2.1. Sepsis-Induced Multiple Organ Dysfunction Syndrome
Sepsis is one of the first illnesses to be described. The term “sepsis” derives from the ancient Greek term “σῆψις, i.e., sipo” (“make rotten”). This term was used by Hippocrates around 400 BCE to describe the process through which infected wounds become purulent
[15]. It took over two millennia to shift our focus onto the role of the host response, and not only on the pathogen itself, in understanding the pathogenesis of sepsis. It is estimated that there are 30 million cases of sepsis and six million sepsis-related deaths worldwide each year
[16][17][18][19][20][21][22]; thus, it is an area of intense medical research.
The dichotomous role of neutrophils in inflammation and infection is well known. These cells play a crucial role in defense against infection; conversely, excessive activation of neutrophils can elicit tissue damage. This is one of the mechanisms that underlies sepsis-induced MODS
[23]. Biomarkers of neutrophil activation may predict MODS in critically ill patients with sepsis
[24]. Plasma concentrations of heparin-binding protein (HBP), myeloperoxidase (MPO), IL-6 and IL-8 appear to be correlated with emergence of the first sepsis-associated organ dysfunction. MPO and HBP become elevated at a median of 12 h before the first organ dysfunction and can remain elevated for up to 24 h, unlike two proinflammatory cytokines with rapid increase and decrease. Therefore, MPO and HBP, as markers of early prolonged neutrophil activation, are not as prone as IL-6 and IL-8 to the effect of sample timing and may be clinically more applicable. Circulating monocytes are also an important limb of innate immunity and are among the first cell-types to respond to pathogens. In a recent study of monocyte subsets in 42 critically ill patients with septic shock,
[25] it was demonstrated that, in early deceased patients, the frequency of classical monocytes (within 12 h of admission) was significantly decreased, while frequency of intermediate monocytes was statistically highly significantly increased, in comparison with patients who survived past the fifth day of ICU stay. Therefore, myeloid cells are a current focus in research and in the clinical setting. Within the framework of complex immune cell function regulation, polarization of macrophage function is an important element. Classically activated macrophages (M1), initially in inflammatory response, can undergo reprogramming to an alternatively activated (M2) phenotype. This contributes to secondary immunosuppression during sepsis. Mitochondria in immune cells are also crucial for immunity. Mitophagy refers to selective degradation of mitochondria by autophagy. Mitophagy in monocytes/macrophages of critically ill patients was investigated by Patoli et al. who demonstrated that, in this patient population, mitophagy was inhibited in blood monocytes of septic in comparison with non-septic patients. They concluded that the inhibition of mitophagy is a physiological process contributing to myeloid cell activation
[26].
MODS is the clinical consequence of a dysregulated, disproportionate immuno-inflammatory response to various stimuli, with a self-perpetuating cycle of neutrophil and macrophage activation
[27]. Regardless of the trigger (sepsis and/or trauma), changes are profound at the genetic, molecular, subcellular and mediator levels. Transcriptome studies have revealed 363 differentially expressed genes during the acute inflammatory response in 36 major trauma patients who did develop MODS and those who did not. The number of differentially expressed genes decreased to 33 by the 24 h timepoint
[28]. The role of T cells in the immune response during MODS development is shown in critically ill patients with sepsis
[29] where Th17 (T helper cell) /Treg (Regulatory T cell) imbalance is related to MODS. Molecular signatures of MODS in critically ill patients reflect cellular dysfunction, hallmarked by mitochondrial dysfunction and endoplasmic reticulum stress
[30]. In defense against microorganisms multiple, complex, and redundant processes are involved. For example, pyroptosis, which promotes release of cytokines
[31]. Leukocytes release inflammatory mediators when activated via PAMPs and DAMPs; in response, there is overproduction of reactive nitrogen species (RNS) and nitric oxide. This is detrimental to electron transfer chain (ETC) function because this crucial pathway becomes irreversibly inhibited, leading to oxidative stress which is compounded mitochondrial DNA damage
[32]. As a result, the energy metabolism of immune cells become defective; and various processes, including oxidative phosphorylation, are inhibited. This cascade of events manifests as immunosuppression
[33]. In sepsis, unfolded or misfolded proteins accumulate in the endoplasmic reticulum (ER), this leads to oxidative and calcium dysregulation, resulting in ER stress
[34].
The SEPSIS 3 definition for sepsis emphasizes the dysregulated host response to infection that leads to life-threatening organ dysfunction. The presence, severity and course of MODS determine the severity of clinical illness in the septic patients
[35]. Cytokine imbalance is not only part of immunopathogenesis of sepsis and septic shock; it can also guide elements of therapy. A recent exploratory analysis of data from the Corticosteroid Therapy of Septic Shock (CORTICUS) trial showed that a low serum interferon-gamma (IFNγ)/IL-10 ratio was associated with increased survival in individuals treated with hydrocortisone whereas a high ratio was associated with better survival in the placebo group
[36]. They concluded that IFNγ/IL-10 may become a suitable molecular maker to help inform the decision to use hydrocortisone in septic shock patients.
