Sepsis is a systemic response to infection commonly found in critically ill patients and is associated with multi-organ failure and high mortality rate. Its pathophysiology and molecular mechanisms are complicated and remain poorly understood. In the present study, we performed a proteomics investigation to characterize early host responses to sepsis as determined by an altered plasma proteome in a porcine model of peritonitis-induced sepsis, which simulated several clinical characteristics of human sepsis syndrome. Haemodynamics, oxygen exchange, inflammatory responses, oxidative and nitrosative stress, and other laboratory parameters were closely monitored. Plasma samples were obtained from seven pigs before and 12 h after the induction of sepsis, and plasma proteins were resolved with two-dimensional gel electrophoresis (n=7 gels/group; before being compared with during sepsis). The resolved proteins were stained with the SYPRO Ruby fluorescence dye and subjected to quantitative and comparative analyses. From approx. 1500 protein spots visualized in each gel, levels of 36 protein spots were significantly altered in the plasma of animals with sepsis (sepsis/basal ratios or degrees of change ranged from 0.07 to 21.24). Q-TOF (quadrupole–time-of-flight) MS and MS/MS (tandem MS) identified 30 protein forms representing 22 unique proteins whose plasma levels were increased, whereas six forms of five unique proteins were significantly decreased during sepsis. The proteomic results could be related to the clinical features of this animal model, as most of these altered proteins have important roles in inflammatory responses and some of them play roles in oxidative and nitrosative stress. In conclusion, these findings may lead to a better understanding of the pathophysiology and molecular mechanisms underlying the sepsis syndrome.
- host response
Sepsis is defined as a systemic inflammatory response syndrome due to presumed or confirmed infection [1,2]. It is associated with multi-organ failure, which in turn serves as a marker for the high mortality rate of sepsis [2,3]. Even with treatments with the currently available regimens of antibiotics, fluid resuscitation, vasoactive compounds, corticosteroids etc. [4–6], the morbidity and mortality rates remain considerably high. Several attempts have been made to define new therapeutic targets, for example IL-12 (interleukin-12) , HMG (high-mobility group) box-1 isoforms , trypsin  and several others ; however, the molecular mechanisms underlying sepsis remain poorly understood. Searching for biomolecules that are involved in the pathophysiology of sepsis would be a significant advance and would facilitate defining new therapeutic targets for better treatment outcomes.
Over the last decade, proteomics has been emerging and used for the high-throughput analysis of proteins. It has been extensively applied to several subdisciplines of biomedical research, particularly for clinical applications, with the ultimate goals to understand normal physiology better, to explore the pathogenic mechanisms of diseases, and to search for novel biomarkers and therapeutic targets for improving treatment outcome [11–13]. In the present study, we applied a gel-based proteomics approach to characterize early responses to sepsis, as determined by changes in the plasma proteome during an early phase of sepsis. A porcine model of peritonitis-induced sepsis, which displays several clinical characteristics resembling those of sepsis syndrome in humans, was employed in our present study. Peritonitis-induced sepsis was initiated by intraperitoneal injection of autologous faeces with careful monitoring of haemodynamics, oxygen exchange, inflammatory responses, oxidative and nitrosative stress, and other laboratory parameters. The plasma proteome at 12 h after the induction of sepsis was compared with the basal plasma proteome (before sepsis induction) using 2-D (two-dimensional) gel electrophoresis. Differential analysis revealed significant differences in levels of 36 protein spots, which were subsequently identified by Q-TOF (quadrupole–time-of-flight) MS and/or MS/MS (tandem MS). Potential roles of these altered proteins induced by sepsis are discussed.
MATERIALS AND METHODS
The present study was performed according to the National Institutes of Health Guidelines on the Use of Laboratory Animals, and the study protocol was approved by the Ethical Committee at Charles University School of Medicine in Plzeň.
