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Circulating mitochondrial DAMPs cause inflammatory responses to injury
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Letter
Nature 464, 104-107 (4 March 2010) | doi:10.1038/nature08780;
Qin Zhang1, Mustafa Raoof1, Yu Chen1, Yuka Sumi1, Tolga Sursal1, Wolfgang Junger1, Karim Brohi2, Kiyoshi Itagaki1 & Carl J. Hauser1
1. Department of Surgery, Division of Trauma, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA
2. Trauma Clinical Academic Unit, Barts and the London School of Medicine & Dentistry, Queen Mary, University of London, Whitechapel Road, London E1 1BB, UK
Correspondence to: Carl J. Hauser1 Correspondence and requests for materials should be addressed to C.J.H. (Email: cjhauser@bidmc.harvard.edu).
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Editor's Summary
4 March 2010
Mitochondrial revolt
Mitochondria are endosymbiotic descendents of bacteria, well tolerated by the eukaryotic cells that they now serve after millions of years of co-evolution. But in extremis it seems strains in the relationship may emerge. Tests on plasma samples from patients who had suffered severe trauma show that mitochondrial DAMPs (or damage-associated molecular patterns) are released into the circulation as a result of tissue damage, where they activate neutrophils via specific formyl peptide receptors. This triggers systemic inflammation, tissue damage and apparent sepsis. These DAMPs interact with receptors that are part of the innate immune response to molecules known as PAMPs (pathogen associated molecular patterns), which are expressed on invading microorganisms, causing bacterial sepsis. This finding appears to explain the apparent sepsis sometimes associated with severe trauma even when no infection is present.
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Abstract
Injury causes a systemic inflammatory response syndrome (SIRS) that is clinically much like sepsis1. Microbial pathogen-associated molecular patterns (PAMPs) activate innate immunocytes through pattern recognition receptors2. Similarly, cellular injury can release endogenous 'damage'-associated molecular patterns (DAMPs) that activate innate immunity3. Mitochondria are evolutionary endosymbionts that were derived from bacteria4 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 Ca2+ 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.
Trauma is a leading cause of premature death5. Injury causes activation of PMNs, organ failure, susceptibility to infection and SIRS1, 6. Bacterial translocation from ischaemic gut to circulation was long thought to cause SIRS7. This was disproven8 although shock may cause gut inflammation9. 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 PAMPs expressed on invading microorganisms. Pattern recognition receptors recognize PAMPs2. Bacterial proteins are N-formylated10, so formyl peptides activate chemoattractant formyl-peptide receptors (FPRs). TLRs respond to many PAMPs, like bacterial DNA that stimulates TLR9. Because mitochondria evolved from saprophytic bacteria to endosymbionts to organelles, the mitochondrial genome (mtDNA) contains CpG DNA repeats and codes for formylated peptides4, 11. Mechanical trauma disrupts cells, so we hypothesized injury might release mitochondrial DAMPs3 into the circulation, activating immunity and initiating SIRS.
To prove trauma releases of MTDs into the circulation, we measured plasma mtDNA in 15 major trauma patients (Injury Severity Score12 >25). Sampling was before resuscitation. Patients had no open wounds or gastrointestinal injuries. The mtDNA of the trauma patients was markedly elevated compared with volunteers. The mtDNA in trauma plasma was 2.7+/-0.94ugml-1 (means+/-s.e.m.), thousands of fold higher than volunteers' levels. The mtDNA was further elevated 24h after injury. Ultracentrifugates of reamed specimens obtained from femurs during clinical fracture repair contained even higher titres of mtDNA. Thus MTDs are mobilized by either external or operative injury to enter the circulation. Bacterial 16S RNA was absent from all specimens.
ondrial formyl peptides can attract PMNs13 and activate related cell lines14. The synthetic peptide N-formyl-Met-Leu-Phe (fMLF) simulates bacterial challenge. However, the role of endogenous formyl peptides in trauma, PMN activation and SIRS is unstudied. Formyl peptides signal through the G-protein-coupled receptors FPR1 and FPRL-1, with high and low affinities respectively. PMN activation by G-protein-coupled receptors causes increased intracellular calcium ([Ca2+]i)15, heterologous and homologous G-protein-coupled receptor desensitization16 and activates MAP kinases (MAPKs)17. MTD from human myocytes induced human PMN [Ca2+]i fluxes equal to 1 nM fMLF (Fig. 1a). MTD from human liver, muscle and fracture haematoma. or from rat muscle or liver produced similar PMN Ca2+ depletion. Whole and fragmented mitochondria had similar potency. Thus release of MTD from all cell types studied activates immunity.
Blocking antibodies to FPR1 abolished Ca2+ depletion (Fig. 1a) and Ca2+ entry (Fig. 1b) responses to MTD. Cyclosporin H (CsH) inhibits FPR118 and abolishes Ca2+ flux to MTD. Isotype control (FPRL-1, matrix metalloproteinase (MMP)-2) antibodies have no effects. Apyrase-treated and untreated MTDs act identically whereas apyrase abolishes [Ca2+]i response to ATP. ATP was undetectable on random assays of MTD (n = 3).
Activating FPR1 desensitizes chemokine receptors, predisposing to infection after trauma16. Human PMNs treated with MTD became insensitive to GRO-α (CXCL1, Fig. 1c). PMNs stimulated by GRO-α, MTD or buffer (Fig. 1d) show identical Ca2+ release by ionomycin. Because Ca2+ stores are equal, suppression by MTD reflects CXCR2 desensitization by FPR1. PMNs also show homologous desensitization when re-challenged with MTD or fMLF. Others have shown that PMN MAPKs are phosphorylated and activated by injury17. Skeletal muscle MTD caused phosphorylation of PMN p38 and p44/42 MAPKs (Fig. 2a, b) with p38 being activated at lower concentrations. Thus muscular injury can liberate mitochondrial DAMPs that activate multiple inflammatory signal pathways.
