Current views on the mechanisms of immune responses to trauma and infection.

According to the World Health Organization, post-traumatic mortality rates are still very high and show an increasing tendency. Disorders of innate immune response that may increase the risk of serious complications play a key role in the immunological system response to trauma and infection. The mechanism of these disorders is multifactorial and is still poorly understood. The changing concepts of systemic inflammatory response syndrome (SIRS) and compensatory anti-inflammatory response syndrome (CARS) early inflammatory response, presented in this work, have been extended to genetic studies. Overexpression of genes and increased production of immune response mediators are among the main causes of multiple organ dysfunction syndrome (MODS). Changes in gene expression detected early after injury precede the occurrence of subsequent complications with a typical clinical picture. Rapid depletion of energy resources leads to immunosuppression and persistent inflammation and immune suppression catabolism syndrome (PICS). Early diagnosis of immune disorders and appropriate nutritional therapy can significantly reduce the incidence of complications, length of hospital stay, and mortality. The study presents the development of knowledge and current views explaining the mechanisms of the immune response to trauma and infection.

Introduction

According to the World Health Organization, injuries are still a major cause of severe disability and death. There are approximately 3-9 million injuries recorded annually in developed countries. It is predicted that in 2020 they will be a major cause of death. According to statistical data, within an hour in the world around 2000 people sustain traffic injuries, including 148 deaths [1-3]. The number of deaths due to injuries has doubled in the last 30 years in Poland and other European countries. The most common cause of death due to injury is damage to the head, chest, and abdomen and associated haemorrhages. Injuries more frequently affect men than women and individuals aged 20-60 years [4-6]. As shown by statistical studies, in 2008 in Poland there were 49,054 traffic accidents, in which 92% of people were injured and 8% died. This was a significant increase in the number of accidents compared to the study conducted in 1985, which recorded 36,100 accidents with a slightly higher mortality (10%) [7]. Overall, in Poland approximately 3 million people are injured annually. Of these, 300,000 require hospitalisation and 30,000 die. The post-traumatic mortality rate in our country is 75/100,000 people a year, which is much higher than in Western Europe or the United States. In Poland, traumatic disability affects approximately 25% of hospitalised patients, and thus 100% more than in the USA and Western Europe [8, 9].

Severe infections (sepsis) and multiple organ failure (multiple organ dysfunction syndrome – MODS) are among the most severe traumatic complications burdened with high mortality [2, 9, 10]. According to the scarce data from intensive care units (ICU), in Poland injuries are still a major cause of severe infections [11, 12]. According to these analyses, severe infections are diagnosed in 50,000 patients a year, including 16% of patients hospitalised in ICUs, approximately half of which die. The largest group are surgical patients (58.9%), and the most common complications are respiratory (88.7%) and cardiovascular failure (82.2%). This is confirmed by studies from other centres elsewhere in the world. Sepsis caused by various factors is the cause of death of every fourth patient [11, 13]. In the United States, 750,000 new cases of sepsis are recorded each year, of which 210,000 die [14]. In turn, in Canada there are about 30,000 cases of sepsis recorded each year, and 35% of these patients die [15]. In summary, 40% of ICU patients are admitted because of sepsis, or they develop its symptoms in a short period of time; of these, 36.7% are patients from the emergency room [11]. As demonstrated by the study conducted in the United States, severe infections are now the cause of more mortalities than stroke, lung cancer, and breast cancer combined [14]. Studies in Polish ICUs showed that severe sepsis is the leading cause of death in 30-60% of patients [16].

An immune response to trauma

As shown in a previous study conducted by E. Faist [17] in a group of 433 young patients with multiple traffic injuries, mortality in the course of multiple organ failure (MOF) was 56%. It was found that 42% of patients died of MOF following concomitant infection. The authors of this study emphasised that in order to improve the results of treatment in these patients, the proper functioning of the respiratory and circulatory systems should be ensured as soon as possible (preferably still at the scene). The results of this study indicated a significant proportion of infections as one of the causes of MOF. The resulting disorders of the immune response after trauma are primarily associated with increased production of inflammatory response mediators (e.g. cytokines) and the dysfunction of gut-associated lymphoid tissue (GALT). According to this hypothesis, translocation of microorganisms from the intestine into the lymphatic system and the peripheral blood occurs due to extensive trauma and ischaemia of intestinal mucosa. This process increases the risk of MOF, mainly respiratory failure [18]. Bone et al. introduced the concept of systemic inflammatory response syndrome (SIRS) (Fig. 1) [19, 20].