Experimental set up and the induction of sepsis
A total of seven pigs were included in the present study and all of them were closely monitored. After an induction of anaesthesia with intravenous atropine (0.5 mg), 2% (v/v) propofol (1–2 mg/kg of body weight) and ketamine (2 mg/kg of body weight), all of the animals were mechanically ventilated with an FiO2 (fraction of inspired oxygen) of 0.4, PEEP (positive end-expiratory pressure) of 5–10 cmH2O, and a tidal volume of 10 ml/kg of body weight. The respiratory rate was adjusted to maintain an arterial PCO2 (partial pressure of carbon dioxide) between 4.0 and 5.0 kPa. Anaesthesia was maintained with continuous intravenous thiopental (10 mg·kg−1 of body weight·h−1) and fentanyl (10–15 μg·kg−1 of body weight·h−1) during surgery and then maintained with continuous intravenous thiopental (5 mg·kg−1 of body weight·h−1) and fentanyl (5 μg·kg−1 of body weight·h−1) thereafter until the end of the study. Muscle paralysis was achieved with pancuronium (0.2 mg·kg−1 of body weight·h−1). The intravenous fluid was Plasma Lyte® solution (Baxter Healthcare) with a rate of 15 ml·kg−1 of body weight·h−1 during surgery and then of 7 ml·kg−1 of body weight·h−1 as a maintenance fluid. Arterial blood glucose levels were maintained at 4.5–7.0 mmol/l using a 20% (w/v) glucose infusion.
A central venous catheter was inserted through the left jugular vein for administration of all of the drugs and fluids. A balloon-tipped thermodilution pulmonary artery catheter was placed via the right jugular vein. A femoral arterial catheter was placed for BP (blood pressure) monitoring and blood sampling. Two tubes were placed through the abdominal wall for the induction of peritonitis and ascites drainage. A cystostomy catheter for urine collection was placed percutaneously under ultrasound guidance.
After all measurements and sample collection at baseline, sepsis was initiated by the induction of peritonitis by inoculating 0.5 g of autologous faeces/kg of body weight suspended in 200 ml of saline into the abdominal cavity through the drainage tubes. A second set of measurements and sample collection were obtained 12 h after the induction of sepsis. In addition to the Plasma Lyte® solution, 6% (w/v) hydroxyethyl starch 130 kDa/0.4 (Voluven® 6%; Fresenius Kabi) at a rate of 10 ml·kg−1 of body weight·h−1 was infused to maintain a cardiac filling pressure ≥12 mmHg [the rate was decreased to 7 ml·kg−1 of body weight·h−1 if the CVP (central venous pressure) or PAOP (pulmonary artery occlusion pressure) ≥18 mmHg]. When the last set of data had been obtained, the animals were killed by KCl injection under deep anaesthesia.
Monitoring of haemodynamics, oxygen kinetics, inflammatory responses, oxidative and nitrosative stress, and other laboratory parameters
Measurements for monitoring systemic haemodynamics included CO (cardiac output), SVR (systemic vascular resistance), intrathoracic blood volume and filling pressures of both ventricles (CVP and PAOP for right and left ventricles respectively). Arterial and mixed venous blood samples were analysed for pH, PO2 (partial pressure of oxygen), PCO2 and haemoglobin oxygen saturation. Systemic DO2 (oxygen delivery) and systemic V̇O2 (oxygen consumption) were derived from the appropriate blood gases and flow measurements [14,15]. Arterial blood samples were obtained for the determination of TNF-α (tumour necrosis factor-α) and IL-6 by immunoassay [14,15]. Oxidative and nitrosative stress was evaluated by measuring concentrations of arterial TBARS (thiobarbituric acid-reacting substances) by spectrophotometry, and arterial NOx (nitrate/nitrite) by colorimetric assay [14,15]. To correct for dilutional effects resulting from volume resuscitation, the levels of NOx, TBARS, IL-6 and TNF-α were normalized to plasma protein content [14,15].