Because mitochondrial DAMPs activated PMN signalling we studied whether they elicit an inflammatory PMN phenotype. MMP-8 is a neutrophil-specific collagenase19 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 PMNs (Fig. 2c). Inhibition by CsH or anti-FPR1 again demonstrates FPR1 dependence (Fig. 2d). Human PMNs synthesized and released IL-8 in response to MTD (Fig. 2e, f) more rapidly than to LPS. This 'bell-shaped' response curve (Fig. 2e) may reflect FPR1 suppression by high concentrations of MTD (see Fig. 1e). In longer incubation studies, LPS was more potent (Fig. 2f).
PMNs use lytic enzymes like MMPs to migrate into bystander organs. We assessed the effects of MTD on PMN migration. Under video-microscopy, PMNs migrated towards MTD from clinical femur fractures (Fig. 2g-j and. Speed and direction of migration were inhibited by CsH. or by antibodies to FPR1. 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 (Fig. 2k). MTD was more active than the FPR agonist W-peptide, and CsH again reduced peritonitis (Fig. 2k).
Mitochondria contain their own genome, but mtDNA resembles bacterial DNA in being circular and having non-methylated CpG motifs20. Mitochondrial DNA has been found in fluids in joints in rheumatoid arthritis and induces inflammation in vivo21. CpG DNA activates TLR9 but activation of PMN by mtDNA is unstudied. TLR9 is expressed by PMN22 and activates p38 MAPK23. So we questioned whether PMN p38 MAPK would be activated by mtDNA at clinical plasma concentrations. We found 1 ·g ml-1 mtDNA caused p38 MAPK phosphorylation (Fig. 3a) but did not activate p44/42 MAPK. Activation of p38 MAPK was blocked by inhibitory oligodeoxynucleotides (TTAGGG, Fig. 3b) that bind CpG motifs and block interactions with TLR9. Looking at downstream signalling, we incubated PMN with CpG DNA (10 ·g ml-1) or mtDNA within the clinical range (1-10 ·g ml-1). Neither released IL-8 effectively alone, but each promoted IL-8 release with low-dose fMLF (1 nM) (Fig. 3c). This is similar to granulocyte-macrophage colony-stimulating factor priming of IL-8 release by CpG DNA22. These data suggest clinically significant activation of PMN secretion by mtDNA/TLR9. In distinction, TLR ligands have no direct effect on PMN chemotoxis.
To determine whether circulating mitochondrial DAMPs could cause neutrophil-mediated organ injury, we injected MTDs 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 3 h after injection (compare Fig. 4a, b). Oxidant lung injury was documented by staining for 4-hydroxy-2-nonenal24 (compare Fig. 4c, d). MTD injection increased lung albumin (Fig. 4e) and wet/dry mass (Fig. 4f), IL-6 (Fig. 4g) as well as elastase accumulation in lung. Bronchoalveolar lavage showed PMN influx into the airways (Fig. 4h), early appearance of tumour-necrosis factor-α (Fig. 4i) and later appearance of IL-6 (Fig. 4j). PMN infiltration was confirmed as increased lung MMP-8 (Fig. 4k). Systemic inflammation was demonstrated as priming of circulating PMNs (Fig. 1f) and their infiltration into liver (Fig. 4l). Control rats showed no evidence of pulmonary or hepatic inflammation.
In conclusion, inflammation occurs after both major trauma and infection16. Recognizing sterile SIRS is critical because empiric antimicrobial use will be ineffective whereas other therapies might be effective. After tissue trauma, MTD circulates and stimulates PMNs, causing systemic inflammation. The molecular similarity of mitochondria to their bacterial ancestors helps explain why traumatic and infective SIRS appear similar3, 25. Mitochondrial DAMPs express at least two molecular signatures (formyl peptides, mtDNA) that act on pattern recognition receptors recognizing bacterial PAMPs. These activate PMN in the circulation (Figs 1f, 2 and 3) rather than at specific targets, inciting non-specific organ attack (Fig. 4) while suppressing chemotactic responses to infective stimuli.
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 pattern recognition receptors that alternatively sense bacteria. This novel model may explain why responses to these ancient 'enemies within' released by injury can mimic sepsis.
Methods Summary
All studies were approved by the institutional review boards of Beth Israel Deaconess Medical Center, Boston, USA, and Queen Mary's University Hospital, London, UK. Animal care was approved by the Institutional Animal Care and Use Committee according to National Institutes of Health guidelines.
Preparation of mitochondria, MTDs and mtDNA
Mitochondria were isolated from resources as per standard protocols.
PMN studies
PMN isolation26, 27, calcium studies15, 16, 26, western blots28, transwell chemotaxis16 and video-microscopy chemotaxis assays29 were performed as previously described.
MTD administration
Male Sprague-Dawley rats were given intravenous MTDs based on mass30. Quantitative PCR of plasma showed mtDNA levels of 122 ± 22 ng ml-1 1 h after injection (normal levels are very much less than 1 ng ml-1). Leukocytes in bronchoalveolar lavages were counted visually. Lungs were inflated gently and fixed in formalin before staining with haematoxylin and eosin or for 4-hydroxy-2-nonenal.
Full methods accompany this paper.
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