1. Introduction
Sepsis is a frequent cause of severe disease and death globally, being a major public health concern [
1,
2,
3,
4]. Sepsis-induced myocardial dysfunction was observed and described for the first time in 1984. Patients suffering from septic shock showed impaired cardiac systolic function by decreased left-ventricular ejection fraction (LVEF) [
5,
6]. Similar courses of reversible myocardial dysfunction due to the systemic inflammatory response syndrome were also shown in other critical illnesses [
7,
8]. Septic patients developing myocardial dysfunction have a significantly higher mortality (70%) [
9]. The prevalence of the disease in septic patients ranges from 10% to 70%. This wide range is the result of a lack of formal diagnostic criteria and under-recognition of septic cardiomyopathy [
10]. For a better understanding of the correlation between systemic inflammatory response and reversible myocardial dysfunction in sepsis, the levels of BCL2 protein, oncogene protein p53, and CD14 surface protein in patients who died from septic shock were investigated [
6].
The human gene CD14 (cluster of differentiation 14) expresses a glycoprotein which is a component of the innate immune system, and it was the first described pattern recognition receptor. The membranous form (mCD14) is anchored mostly to the membrane of macrophages, but also that of neutrophils and dendritic cells. The soluble form (sCD14) appears after shedding from the membrane or as a result of direct secretion from intracellular vesicles [
11], especially from the liver and monocytes. Both mCD14 and sCD14 are present in enterocytes [
12]. Along with TLR4 (Toll-like receptor 4) and MD-2 (lymphocyte antigen 96), CD14 is a coreceptor for the detection of bacterial lipopolysaccharide (LPS) [
13,
14]. In the presence of lipopolysaccharide-binding protein (LBP), it binds to LPS. Additionally, it is able to recognize other pathogen-associated molecular patterns such as lipoteichoic acid [
15].
Basically, the Toll-like Receptor (TLR) family recognizes several microbial products, including bacterial cell wall components and DNA [
16]. Both preclinical studies (animal models) and clinical ones involving human subjects have proven an association between TLR4 mutations and a diminished degree of responsiveness to LPS [
17]. Myocardial dysfunction after endotoxin exposure depends on the presence of cell-wall receptors such as TLR4 and CD14 [
18,
19]. It was demonstrated that CD14-deprived mice showed a normal cardiac function, being protected by LPS-induced shock [
19], while others showed a decreased ventricular fraction [
20]. However, the exact mechanism of action remains unclear. It can be assumed that cardio-depression may be induced by secondary cytokines such as TNF-α or endotoxins, as a result of CD14-mediated pathways [
21,
22].
In this context, the aim of this our study was to determine any significance of the mCD14 and sCD14 levels in the septic cardiomyopathy, and to evaluate the correlation with lipopolysaccharide-binding protein (LBP) by means of immuno-histological examinations.
3. Results
Septic cardiomyopathy is a common feature of severe sepsis syndromes and results in impaired intrinsic cardiac contractility. According to the particular infection site, the distribution of the study group is presented in
Table 1, while the epidemiological data including the age, gender, and social provenance are displayed in
Table 2. The mean value of the age for the study group was 60.3 years (±16.5). The mean value of the age (in months) for the control group was 4.2 months (±2.6). The myocardial cell expression of CD14 protein was significantly increased in the study group, as compared with control cases, where the expression rate remained under 10% (
p = 0.0013) (
Table 3). The patients with a higher expression of CD14 were older, but the difference did not reach the limit of statistical significance (
p = 0.1204) (
Table 4). There was a positive correlation between cellular (membranous, mCD14) and intravascular (soluble, sCD14) CD14 values (
p = 0.0077) (
Table 5). Females showed a higher incidence of increased intravascular CD14, but this difference did not reach the statistically significant threshold (
Table 6). There was no difference in sCD14 expression rates related to the provenance (
p = 0.9271), infection site (
p = 0.7301), and age (
p = 0.4976, with a tendency toward older age (
Table 7).
Evaluation of microscopic sections using only conventional H&E staining does not reveal the presence of LPS. H&E staining emphasizes interstitial edema, muscle fiber suffering, myocardial cytolysis, and the presence of inflammatory cells (
Figure 1A). On the other hand, the use of auxiliary immunohistochemistry techniques provides valuable, additional information. The microscopic image shows myocardial cells, among which numerous inflammatory cells with a membranous, brown color indicate immune expression of CD14 as a cluster of differentiation. mCD14 is a multifunctional glycoprotein expressed mainly on the membrane surface of macrophages. According to
Figure 1B, CD14 is also highly distributed on the cell surface of neutrophils (mCD14). Analysis of blood vessels revealed how inflammatory cells such as neutrophils/monocytes (mCD14) migrate into the myocardium through the vascular wall. The soluble part sCD14 (LPS) remains strictly confined in the blood vessel lumen (
Figure 1C). The blood vessels are located intramuscularly, as mCD14 can also be seen with sCD14. In the lumen of this vessel, several granular extracellular brown areas (soluble CD14), as well as monocytes and neutrophils (mCD14), are noticeable (
Figure 1D).