2-D Gel electrophoresis
Plasma samples were diluted 1:5 with deionized water, and protein concentrations in individual samples were measured using the Bradford method . Protein solutions (each with 200 μg of total protein) were then pre-mixed with a rehydration buffer containing 7 mol/l urea, 2 mol/l thiourea, 2% (w/v) CHAPS, 120 mmol/l DTT (dithiothreitol), 40 mmol/l Tris base, 2% ampholytes (pH 3–10) and a trace of Bromophenol Blue to make the final volume of 150 μl/sample. The mixtures were rehydrated on to Immobiline™ DryStrips (7 cm long IPG strips; linear pH gradient of 3–10 and of 4–7; GE Healthcare) at room temperature (25 °C) for 10–15 h. The first-dimensional separation or IEF (isoelectric focusing) was performed in the Ettan IPGphor II IEF System (GE Healthcare) at 20 °C, using a stepwise mode to reach 9083 Vh. After completion of the IEF, the strips were first equilibrated for 15 min in an equilibration buffer containing 6 mol/l urea, 130 mmol/l DTT, 112 mmol/l Tris base, 4% (w/v) SDS, 30% (v/v) glycerol and 0.002% Bromophenol Blue, and then in another similar buffer, which replaced DTT with 135 mmol/l iodoacetamide, for a further 15 min. The second-dimensional separation was performed on a 12% (w/v) polyacrylamide gel using a SE260 mini-vertical electrophoresis unit (GE Healthcare) at 150 V for approx. 2 h. Separated proteins were visualized with SYPRO Ruby fluorescence staining (Invitrogen/Molecular Probes). Gel images were taken using a Typhoon laser scanner (GE Healthcare).
Matching and analysis of protein spots
Image Master 2D Platinum (GE Healthcare) software was used for matching and analysis of protein spots in 2-D gels. Parameters used for spot detection were (i) minimal area=10 pixels; (ii) smooth factor=2.0; and (iii) saliency=2.0. A reference gel was created from an artificial gel combining all of the spots presenting in different gels into one image. The reference gel was then used for matching the corresponding protein spots between gels. Background subtraction was performed and the intensity volume of each spot was normalized with the total intensity volume (summation of the intensity volumes obtained from all spots within the same 2-D gel). Significant differences in intensity levels of protein spots were defined as changes with all of the followings: (i) sepsis/control ratios ≥2-fold or ≤0.5-fold; (ii) P<0.05; and (iii) consistent presence (or absence) in all gels within the group.
In-gel tryptic digestion
The protein spots whose intensity levels significantly differed between groups were excised from the 2-D gels, washed twice with 200 μl of 50% (v/v) ACN (acetonitrile)/25 mmol/l NH4HCO3 buffer (pH 8.0) at room temperature for 15 min, and then washed once with 200 μl of 100% ACN. After washing, the solvent was removed, and the gel pieces were dried using a SpeedVac concentrator (Savant) and rehydrated with 10 μl of 1% (w/v) trypsin (Promega) in 25 mmol/l NH4HCO3 (pH 8.0). After rehydration, the gel pieces were crushed with a siliconized blue stick and incubated at 37 °C for at least 16 h. Peptides were subsequently extracted twice with 50 μl of 50% (v/v) ACN/5% (v/v) TFA (trifluoroacetic acid); the extracted solutions were then combined and dried with a SpeedVac concentrator. The peptide pellets were resuspended with 10 μl of 0.1% TFA and purified using ZipTipC18 (Millipore). The peptide solution was drawn up and down in the ZipTipC18 ten times and then washed with 10 μl of 0.1% formic acid by drawing up and expelling the washing solution for three times. The peptides were finally eluted with 5 μl of 75% (v/v) ACN/0.1% formic acid.
