Background
Trauma and hemorrhagic shock (HS) stimulate a systemic release of endogenous molecules that are known to activate the innate immune system [
1]. These inflammatory mediators interact with Toll-like receptors (TLR), initiate the expression of transcription factors (e.g. NFκB) and provoke local and systemic liberation of pro- and anti-inflammatory cytokines [
1,
2]. Toll-like receptor 2 (TLR-2) is expressed on the surface of immune cells (leukocytes) and in the lung and liver. It has been shown that bacterial cell wall components are able to stimulate the systemic immune response through the TLR-2 pathway [
3]. The associated imbalance between the pro- and anti-inflammatory immune system may result in either self-destructive hyper-inflammation or immune paralysis and sepsis [
4]. Both are associated with the development of multiple organ failure (MOF) and high mortality rates [
5,
6]. Therefore, numerous studies have aimed to identify protective mechanisms that modulate the immune response following trauma or sepsis [
7,
8].
Macrophage-activating Lipopeptide-2 (MALP-2) is a lipopeptide and was primarily isolated from the cell wall of
Mycoplasma fermentans and was synthetized chemically [
9]. It is known to activate macrophages and other immune cells (e.g. B-cells) via TLR-2 / TLR-6 heterodimers [
10]. The initial contact of MALP-2 and the immune system initiates a pro-inflammatory immune response [
11‐
13]. However, desensitization of signal cascades was also observed in studies [
3]. Pre-treatment with MALP-2 in mice with sepsis and peritonitis was associated with an attenuated immune response and reduced mortality rates [
3]. Moreover, MALP-2 administration into the respiratory tract resulted in beneficial effects in murine pneumonia model with
Streptococcus pneumoniae[
14]. All these studies documented protective effects after MALP-2 treatment in infection models. The role of MALP-2 therapy in sterile inflammation has not been studied so far. To this end, we analyzed whether treatment with MALP-2 prior and after to an inflammatory stimulus attenuates the systemic inflammatory response and liver and lung inflammation in a murine hemorrhagic shock model.
Discussion
Numerous substances, including hormones (e.g. DHEA) [
8], cytokines (e.g. IL-10) [
26,
27] and diets [
28] have been identified to have immuno-modulatory effects. The administration of the gram-negative bacterial cell wall components prior to a second hit has been shown to diminish the severity of the inflammatory response [
29]. This phenomenon was termed endotoxin tolerance. Similar to lipopolysaccharide (LPS), the injection of TLR-2 ligand (MALP-2) was associated with a decrease of pro-inflammatory mediators and immunomodulation [
3]. It has been postulated that MALP-2 attenuates the immune response with a sufficient response for protective pathogen defense [
3]. The consequences of MALP-2 treatment on the immune response were only studied in animals with bacterial infection [
3,
14,
30]. The aim of these experiments was to analyze the role of MALP-2 treatment on sterile inflammation in response to HS.
This study revealed the following results: 1) Despite single and lowest administration dose the injection of MALP-2 prior to the induction of HS is feasible to attenuate the systemic IL-6 response. 2) Organ specific inflammation has been observed after the pre-exposure to MALP-2. While MPO activity within the lung tissue was tendentially decreased in pre-treated animals, the highest hepatic MPO levels were measured in the same group.
