Various life-threatening medical conditions may cause critical illness in patients requiring admission to an ICU. Despite the vast heterogeneity of admission causes including cardiopulmonary resuscitation, various shock states, and major surgery, patients are often characterized by a strong inflammatory reaction despite an absence of microbial pathogens.
This condition has been described as systemic inflammatory response syndrome (SIRS) which affects the majority of patients admitted to medical ICUs (1, 2). Tissue hypoperfusion and injury may result in cell death with subsequent release of strictly intracellular components into the circulation. These so-called damage-associated molecular patterns (DAMPs) may be recognized as intracellular molecules, they may be recognized by the innate immune system as “non-self” through pattern recognition via Toll-like receptors (TLRs) expressed in monocytes and neutrophils, the key cell lines of innate host defense. Upon activation, a pronounced inflammatory response followed by subsequent cytokine release may provide an explanation for the similarity of pathogen-induced and sterile-immune activations, as seen in critically ill patients (3).
Mammalian TLRs were first described and characterized during the end of the 20th century (4). Toll-like receptor 4 (TLR4) was found to be the counterpart of the Drosophila's Toll receptor, which plays a crucial role in host defense against fungal infection (5). Additional proteins with structural similarity were identified and categorized as the TLR family. A total of ten human TLRs have been identified so far (6). Within its physiologic role, TLR-2 is responsible for detecting specific pathogen-associated molecular patterns found on Gram-positive bacteria. TLR-9 recognizes bacterial DNA (unmethylated oligodeoxynucleotide [CpG] DNA) whereas TLR-5 binds to bacterial flagellin (6, 7). TLR-4 functions as a fundamental receptor for the recognition of lipopolysaccharide (LPS) while the third TLR-family consisting of TLR-3, TLR-7, TLR-8, and TLR-9 are generally localized intracellularily for activation by bacterial and viral nucleic acids (6).
A great variety of DAMPs may be released during critical illness and recognized by TLRs including heat shock proteins, uric acid, mitochondrial DNA (mtDNA), components of the extracellular matrix and high mobility group box-1 protein (8, 9). Although the importance of TLR signaling was clearly demonstrated in acute infection and severe sepsis, accumulating evidence suggests an equally important role of TLR signaling in critically ill patients suffering from various critical disease states including trauma and burns, acute myocardial infarction, cardiopulmonary resuscitation, cardiogenic shock, or acute kidney injury (10–15).
However, whether TLR upregulation may be used to identify patients at risk for worse outcome independent of their ICU admission cause has not been prospectively tested yet. Therefore, the aim of this study was to analyze expression patterns of TLR-2, TLR-4, and TLR-9 on circulating neutrophils and monocytes and their association with 30-day mortality.
MATERIALS AND METHODS
Patients and study design
This is a single-center, prospective observational study including all consecutive patients above the age of 18 years that were admitted between August 2012 and August 2013 to the cardiovascular ICU within the Department of Internal Medicine II of the Medical University of Vienna at the General Hospital of Vienna. This is a tertiary care medical ICU; therefore, the entire spectrum of critically ill medical patients is being treated. Our ICU specifically focuses on cardiovascular diseases; also patients after heart and thoracic surgery are being admitted. This study complies with the Declaration of Helsinki and was approved by the ethical committee of the Medical University of Vienna (EK 1101/2012). In addition, we included 40 age and sex-matched hospital controls that had no signs of critical illness and were included at the general cardiology ward. Conscious patients were required to give informed consent, whereas for unconscious patients the need for informed consent was waived by the ethical committee before inclusion of the patient, but informed consent was obtained later when the patient was able to provide it.
