Damage-associated molecular patterns in trauma

Traumatic injury is one of the major contributors to worldwide mortality as well as one of the world’s most relevant but neglected health concerns [1, 2]. Multiply injured or polytraumatized patients most frequently die either immediately or early within only of a few hours after the traumatic insult due to their injury severity, severe traumatic brain injuries (TBI), and/or exsanguination, or in the later post-injury phase due to inflammation-related complications, which affect the immune system homeostasis, resulting in, e.g., sepsis, septic shock, or multiple organ failure (MOF) [2‐6]. Adjacent to physical factors, which are intensely treated in the intensive care units (ICU), in case of survival, the multiply injured patient may suffer from serious impairments in cognitive, psychological, or psychosocial issues [7‐9].

Over the last decades, several experimental (poly)trauma models which combine different injury patterns have been developed and applied to study and understand the basic pathophysiology of the posttraumatic sequelae in polytraumatized patients. While the primary impact can only be addressed by injury prevention, the later detrimental sequalae may be prevented by an abrogation of the posttraumatic course [10, 11]. The typical pathophysiological course of traumatized patients under ICU treatment is characterized by a systemic inflammatory response to severe injury that begins immediately after trauma, and which involves complex interactions across the hemostatic, inflammatory, endocrine, and neurological systems [3, 12, 13]. Modern notions hold that this clinically observed so-called systemic inflammatory response syndrome (SIRS) reflects an uncontrolled overshooting systemic innate immune response caused by polytrauma-induced emission of large amounts of so-called damage-associated molecular patterns (DAMPs) which circulate systemically to affect the whole body of the patient [3, 12‐15]. The term DAMP was coined in 2003 by Land [15]. And it is apparently the DAMPs which lead to the catastrophe of the sterile hyperinflammatory, sepsis-like, life-threatening SIRS, which is most often associated with organ failure, MOF, or multiple organ dysfunction syndrome (MODS) [12, 13, 16]. Typically, this DAMP-induced systemic cytokine/chemokine-mediated hyperinflammatory response to severe trauma is associated with an intense and long-lasting counterbalancing compensatory anti-inflammatory response syndrome (CARS) resulting in posttraumatic immunosuppression (IS) [3, 5, 17‐19]. CARS can be regarded as a disorder caused by a hyper-resolution of hyperinflammation in terms of a mirror-imaged counter-regulation of SIRS. Thus, for several decades research on posttraumatic complications was based on a biphasic posttraumatic inflammation model. The central pathophysiological principle there was to find the balance between a qualitative defense against invasive putative pathogens and additionally reducing collateral damage by immune cells [20‐22]. Of note, however, instead of a previously assumed biphasic inflammatory response consisting of an initial proinflammatory and a subsequent counterbalancing anti-inflammatory response, hyper-resolving processes are now considered to occur simultaneously with hyperinflammation. There is presumably only a slight delay between initial hyperinflammation and the following hyper-resolution, which is possibly caused by the production of suppressing/inhibiting inducible DAMPs (SAMPs) by already DAMP-activated/initialized responses of innate immune cells [23]. Recently, this novel term accomplishing the DAMPs/SAMPs post-injury response was coined by Land [23]. The posttraumatic course of this inflammatory response is briefly represented in Fig. 1.

