Cytokine 92 (2017) 83–92

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Immunoparalysis: Clinical and immunological associations in SIRS and severe sepsis patients Panagiotis Papadopoulos a, Aikaterini Pistiki b,1, Maria Theodorakopoulou a, Theodora Christodoulopoulou a, Georgia Damoraki b, Dimitris Goukos c, Efrossini Briassouli c, Ioanna Dimopoulou a, Apostolos Armaganidis a, Serafim Nanas d, George Briassoulis e, Sotirios Tsiodras b,⇑,1 a

2nd Department of Critical Care, Attikon University Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece 4th Department of Internal Medicine, Attikon University Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece First Department of Propaedeutic Internal Medicine, Laikon University Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece d First Critical Care Department, Evangelismos Hospital, University of Athens, Athens, Greece e Pediatric Intensive Care Unit, University Hospital, University of Crete, Heraklion, Greece b c

a r t i c l e

i n f o

Article history: Received 25 February 2016 Received in revised form 6 January 2017 Accepted 13 January 2017

Keywords: Sepsis SIRS Heat shock proteins SOCS HLA-DR Immunoparalysis

a b s t r a c t Introduction: This study was designed to identify changes in the monocytic membrane marker HLA-DR and heat shock proteins (HSPs) in relation to T-regulatory cells (T-regs) and other immunological marker changes in patients with systemic inflammatory response syndrome (SIRS) or sepsis/septic shock. Methods: Healthy volunteers, intensive care unit (ICU) patients with SIRS due to head injury and ICU patients with severe sepsis/septic shock were enrolled in the current study. Determination of CD14+/ HLA-DR+ cells, intracellular heat-shock proteins and other immunological parameters were performed by flow cytometry and RT-PCR techniques as appropriate. Univariate and multivariate analysis examined associations of CD14/HLA-DR, HSPs, T-regs and suppressor of cytokine signalling (SOCS) proteins with SIRS, sepsis and outcome. Results: Fifty patients (37 with severe sepsis and 13 with SIRS) were enrolled, together with 20 healthy volunteers used as a control group. Compared to healthy individuals, patients with SIRS and severe sepsis showed progressive decline of their CD14/HLA-DR expression (0% to 7.7% to 50% within each study subpopulation, p < 0.001). Mean fluorescent intensity (MFI) levels of HSP70 and HSP90 on monocytes and polymorphonuclear cells were significantly higher in SIRS patients compared to controls and fell significantly in severe sepsis/septic shock patients (p < 0.05 for all comparisons). There was no statistically significant difference between subgroups for levels of T-regulatory cells or relative copies of Suppressor of Cytokine Signalling 3 (SOCS3) proteins. In univariate models percent of CD14/HLA-DR was associated with mortality (OR: 1.8 95%CI 1.02–3.2, p = 0.05), while in multivariate models after adjusting for CD14/HLA-DR only younger age and lower Acute Physiology and Chronic Health Evaluation II (APACHE II) scores were associated with increased chances of survival (beta
0.05, OR 0.9, 95% CI 0.9–0.99, p = 0.038 for age and beta
0.11, OR 0.89, 95% CI 0.8–0.99, p = 0.037 for APACHE II score). Conclusions: Significant associations with SIRS and sepsis were found for CD14/HLA-DR expression and monocyte and polymorphonuclear cell levels of HSP70 and 90. The role of these biomarkers in assessing the prognosis of sepsis needs to be further explored and validated in prospective studies. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction

⇑ Corresponding author at: National and Kapodistrian University of Athens Medical School, Attikon University Hospital, 1 Rimini street, 12462 Athens, Greece. E-mail addresses: [email protected], [email protected] (S. Tsiodras). 1 The two first authors contributed equally to this work. http://dx.doi.org/10.1016/j.cyto.2017.01.012 1043-4666/Ó 2017 Elsevier Ltd. All rights reserved.

Treatment of sepsis is currently mainly based on prompt optimisation of physiological parameters and antibiotic use since the early goal-directed therapy has been shown to increase survival considerably [1]. In contrast, despite significant efforts our understanding of the pathophysiology of sepsis is still evolving. Moreover, no significant improvement in reducing death rates has