Another key component of immuno-inflammatory dissonance in critical care setting is activation of endothelial cells which may lead to endothelial dysfunction and glycocalyx destruction. This represents an incremental assault on virtually all organ systems. When this occurs, microvascular and tissue perfusion dysfunction, arteriovenous blood shunting, loss of vascular tone and procoagulant state are, nearly always inevitable
[37]. Vasodilatation is the multifactorial consequence of acidosis on vascular smooth muscle, which leads to induction of nitric oxide production (NO). Loss of systemic vascular resistance may lead to complete vasoplegia; this might be initially compensated for some time by increased cardiac output but will be exacerbated by myocardial depression
[38]. Fluid leak and tissue edema are ubiquitous: in the lungs, gas exchange is impaired, leading to acute respiratory distress syndrome (ARDS). Apart from microvascular derangements and tissue hypoperfusion, acute kidney injury may occur because of direct cytokine effects. Hepatic injury also occurs and this can contribute to coagulopathy. Specific forms of DAMPs, for example, bacterial and mitochondrial N-formyl peptides (NFPs) activate the innate immune system via formyl peptide receptors (FPR) which are present on immune and non-immune cells such as vascular endothelial cells
[39]. The subsequent inflammatory response leads to endothelium barrier breakdown and the consequences described above. Novel approaches to try to protect the host from deleterious effects of imbalance between pro- and anti-inflammatory mediators include blood-filtering devices such as antibody-modified conduits (AMCs) that can remove specific cytokines in vitro. AMCs that use antibodies against human vascular endothelial growth factor A (VEGF-A) or TNF-α are able to remove specific cytokines from the blood in vitro
[40].
The macrocirculation and microcirculation must be coherent to allow effective systemic hemodynamic-driven resuscitation that corrects organ perfusion and oxygenation. To further complicate matters, hemodynamic coherence is often lost during inflammation and infection
[41]. Therefore, physiologic approaches are needed that allow the monitoring of the hemodynamic parameters and allow optimal oxygen delivery in shock resuscitation
[42]. The importance of this principle is illustrated in the use of fluid challenge in critically ill patients
[43]. Where there can be limited correlation between absolute changes in cardiac macrocirculation and small diameter vessels (microcirculation) in response to administration of a fluid challenge.
Host–pathogen interaction is complex, starting with recognition of pathogens by the host with consequent induction of inflammatory response via various Pattern Recognition Receptors (PRR) such as Toll-like receptors (TLR)
[44][45]. Bacteria and cytokines have a multifaceted and intertwined relationship. One of the most interesting aspects of this relationship has been investigated by Meduri et al. for over two decades
[46][47][48]. They demonstrated that IL-1β, TNF-α and IL-6 enhance bacterial growth in patients with sepsis-associated ARDS. They also found that at the onset of ARDS and over time, the level of these cytokine, both in plasma and bronchoalveolar lavage fluid—BAL, were significantly higher in nonsurvivors. They investigated extracellular and intracellular growth of
Staphylococcus aureus,
Pseudomonas aeruginosa and
Acinetobacter species obtained from patients with ARDS, in response to graded concentrations of IL-1β, TNF-α and IL-6 in vitro. When the various bacteria were exposed to low concentrations of each proinflammatory cytokine (10–250 pg values equivalent to those in ARDS survivors), bacterial growth was not promoted, and monocytes were efficient in killing ingested bacteria. Conversely, when the bacteria were exposed to higher concentrations of cytokines (values equivalent to those in ARDS nonsurvivors), bacterial growth showed a dose-dependent enhancement. It was then demonstrated that impaired intracellular bacterial killing in activated monocytes was associated with increased expression of cytokines, and enhanced monocyte killing function on exposure to methylprednisolone was associated with decreased IL-1β, TNF-α and IL-6 expression
[49]. The downregulation of dysregulated systemic inflammation is important in accelerating disease resolution and in decreasing the risk of nosocomial infections
[50]. A similar study investigated the association of IL-6 and IL-10 levels with mortality in patients with sepsis and septic shock
[51]. They found that IL-6 and IL-10 levels were both independently associated with mortality, but that the balance of these inflammatory mediators (IL-6/IL-10 interaction) does not seem to impact either early, intermediate or late mortality in ICU patients with sepsis. However, the balance of proinflammatory and anti-inflammatory mediator response may not be reflected by analyzing two pleiotropic cytokines
[52], as IL-6 can be both proinflammatory and anti-inflammatory, depending on the context.