Fig. 1

The hypothetical model of pro-inflammatory (SIRS) and anti-inflammatory response (CARS) to trauma and infection, which can lead to multiple organ failure (according to Bone et al.) [19]

According to this theory, the immune response to tissue injury and associated infection starts at the site of the injury and is related to the production of pro- and antiinflammatory mediators, primarily by cells of the innate immune response (e.g. neutrophils). This part of the immune response is directed at restoring the homeostasis, and may be considered as an early physiological inflammatory reaction. The local inflammatory response and the associated immune response is a favourable phenomenon in the process of wound healing. It is known, however, that persistent infection in the wound or the existence of any additional source of infection (e.g. in the respiratory tract) increases the local and systemic immune disorders, which can lead to sepsis and multiorgan failure. According to this concept, exacerbation of immune disorders through increased activation of the immune system can lead to “immune paralysis” syndrome (CHAOS), which increases the number of complications and mortality [19, 21]. An important element of this hypothesis was the attempt to link immune disorders with clinical signs of disease severity. The authors stressed the importance of apoptosis of the immune system cells (macrophages, neutrophils, lymphocytes), which may exacerbate immunosuppression and is an important element of response to injury/infection, and they indicated the importance of mediators involved in the inflammatory response. A better understanding of the impact of pathological response of SIRS/CARS type (compensatory anti-inflammatory response syndrome) on the occurrence of MODS was hindered by poorly understood cooperation mechanisms of immune cells involved in direct organ damage and the influence of other effects (e.g. the size of the injury, previous comorbidity, type of infection) [18]. Attempts to simplify the classical molecular model have had an adverse impact on the diagnostics and have given a better understanding of the immune response to injury and infection. The above-presented concept of immune disorders has not been widely adopted by clinicians because of the complicated course of SIRS/CARS response and difficulties in the interpretation of the results of immunological studies in severely ill patients with infections. Nevertheless, a simple clinical model of SIRS, sepsis, and severe sepsis diagnostics has been introduced (Table 1) [22, 23].

Table 1

Clinical diagnosis of SIRS, sepsis and severe sepsis [22]

SIRS	Sepsis	Severe sepsis
Body temperature < 36°C or > 38°CHR > 90/minRespiratory rate > 20/min or Hyperventilation PaCO2 < 32 mmHgWBC <4000 or > 12000/mm3Immature granulocytes > 10%	SIRS + suspected or confirmed infection	Sepsis + organ dysfunction(s) Oliguria Acute alteration of mental statusAcute respiratory distressCirculation dysfunction(s) Hypotension or hypoperfusion Requirement of inotropic or vasopressor agents

A lack of conviction of clinicians to perform immunological tests among the most seriously ill patients had a significant impact on reducing early molecular diagnostics in the ICU, although numerous studies have indicated that the immune dysfunction observed in these patients preceded the occurrence of organ complications [24]. Thus, a routinely applied scheme of SIRS, sepsis, and severe sepsis diagnosis still does not include the evaluation of selected immunity parameters (e.g. testing the levels of cytokines, chemokines, or alarmins) that could give information about the dynamics of the immune response to trauma and infection, and potential complications. This scheme does not include immune disorders caused by injury and infection, which are a direct cause of multiple organ failure. It is known that the mechanism of these disorders is multifactorial. The diagnosis of sepsis in the initial stage can be difficult. Clinical signs of MODS usually occur late, and routine laboratory diagnostics markers (e.g. levels of CRP, PCT, lactates, D-dimers, albumins) are still of little help in the early diagnosis of threatening complications. The increase in the concentration of these indicators is most often observed at the onset of clinical symptoms. As numerous studies have shown, the levels of immune mediators of pro- and anti-inflammatory responses are significantly elevated in patients with MODS and usually precede the occurrence of clinical symptoms by dozens of hours. This phenomenon has been termed “immunological diagnostic window” (Fig. 2) [24-26].