4. Discussion
CD14 acts as a coreceptor for TLR4 and interferes with the LPS response. CD14 forms a complex with LPS and the LPS-binding protein (LBP), and this complex binds to the TLR4, inducing NF-κB-associated immune responses [
24]. The onset of the immune responses induces the release of tumor necrosis factor alpha (TNF-α), IL-1, IL-6, and IL-8 [
25]. Many studies concluded that a higher level of LPS associated with an overexpression of CD14 acts as a trigger for the release of a broad spectrum of cytokines. A higher cytokines level indicates a higher probability of an adverse immune response.
To the best of our knowledge, this is the first paper in which the expression of CD14 surface protein was evaluated in septic shock and septic cardiomyopathy in humans. Our study showed a positive association between markedly increased values of CD14 and an adverse patient evolution. A statistically significant increase in the level of membranous and soluble CD14 surface protein was found in the study group. Overall, there was a tendency for higher values in relation to older patients, but without statistical significance. The increasing values with age (despite not statistically significant) might be a clue for the elevated mortality level in older patients. There was no statistically relevant difference regarding the patient’s gender (
p = 0.3990), provenance (
p = 0.9892), or infection site (
p = 0.3803). The results of our study regarding patient gender with septic shock are in contradiction with other studies [
26]. One explanation could be the relatively small number of studies worldwide. Most studies revealed that female sex hormones exhibit protective effects in septic shock. Estrogens and their precursors might attenuate the levels of proinflammatory agents and reduce the effects for septic patients [
26,
27].
It is very well known that there are two types of LPS coreceptors: membrane-bound CD14 (mCD14) complexed with TLR4 and the LBP–LPS complex, and soluble CD14 (sCD14) complexed with LBP–LPS located in the blood. The expression of mCD14 on many epithelial cells such as human endometrial stromal cells may explain the cytokine storm [
28]. The soluble form of CD14 is able to bind LPS and activate cells that otherwise do not express membrane-bound CD14 (e.g., endothelial, epithelial, and smooth muscle cells) [
29,
30]. The role of sCD14 was revealed by previous studies, suggesting that the CD14–TLR4–MD2 complex is involved in the phagocytosis of Gram-negative bacteria and intracellular signaling by LPS [
31,
32,
33].
Our study revealed a positive association between cellular expression (membranous, mCD14) and intravascular (soluble, sCD14) levels. Despite the fact that the patient’s gender did not significantly influence the intravascular CD14 expression rate in the study group (
p = 0.4612), the females showed a higher incidence. CD14 plays a double role: (1) as a component of the innate immune system, a pattern recognition receptor (PRR) proposed to bind conserved molecular structures on microbes (pathogen-associated molecular patterns, PAMPs, e.g., LPS) [
34]; (2) as an active agent involved in the apoptotic cell cleaning process. Apoptotic cell-associated molecular patterns (ACAMPs) interfere with PRR and LPS-like structures, as revealed on apoptotic cells [
35]. Increased values of BCL2 and p53 in septic cardiomyopathy were associated with increased mortality [
36]. Thus, CD14 ligation with different ligands (PAMP or ACAMP) leads to an opposite response. CD14 binds LPS to generate proinflammatory responses or binds ACAMP for the clearance of apoptotic cells in a noninflammatory way [
37,
38]. It would be interesting to evaluate the level of CD14 in survivors of septic shock of comparable severity. This would prove the association between increased expression rates and poor outcome in septic shock. Various studies attempted to identify a role of complement, coagulation, and inflammation pathways in the pathophysiology of myocardial injury [
39].
The microcirculatory disorders induced by the microorganism associated with destruction of myocardial fibers generate the release of oxygen species and may also contribute to the severity of septic myocarditis [
40,
41]. Myocardial biopsies cannot be obtained in critically ill patients. However, serum levels of mCD14 and sCD14 could be compared, considering their strong relationship. A comparison with different control groups, such as patients who died of trauma, could prove their specific significance in septic shock [
41], and this is our goal for future work. Our current study was a quantitative retrospective study, but further investigations are needed to determine the minimum levels required of mCD14 and sCD14 to trigger death from septic cardiomyopathy [
42].
A comparison with different control groups, i.e., patients who died of trauma, could prove of valuable significance in better understanding septic shock.
This was a quantitative retrospective study, and further studies are needed to determine the minimum amount of LPS required to trigger death from septic cardiomyopathy.