MODS can be considered a heterogeneous syndrome. Another important player in both organ and immune dysfunction is activated complement protein C5a, which exerts deleterious effects on organ systems as well as suppressing antimicrobial functions of key immune cells
[53]. In polymicrobial sepsis, plasma products of complement activation in plasma including C5a anaphylatoxin and its receptors C5aR1 and C5aR2, are closely followed by extracellular histones that carries strong proinflammatory and prothrombotic activity
[54]. In animal model of sepsis, both complement activation products and extracellular histones cause cell injury and multiple organ dysfunction. Neutralization of C5a through antibody or knockout prevents extracellular histones and the subsequent organ failure in septic mice.
An intricate network of immune cells is activated by PAMPs and DAMPs. Impaired macrophage function is considered to be one of the most important causes of immune paralysis and can contribute to organ dysfunction and lethal outcome in sepsis. One in vitro study demonstrated that endogenous purine ATP facilitates the killing of bacteria that cause sepsis by macrophages via P2X4 receptors (P2X4Rs)
[55]. Given that extracellular levels of ATP are increased in sepsis, P2X4Rs might be promising therapeutic targets.
B cell responses are altered and are oriented toward an exhausted-like/immunoregulatory profile during sepsis-induced immunosuppression
[56]. Natural killer (NK) cells are large granular lymphocytes, acting as coordinators of early responses to bacteria through production of interferon (IFN)-γ which amplify the antimicrobial functions of myeloid cells. Conversely, if excessive NK cell activation occurs, production of IFN-γ will increase and this can result in organ injury and dysfunction
[57]. Extracellular vesicles (EVs) which are 30 nm to several µm in size, are released from immune cells on activation and apoptosis. EVs express membrane epitopes that are specific to their parental cells. There is speculation that EVs act as mediators in sepsis, both as friends and foes. Their role in systemic inflammation mostly depends on origin and the cargo they carry, which makes them potential candidates for drug delivery
[58].
Inflammasomes are large, intracellular multiprotein complexes and may play a role in sepsis. They detect and respond to a number of PAMPs, including bacterial flagellin, and DAMPs, such as uric acid crystals. Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ACS) is a key component of the inflammasome. When inflammasomes are activated and assembled, ASC moves from its diffuse distribution in the cytoplasm into a single speck that serves as a supramolecular signaling platform. These interesting structures promote the maturation of the pro-inflammatory cytokines IL-1β and IL-18. When evaluating ASC–speck formation in monocytes during the first week of sepsis in patients, the highest number of ASC–speck
+ monocytes can be detected on day 6–7. Survival analysis shows that patients with lower numbers of ASC–speck
+ monocytes (<1650 cells/mL) on day 6 has greater risk of mortality
[59].
One of the mediators of chronic immunosuppression in sepsis is vagal activation
[60]. Enhanced vagus nerve tonic activity results in an immunosuppressed phenotype in patients who survive sepsis. Since cholinergic tone can be pharmacologically modulated, targeting this process may be a novel therapeutic approach to prevent latter infections in these immunocompromised patients.
Long-term sequelae of sepsis immunology are becoming the focus of attention. In a study by Rodriguez-Rosales et al., long-term immune effects of human experimental endotoxemia were investigated when healthy subjects were challenged with endotoxin (1 ng/kg)
[61]. Twenty days post-endotoxin, flow cytometry revealed, among other things, increase in absolute numbers of intermediate monocytes with lower human leukocyte antigen–DR isotype—HLA-DR expression. Long-term host immune response trajectories (up to 12 months) were investigated in a cohort of 483 hospitalized sepsis survivors
[62]. Approximately 25% of these individuals had elevated C-reactive protein (CRP) at 12 months and about 50% of them had elevated soluble PD-L1 (a marker of immunosuppression). This suggest that persistent elevation of inflammation and immunosuppression markers is common up in sepsis survivors and may be associated with poor long-term outcomes. A recent review discussed syndrome of chronic critical illness (CCI) which encapsulate sepsis patients who survive the early “cytokine or genomic storm”, but then fail to recover fully, and progress to a persistent manageable organ dysfunction state that requires prolonged intensive care
[63]. It is suspected that as many as one third of sepsis survivors develop CCI which is in part due to a maladaptive host response to processes mediated by PRRs. CCI is characterized by exhaustion and atrophy of T cells, expansion of suppressor cell function as well as chronic inflammation and dysregulated myelopoiesis. Authors proposed that PICS in survivors of critical illness represent a unique immune endotype, with persistent release of DAMPs and PAMPs from secondary infections.
Aging of the population predisposes to the development of both CCI and PICS. The elderly patients are more susceptible to sepsis and are at greater risk of mortality. This is, in part, result of immunosenescence and a marked decline in cell-mediated and humoral immunity that is seen with increasing age
[64][65].