The recognition of DAMPs by immune cells initiates an acute hyperinflammatory condition of SIRS that induces physiological changes such as hypothermia or hyperthermia, elevated heart rate, and leukocytosis/leukocytopenia [24, 25]. The uncontrolled or protracted clinical signs of SIRS constitute a risk factor for organ failure, delayed hospital-acquired infections as well as occasionally MOF after trauma [26‐28]. The concept of SIRS was introduced in early 1990s and charged with the task of developing an easy-to-apply set of clinical parameters, as well as biomarkers, to improve the early identification of potential candidates for clinical trials and new treatment strategies for sepsis, as well as for the identification of patients at risk of infectious complications, respectively [25]. Thus, since three decades, the knowledge on the complex pathophysiological and posttraumatic inflammatory response contributing to the development of post-injury complications is growing [29‐32]. Numerous cellular and humoral proinflammatory mediators including complement factors are markedly elevated in patients suffering from SIRS and associated complications on the other hand. In general, SIRS is classically dominated by overwhelming hyperinflammatory processes which are characterized by elevated circulating levels of multiple powerful cellular and humoral proinflammatory mediators. Of those, notably in the experimental and clinical sepsis-associated SIRS, cytokines (e.g., tumor necrosis factor (TNF), interleukin (IL)-1α, IL-1β, and IL-6), chemokines, growth factors, adhesion molecules, gaseous substances, vasoactive peptides, and cell stress markers have been identified and analyzed [33]. After the concept of SIRS was created, a number of publications emerged heralding the efficacy of SIRS in predicting outcomes after trauma [34, 35]. Accumulating evidence indicates that SIRS associated with MOF is the result of traumatic injury-induced either accidental or regulated cell death such as necroptosis, serving as a robust source for emission of DAMPs, while the impact of apoptosis may be presumably less significant here [36‐39]. Today, it is widely accepted that DAMPs are the key predominant molecules which trigger the biological host response to trauma including SIRS, by activating and recruiting effector cells including antigen-presenting cells of the immune system [12, 40‐46]. Thus, basically, every DAMP proven to promote efferent proinflammatory pathways can be considered to be involved in the development of SIRS. In 2012, in accordance with the newly recognized “sterile SIRS” as a severe complication form in polytraumatized patients, first clues to a critical role of DAMPs in the promotion of those hyperinflammatory responses have been reported [47]. Among those molecules, endogenous DAMPs including high-mobility group box (HMGB) protein 1, heat shock proteins (HSPs), certain members of the S100A family, histones, free nucleic acids, members of the IL-1 cytokine as well as complement family as bona fide intracellular effectors, which upon abundant rapid release alert the environment about cell stress and danger, have already been reported to activate the posttraumatic innate immune responses and drive organ dysfunction(s) after trauma [12, 13, 46, 48].

Although a large number of endogenous nuclear or cytosolic DAMPs have been described in the context of the local/systemic posttraumatic and/or noninfectious “sterile” inflammatory response, which represents the key driver of the late occurring post-injury complications and fatal outcome rates, the knowledge on their variety and underlying mechanisms still remains unknown [49‐51]. In particular, it is unknown whether there is a distinct pattern of DAMPs emission that may be helpful in differentiating between, i.e., sterile and infectious (bacterial or viral) sepsis form. The knowledge on posttraumatic DAMPs pattern may allow to further define the exact roles of DAMPs in hyperinflammation and develop therapeutic inhibitors targeting specific DAMPs to improve outcomes. Thus, some of most critically involved DAMPs in both sterile and infectious posttraumatic disorders will be briefly described in this review.

The recognition of an overshooting SIRS which may be associated with septic complications has provided several attractive targets for new anti-inflammatory drugs which were designed to prevent further propagation of inflammation in past. Yet, nowadays, treatment of sepsis in critically ill patients is mainly based on the prompt intravenous administration of adequate antimicrobial agents and support of organ functions. For the last four decades, in theory, many different targets for treatment were designed to inhibit, bind, or neutralize the proinflammatory mediators that were assumed to be responsible for the network of events that culminated in detrimental clinical outcomes. Disappointingly, however, targeted clinical trials have shown that most of these agents failed to cure the septic disease [52‐56]. Even the promising US FDA-approved drug, recombinant human-activated protein C or drotrecogin-α failed in clinical trials and showed similar effects as placebo. Intriguingly, these observations paved the way to another typical symptomatic feature of the disorder: the CARS [24]. The high susceptibility to secondary infections after the initial phase of sepsis or after trauma has been attributable to CARS causing the posttraumatic IS [17, 19, 57]. Interestingly, for a long time, intensive care physicians have observed that patients with SIRS were prone to severe, sometimes lethal infectious diseases. However, in search of the causes during the 1970s and 1980s, researchers found that many of those patients had acquired an impaired immune state characterized by various markers of IS. The concept was actually underlined from the beginning as the treatment strategies with anti-inflammatory agents failed in clinical trials on septic patients.