At study inclusion, cause of admission as well as baseline demographics, clinical history, and parameters were noted. Major interventions that were undertaken before admission or within the first 72 hours after admission were recorded (including mechanical ventilation, surgery, intra-aortic balloon counterpulsation, extracorporeal membrane oxygenation, extracorporeal renal replacement therapy, and use of catecholamines). The main outcome parameter was defined as 30-day survival. For the quantification of disease severity, the Simplified Acute Physiology Score (SAPS) II (16), the Acute Physiology and Chronic Health Evaluation II (APACHE II) (17), and the Sepsis-related Organ Failure Assessment (SOFA) score (18) were used.
For flow cytometry analysis, blood was drawn within the first 24 hours after ICU admission into an ethylenediaminetetraacetic acid (EDTA) tube after discarding the initial 3 ml of blood. After the initial 3 ml of blood had been discarded, blood was drawn into an EDTA tube for immediate flow cytometry. In addition, a serum separator tube, a 3.8% sodium citrate vacuette tube and an EDTA tube (all Greiner Bio-One) were collected, immediately centrifuged (4°C, 3000 RPM, 15 min) and stored at −80°C for later analysis.
Whole blood flow cytometry (FACS) for the determination of monocyte and neutrophil TLR expression was performed using a FACS Canto II with the FACS Diva Software (both Becton Dickinson). Briefly, 100 μl of EDTA-anticoagulated blood was stained with respective saturating concentrations of the following fluorochrome-conjugated monoclonal antibodies (mAbs): Phycoerythrin-Cy7 (PE-Cy7) mAb for CD45, peridinin chlorophyll protein (PerCP)-labeled mAb for CD14 (BD Biosciences), allophycocyanin (APC)-H7-labeled mAb for CD16 (BD Biosciences), fluorescein isothiocyanate (FITC)-labeled mAb for TLR-2 (eBioscience), APC-labeled mAb for TLR-4 (eBioscience), PE-labeled mAb for TLR-9 (BD Biosciences) and corresponding isotype controls. For the detection of TLR-9, which is expressed intracellularly, cells were fixed and permeabilized (Invitrogen Fix&Perm kit, life technologies) and incubated with anti-TLR-9 antibody and isotype control. After incubation for 15 minutes in the dark, 1.5 ml lysing solution (BD FACS Lysing solution, BD Biosciences) was added. After another 15 minutes of incubation in darkness, cells were washed 3 times by adding 1 ml PBS and centrifugating at 820 rpm for 5 minutes each. Cells were then resuspended in 1 ml PBS for FACS analysis.
Monocytes were defined as CD45+ cells exhibiting a typical forward and sideward scatter profile as well as CD14/CD16 expression, whereas neutrophils were defined as CD45+ and CD16+ as well as CD14− cells exhibiting a typical forward and sideward scatter profile (Figure 5). Monocyte subsets were defined as classical monocytes (CD14++/CD16−), intermediate monocytes (CD14++CD16+; IM) and nonclassical monocytes (CD14+/CD16++) in line with the 2010 nomenclature (19).
Measurement of interleukin-6
Interleukin-6 was measured by enzyme-linked immunosorbent assay according to the instructions of the manufacturer (R&D Systems, Minneapolis, MN). Limit of detection was 0.7 pg/ml. Intraassay coefficient of variation (CV) was 4.2% and interassay CV was 6.4%.
Sample size calculation analysis revealed that in a cohort with a mortality rate of 25%, given a power of 0.8 and a significance level of 0.05, we would need 208 patients to detect a difference of 20% between TLR-mean fluorescence intensity (MFI) between survivors and nonsurvivors. Categorical variables are summarized as counts and percentages and are compared by the χ2 or by Fisher's exact test as appropriate. Kolmogorow–Smirnow test was used to test for normal distribution and Spearman's rank-order correlations were calculated as TLR expression showed a skewed distribution. Continuous variables are expressed as median and interquartile range (IQR). Data were compared by Mann–Whitney test and the method of Bonferroni–Holm was used to adjust for multiple testing. Cox proportional hazard regression analysis was performed to assess the effect of TLR expression on survival. Interaction terms were included in the Cox regression model to assess interactions between variables. Cox regression models were adjusted for APACHE II score to account for clinical parameters and for all laboratory variables that were imbalanced between survivors and nonsurvivors indicated by a P value <0.05. Receiver-operating characteristic (ROC) curves were calculated for 30-day survival utilizing neutrophil TLR-2 expression, neutrophil TLR-9 expression, APACHE II, SAPS II, and SOFA score as test variables. Kaplan–Meier analysis (log-rank test) was applied to verify the time-dependent discriminative power of monocyte subsets below or above the median. Two-sided P values of <0.05 indicated statistical significance. SPSS 18.0 (IBM Corporation, Armonk, NY, USA) was used for all analyses.