For the past two decades, attention to sepsis has been intensified because of growing recognition that it is one of the most common and lethal conditions we face (approximately 50 million people worldwide annually), whether as a patient, provider, hospital or public health agency
[66]. Therefore, early prediction of sepsis is of utmost importance in order to provide optimal care at an early stage. Recent implementation of soft-computing and machine learning techniques can illustrate how complex and difficult this task is
[67]. Both researchers and clinicians are acutely aware of the complexity and heterogeneity of sepsis which is a significant impediment to adequate treatment. Thus, quite an effort has been undertaken to identify subgroups of sepsis patients who represent distinct functional endotypes based on measurable genetic and biologic differences
[68]. Endotyping may also identify individuals unlikely to benefit, or more likely to be harmed, by specific therapies. Sepsis is heterogeneous syndrome, characterized by a vast set of clinical and biological features
[69], combinations of these features may represent previously unrecognized groups, or sepsis subclasses with different risks of outcome and response to a given treatment. The authors of one study established a machine learning model to classify sepsis into different immune endotypes based on transcriptomics data
[70]. They identified two immune subphenotypes associated with sepsis and termed them immunoparalysis and immunocompetent endotypes. They also found that percentages of M0 macrophages, M2 macrophages, naïve B cells and naïve CD4 T cells were associated with cumulative mortality at 28 days. More than a decade ago, we investigated polymorphisms of genes encoding tumor necrosis factor-alpha, interleukin-10, cluster of differentiation-14 (CD14) and interleukin-1ra in critically ill patients
[12]. Some of the polymorphisms were significantly associated with outcome, for instance. Epigenome-wide methylation analysis of whole blood DNA samples from a cohort of 66 septic and 68 non-septic critically ill patients on day 1 of ICU admission
[71] and weighted gene co-expression network analysis was performed. It showed DNA co-methylation modules associated with severity of illness, need for vasopressors, and length of stay.
Understanding of the heterogeneity in the individual host response to infection is necessary for effective targeted sepsis therapy. Other studies investigated this heterogeneity by defining the variation between individuals in the transcriptome of patients with sepsis
[72]. Transcriptomic analysis of peripheral blood leukocytes suggests the presence of two distinct sepsis response signatures (SRS1 and SRS2). SRS1 (detected in 41% patients) identifies individuals with an immunosuppressed phenotype that include features of impaired antigen processing ability and endotoxin tolerance, T cell exhaustion, as well as downregulation of human leucocyte antigen (HLA) class II. SRS1 was associated with significantly higher mortality in comparison with SRS2.
NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex with a number of functions including control of DNA transcription, production of cytokines and cell survival
[73]. This mediator was initially discovered in 1968 by Sen and Baltimore who identified its role in the transcription of immunoglobulin κ-light chains in B lymphocytes. One study demonstrated that sepsis patients display a reduced ability to activate NF-κB in multiple cell types
[74]. Intensive care unit (ICU)-acquired infections (IAI) result in a longer hospital and ICU stay, as well as increased costs and mortality. To identify patients at risk of IAI, authors of one study evaluated the association of the systemic mRNA expression of two biomarkers of host response, CD74 (cell surface receptor for the cytokine macrophage migration inhibitory factor—MIF) and IL-10, with IAI in a large number of ICU patients
[75]. They found that immune monitoring using these two immune biomarkers could be appropriate for the identification of IAI risk in ICU patients. This suggested that immune profiling of critically ill patients can be integrated through a multimodal real-time diagnostic work-up of IAI
[76]. Investigating features of the immune response in sepsis, as potential biomarkers, is challenging because of the temporal effects: over time there are differences between patients (interindividual) as well as within the same patient (intraindividual)
[77]. Functional immunity changes cannot be adequately assessed by routine non-specific inflammatory biomarkers (CRP, procalcitonin). Thus, sepsis biomarkers are still much in focus of numerous investigations
[78][79][80][81][82][83][84].