Fig. 2

Cascade inflammatory response to trauma and infection, including immunological indicators and “diagnostic window”

Previous studies have highlighted the disorders of cellular immunity and immunosuppression associated with trauma, stressing the importance of cytokines (IL-2, IL-13, TGF-β), prostaglandins, thymus hormones, complement, NK cells, and granulocytes [27]. The relationship has been found between the severity of SIRS/CARS and MOF and impaired apoptosis of cells involved in pro- and anti-inflammatory response (mainly neutrophils), which increases the production of cytokines and free radicals. It has been suggested that uncontrolled immune response to trauma or infection can lead to the induction of self-destruction of tissues and organ dysfunction distant from the site of injury (e.g. in the lungs). It was necessary to modulate immune cell function in order to improve the resistance in severely ill patients. This concept has survived to this day. The proposed treatment with antibodies neutralising the action of mediators of the inflammatory response (anti- TNF-α, anti-endotoxin, IL-1antagonist, NO inhibitor, eicosanoids) [28], thymus hormones [29], indomethacin [30], thymopentin in combination with indomethacin [31], stimulation of IL-2 production by lymphocytes [32, 33], and regulation of macrophage activity (including blocking the migration of macrophages) [34]. In addition, modulation of the immune response was attempted by endotoxin removal (hemofiltration) or the blocking action of the complement system activity and neutralisation of active oxygen species [10, 35]. As demonstrated by numerous clinical studies, these methods have not caused a significant decrease in mortality in the most severely ill patients [10]. Although preliminary experimental results were promising, they have not been confirmed in randomised clinical trials. Some of these failures resulted from improper use of animal models or from difficulties in precise classification of clinically septic patients. Additionally, there was no correlation between the inflammatory response and the severity of the clinical status of a heterogeneous group of septic patients. It should be assumed that the more precise linking of the inflammatory response with the severity of the disease could be helpful to give a better understanding of the contribution of immune disorders in severe infections. It has been found that the immune response in sepsis is one of the causes of multiple organ failure, but changes in the inflammatory response do not always correlate with the clinical status of the patient. Probably, monitoring changes in immunity during treatment could provide an answer to the question of to what extent a given immunological indicator can be used in practice as an early marker of impending organ failure.

A division into early and late multiple organ failure has been introduced, depending on the dynamics of changes in the inflammatory response [36]. Early organ failure was caused by pathological immune disorders occurring during the SIRS response, while late organ failure was caused by pathologic response of the CARS type (Fig. 3). A concept of physiological SIRS and CARS response was also presented, which may develop in patients with extensive trauma and does not lead to severe septic complications and multiple organ failure [36]. An unquestionable problem of this research was the determination of specific numerical values for inflammatory response markers that allow clinicians to distinguish early physiological SIRS reaction from pathological response. Similar problems were related to CARS, in which the increased production of IL-4 was assumed to be an “early” indicator of immunosuppression [28].

Fig. 3

Dynamics of changes in SIRS and CARS response to trauma and infection, including the physiological and pathological reactions (according to Moore et al.) [36]

According to a newer concept developed by Murphy et al. [37] (Fig. 4), CARS occurs in parallel SIRS-compensating response, mainly associated with increased production of anti-inflammatory cytokines (e.g. IL-4, IL-10) and the development of immunosuppression.

Fig. 4

Changes in the inflammatory response to trauma and infection, including the parallel course of SIRS and CARS response (according to Murphy et al.) [37]

According to these authors, an additional factor (e.g. infection, re-injury) may be the cause of another SIRS type reaction (second hit SIRS), and a pathological increase in this response can lead to MODS. Thus, a new element of this scheme is made up of parallel reactions of SIRS and CARS and disorders of the innate and acquired immune response, which increase the risk of severe septic complications. Despite the simplification, the scheme presented indicates a complex mechanism of immune disorders that occur as a result of severe trauma and infection. According to the hypothesis of the authors, the largest immune disorders, as a result of extensive trauma, occur during the first seven days after injury, and then physiological “extinction” of inflammatory response should occur after fourteen days from the uncomplicated trauma. A certain novelty was the introduction of the second hit concept of SIRS response, which can also lead to MODS. In addition, the key role of the immune response self-adaptation to trauma was stressed. This scheme did not include the important parameters of innate antimicrobial response, particularly the participation of Toll-like receptors (TLR2, TLR4), although the authors of this study pointed out the significant role of these receptors in response to trauma and infection. In patients after injury with infection, increased expression was emphasised of TLRs located on macrophages and neutrophils, as a defence mechanism enhancing the innate immune response. Lack of increased expression of TLRs favoured the occurrence of severe infection [37].