Protein identification by MALDI (matrix-assisted laser-desorption ionization)–Q-TOF MS and MS/MS analyses
The proteolytic samples were premixed 1:1 with the matrix solution [5 mg/ml CHCA (α-cyano-4-hydroxycinnamic acid) in 50% (v/v) ACN, 0.1% (v/v) TFA and 2% (w/v) ammonium citrate] and spotted on to the 96-well sample stage. The samples were analysed using a Q-TOF Ultima™ mass spectrometer (Micromass), which was fully automated with a pre-defined probe motion pattern and the peak intensity threshold for switching over from MS survey scanning to MS/MS, and from one MS/MS to another. Within each sample well, parent ions that met the pre-defined criteria (any peak within the m/z 800–3000 range with an intensity above 10 counts±include/exclude list) were selected for collision-induced dissociation MS/MS using argon as the collision gas and a mass dependent ±5 V rolling collision energy until the end of the probe pattern was reached. The low-mass and high-mass resolution of the quadrupole were both set at ten to give a precursor selection window of approx. 4 Da wide. Manual acquisition and optimization for individual samples or peaks was also possible.
The instrument was externally calibrated to a <5 p.p.m. accuracy over the mass range of m/z 800–3000 using sodium iodide and PEG [poly(ethylene glycol)] 200, 600, 1000 and 2000 mixtures and adjusted further with Glu-fibrinopeptide B as the near-point lock mass calibrant during data processing. At a laser firing rate of 10 Hz, individual spectra from a 5 s integration period acquired for each of the MS survey and MS/MS performed were combined, smoothed, de-isotoped (fast option) and centroided using the ProteinLynx™ GlobalSERVER 2.0 data processing software (Micromass). This entailed the identification of the monoisotopic carbon-12 peaks for MS data and deconvolution of multiply charged spectra to their singly charged equivalents for MS/MS data. MaxEnt 3™, a maximum-entropy-based technique, has been designed for this purpose and is an integral part of ProteinLynx™ GlobalSERVER 2.0 . The combined MS and MS/MS ion meta data were searched in concert against the NCBI (National Center for Biotechnology Information) mammalian protein database using the ProteinLynx™ GlobalSERVER 2.0 workflow. The search algorithm employed a Hidden Markov Model that incorporates empirically determined fragmentation characteristics to increase the efficacy of the search. Additionally, the MS and MS/MS data were extracted and outputted as the searchable .txt and .pkl files respectively, for independent searches using the MASCOT search engine (http://www.matrixscience.com), assuming that peptides were monoisotopic. Fixed modification was carbamidomethylation at cysteine residues, whereas variable modification was oxidation at methionine residues. Only one missed trypsin cleavage was allowed, and peptide mass tolerances of 100 and 50 p.p.m. were allowed for the peptide mass fingerprinting and MS/MS ion search respectively.
Pathway analysis was performed using the Pathway Tools software version 12.5 (http://bioinformatics.ai.sri.com/ptools/). This bioinformatic tool is a comprehensive symbolic systems biology software that supports several applications in bioinformatics and systems biology [18,19]. Biological processes, molecular functions, subcellular localizations and MetaCyc pathways of the altered proteins were obtained by querying the protein or gene ID to the Pathway databases .
All values are shown as means±S.E.M., unless otherwise stated. Comparisons between groups (basal control compared with 12 h after the induction of sepsis) were performed using either a paired Student's t test or Wilcoxon signed rank test. P values <0.05 were considered statistically significant.
Haemodynamic and oxygen exchange parameters, inflammatory responses, oxidative and nitrosative stress, and other laboratory parameters are shown in Table 1. All animals developed normotensive hyperdynamic circulation with reduced SVR. Adequate fluid resuscitation was ensured by monitoring cardiac filling pressures (both CVP and PAOP were monitored for right and left ventricles respectively), which were significantly increased over time. The increased CO resulted in a significant rise in systemic DO2, whereas systemic V̇O2 remained unchanged. The peritonitis-induced sepsis caused a significant fall in arterial pH and a marked increase in plasma levels of TNF-α and IL-6. Overproduction of NO in this model was documented by a significant increase in arterial NOx levels. These changes were accompanied by a remarkable increase in TBARS levels, providing the evidence for oxidative stress.