2.2. Trauma-Induced Multiple Organ Dysfunction Syndrome
Trauma is the third leading cause of mortality worldwide as well as the first cause of fatality and disability in those younger than 45 years of age
[85]. Later deaths are result of development of MODS and infections in trauma patients who are profoundly immunosuppressed; that occurs in 45% of severe trauma patients. Trauma-induced MODS and immunosuppression are, for the most part, result of innate immunity activation. DAMPs, normally hidden from the immune system, are abundantly released after severe musculoskeletal injury; through binding to cell surface and intracellular neutrophil receptors (PRRs for instance), they generate systemic inflammation. Mitochondria, organelles of bacterial origin, are important regulators of inflammatory response and are a platform for PRR signal transduction
[86]. Spatial proximity of mitochondria and inflammasomes in perinuclear regions enables modulation of inflammasome by these organelles. Mitochondria share some molecular traits with bacteria; normally these components are not ligands for PRRs, but during cellular damage they are released and do act as DAMPs. These include mitochondrial DNA, large quantities of extracellular ATP, cardiolipin (normally found only in the inner mitochondrial membrane) and formyl-peptides. When innate immunity is unsuccessful in clearing DAMPs and/or PAMPs, adaptive immunity is activated. Mitochondrial metabolic states within synapse between dendritic cells and lymphocytes are able to polarize adaptive immunity: glycolytic metabolism is associated with proinflammatory, whereas oxidative metabolism is associated with anti-inflammatory response. The role of mitochondrial metabolism in dendritic cells therefore induces either pro- or anti-inflammatory differentiation of T helper cells. Given the fact that underlying mechanisms of trauma-induced MODS are not yet fully elucidated. Aswani et al., investigated whether mitochondrial (mt) DNA, released after various degrees of tissue damage and hemorrhagic shock, is sufficient to induce MODS in a rodent model
[87]. mtDNA, similar to bacterial DNA, has large quantity of highly stimulatory unmethylated CpG DNA motifs, which are ligands for TLR-9 and will trigger inflammation. The authors demonstrated that release of mtDNA is sufficient for MODS development and they showed that neutralizing this mediator, as well as nuclear DNA, with the nucleic acid scavenging polymer, hexadimethrine bromide (HDMBr) is able to rescue from MODS. They concluded that it could have utility in treatment of human trauma-induced MODS.
Alarmins, which are DAMPs released after trauma include all nucleic acids, HMGB1, HSPs and S100 proteins. These mediators activate multiple receptors and signaling systems such as PRRs, Receptor for Advanced Glycation Endproducts (RAGE) and Triggering Receptor Expressed on Myeloid cells-1 (TREM-1). DAMPs activate neutrophils and dendritic cells; thus, both the innate and adaptive immunity are set in motion. Posttraumatic immunosuppression renders trauma patients susceptible to secondary infection. The role of DAMPs in that process have been investigated in adult trauma patients. A study reported an inverse relationship between levels of HSP70 and nuclear DNA on one hand and HLA-DR expression conversely. DAMPs also induce long-term endotoxin tolerance. Via TLRs, DAMPs may also induce epigenetic alterations
[88]. These gene-specific chromatin modifications are associated with transient silencing of various classes of genes, including pro-inflammatory mediators
[89]. HMGB1-RAGE signaling results in functional exhaustion of mature monocytes and lymphopenia; this is the hallmark of immune suppression following extensive brain ischemic injury
[90]. DAMPs can induce immunosuppression without a preceding inappropriate inflammatory response. The endogenous purine nucleotides are major regulators of the inflammatory response
[91]. Adenosine is a catabolite of ATP, and during inflammation it signals by binding and activating purinergic receptor. High amount of adenosine released after trauma may directly induce Th2 response
[92].
Patients suffering from multiple traumas often require massive blood transfusion; thus, it is important to bear in mind that red blood cells contain DAMPs and promotes the formation of the inflammasome
[91]. Potent DAMPs that may be released by red blood cell lysis include haem, HSPs, such as Hsp70, IL-33 and Adenosine 5’ triphosphate. Hemolysis represents a major inflammatory trigger
[93]. IL-33 is expressed in the nucleus of epithelial cells and is released into the extracellular space following tissue damage. It has been shown to initiate the Th2-polarizing function of dendritic cells and stimulates the secretion of anti-inflammatory cytokines
[94]. After tissue injury, massive DAMPs release leads to overwhelming systemic inflammation and early MODS; in addition, these mediators may lead to immunosuppression; thus, the severely injured are prone to secondary infection and late MODS. Plasma mtDNA is associated with the evolution of systemic inflammation, MODS, and increased mortality in severely injured patients
[95]. HMGB-1 can activate alveolar macrophages to produce proinflammatory cytokines and induce acute lung injury (ALI) through TLR-4. Alteration in tight junction and increased permeability leads to interstitial lung edema
[96]. DAMPs and EVs can activate innate immune receptors and coagulation cascades, and this leads to an inflammatory response and blood coagulation. Several immunothrombotic agents play a role in promoting inflammation and activation of coagulation, these include extracellular DNA, HMGB1, the S100 family of intracellular low-molecular-weight calcium-binding proteins and histones
[97]. Histones are cationic nuclear proteins that packages DNA into nucleosome. Extracellular, circulating histones, released as DAMPs after trauma, express direct cytotoxicity to both epithelial and endothelial cells by altering membrane permeability and causes calcium influx. This is associated with post-traumatic ALI
[98]. Another DAMP, N-formyl peptide is released from the from mitochondrial matrix and is a well-known leukocyte chemoattractant which promotes chemotaxis of neutrophils to regions of sterile inflammation. EVs contain cellular cargo-like proteins, DNA and RNA and play an important role in intercellular communication. However, these interesting structures also carry various immunothrombotic mediators such as mtDNA, HMGB1 or HSP, depending on their origin. Exosomes are smaller than 0.1 µm in size and originate from multivesicular bodies (MVBs)
[99]. EVs can transfer their cargo by endocytosis, phagocytosis, and micropinocytosis as well as membrane fusion
[100]. Microparticles, released from endothelial and circulating cells following sepsis-induced microvascular injury, can contribute to endothelial dysfunction, immunosuppression and MODS
[101]. Polytrauma or invasive surgery will produce DAMPs and EVs that cause microinjury and de novo release of immunothrombotic DAMPs and EVs in distant organs, thereby promoting post-traumatic MODS
[97].