We included 233 consecutive patients and flow cytometry data was available for 215 patients. Patients for whom cytometry data was not available did not differ with respect to baseline characteristics to patients with flow cytometry data (data not shown). No patients were lost to follow-up. Baseline characteristics are given in Table 1. About 60% of patients were male and the median age was 65.8 (55.0–76.5) years. Among our medical patients, the most common causes of admission comprised cardiopulmonary resuscitation, acute heart failure or cardiogenic shock followed by sepsis and respiratory failure. From our 17 septic patients, 6 suffered from gram-positive, 8 from gram-negative and 3 from fungal sepsis, the primary focus was the abdominal tract in 6, the lung in 4, wound infections in 3 and the urogenital tract in 2 patients, while 2 patients suffered from endocarditis. The diagnostic category “respiratory failure” included 7 patients with pneumonia, 5 patients with exacerbation of chronic obstructive pulmonary disease, 3 patients were admitted after lung transplantation, 2 cases of severe acute pulmonary embolism and one patient with lung fibrosis. All our surgical patients (n = 45; 20.9%) underwent cardiac surgery using cardiopulmonary bypass (75% elective, 25% emergency; coronary artery bypass grafting (CABG) in 31.8%; heart valve surgery in 20.5%, combined CABG and heart valve surgery in 22.7%, other surgeries in 25%).
Median APACHE II score was 19 (12–25) median SAPS II score was 44 (31–57). 58.1% of patients were mechanically ventilated and 57.2% needed vasopressor treatment. About 33% of patients underwent heart surgery or heart valve intervention and 67% were medical patients; 30-day mortality was 26%.
Toll-like receptor expression on monocytes and neutrophils
Out of total leucocytes 69.8% (58.5–79.0%) were neutrophils and 5.8% (3.5–7.8%) were monocytes. Although TLRs on neutrophils and monocytes show good correlations (Table 2), patients after cardiac surgery or heart valve intervention showed significant lower TLR-2 expression on neutrophils as compared with medical patients (MFI: 3064 IQR 2716–3532 versus 3710 IQR 2720–4431; P < 0.005), but expression of TLR-4 and TLR-9 on neutrophils as well as expression of TLRs on monocytes were not different between the two patient groups. In patients with sepsis (n = 17), TLR-4 expression on monocytes was significantly increased (MFI: 1011 IQR 771–1133 versus 766 IQR 683–865; P < 0.05). TLR-2 and TLR-9 on monocytes as well TLR expression on neutrophils were not associated with the presence of sepsis at admission.
TLR-2 expression on neutrophils showed a borderline correlation with APACHE II score (R = 0.26; P < 0.001), SAPS II (R = 0.30; P < 0.001), and SOFA score (R = 0.18; P < 0.01). In contrast, neutrophil TLR-4 and TLR-9 expression as well as TLR expression on monocytes were not associated with disease severity scores (data not shown). TLR-2 was also weakly associated with serum lactate (R = 0.20; P < 0.01). Interestingly, TLR-2 (R = 0.44; P < 0.001), TLR-4 (R = 0.32; P < 0.001), and TLR-9 (R = 0.20; P < 0.005) on neutrophils, as well as TLR-4 expression on monocytes (R = 0.15; P < 0.05) were correlated with serum creatinine. Additionally, out of TLR-2, TLR-4, and TLR-9 on monocytes and neutrophils, only TLR-2 expression on neutrophils showed a weak association with the inflammatory parameters C-reactive protein (CRP; R = 0.15; P < 0.05) and procalcitonin (R = 0.18; P < 0.01) and TLR-4 expression on neutrophils showed a weak but negative association with CRP (R = −0.17; P < 0.05) and interleukin-6 (R = −0.16; P < 0.05).