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been achieved over the last decade. It is clear that further elucidation of the biology of sepsis will lead future diagnostic strategies and guide therapeutic approaches. A profound activation of the innate, non-specific immunity characterizes the onset of septic shock. An overwhelming release of pro-inflammatory mediators accompanies this early response, followed immediately by an immunosuppression state that further contributes to the adverse outcomes associated with sepsis [2]. The exact starting point, the intensity and extent of the hyperinflammatory state versus a subsequent anti-inflammatory state are difficult to identify, above all in the clinical setting [3]. The discovery of novel biomarkers that could assist in the differentiation of these stages of sepsis as well as contribute to better understanding of the course of the sepsis syndrome will assist in guiding interventions and in the prediction of the overall outcome. Different molecules have been studied over the years to this effect. The expression of the monocytic membrane marker HLA-DR has been shown to be negatively influenced by the presence of systemic inflammatory response syndrome (SIRS) and septic shock and has been suggested to correlate with sepsis mortality in the second (immunosuppressive) stage of sepsis [4,5]. Heat shock proteins (HSP) are cellular proteins that are highly upregulated to protect cells against elevated temperatures [6]. The heat shock proteins 70 and 90 (HSP70, HSP90) are ubiquitous chaperones with numerous roles, including cell protection against stress, anti-apoptotic effects independent of the Fas-ligand mediated pathway and other not fully understood, immunomodulatory functions [6–8]. HSP90 and HSP70 have been identified as functional receptors for the bacterial lipopolysacharide (LPS) on the surface of human monocytes/macrophages acting in a CD14independent fashion [9]. They can both serve as novel biomarkers for the characterization of the different stages of sepsis [10–12]. The regulatory T cells, a specific CD4+, CD25+ T cell minor subpopulation, with important immunosuppressive regulatory effects, are also gaining increasing interest regarding their role during the second phase of sepsis [13–15]. Dysregulation of cytokine signalling may have a significant role in the immune modulation observed in all the phases of sepsis [16,17]. The suppressors of cytokine signalling (SOCS) proteins have a key negative regulatory role in the Janus kinase/signal transducer and activator of transcription pathway and are important regulators of adaptive immunity [18–21]. The downregulation of proinflammatory cytokines by SOCS proteins may be desirable to boost the immune responses [22,23]. In the current study, we attempted to identify the relation between the state of immunoparalysis and the expression of the above named biological markers, namely the HLA-DR surface marker, the HSP70, HSP90 and SOCS3 intracellular proteins, and the population of the T-reg cells. For this purpose, these immunological markers were studied both in healthy volunteers and patients with SIRS or severe sepsis/septic shock. The aim was to further identify and understand patterns of immune response in patients with SIRS and severe sepsis/septic shock.

2. Methods 2.1. Clinical criteria Intensive care unit (ICU) hospitalized patients fulfilling the criteria of severe sepsis/septic shock and ICU patients with SIRS due to head injury were enrolled in the current study. Inclusion criteria for the participating patients were: (a) age P18 years; (b) presence of early severe sepsis/septic shock (48 h or less after the first symptoms) and (c) presence of SIRS in patients with head trauma. Severe sepsis/septic shock or SIRS were defined

according to guidelines of the surviving sepsis campaign [24]. More specifically, presence of two or more of the following criteria at day 0, defined SIRS: (1) pyrexia >38 °C or temperature below 36 °C; (2) leukocytosis (>10,000/lL) or leukopenia (10% bands; (3) tachycardia (>90 beats/min); and (4) increased respiratory rate (>20 breaths/min) or mechanical ventilation. Presence of an infection (either probable or proven) together with SIRS defined sepsis. Sepsis-induced hypoperfusion or organ dysfunction thought to be due to the infection defined severe sepsis. Shock was defined as presence of hemodynamic instability for at least 1 h despite adequate fluid resuscitation or requirement of vasopressors [24]. Shock was defined as septic shock if sepsis was present. Exclusion criteria were: (a) presence of malignancies; (b) presence of autoimmune diseases; (c) prior use of corticosteroids; (d) immunosuppressive illness; (e) late sepsis or SIRS. In addition blood was sampled from age- and gender-matched healthy volunteers. Twelve milliliters of blood were collected from every patient and control in a heparin tube (Becton Dickinson, Cockeysville, MD, USA) under sterile conditions at the time of study inclusion. Complete blood count, serum creatinine levels and maximum glucose levels at the day of observation were recorded. All blood samples were collected within 24 h of the development of sepsis. The reasons for this were: (a) to have comparable data reflecting early response to sepsis, since comparison of sepsis to SIRS/trauma patients is also more reasonable in an early time-window; (b) to have fewer external factors affecting patients’ condition e.g. hospital-related events or complications. The classification systems for severity and prognosis in intensive care patients APACHE II and SOFA (Sequential Organ Failure Assessment) scores were calculated for each participating patient. For head injury/trauma patients we calculated the injury severity score (ISS). The study protocol was approved by the Ethics Committee of the ATTIKON University hospital (822/13-12-2011). Written inform consent was signed by the patient or their first-degree relative of each patient and by each healthy volunteer.