Trauma-induced DAMPs, as well as PAMPs, may trigger assembly of inflammasomes that are intracellular multiprotein complexes. These were initially described in 2002 as caspase-1 activating multiprotein complexes
[102]. Initial tissue damage, blood loss and subsequent secondary injuries will lead to local and systemic release of DAMPs. Recognition of these mediators by the innate immunity triggers both excessive inflammation (which propagate remote, secondary tissue damage) and immunosuppression (which may contribute to secondary post-traumatic infection and sepsis); contributing to MODS and increased mortality. In trauma, mechanical tissue injury and blood loss are associated with secondary ischemia/reperfusion (I/R), hypothermia, hypoxia, coagulopathy and neuroendocrine disorders. The consequences of these range from cell stress to cell death
[103][104]. Inflammasomes are named after their intracellular receptor, including nucleotide-binding oligomerization domain-like receptors, or NOD-like receptors (NLR), Absent in melanoma 2 (AIM2)-like receptors—ALR, retinoic acid-inducible gene-I-like receptors (RLR) or pyrin
[105]. Specific roles of inflammasomes, for example as an intra-cytosolic sensor detecting mostly intracellular stimuli, provide another means of activation, through recognition of specific ligands to the sensing of intracellular disturbances. Regardless of the stimulus, activated inflammasome allows the caspase-1 dependent cleavage of pro-IL-1β and pro-IL-18
[106]. DAMPs are crucial part of the pathogenesis of trauma-induced MODS and support a vicious cycle of injury
[107]. Both inflammasome-mediated pro-inflammatory release and pyroptotic cell death promote the initiation, enhancement and propagation of trauma-induced inflammation
[108]. Pyroptosis features include cytoplasm swelling and cell membrane destruction as well as release of intracellular contents into extracellular space, thus contributing to sterile inflammation
[109]. Diffuse activation of endothelium in an organ-specific manner is set in motion after systemic release of DAMPs
[102]. Investigation of in vivo mouse model of hemorrhagic shock demonstrated NLRP3 activation in lung vascular endothelial cells, enhancing the proinflammatory response via pyroptosis and IL-1β release
[110]. Although anucleate, platelets have functional translational material associated with mRNA transcripts, including IL-1β; thus, these cells can assemble functional NLRP3
[102]. Platelets express various immune receptors, cell surface adhesion molecules and many immunomodulatory mediators contained in preformed granules. these cells adhere to endothelial cells and leukocytes to form aggregates when activated by circulating DAMPs
[102][111]. In the setting of tissue damage, platelets facilitate leukocyte activation and adhesion to post-ischemic microvessels. They also modulate degranulation and phagocytosis of neutrophils
[102]. Ischemia-reperfusion will compound tissue injury; at cellular level, reperfusion triggers enormous production of ROS, calcium overload and mitochondrial dysfunction. This chain of events can end in cell death. During I/R, the NLRP3 inflammasome–IL-1β–IL-18 axis is crucial in organ-specific tissue injury, such as myocardial injury and renal necroinflammation, for example
[112]. The NLRP3 and AIM2 inflammasomes are also important in hepatic I/R injury. Locally released DAMPs such as ROS, ATP or extracellular histones will activate inflammasomes in Kupffer cells
[113]. Traumatic brain injury (TBI) will immediately damage tissue, while the pro-inflammatory innate immune response to neuro-injury, termed neuroinflammation, will additionally extend lesions via secondary cellular damage
[114]. The NLRP1 inflammasome is assembled before neuron and other CNS cells stimulation; thus, it is of special interest in TBI as a crucial factor of induction and propagation of neuroinflammation
[115]. TBI impacts peripheral cellular immune response via the hypothalamic–pituitary–adrenal axis, thus contributing to secondary damage to distant organs and susceptibility to infection
[116]. The lungs are particularly susceptible to trauma-related ALI, being exposed to central venous blood conveying systemic DAMPs from injured tissues through pulmonary vasculature. ALI and/or ARDS leads to systemic and local activation of NLRP3 inflammasome
[117]. Critically ill trauma patients often need mechanical ventilation (MV) and massive blood transfusion. MV-induced inflammatory lung injury may be consequence of cyclic alveolar stretch-induced activation of NLRP3 inflammasome; mechanism involves DAMPs: mitochondrial ROS generation and uric acid release
[102]. The priming step of transfusion-related ALI—TRALI may be associated with NLRP3 inflammasome expression in various types of lung immune and endothelial cells, which constitute what is known as the “first hit”, while DAMPs from stored blood units with some degree of hemolysis, leading to the presence of heme or extracellular ATP may induce inflammasome activation and subsequent inflammation, resulting in a “second hit”
[93]. Finally, dysfunctional inflammasomes in immune cells may be involved in post-trauma immunosuppression
[118]. Authors of one study reported that NLRP1 gene expression following LPS stimulation is reduced in trauma patient monocytes. The decrease in mRNA levels of NLRP1 persisted over 10 days from admission to the emergency department
[119]. In monocytes isolated from non-trauma patients, who had undergone cardiopulmonary resuscitation, levels of AIM2 gene expression as well as ability to release IL-1β were downregulated
[120].