Of note, monocyte subsets showed a distinct TLR expression profile, with the proinflammatory subtype of intermediate subsets exhibiting the strongest expression of all three TLRs analyzed (Figure 1). However, subset specific TLR expression was not associated with clinical parameters or disease severity scores.
Toll-like receptor expression and 30-day mortality
Neutrophil expression of TLR-2 was significantly higher in nonsurvivors as compared with survivors (MFI: 4026 IQR 3344–4609 versus 3254 IQR 2667–4002; P < 0.001). In the CPR subgroup, patients that died within 30 days showed higher TLR-2 expression (MFI: 4007 (3053–4736) as compared with those that survived 2714 (2527–3462); P = −0.008), whereas in the subgroup of patients with heart failure, TLR-2 expression did not differ according to mortality (data not shown). Furthermore, nonsurvivors showed increased TLR-9 expression on neutrophils as compared with survivors (MFI: 3268 IQR 2322–5817 versus 2697 IQR 2131–3408; P < 0.05). In contrast, TLR-4 expression on neutrophils and TLR expression on monocytes were not associated with survival, respectively (Figure 2). Similarly, monocyte subset-specific expression of TLRs was also not associated with outcome (data not shown).
In addition, we included 40 age and sex-matched stable hospital controls (age: 69.0 IQR 62–74 years, 57.5% male) and compared TLR expression to that of the critically ill patients (Figure 2). Controls showed significantly lower expression of TLR-4 (P < 0.01) and TLR-9 (P < 0.001) on neutrophils as compared with survivors and lower neutrophil expression of TLR-2 (P < 0.001), TLR-4 (P < 0.01) and TLR-9 (P < 0.001) as compared with nonsurvivors, respectively. TLR expression on monocytes did not differ between controls and critically ill patients, with the exception of higher TLR-4 expression of hospital controls compared with survivors (P < 0.01).
Neutrophil TLR-2 (odds ratio 6.1, 95% confidence interval [CI] 2.5–14.6; P < 0.001) and TLR-9 (odds ratio 2.0, 95% CI 1.1–3.8; P < 0.05) expression in the third tertile were associated with mortality (Figure 3). Cox regression analysis revealed that neutrophil TLR-2 and TLR-9 expression predicted mortality independently from APACHE II, serum lactate, serum creatinine, and procalcitonin (Table 3). ROC curves were created to compare the predictive value of neutrophil expression of TLR-2 (AUC = 0.67; P < 0.001) and TLR-9 (AUC = 0.61; P < 0.05) with the severity scores APACHE II (AUC = 0.87; P < 0.001), SAPS II score (AUC = 0.84; P < 0.001), and SOFA score (AUC = 0.81; P < 0.001), respectively (Figure 4).
In this single-center observational study including 215 consecutive patients admitted to a cardiovascular medical ICU at a tertiary university hospital, expression of TLR-2 and TLR-9 on circulating neutrophils at ICU admission was predictive for short-term mortality within 30 days. Neutrophil TLR-2 and TLR-9 expression predicted outcome independent from several well-established prognostic markers including lactate and serum creatinine as well as the APACHE II score. As expected, patients were severely ill reflected by an APACHE II score of 19 (IQR 12–25), as well as a high percentage of patients requiring mechanical ventilation and vasopressors. This was reflected by a 30-day mortality rate of about 25%.
