Injury causes a systemic inflammatory response syndrome (SIRS) clinically much like sepsis 1. Microbial pathogen-associated molecular patterns (PAMPs) activate innate immunocytes through pattern recognition receptors 2. Similarly, cellular injury can release endogenous damage-associated molecular patterns (DAMPs) that activate innate immunity 3. Mitochondria are evolutionary endosymbionts that were derived from bacteria 4 and so might bear bacterial molecular motifs. We show here that injury releases mitochondrial DAMPs (MTD) into the circulation with functionally important immune consequences. MTD include formyl peptides and mitochondrial DNA. These activate human neutrophils (PMN) through formyl peptide receptor-1 and TLR9 respectively. MTD promote PMN Ca2+ flux and phosphorylation of MAP kinases, thus leading to PMN migration and degranulation in vitro and in vivo. Circulating MTD can elicit neutrophil-mediated organ injury. Cellular disruption by trauma releases mitochondrial DAMPs with evolutionarily conserved similarities to bacterial PAMPs into the circulation. These can then signal through identical innate immune pathways 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.

There is limited understanding of the mechanisms by which tissue trauma, shock, and inflammation initiate trauma coagulopathy. Acute Coagulopathy of Trauma-Shock should be considered distinct from disseminated intravascular coagulation as described in other conditions. Rapid diagnosis and directed interventions are important areas for future research.

Bacterial DNA (bDNA) can activate an innate-immune stimulatory "danger" response via toll-like receptor 9 (TLR9). Mitochondrial DNA (mtDNA) is unique among endogenous molecules in that mitochondria evolved from prokaryotic ancestors. Thus, mtDNA retains molecular motifs similar to bDNA. It is unknown, however, whether mtDNA is released by shock or is capable of eliciting immune responses like bDNA. We hypothesized shock-injured tissues might release mtDNA and that mtDNA might act as a danger-associated molecular pattern (or "alarmin") that can activate neutrophils (PMNs) and contribute to systemic inflammatory response syndrome. Standardized trauma/hemorrhagic shock caused circulation of mtDNA as well as nuclear DNA. Human PMNs were incubated in vitro with purified mtDNA or nuclear DNA, with or without pretreatment by chloroquine (an inhibitor of endosomal receptors like TLR9). Neutrophil activation was assessed as matrix metalloproteinase (MMP) 8 and MMP-9 release as well as p38 and p44/42 mitogen-activated protein kinase (MAPK) phosphorylation. Mitochondrial DNA induced PMN MMP-8/MMP-9 release and p38 phosphorylation but did not activate p44/42. Responses were inhibited by chloroquine. Nuclear DNA did not induce PMN activation. Intravenous injection of disrupted mitochondria (mitochondrial debris) into rats induced p38 MAPK activation and IL-6 and TNF-alpha accumulation in the liver. In summary, mtDNA is released into the circulation by shock. Mitochondrial DNA activates PMN p38 MAPK, probably via TLR9, inducing an inflammatory phenotype. Mitochondrial DNA may act as a danger-associated molecular pattern or alarmin after shock, contributing to the initiation of systemic inflammatory response syndrome.

Efficient activation of neutrophils is a key requirement for effective immune responses. We found that neutrophils released cellular adenosine triphosphate (ATP) in response to exogenous stimuli such as formylated bacterial peptides and inflammatory mediators that activated receptors for Fcγ, interleukin-8, C5a complement, and leukotriene B4. The release of ATP in response to stimulation of the formyl peptide receptor (FPR) occurred through pannexin-1 hemichannels that colocalized with FPR and P2Y2 nucleotide receptors on the cell surface to form a purinergic signaling system that facilitated the activation of neutrophils. Disruption of this purinergic signaling system by inhibiting or silencing pannexin-1 hemichannels or P2Y2 receptors blocked the activation of neutrophils and impaired innate host responses to bacterial infection. Thus, purinergic signaling is a fundamental mechanism that is required for neutrophil activation and immune defense.

rFVIIa reduced blood product use but did not affect mortality compared with placebo. Modern evidence-based trauma lowers mortality, paradoxically making outcomes studies increasingly difficult.