Trauma accounts for 10% of deaths and 16% of disabilities worldwide
[121]. After major trauma, massive release of neutrophils occurs. Circulating neutrophils are dormant until activated by PAMPs and/or DAMPs., and when activated they carry out various functions including phagocytosis, degranulation, release of neutrophil extracellular traps (NETs), ROS and cytokines. These cells have altered functions and phenotypic markers because banded and even immature cells, such as metamyelocytes, enter circulation from bone marrow
[122]. Authors of one study reported that major trauma is associated with subsequent delay of neutrophil apoptosis for at least 10 days, whether trauma victims developed sepsis or not
[123]. Accumulation of activated neutrophils will lead to NETs formation in response to injury
[124]. Surgery, as a form of controlled trauma, can, by itself, induce formation of NETs (elective total hip replacement for example). In these patients, it is a part of sterile inflammatory response mounted by innate immunity
[125]. Trauma modulates neutrophil phenotypes and can lead to increased cell size and membrane plasticity, as well as modified shape (elongation). Neutrophil cell size can be significantly different between trauma survivors and nonsurvivors
[126]. Distinct neutrophil subsets have been suggested to exist in trauma patients and in a human acute inflammation model, in which the hypersegmented CD62L
DIM/CD16
POS subset can be separated from mature segmented neutrophils by multiplex proteomics comparison and immunosuppressive capacity
[127][128]. Subset of neutrophils, predominantly CD11b
(+)/Gr-1
(+)/CXCR4
(hi) neutrophils recruited by vascular endothelial growth factor A-VEGF-A might be beneficial to repair the initial trauma impact. This subset of neutrophils delivers large amounts of the effector protein matrix metalloproteinase-9 (MMP-9), required for revascularization and functional reintegration
[129]. Trauma has an impact on neutrophil migration; high levels of the neutrophil chemotactic factor IL-8 have been found in trauma patients
[130]. Increased oxidative burst in neutrophils correlates with more extensive brain tissue injury by ROS
[131]. Neutrophils are major producers of ROS which have been recognized as a component of NLRP3 activator in hepatic I/R injury
[132]. NLRP3 is essential for acute sterile inflammation
[133]. Newly formed ROS in injured tissue results in the migration and activation of more neutrophils
[121], thus creating a vicious circle.
In the early phase after major trauma, surgical procedures should be carefully planned. Surgical procedures can be viewed as additional trauma load and constitutes a “second hit”. Authors of a recent pilot study investigated the immune status of trauma patients
[134]. They used highly standardized systems to draw peripheral whole blood from seven polytraumatized patients with high injury severity score (ISS ≥ 32) and challenged it with bacterial LPS. In comparison with samples from healthy volunteers there was a significant decrease in the release of monocyte-derived mediators and surprisingly stable, unaltered or even increased concentrations of cytokines related to T cell maturation and function (IFN-γ, IL-2, IL-4 and IL-9). Levels of pro-inflammatory cytokines were reduced in response to LPS early after severe trauma. However, 24 h after injury, TNF response was not profoundly impaired. This led the authors to conclude that functional immune monitoring may be used to optimize the timing of necessary surgical interventions in severely injured patients. Another recent study focused on trauma-induced long-term alterations of immune response six months after major trauma event in 12 survivors
[135]. CD4, CD8, CD14, PD-1, B and T lymphocyte attenuator (BTLA) cytotoxic T-lymphocyte-associated protein 4, TLR-2, -4, and -5, Dectin-1, PD-1L and HLA-DR expression were determined by flow cytometry. Cytokine release (IL-2, -4, -6, -10, and 17A, TNF-α, IFN-γ) was determined after stimulation of whole blood with LPS-, α-CD3/28, or zymosan. At the time-point of six months post-trauma, the overall immune responses were toward immunosuppression. They reported monocyte TLR-2 and TLR-4 suppression, for the first time six months after trauma which can also be observed during severe trauma
[136]. It was also found that 6 months after trauma, there was no longer HLA-DR suppression, which is a feature characteristic of the early response to polytrauma. This may be explained by the short life span of classical and non-classical monocytes
[137]. Following hospital discharge following traumatic injury, hospital-acquired infections remain a cause for post-discharge mortality
[138][139]. An immunosuppressive phenotype, observed in neutrophils and monocytes, with impaired cytokine production after LPS challenge, is a common mechanism for trauma-induced MODS
[140]. Therefore, it is important to explore ways of predicting the development of trauma-induced MODS, to allow detection as early as possible. Authors of one study measured a variety of inflammatory mediators from blunt trauma victims almost immediately after the event (within 24 h) to derive patient-specific “inflammation barcodes”. These barcodes can be used to predict development of MODS much more reliably than individual inflammatory mediators