In our prospective analysis, we measured TLR-2, TLR-4, and TLR-9 expression on monocytes and neutrophils, the main cellular players of the innate immune response. Besides their physiologic role in detecting gram-positive (TLR-2) and gram-negative (TLR-4) bacteria as well viral nucleic acids (TLR-9), those three TLRs have been implicated in the pathophysiology of sterile inflammation as seen in critical illness (6, 8). Possible endogenous TLR-2 and TLR-4 ligands include the heat shock protein family, high-mobility group box protein-1 and several others (8, 9, 20). TLR-9 is described to recognize mitochondrial DNA as DAMP because of its strong resemblance of bacterial DNA (21). The most other DAMPs studied are TLR-2 and TLR-4 dependent (8, 9, 22).
We have decided to include all consecutive patients admitted to our ICU, a medical ICU at a tertiary care university hospital treating the entire spectrum of critically ill medical patients with a focus on cardiovascular diseases, including also patients after cardiac or thoracic surgery. By doing so we were able to analyze a possible participation of the innate immune system in critical illness irrespective of the underlying pathophysiology as seen on a daily basis in clinical routine and acknowledged decades ago as SIRS (1).
One remarkable finding within our study population is that neutrophil but not monocyte TLR expression is associated with outcome. One possible explanation might be the staged innate immune response described in various sterile and infectious diseases where neutrophils take the lead with a quick and strong activation upon injury or infection, whereas monocytes are characterized by a delayed response after acute injury and are implicated in repair mechanisms (23). This differential response has been described for different critical illness triggering diseases, including myocardial infarction, stroke, and systemic ischemia reperfusion, respectively (24–26). Therefore, our observations might be explained by the early analysis after ICU admission.
Approximately, one-third of our patients were admitted after cardiac surgery or interventional heart valve procedures. These patients were characterized by a lower neutrophil TLR-2 expression as compared with medical patients, whereas TLR-4, TLR-9 expression on neutrophils did not differ between the groups. These findings are of interest, as they might suggest reduced inflammatory activation in patients after major surgery as compared with medical critical illness. One explanation may be that medical critical illness causes a diffuse, albeit more pronounced tissue damage and subsequent DAMP release as opposed to the surgical trauma, even when undergoing major cardiovascular surgery (27). We could previously show that within the same study population, levels of intracellular DAMPs, in this case mitochondrial DNA, could be found in significantly higher levels within the circulation in those patients with medical critical illness as compared with those admitted after cardiac surgery (27).
Of interest is the finding that TLR-4 expression on monocytes was increased in patients with sepsis (n = 17), in line with previous studies and the widely accepted role of TLR-4 in LPS signaling (28). Although TLR-2 indisputably plays a major role in pathogen recognition and inflammatory activation during septic conditions, TLR-4 is the essential pattern recognition receptor causally involved in the overwhelming immune activation seen in sepsis and septic shock (28, 29). Interestingly, TLR-4 was not associated with mortality in the whole study cohort, which might be explained by the low number of patients with gram-negative sepsis as the primary reason for ICU admission.
In our patients, neutrophil TLR-2 expression was weakly associated with serum lactate, a finding that has already been described on monocytes in patients with cardiogenic shock (11). Of additional interest is the observed, albeit weak correlation between neutrophil TLR-2 expression and several acknowledged prognostic scores for critical illness including APACHE II, SAPS II, and SOFA. Interestingly, TLR-9 was not associated with these scores, but was an independent predictor of outcome, suggesting TLR-9 expression as a marker of innate immune activation not reflected by disease severity. Our findings of an overactive immune system and high levels of circulating intracellular DAMPs (27) suggest a different and complementary pathophysiologic pathway of patient deterioration in critical illness that is not being covered by common disease severity scores.
Another interesting finding within this analysis is that we could only show weak correlations between TLR-2 on neutrophils and CRP as well as procalcitonin, whereas there were no associations between TLR-9 and circulating inflammatory markers, and the proinflammatory cytokine interleukin-6 was only weakly negatively associated with TLR-4 expression. These findings may be interpreted as an early, unspecific upregulation of TLRs in response to circulating DAMPs which may precede interleukin-6 production (30).