2.2. Determination of intracellular heat-shock proteins Whole blood (100 ll) was incubated in the dark with monoclonal antibodies anti-CD33 (5 ll) at the flurochrome phycoerythrin-Cy5 (PeCy5, emission 667 nm, Biolegned, SanDiego, CA, USA) and anti-CD45(5 ll) at the fluorochrome phycoerythrinCy7 (PeCy7, emission 767 nm, Biolegend, London, UK). White blood cells were then fixed (Fixation Buffer, Biolegend) and permeabilized (Permeabilization Wash Buffer, Biolegend) and incubated with monoclonal antibodies anti-Hsp70/Hsp72 at the fluorochrome fluorescein isothiocyanate (FITC, emission 525 nm, Enzo Life sciences, NY, USA) and anti-Hsp90a at the fluorochrome phycoerythrin (PE, emission 575 nm, Enzo) for intracellular staining at a 1:10 dilution according to manufacturer’s instructions. Afterwards blood was washed and red blood cells were lysed with NH4Cl solution (Lysing solution, Beckman Coulter Co, Miami, FL, USA). White blood cells were washed, reconstituted in PBS and analyzed after running through the CYTOMICS FC500 flow cytometer (Beckman Coulter Co); 100,000 events were analyzed. Separate gating was used to determine the mean fluorescence intensity (MFI) of HSP70 and HSP90 on monocytes and polymorphonuclear cells according to their characteristic CD33/SS and CD45/SS scattering. Monocytes were defined as the cell population with lower granulation and high expression of CD33 and polymorphonuclear cells as the population with higher granulation and dim expression of CD33. CD45 was used as an internal control to verify the separation of the two populations. IgG isotype controls were used for each patient.

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2.3. Determination of T-regulatory cells Peripheral blood mononuclear cells (PBMC’s) were isolated from blood after gradient centrifugation over Ficoll (Biochrom, Berlin, Germany). Isolated cells were washed three times and counted using a Neubauer chamber with trypan blue exclusion of dead cells. Cells were reconstituted in RPMI 1640 enriched with 2 mM of L-glutamine, 500 mg/ml of gentamicin, 100 U/ml of penicillin G and 10 mM of pyruvate (Biochrom). 1x106 PBMC’s were stained with monoclonal antibodies anti-CD4 at the fluorochrome fluorescein isothiocyanate (Immunotech, Marsailles, France) and antiCD25 at the fluorochrome phycoerythrin-Cy5 (Immunotech). Tregulatory cells were stained using the PE anti-human Foxp3 staining set (eBioscience Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Cells were analyzed through the cytometer and CD4 high/CD25 high/Foxp3 positive lymphocytes were considered T-regulatory cells.

2.4. Determination of Suppressor of cytokine signalling protein 3 mRNA levels 5x106 PBMC’s were lysed with Trizol (Applichem, Darmstadt, Germany). The cell pellets were treated with chloroform, and contaminating DNA was removed by treatment with 0.04 U/ll DNAase (New England BioLabs, Ipswich, MA, USA) for 30 min at 37 °C. Total RNA was isolated and checked for purity. Concentrations were quantified spectrophotometrically. To produce cDNA, 1 lg of RNA was incubated with 0.4 mM of dNTPs (New England BioLabs, Herts, UK), 1 U of RNA-sin (New England BioLabs), 10 mM DTT (New England BioLabs) and 5 of the reverse transcriptase buffer in a thermocyler (SensoQuest, Göttigen, Germany). Samples without reverse transcriptase were used as blanks and cDNA was kept at
80 °C until assayed. Quantitative RT-PCR was performed on duplicate samples in 96-well plates with an iQ5 Real Time PCR Detection System (BioRad Laboratories Inc, Hercules CA, USA). Quantification of SOCS3 mRNA levels was calculated relative to the reference gene encoding b2-microglobulin (b2-m), which was stably expressed. SOCS3 is one of the most well-studied molecules of its family and plays a pivotal role in down-regulating inflammation, by interfering to, amongst others pathways, IL-6 cytoplasmic signalling [21]. Reactions were performed in a final volume of 20-ll containing 10 ll FluocycleTM II SYBR Master Mix (EuroCloneS.p.a, Milan, Italy), 7.0 ll PCR-grade water (AppliChem), 2.0 ll cDNA, and 1.0 ll each of the 20 lM forward and reverse gene-specific primers. The cycling conditions comprised 95 °C for 5 min, followed by 45 PCR cycles. Each cycle consisted of three steps: denaturation (95 °C for 10 s), annealing (60 °C for 30 s), and extension (75 °C for 30 s). Blanks were subjected to this protocol as well. Gel electrophoresis 3% w/v stained with ethidium bromide (AppliChem) and melt curve analysis was used to verify the formation of specific products. PCR primers were: SOCS3 sense 50 -TGC GCC TCA AGA CCT TCA G-30 ; antisense 50 -GAG CTG TCG CGG ATC AGA AA-30 ; b2-microglobulin, sense 50 -ATG AGT ATG CCT GCC GTG TG-30 ; antisense, 50 -CCA AAT GCG GCA TCT TCA AAC-30 . To evaluate b2-m stability for this study we have used the GeNorm algorithm [25]. The relative fold expression was measured using the formula 2
DDCT in which CT is the threshold cycle number [26]. We also checked amplification efficiencies of target and reference gene (doubling every cycle 100% efficiency) by dilution curve (10-fold dilution series from cDNA and by plotting the Ct as a function of log [10]). cDNA was diluted to determine efficiency for all genes. The absolute value of the slope of DCT vs. log input was