The phenomenon of impaired immunity that was frequently observed in posttraumatic as well as in septic conditions essentially reverses many of the typical events encountered in SIRS. However, research over the last several decades uncovered the anti-inflammatory and immunosuppressive mechanisms that govern the clinical post-injury disorders. Initially, IS corresponds to a homeostatic phenomenon to avoid the remote organ injuries caused by the early SIRS; however, it becomes deleterious when it persists, rendering patients prone to secondary infections [17, 58]. Nowadays, CARS is not regarded as part of a biphasic inflammatory response consisting of an initial SIRS with a subsequent CARS. It became evident that the hyper-resolving processes of inflammation (CARS, IS) occur nearly in parallel with the hyperinflammatory response, but also that they can exist separately from the SIRS [28, 59]. Moreover, CARS is not only a simplified anti-inflammatory process, but rather a disorder caused by a hyper-resolution of inflammation.

Remarkably, next to alterations of the innate immune system, posttraumatic IS has been closely linked to modulations of the adaptive immune system. Here, among others, i.e., a shift from a T-helper cell type (Th)1- to a Th2-mediated immune response, T cell anergy in traumatized patients, suppression of T cell effector functions, modulated frequency of T regulatory cells have been observed [19, 21, 22, 60‐64]. This posttraumatic response suggests that in reverse with the proinflammatory action of DAMPs in SIRS, SAMP triggers the proresolving pathways in CARS and IS. Besides activating DAMPs, potentially suppressing DAMPs, that is SAMPs, are mainly produced by activated leukocytes and macrophages upon stress and injury, which—in parallel to proinflammatory responses, convey anti-inflammatory processes and shape immunosuppressive responses. In 2015, it was shown that the gene expression of proresolving lipid mediator pathways was closely associated with clinical outcomes in traumatized patients [65]. Patients with uncomplicated recoveries had differential and higher resolving scores [65]. The SAMPs prostaglandin E2 or AnxA1 plays significant roles in proresolving CARS [66‐68]. Accordingly, DAMPs and SAMPs constitute valuable tools in diagnosis and therapy of severely injured and/or infected patients that are expected to find their way into clinical routine at the ICUs. Hyper-resolution with the following IS may be the most critical factor for the immune status of patients with either sterile or infectious SIRS. Thus, here as well, measurement of DAMPs with their pattern, but also continuous determination of SAMPs with their pattern, respectively, may be used as hospital admission criteria for decisions supporting further diagnostic, prognostic, and therapeutic modalities. Although a closer look into both DAMPs and SAMPs is necessary to understand the posttraumatic response, in the underlying review only DAMPs will be addressed.

DAMPs are released from a damaged or diseased cell, and upon their release, they can stimulate a sterile immune or inflammatory response [13, 49, 69]. Across the tree of life, DAMP-induced immune responses serve as defense strategies aimed at maintaining and restoring homeostasis. However, despite their initially beneficial character, when dysregulated, uncontrolled, and exaggerated, the inflammatory and tissue repairing processes may become pathogenic and can lead to serious pathologies, i.e., sepsis, cardiovascular diseases, neurodegenerative diseases, diabetes, obesity, and asthma, as well as classic inflammatory diseases. Notably in the last two decades, the biological host response to trauma, which initially has been characterized by massive cytokine release as well as the activation and recruitment of effector cells including antigen-presenting cells, has further employed a large number of both microbial and host alarmins [12, 40‐44]. In general, on the biochemical level, the posttraumatic immune response is not only activated by foreign non-self material, but includes also endogenous factors, DAMPs, which are released from necrotic or physiologically “stressed” cells, with the aim of initiating and recruiting effector cells of the immune system [70‐72]. A large number of those endogenous nuclear or cytosolic triggers have been described to initiate and perpetuate the systemic posttraumatic and/or noninfectious inflammatory response; however, the knowledge on their precise role still remains unknown [13, 49‐51]. They signal “danger” to the host and trigger an inflammatory response, which in physiological healing leads over to tissue regeneration, but when dysregulated can cause pathologic inflammation and related disorders.