Undoubtedly, discrimination between normal and pathological inflammatory response is a difficult element of diagnosis [38, 39]. According to the current concept of the inflammatory response to trauma and infection concomitant to SIRS, in which the cells of the innate immune response are primarily involved, CARS inflammatory response develops, which may lead to immunosuppression. This part of the immune response involves adaptive immune cells including lymphocytes producing anti-inflammatory cytokines, immunosuppressive TReg cells (CD4+/ CD25+), and antigen-presenting dendritic cells [40]. More recent studies have confirmed that CARS can lead to a reduction in the severity of SIRS proinflammatory response in order to achieve balance, but in some patients it led to increased immunosuppression [41]. It should be assumed that the adoption of the hypothesis of a parallel course of SIRS and CARS was another factor that hindered the designation of appropriate immunological markers that would allow the differentiation of physiological reaction and pathologic post-traumatic response.

It should be recalled that based on an extensive review of the literature, the control of the dynamics of inflammatory response in severe infection, on the basis of selected SIRS and CARS parameters (e.g. TNF-α, IL-1, IL-6), has repeatedly been proposed [10]. The authors have also suggested other prognostic factors, including genetic predisposition, the type and source of infection, and the degree of multiple organ failure, and formulated on this basis a classification system for sepsis (PIRO scoring system – ) (Table 2) [10, 42]. This system attempted to link the assessment of selected genetic, immunological, and bacteriological markers with clinical signs of infection.

Table 2

Classification of sepsis based on the PIRO system [42]

Diagnostics	P	I	R	O
	Predisposition	Infection	Response	Organ failure
Clinical	AgeComorbidityGeneral conditionCause of trauma/infection (degree and severity)	Susceptibility to infectionSite of infectionPathogen identificationSource of infection (hospital or non-hospital)Extent of the infection	SIRSOther clinical signs ofsepsisshockCRP	Organ failure according to the scale: MODS, SOFA, LODS, PEMOD, PELOD
Molecular	Genetics (Polymorphism TLR, TNF, IL-1, CD14)	Gene transcript profilesAnalysis of microbial products (LPS, mannan, bacterial DNA)Assessment of virulence factors	BiomarkersNon-specific markers of inflammation (e.g. PCT, IL-6)or markers of immune disorders (e.g. HLA-DR)	Monitoring of cell-mediated immunity (abnormal apoptosis, Mitochondrial and endothelial damage and activation of adhesion molecules, cell hypoxia)

MODS – multiple organ dysfunction syndrome, SOFA – sequential organ failure assessment, LODS – logistic organ dysfunction system, PEMOD – paediatric multiple organ dysfunction, PELOD – paediatric logistic organ dysfunction

The most recent studies, conducted in the Netherlands, Portugal, and the United States on a large group of patients (about 3,500), compared the PIRO system with the scales routinely applied in the evaluation of the severity of the patients’ status (APACHE – Acute Physiology and Chronic Health Evaluation, SOFA – Sequential Organ Failure Assessment, MEDS – Mortality in Emergency Department Sepsis). Based on these studies, it was found that this system had a higher prognostic value in the assessment of severity of infection, MODS and mortality risk. Moreover, it was found that the point value in this system correlated with clinical symptoms [43-45].

The next scheme (Fig. 5), which illustrates the dynamics of pro- and anti-inflammatory response to trauma and infection, emphasised the influence of other factors on the inflammatory SIRS response (mechanism of injury, ischaemia, reperfusion, medical procedures, ATLS – Advanced Trauma Life Support, type of surgery, anaesthesia, additional infection, and possible reoperation) [46].

