibodies to FPR1 (, Video 4
). Last, we showed in vivo
PMN infiltration in response to clinical concentrations of MTD by placing enough liver-derived MTD into mouse peritoneum to model traumatic necrosis of 10% of the mouse’s liver. Neutrophilic peritonitis developed quickly (). MTD was more active than the FPR agonist W-peptide and CsH again reduced peritonitis ().
Mitochondria contain their own genome, but mtDNA resembles bacterial DNA in being circular and having nonmethylated CpG motifs 20
. mtDNA has been found in joint fluids in rheumatoid arthritis and induces inflammation in vivo
. CpG DNA activates TLR9 but activation of PMN by mtDNA is unstudied. TLR9 is expressed by PMN 22
and activates p38 MAPK 23
. So we questioned whether PMN p38 MAPK would be activated by mtDNA at clinical plasma concentrations (Supplementary Fig. 1d
). We found 1μg/ml mtDNA caused p38 MAPK phosphorylation () but did not activate p44/42 MAPK. p38 MAPK activation was blocked by inhibitory oligodeoxynucleotides (ODN TTAGGG, ) that bind CpG motifs and block interactions with TLR9. Looking at downstream signaling, we incubated PMN with CpG DNA (10μg/ml) or mtDNA within the clinical range (1–10μg/ml). Neither released IL-8 effectively alone, but each promoted IL-8 release with low-dose fMLF (1nM) (). This is similar to GM-CSF priming of IL-8 release by CpG DNA. 22
These data suggest clinically significant activation of PMN secretion by mtDNA/TLR9. In distinction, TLR ligands have no direct effect on PMN chemotaxis (Supplementary Fig. 5
mtDNA activates PMN via CpG/TLR9 interactions
To determine whether circulating mitochondrial DAMPs could cause neutrophil-mediated organ injury, we injected MTD equivalent to 5% of the rat’s liver intravenously and examined whether that recreated organ injury in vivo
. Animals demonstrated marked inflammatory lung injury as early as 3h post-injection (). Oxidant lung injury was documented by staining for 4-hydroxy-2-nonenal (4-HNE) 24
(). MTD injection increased lung albumin () and wet/dry weight (), IL-6 () as well as elastase accumulation in lung (Supplementary Fig. 6
). Bronchoalveolar lavage showed PMN influx into the airways (), early appearance of TNF-α () and later appearance of IL-6 (). PMN infiltration was confirmed as increased lung MMP-8 (). Systemic inflammation was demonstrated as priming of circulating PMN () and their infiltration into liver (). Control rats showed no evidence of pulmonary or hepatic inflammation.
MTD cause systemic inflammation and organ injury in vivo
In conclusion, inflammation occurs after both major trauma and infection16
. Recognizing sterile SIRS is critical since empiric antimicrobial use will be ineffective whereas other therapies might be effective. After tissue trauma MTD circulates and stimulates PMN, causing systemic inflammation. The molecular similarity of mitochondria to their bacterial ancestors helps explain why traumatic and infective SIRS appear similar 3,25
. Mitochondrial DAMPs express at least two molecular signatures (formyl peptides, mtDNA) that act on PRRs recognizing bacterial PAMPs. These activate PMN in the circulation (, , ) rather than at specific targets, inciting non-specific organ attack () while suppressing chemotactic responsess to infective stimuli (, and Supplementary Fig. 4
Formyl peptides and mtDNA are likely only a subset of the DAMPs released by trauma, but they appear important at clinical concentrations. Other intracellular ‘alarmins’ may similarly be important after injury and other immune cells probably respond to mitochondrial DAMPs. Injury-derived mitochondrial DAMPs however, are clearly recognized by innate immunity using PRR that alternatively sense bacteria. This novel paradigm may explain why responses to ancient ‘enemies within’ released by injury can mimic sepsis.