In the pathology of severe trauma, trauma-induced generation/emission of DAMPs can refer to cell-intrinsic modified DAMPs such as cell-free deoxyribonucleic acid cfDNA or DAMPs passively released from dying cells. Thus, any kind of traumatic cell stress/tissue injury provoked by mechanical trauma (e.g., blow, crush, penetrating wound, or surgical procedures usually related to fracture, hemorrhage, and/or infections), thermal trauma, or metabolic trauma such as promoted by ischemia/reperfusion injury, acidosis, hypoxia/hypoxemia, provokes a DAMP release [23]. Clinically, SIRS is characterized by local and systemic release of proinflammatory cytokines produced by DAMP-activated innate immune cells including IL-1β, TNF, and IL-6, arachidonic acid metabolites, proteins of the contact phase and coagulation systems, and complement factors as well as hormonal mediators [3, 34, 73, 74]. Among other factors, proinflammatory cytokines also activate coagulation pathways, whereby enhanced thrombin formation increases fibrinogen cleavage and reduces fibrin monomers from polymerizing to form stable fibrin clots (hypercoagulability), which is why fibrinogen depletion is observed [75]. Disseminated intravascular coagulation (DIC) is a heterogeneous group of disorders, which manifests as a spectrum of hemorrhage and thrombosis complicating many primary conditions including, i.e., sepsis and trauma [76]. Although its pathophysiology is complex, there is growing evidence that DAMPs emitted in excess play a crucial role in the pathogenesis of DIC [76]. Here, i.e., cfDNA, also known as extracellular DNA, which can be released by various host cells including neutrophils, macrophages, eosinophils, and tumor cells, as well as by certain strains of bacteria plays a significant role [77‐82]. Elevated levels of cfDNA have been found in various pathologic conditions including trauma [83, 84]. Interestingly, cfDNA that appears to exert both pro- and anti-fibrinolytic effects is a good predictor of trauma patient’s outcome in the ICU [84]. Activation of neutrophils with microbial or inflammatory stimuli results in the release of neutrophil extracellular traps (NETs) [77]. Upon cell death or specific cell activation of hematopoietic and parenchymal cells, extracellular cfDNA as well as DNA-binding proteins (e.g., histones and HMGB1) are released into circulation [13]. Those DNA-binding proteins are also strongly procoagulant and are involved in the pathogenesis of DIC [76, 85]. Another factor that can be directly influenced by DAMPs and contribute to DIC is the cellular migratory behavior with barrier loss. DAMP-induced endothelial expression of adhesion molecules facilitates leukocyte adhesion, promoting extravasation of leukocytes from the circulation into damaged tissue [86]. Mitochondrial (mt) DAMPs like mitochondrial DNA (mtDNA) and peptides appear in the blood after injury or shock, activate human leukocytes and might contribute to increased endothelial permeability during systemic inflammation [87]. The various DAMP motifs from mitochondria can act on endothelia and/or leukocytes via multiple pathways by enhancing leukocyte adherence to endothelial cells, activating cell–cell interactions, and subsequently increasing systemic endothelial permeability [87]. Mitochondrial DAMPs may be important therapeutic targets in conditions where inflammation pathologically increases endothelial permeability [87]. The mechanisms of a concerted action between endothelial cells and leukocytes, endothelial cell damage, leukocytes extravasation, microcirculatory disturbances, or DIC frequently lead to cell loss of parenchymal cells with the following organ failure. Increased systemic levels of DAMPs have been positively correlated with mortality and morbidity of patients or animals with trauma or surgical insults, while blocking or neutralizing DAMPs with specific small molecules or antibodies ameliorated sepsis and SOF in vivo [88]. As briefly introduced above, the clinical manifestation of severe multiple tissue injury including burn damage is characterized by a systemic cytokine/chemokine-induced hyperinflammatory response resulting in SIRS which is accomplished by an intense and long-lasting CARS associated with posttraumatic immunosuppression that predisposes patients to infectious sepsis and MOF [3, 17, 19, 57]. For example, in a murine burn injury model, IL-10 levels were significantly increased at 24 h, 7 days, and 21 days post-injury as compared with IL-10 levels in sham mice [89]. Thus, accumulating evidence suggests that clinically observed infectious complications are associated with high susceptibility of the patient to secondary bacterial, fungal, and viral infections and that this state is mediated by suppressing DAMPs by SAMPs which control homeostasis following injury. Yet, in the following, a closer look at the role of exemplary DAMPs in traumatic setting is taken. Since DAMPs are a culmination of highly heterogenous mediators, it is nearly impossible to choose the most important ones. Thus, in this review, we focus on the most prominent DAMPs involved in the posttraumatic pathophysiology, but other DAMPs of equal importance, which have been neglected here, may be addressed in further studies. This review sought to primarily describe the exemplary variety of DAMPs in their preclinical and clinical context.