First, in contrast to KC values, the systemic release of IL-6 did not increase after i.p. injection of TLR-2 agonists (
Group MALP T) when compared with levels in non-treated animals with HS (
Group HS). This observation is in line with previous reports [
3]. Feterowski et al. reported a slight increase in TNF-α levels and significant elevation of KC within 2 hours after MALP-2 injection [
3]. Moreover, Reppe et al. reported comparable cytokine patterns within the lung tissue following inhalative MALP-2 administration [
14]. Pre-exposure (12 hours) to MALP-2 prior to the HS (
Group MALP PT) was associated with a significant reduction of plasma IL-6 levels in our study. Protective effects on the immune response after MALP-2 treatment have been already demonstrated in a murine sepsis model [
30,
31]. The administration of MALP-2 did not affect the levels of anti-inflammatory IL-10 in our study. These results are in line with finding from Feterowski et al., which described no elevation of IL-10 after systemic injection of MALP-2 in mice [
3]. In addition, i.p. MALP-2 injection was associated with significantly elevated MCP-1 levels when compared to levels in mice without MALP-2 treatment. Our results are in line with findings reported by Kaufmann et al. [
32]. Increased MCP-1 production accompanied by TNF-α was reported in human monocytes after MALP-2 administration. Moreover, our results indicate that the TLR-2 agonist appears to affect sterile inflammation pathways. TLR’s recognize gram-positive and gram-negative microbial components [
33], which have been termed Pathogen-Associated Molecular Patterns (PAMPs), and generate a complex immune response [
33]. In addition, endogenous tissue ligands (Damage-Associated Molecular Patterns (DAMPs)) were also identified and are known to induce systemic inflammation [
1]. Authors have hypothesized that the immune system is mainly designed to identify threatening signals of either infection or injury, rather than differentiate between self and non-self [
34]. However, it is still controversial whether the acute inflammatory response is activated by PAMPs or DAMPs through a universal pathway system. A similar pattern of acute inflammation after infectious and non-infectious stimulus has been described [
18]. Moreover, there is increasing evidence that DAMP-mediated inflammation is generated by the TLR mechanisms [
35]. In contrast, other studies pointed out DAMP specific pathways of the TLR activation [
1,
36].
Second, we identified an organ specific inflammation following MALP-2 administration. Intraperitoneal stimulation with MALP-2 12 hours prior to the initiation of HS was associated with tendentially decreased pulmonary MPO activity. This finding might be a consequence of the reduced systemic pro-inflammatory response recorded in our experiment. Moreover, this might be associated with changes in expression and sensitivity of TLRs within peritoneal cavity. Studies identified slight not significant increased TLR-4 expression within the spleen tissue after i.p. MALP-2 administration [
3]. Moreover, TLR-2 up-regulation in the lung after inhalative MALP-2 application has been demonstrated [
14]. The reduction of the pro-inflammatory mediators and pulmonary MPO activity might be accompanied by increased peritoneal inflammation and infiltration of neutrophils to a different compartment [
14]. We observed a pronounced MPO activity within the liver in mice with MALP-2 treatment. MALP-2 is known to stimulate neutrophil recruitment to the site of application [
10,
37]. High liver MPO activity might be also related to increased influx of leukocytes into the peritoneal cavity and splanchnic circulation.
Treatment with TLR-2 agonists following HS does not attenuate the initial sterile inflammatory response in our study. In comparison to untreated mice, treatment with MALP-2 following HS was associated with increased levels of KC and MCP-1 and unchanged levels of IL-6. Moreover, the MPO activity could not be reduced. However, the therapeutic approach might be interesting in a presence of second hit (additional surgery) or infection (pneumonia/sepsis). Further studies are necessary to prove the therapeutic possibilities. In addition, in this study we focused on local and systemic inflammatory response. We did not determine the function or damage of organs such liver and kidney. Therefore, there is a lack of evidence of early multiple organ failure in this study.
Our analysis should be interpreted with respect to the following limitation: Only one time point and one treatment dosage have been assessed in our study. Therefore, no conclusions on dynamics of inflammation can be drawn out of these data. MALP-2 is dissolved in 30% 2-propanol / water for stock solution. However, we did not include 2-propanol to PBS in our control groups. It has to be considered that 2-propanol may influence the systemic inflammatory response.
Competing interests
This work has been supported by the Else Kröner-Fresenius Foundation. There are no further financial and non-financial competing interests.
Authors’ contributions
All authors were involved in the research project and preparation of the manuscript. TT and HCP: They made a substantial contribution to the conception and design, and gave a critical and final approval. RP, PL, DD, and PK: They have performed the study, have collected the data and made an analysis and interpretation of these data. They also made a draft of the manuscript and revisions. All authors read and approved the final version of the manuscript.