One counterintuitive result within our study is the seemingly diverging role of TLR-2 and TLR-4, the two TLRs activated by most DAMPs described to date (22). Although variable TLR-2 and TLR-4 expression were shown in septic conditions (31), data for sterile inflammatory states are scarce. However, similarly to our study, two previously published articles reported diverging actions of TLR-2 and TLR-4 in sterile inflammation, one in patients after successful CPR admitted to a medical ICU (10) and the second one in an experimental burn mouse model (32). As outlined above, a TLR-4 upregulation was only observed in septic patients, in line with its central role in LPS signaling. It has to be noted, however, that no data is available whether increased TLR surface expression on circulating leukocytes translates into increased activation of TLR signaling pathways.
As already mentioned, neutrophil TLR-9 expression at ICU admission was predictive of 30-day mortality in the study population presented herein. TLR-9 constitutes an intracellular member of the TLR-family activated by unmethylated CpG sequences usually found on bacterial DNA, while relatively rare on the human genome. According to the endosymbiont theory, mitochondria originated from bacteria more than a billion years ago that entered into unicellular anaerobes. Interestingly, they still resemble bacteria as they exhibit a double-membrane and a circular genome containing CpG sequences. Release of mitochondrial DNA in critical illness via cell and tissue damage may activate TLR-9 and thus may be partly responsible for the inflammatory response observed in critical illness (9, 21). In the here presented study collective, we were already able to demonstrate that circulating mtDNA levels at ICU admission strongly predict 30-day mortality (27). In this regard, it is of great interest that the represented neutrophil TLR-9 expression is strongly and independently predictive of mortality, suggesting mtDNA being a possible ligand and carrier of the strong and detrimental immune response.
Another novelty presented within this article is the diverse TLR expression of circulating monocyte subsets. Although subset specific TLR expression was not associated with outcome in our patient cohort, we describe a subset specific TLR expression with intermediate monocytes exhibiting the highest expression of TLR-2, TLR-4, and TLR-9. This is of interest, as we have described an association of an increased proportion of intermediate monocytes, the monocyte subset with the highest TLR expression, and outcome (33). The described finding of an increased TLR expression of intermediate monocytes in critically ill patients provides further evidence for the proinflammatory phenotype of this monocyte subset.
Potential limitations of this investigation include the single-center study design as well as the heterogeneity of the study population included, suffering from various underlying diseases, but also including postsurgical patients. On the contrary, the nature of an all-comer study may be seen as a strength of this study as it accurately reflects the patient population ICU physicians are confronted with on a daily basis and demonstrates TLR-upregulation as a disease-independent marker of inflammatory activation as well as a prognostic marker for 30-day mortality. Furthermore, because of the nature of an observational clinical study, we cannot draw any functional insights regarding TLR signaling in these patients. Therefore, further studies are required to elucidate the pathophysiological mechanisms behind our observation. Blood for flow cytometry was only obtained early after ICU admission; therefore, a longitudinal study would be needed to assess whether serial flow cytometry measurements may add prognostic value.
In conclusion, this study provides evidence for prognostic properties of neutrophil TLR-2 and TLR-9 expression with respect to 30-day mortality in an unselected cohort of critically ill patients, independent from APACHE II, serum lactate, creatinine, and procalcitonin levels. Of note, neutrophil TLR-4 as well as monocyte TLR expression was not associated with outcome in our population. These findings provide evidence for a leukocyte and TLR-subtype-specific role of innate immune activation in critically ill patients. Further studies are needed to analyze pharmacological interference with TLR signaling as a therapeutic means.
This work was supported by the FWF Austrian Science Fund, Grant Number SFB-54, Cellular Mediators Linking Inflammation and Thrombosis, by the Association for the Promotion of Research on Arteriosclerosis, Thrombosis and Vascular Biology (ATVB), and the Ludwig Boltzmann Cluster for Cardiovascular Research.
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