[141]. Trauma causes an abrupt transition from health to systemic physiological crisis. A recent study applied single-cell RNA sequencing to mononuclear cells from the peripheral blood and bone marrow in injured mice and trauma patients
[142]. Transcriptomic analysis of leukocytes from severe trauma patients revealed a “genomic storm” with more than 80% of the leukocyte transcriptome altered during the first 28 days after major trauma. The greatest changes in gene expression in mice was seen in monocytes. After systemic injury, the monocytes gene expression pattern deviated from a steady state with similar changes in critical transcription factors. The changes in human CD14+ monocytes can be generalized into six signatures (SGs) with two trauma patient subtypes (SG1 vs. SG2) in the whole-blood leukocyte transcriptome in the first 12 h following injury. SG1 patients showed a longer recovery, more severe dysfunction in organs, and a higher number of complications compared with SG2. The two subtypes were also repeated for burn and sepsis patients suggesting a common immune response pathway.
Most clinicians consider medical and surgical patients to represent two varied groups, and that infection greatly affects the mortality in surgical patients
[143]. Tissue damage and blood loss during surgical procedures will induce systemic inflammation. Contribution of anesthesia to immune modulation must also be considered. Opioids, such as the widely used remifentanil, are immunosuppressants and act via opioid receptors on leukocytes. One review
[143] concluded that surgical infections are different from medical infections for a variety of reasons specific to surgical patients for example, due to a primed systemic inflammatory response caused by surgical insult, immediate postoperative immune suppression, anesthesia-induced immunomodulation, blood transfusion, I/R injury, etc. Thus, the course of surgical infections is more complex than medical ones. Authors of one study investigated immune response in a specific surgical setting, cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). They found that increased plasma levels of DAMPs (HSP70, HMGB1, S100A8/S100A9, S100A12, nuclear DNA, lactate dehydrogenase—LDH, which is a nonspecific marker of unscheduled cell death), were associated with immune suppression and postoperative infections
[144]. CRS-HIPEC procedure caused excessive DAMP release. An increase in plasma HMGB1 levels was found to be associated with the decrease in HLA-DR expression in the aforementioned study. This is consistent with the findings of another study which included blunt chest trauma patients, where HMGB1 concentrations were associated with a higher risk for sepsis
[145]. The amount of cell-free DNA, another important DAMP, is a prognostic tool for mortality as well as trauma severity and post-traumatic complications
[146]. Burn victims are especially susceptible to infection. Neutrophil phagocytosis, oxidative burst capacity NET generation (NETosis), immature granulocyte (IG) count, plasma cell-free DNA (cfDNA) and plasma citrullinated histone H3 (Cit H3—a specific marker of NETosis) levels were measured up to one year following burn injury in 63 patients with burns to ≥ 15% total body surface area in an interesting study
[147]. In addition, were measured. Neutrophil dysfunction, elevated IG counts as well as elevated plasma cfDNA and Cit-H3 levels were reported during septic episodes. All of the aforementioned measurements demonstrated potential as biomarker(s) of sepsis following burn injury. Neutrophil dysfunction may also actively contribute to the development of sepsis. Another interesting, recent study focused on surgical patients. Fresh blood samples revealed leukocytes with reduced viability in critically ill surgical patients. The authors investigated decreased leukocyte viability, the implications for leukocyte functioning and its clinical implications
[148]. Non-viable neutrophils in vitro are referred to as fragile neutrophils in vivo. Overall neutrophil function was found not to be impaired in patients with fragile neutrophils, but these cells were associated with critical illness. Of the 11,871 patients, 75 (0.63%) had fragile neutrophils during hospitalization, and75.7% of these developed an infection, 70.3% required ICU admission and 31.3% died in hospital. Therefore, fragile neutrophils were mostly detected in surgical patients with recurrent or serious infections. Conversely, these cells were also observed in the absence of infection in patients who sustained high energy trauma and in patients with multiple or major surgeries (second hit). An advantage of this study is the usage of standardized, routine hematology analyzer
[149], because manual work-up of blood samples leads to high number of apoptotic and necrotic neutrophils (up to 99%) due to in vitro manipulation which can easily affect results.