Fig. 5

An example of changes in the pro- and anti-inflammatory response to injury and infection (according to Brochner et al.) [46]

These factors also affect the variability of CARS response. According to these authors, the presented scheme should be reflected in the treatment of post-traumatic patients, which should be supported by monitoring the levels of IL-6 in peripheral blood, coagulation markers, lactates, changes in pH, temperature, and diuresis. In the first hours of the occurrence of trauma, life-saving treatments should be primarily implemented (e.g. chest drainage). The authors of this study emphasise that every surgical procedure may be another stimulus that increases the immune disorders, therefore, subsequent re-operations should be performed only after normalisation of the indicators examined [46].

In the latest studies, the concept of SIRS and CARS has been complemented with genetic studies. The results of these studies have shown that the concept of the socalled second hit SIRS response to trauma and infection is unlikely (Fig. 6A) [47]. In turn, based on the expression of genes (mediators of the immune response), it was confirmed that SIRS and CARS occur simultaneously (Fig. 6B).

Fig. 6

Dynamics of changes in SIRS and CARS response in trauma patients based on genetic studies (according to Xiao et al.) [47]

The concept presented covers a fairly wide range of genetic studies. The following genes have been studied: TLRs (except for TLR3 and TLR7), haptoglobins, collagenases, cytokines (IL-1Ra, IL-4, IL-6, IL-8, IL-10) expressed in T and B cells, leukocytes, neutrophils, and genes encoding proteins responsible for apoptosis [47]. Attention has been drawn to the dynamics of the selected signalling pathways activation or their suppression in conjunction with the degree of the severity of multiple organ failure. The authors introduced the term „gene storm” stemming from the involvement of virtually all genes in response to external and internal stimuli. It was found that the trauma can induce the production of various inflammatory mediators and activate the receptor protein genes involved in the recognition of molecular patterns of microorganisms (PRR), suppressing at the same time receptor genes responsible for antigen presentation, proliferation of T cells, apoptosis, receptor function or activity of NK cells. “Gene storm” is a highly coordinated and repeatable response, so that the immune system undergoes rapid adaptive changes in response to trauma. In this work, it was found that the quantitative and not qualitative changes in genes’ expression are the cause of post-traumatic complications. In patients without complications, silencing of gene transcription occurs within 7-14 days, whereas gene activation in patients with complications lasts significantly longer (over 28 days) [47].

Thus, the previous concepts assuming that SIRS is short term and transient were wrong. The expression of genes involved in the immune response can be prolonged in severely ill patients after the injury, resulting in the production of immature bone marrow cells (MDSCs – myeloid derived suppressor cells), which exhibit a potent immunosuppressive action and do not differentiate into the cells of the early immune response [48]. The main mediators of myeloid cell activation include cytokines and prostaglandins, which are involved in both pro- and anti- inflammatory response [47, 48]. Appropriate myeloid stem cells with the ability of self-renewal differentiate successively into CMP cells (common myeloid progenitor) and MDSCs, which are important in the formation of early immune response cells (granulocytes, macrophages, dendritic cells). Trauma excessively increases the production of MDSCs, the result of which is that the immature cells cannot activate subsequent immune response pathways [48].

It is known that in severely ill patients after trauma, treated long-term in the ICU, the cells of the immune response to injury and infection that produce nitric oxide (NO), reactive oxygen species (ROS), and other mediators of immunosuppressive action consume large amounts of energy (e.g. arginine). Rapid depletion of energy resources may give rise to so-called persistent inflammation and immune suppression syndrome catabolism (PICS) (Fig. 7) [49].

Fig. 7

A new concept of the immune response to trauma and infection, involving PICS and disorders of cell-mediated immunity (according to Gentile et al.) [49]

There is a need to change the therapeutic approach directed at balancing the multi-level defects of immune response and reducing protein catabolism, which in turn requires appropriate monitoring of immunity changes. A wider introduction of testing the concentrations of selected cytokines of pro- and anti-inflammatory action (IL-6, IL-10, IL-1Ra, or sTNFRI) to the routine diagnostics of inflammatory response to trauma and infection raises some hopes. These indicators give earlier information about the pathological changes in the innate immune response to injury and infection, and their concentration can be routinely assayed using a simple ELISA [49]. On the other hand, the routine use of flow cytometry allows rapid evaluation of disorders of cell-mediated immunity, including the examination of the current activity of monocytes involved in the later stage of the immune response to trauma and infection (analysis of HLA-DR or CD80/CD86 expression in CD14+ cells) [50]. More advanced techniques enable evaluation of the expression of selected genes in the MDSC responsible for persistent immunosuppression, but this part of diagnostics is still in the experimental phase [51].