We initially resolved the plasma proteome using the broad-range IPG strips (with a linear pH gradient of 3–10) and observed that most of the porcine plasma proteins were resolved at a pH range of 4–7 (results not shown). We therefore used the narrow-range IPG strips (with a linear pH gradient of 4–7) for the differential proteomics study. Approx. 1500 protein spots were visualized in each 2-D gel (Figure 1). There were seven gels derived from basal plasma samples of seven animals, and another seven gels derived from these animals but at 12 h after the induction of sepsis. Quantitative intensity analysis and statistics revealed significant differences in levels of 36 protein spots during sepsis (Figure 1 and Table 2). These differentially expressed protein spots included 30 spots with increased levels and six spots with decreased levels. All of these altered proteins were then successfully identified by Q-TOF MS and/or MS/MS analyses. Some of the altered proteins (e.g. albumin, haptoglobin and inter-α-trypsin inhibitor family heavy chain-related protein) were identified as multiple isoforms of the same proteins, most probably due to post-translational modifications. As a consequence, a total of 22 unique proteins were identified from the 30 up-regulated spots, whereas five unique proteins were identified from the six down-regulated spots. Identities, quantitative data, degrees of changes and other related information of all of the altered proteins are summarized in Table 2. Functional analysis using the Pathway Tools software revealed their biological processes, molecular functions, subcellular localizations and involved metabolic pathways, which are summarized in Supplementary Table S1 (see http://www.ClinSci.org/cs/116/cs1160721add.htm).
The pathophysiology of sepsis is considerably complex and understanding it could lead to identification of novel therapeutic targets with better treatment outcome. In the present study, we applied a proteomics approach to characterize changes in the plasma proteome during an early phase of sepsis in a porcine model of peritonitis-induced sepsis. Clinical features of this model simulated to a very large extent the clinical presentations of early sepsis syndrome in humans, including normotensive hyperdynamic circulation with reduced SVR, development of metabolic acidosis and increased plasma levels of inflammatory mediators (i.e. TNF-α and IL-6), accompanying nitrosative and oxidative stress.
The present study is the first to report the application of proteomics to the study of altered plasma proteome in a large animal model of sepsis, as previously available, but limited, findings had only been obtained from rodent models . These small animal models are often criticized for their limited clinical relevance. Indeed, many emerging strategies, which have been found effective in these models, fail to show any benefit in large animal or human studies. This might be even more important in the context of proteomic analysis, as there are marked differences between species. The fundamental differences include different regulation of inflammation, cardiovascular response to endotoxin or bacteria etc. Rodents differ markedly from humans with respect to their tissue antioxidative capacity and susceptibility to oxidative stress [21–24]. In the present study, differential proteomics analysis revealed altered plasma levels of 36 protein forms representing 27 unique proteins in our porcine sepsis model. The roles of some of these altered proteins are highlighted below.
CD14 is a receptor for bacterial LPS (lipopolysaccharide) that co-ordinates with TLR4 (Toll-like receptor 4) and MD-2 (myeloid differentiation-2) to mediate the innate immune response to bacterial LPS [25,26]. Activation of this upstream signalling pathway leads to the activation of the downstream factor NF-κB (nuclear factor κB), secretion of several inflammatory cytokines and, ultimately, inflammatory responses. Some bacterial infections use CD14 to enhance its invasion into hosts . Moreover, monocyte CD14 and soluble CD14 can be used as markers for predicting mortality in patients with severe community-acquired infection . In the present study, we identified an increased level of plasma CD14 in our porcine model of early sepsis, consistent with the findings published by Brunialti et al. , who reported an increased serum level of CD14 in patients with sepsis. These results indicate that CD14 is an important upstream molecule in the inflammatory cascade of sepsis.