Release of immature white blood cells into the circulation may also contribute to a failure to clear infection and an increased propensity to organ failure. Concomitantly, profound changes occur within the bone marrow, which include the increased release of erythroid and myeloid progenitors into the circulation, a decrease in progenitor cell growth within the bone marrow, and an impaired growth of the bone marrow stroma. Erythropoietin levels are preserved following trauma, implying that the persistent anemia of injury is related to the failure of the bone marrow to respond to erythropoietin.

Trauma and sepsis can cause acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) in part by triggering neutrophil (PMN)-mediated increases in endothelial cell (EC) permeability. We had shown that mitochondrial (mt) damage-associated molecular patterns (DAMPs) appear in the blood after injury or shock and activate human PMN. So we now hypothesized that mitochondrial DAMPs (MTD) like mitochondrial DNA (mtDNA) and peptides might play a role in increased EC permeability during systemic inflammation and proceeded to evaluate the underlying mechanisms. MtDNA induced changes in EC permeability occurred in two phases: a brief, PMN-independent ‘spike’ in permeability was followed by a prolonged PMN-dependent increase in permeability. Fragmented mitochondria (MTD) caused PMN-independent increase in EC permeability that were abolished with protease treatment. Exposure to mtDNA caused PMN-EC adherence by activating expression of adherence molecule expression in both cell types. Cellular activation was manifested as an increase in PMN calcium flux and EC MAPK phosphorylation. Permeability and PMN adherence were attenuated by endosomal TLR inhibitors. EC lacked formyl peptide receptors but were nonetheless activated by mt-proteins, showing that non-formylated mt-protein DAMPs can activate EC. Mitochondrial DAMPs can be released into the circulation by many processes that cause cell injury and lead to pathologic endothelial permeability. We show here that mitochondria contain multiple DAMP motifs that can act on EC and/or PMN via multiple pathways. This can enhance PMN adherence to EC, activate PMN-EC interactions and subsequently increase systemic endothelial permeability. Mitochondrial DAMPs may be important therapeutic targets in conditions where inflammation pathologically increases endothelial permeability.

Neutrophil extracellular traps (NETs) are critical for anti-bacterial activity of the innate immune system. We have previously shown that mitochondrial damage-associated molecular patterns (mtDAMPs), including mitochondrial DNA (mtDNA), are released into the circulation after injury. We therefore questioned whether mtDNA is involved in trauma-induced NET formation. Treatment of human polymorphoneutrophils (PMN) with mtDNA induced robust NET formation, though in contrast to phorbol myristate acetate (PMA) stimulation, no NADPH-oxidase involvement was required. Moreover, formation of mtDNA-induced NETs was completely blocked by TLR9 antagonist, ODN-TTAGGG. Knowing that infective outcomes of trauma in elderly people are more severe than in young people, we measured plasma mtDNA and NET formation in elderly and young trauma patients and control subjects. MtDNA levels were significantly higher in the plasma of elderly trauma patients than young patients, despite lower injury severity scores in the elderly group. NETs were not visible in circulating PMN isolated from either young or old control subjects. NETs were however, detected in PMN isolated from young trauma patients and to a lesser extent from elderly patients. Stimulation by PMA induced widespread NET formation in PMN from both young volunteers and young trauma patients. NET response to PMA was much less pronounced in both elderly volunteers’ PMN and in trauma patients’ PMN. We conclude that mtDNA is a potent inducer of NETs that activates PMN via TLR9 without NADPH-oxidase involvement. We suggest that decreased NET formation in the elderly regardless of higher mtDNA levels in their plasma may result from decreased levels of TLR9 and/or other molecules, such as neutrophil elastase and myeloperoxidase that are involved in NET generation. Further study of the links between circulating mtDNA and NET formation may elucidate the mechanisms of trauma-related organ failure as well as the greater susceptibility to secondary infection in elderly trauma patients.