The “danger model” was proposed as an alternative to the “self versus non-self recognition model” [364, 365]. Briefly, the immune system discriminates not only between self and non-self, but also between safe and dangerous as well, as clinically proved by Walter Land and presented by Polly Matzinger in the “Danger Model,” expanding the work of Janeway and others [70, 71, 364‐367]. This complex response to stress employs numerous equivalent or comparable components of PAMPs, DAMPs, or newly introduced SAMPs, which can be found in most vertebrates, invertebrates, and even plants [23, 366‐369]. These molecules are sensitized and recognized via PRR mediating very potent immune responses [370, 371]. Several classes of PRR have been identified so far, including the most prominent group of toll-like receptors (TLR), nucleotide oligomerization domain (NOD)-like receptors (NLR), members of the c-type lectin receptors (CLR) like mannose binding lectine (MBL) or receptor for advanced glycation end products (RAGE) among others [187, 370, 372, 373].

This review discusses only a few of the currently discussed DAMPs that play important roles in the inflammatory or regenerative responses upon trauma. Although the provided list is certainly both incomplete and provides only a limited overview to the concept of trauma-induced DAMPs, it remains evident that due to the bivalent character and often pleotropic effects of a DAMP, its functions in the posttraumatic inflammation and regeneration still remain to be studied in detail. However, the new insights into the contribution of DAMPs to the sepsis-like inflammation and immune paralysis-like states that can be observed in severely injured patients will be helpful in further specifying the clinical picture presented by, e.g., polytrauma. Considering the “SIRS and CARS paradigm,” and the terms mixed antagonist response syndrome and persistent inflammation, immunosuppression and catabolism syndrome, the role of these injury-induced activating and suppressing molecules clearly marks their predominant role in posttraumatic pathologies. It is indisputable that both systemic as well local presence of DAMPs is obligatory for the immune response upon traumatic insult. Yet, adjacent to their ability to be used as biomarkers to indicate or monitor disease or injury severity, and for eventual improving the timing of secondary surgery, they remain either negative or positive contributing factors for the disease development. In polytrauma, there is tentative evidence of a model suggesting that, simultaneously or somewhat retardedly with uncontrolled overshooting emission of proinflammatory DAMPs in excess (resulting in hyperinflammatory SIRS and MOF), uncontrolled overshooting generation/emission of SAMPs in excess takes place. Those “suppressing molecules” may lead to an exaggerated and long-lasting CARS associated with a stage of immunosuppression—still aimed at maintaining tissue homeostasis. Considering this scenario, it certainly remains to be further discussed if the biologically natural attempt of the innate immune defense system to reach homeostasis via emission of SAMPs in case of severe injury-induced SIRS potentially and tragically ends up with the creation of an overshooting anti-inflammatory/proresolving and immunosuppressive milieu rendering the patient susceptible to secondary life-threatening, often lethal infections. Although the data from SAMPs involved in polytrauma are sparse and, though, the various suppressing molecules are not systemically investigated, there is preliminary evidence suggesting a role of this class of suppressing DAMPs in posttraumatic inflammation. Therefore, increased knowledge exploring the dual role of DAMPs and the interplay with SAMPs after trauma, and with their nuclear functions, transcriptional targets, release mechanisms, cellular sources, and other functions and interactions in traumatized patients is warranted. Adjacent to in vivo and in vitro studies, and furthermore, based on some contradictory findings, which often simply originate from differences between the immune system of animals and human, further clinical research is necessary to resolve this puzzle.