Haptoglobin is one of the acute-phase reaction proteins [30,31] that also binds to haemoglobin with a potent affinity and thus can prevent renal iron loss and oxidative damage mediated by free haemoglobin [32–34]. Hence it serves not only as an acute-phase reactant, but is also involved in oxidative stress pathways. Several lines of evidence have demonstrated the increased level of haptoglobin as a scavenger system in a number of models of oxidative stress [35–37]. Another protein involved in oxidative stress is haemopexin, which binds to haem and transports it to the liver for its breakdown and iron recovery. As haem is highly toxic to cells due to pro-inflammatory and oxidative effects, haemopexin thus serves as an anti-inflammatory molecule and an oxidative scavenger [35,38]. Our present results are consistent with the findings in previous studies by Kalenka et al.  and Ren et al. , who reported increased plasma haptoglobin levels in patients with sepsis and increased plasma haemopexin in a murine model of sepsis respectively. The increased levels of both haptoglobin and haemopexin identified in our present study thus strengthen the important roles of these two molecules in mediating inflammatory processes and oxidative stress during an early phase of sepsis.
There were several altered proteins whose roles in sepsis remain unclear. For example, microfilament and actin filament cross-linker protein isoforms 3 and 4 (microtubule-actin cross-linking factor 1, isoforms 3 and 4) and plectin 1, which are involved in cytoskeletal assembly [40–44]. These proteins interlink intermediate filaments with microtubules and microfilaments, and also anchor intermediate filaments to desmosomes or hemidesmosomes [43–46]. The precise roles of these altered proteins in sepsis are somewhat interesting and deserve further investigation.
It should be noted that there are some limitations in our present study. First, 2-D gel electrophoresis is generally not a very sensitive technique to detect all components in the proteome. Therefore several low-abundance proteins and their subtle changes could not be detected. This fact was reflected in our present study as we did not find some of the proteins whose roles in sepsis and inflammatory responses have been established, for example CRP (C-reactive protein), serum amyloid A, fibrinogen and HSPs (heat-shock proteins). Secondly, we evaluated changes in the plasma proteome at only a single time point. Assessing dynamic changes over time would probably yield even more important results compared with the ‘before–after’ approach. This could serve as a platform or the starting point for our subsequent studies (i.e. sepsis compared with septic shock).
In summary, in the present study we have identified a set of plasma proteins with significantly altered levels during the early phase of sepsis in a porcine model of peritonitis-induced sepsis using a proteomics approach. The proteomics results could be related to the clinical features of this animal model, as most of these altered proteins have important roles in inflammatory responses and some of them play roles in oxidative and nitrosative stress. Some findings are considerably novel and exploring their roles in association with the pathophysiology of sepsis may lead to the identification of new therapeutic targets for better treatment outcome in sepsis. Additionally, some altered proteins may serve as potential markers for early sepsis.
This work was supported by the Ministry of Education, Czech Republic [research project MSM 0021620819 (to M. M.)]; by The Thailand Research Fund, Commission on Higher Education, Mahidol University (to V. T.); the National Research Council of Thailand (Siriraj Grant for Research and Development; to V. T.); and by the National Center for Genetic Engineering and Biotechnology (to V. T.).
We are grateful to the Core Facilities for Proteomics and Structural Biology Research, Institute of Biological Chemistry, Academia Sinica, Taiwan.
Abbreviations: 2-D, two-dimensional; ACN, acetonitrile; BP, blood pressure; CO, cardiac output; CVP, central venous pressure; DO2, oxygen delivery; DTT, dithiotheitol; FiO2, fraction of inspired oxygen; IEF, isoelectric focusing; IL, interleukin; LPS, lipopolysaccharide; Q-TOF, quadrupole–time-of-flight; MS/MS, tandem MS; NCBI, National Center for Biotechnology Information; NOx, nitrate/nitrite; PAOP, pulmonary artery occlusion pressure; PCO2, partial pressure of carbon dioxide; PEEP, positive end-expiratory pressure; PO2, partial pressure of oxygen; SVR, systemic vascular resistance; TBARS, thiobarbituric acid-reacting substances; TFA, trifluoroacetic acid; TNF-α, tumour necrosis factor-α; V̇O2, oxygen consumption
- © The Authors Journal compilation © 2009 Biochemical Society