An important element in the treatment of these patients is the need to rapidly provide energy substances required for the proper functioning of the immune cells in a suitable nutritional therapy. This applies in particular to patients with PICS requiring longer treatment in the ICU [47, 48]. These patients are diagnosed with difficult to balance catabolism, exacerbating malnutrition, impaired wound healing, and recurrent infections. PICS is characterised by simple parameters assessing the nutritional status: ICU stay > 10 days, weight loss >10% during hospitalisation, or BMI < 18%, CRP level > 150 mg/dl, total lymphocyte count (TLC) < 0.8 × 109/l, albumin <3.0 g/dl, prealbumin < 10 mg/dl, retinol binding protein (RBP) < 10 mg/dl [49]. The authors emphasise that the clinical image is accompanied by chronic inflammation, increasing immunosuppression with macrophage function paralysis, and a decreased count and activity of lymphocytes. This group of patients usually requires long-term treatment in the ICU and is subject to the highest risk of secondary complications (respiratory failure, traumatic coagulopathy syndrome, hypothermia, hypocalcaemia, acidosis, infection) and death [49, 52]. Previous therapy attempts using antibodies, factors stimulating the activity of immune cells, or immunomodulatory preparations did not yield any significant breakthrough in reducing the high mortality of patients with severe sepsis treated in the ICU. Some hope for improving the results of treatment is associated with a more accurate assessment of nutritional status and with the wider introduction of new immunomodulatory preparations, e.g. an appropriate nutritional treatment of immunonutrition type – agents blocking immune response by affecting the TLRs (e.g. Eritoran) [53, 54]. The clinical usefulness of novel survival biomarkers and severity of infection has also been tested (e.g. Endocan) [55]. Patients in metabolic stress after major trauma, surgery, or sepsis often require supply of a modified diet composition enriched with specific immune- modulating nutrients such as glutamine, arginine, omega-3 fatty acids, or nucleotides (Impact, Reconvan, Intestamin) [54-58]. Opinions on the use of immunonutrition in severely ill patients with post-traumatic sepsis, treated in the ICU, are still controversial. As recent studies have shown, the use of glutamine in patients with multiple organ failure may increase mortality [59, 60]. Characteristic in these studies are very high doses of glutamine (0.35 g/kg bw/d i.v. + 30 g/d enterally) that may exacerbate organ failure (according to ESPEN, glutamine dose should not exceed 0.5 g/kg bw/d). It should be emphasised that these studies were performed in a heterogeneous group of patients, which could also have influenced the obtained results.

Summary

In conclusion, one of the important factors that increase mortality in posttraumatic patients is pathological pro- and anti-inflammatory response occurring in the first hours after extensive trauma with massive infection, which is still difficult to control and to discriminate from physiological immune response. Interpretation of the results of studies is hindered by the parallel course of SIRS and CARS response, and the significant heterogeneity of the patient groups investigated. Complementing the SIRS and CARS concept with genetic research partly explains this problem. As demonstrated by the most recent studies, during the so-called gene storm, which occurs immediately after the injury and infection, a large number of genes are induced, which are ultimately responsible for the production of many inflammatory response mediators and the expression of receptors involved in the recognition of molecular patterns associated with pathogens. In parallel, the expression of genes responsible for the proper presentation of antigens, lymphocyte proliferation, and apoptosis is decreasing. These are primarily qualitative rather than quantitative changes in the expression of genes that are one of the causes of severe post-traumatic complications. Prolonged activation of genes is another factor that increases the risk of complications. Changes in gene expression detected early after injury precede the occurrence of later complications with a typical clinical picture. Furthermore, rapid depletion of energy resources increases the immunosuppression, burdened with the highest mortality rate. Early diagnosis of PICS and appropriate treatment, nutritional therapy in particular, can reduce the length of stay of patients in the ICU and decrease mortality.