ATPergic signaling disruption in human sepsis as a potential source of biomarkers for clinical use

Sepsis is a life-threatening organ dysfunction caused by a dysregulated inflammatory response to infection. To date, there is no specific treatment established for sepsis. In the extracellular compartment, purines such as adenosine triphosphate (ATP) and adenosine play essential roles in the immune/inflammatory responses during sepsis and septic shock. The balance of extracellular levels among ATP and adenosine is intimately involved in the signals related to immune stimulation/immunosuppression balance. Specialized enzymes, including CD39, CD73, and adenosine deaminase (ADA), are responsible to metabolize ATP to adenosine which will further sensitize the P2 and P1 purinoceptors, respectively. Disruption of the purinergic pathway had been described in the sepsis pathophysiology. Although purinergic signaling has been suggested as a potential target for sepsis treatment, the majority of data available were obtained using pre-clinical approaches. We hypothesized that, as a reflection of deregulation on purinergic signaling, septic patients exhibit differential measurements of serum, neutrophils and monocytes purinergic pathway markers when compared to two types of controls (healthy and ward). It was observed that ATP and ADP serum levels were increased in septic patients, as well as the A2a mRNA expression in neutrophils and monocytes. Both ATPase/ADPase activities were increased during sepsis. Serum ATP and ADP levels, and both ATPase and ADPase activities were associated with the diagnosis of sepsis, representing potential biomarkers candidates. In conclusion, our results advance the translation of purinergic signaling from pre-clinical models into the clinical setting opening opportunities for so much needed new strategies for sepsis and septic shock diagnostics and treatment.


Introduction
Sepsis is a life-threatening organ dysfunction caused by a dysregulated inflammatory response to infection [1].The systemic inflammatory response syndrome (SIRS) and the events that trigger multi-organ failure during sepsis involve both exaggerated inflammation and immune suppression [2] and are yet to be fully understood.The literature regarding sepsis research has advanced substantially in topics such as growing alertness, early identification, and rapid onset with antibiotics and organ supportive care [3], that although important, are not specific targets to treat the disease.Sepsis is a condition with no specific pharmacological treatment strategies established, leading to unacceptably high mortality and morbidity rates [4], still now, all over the world.Studies on purinergic signaling related to the pathophysiology of sepsis have suggested this cellular/molecular pathway as a potential target for treatment, but still most evidence is centered in pre-clinical research [5][6][7][8][9].
In the extracellular compartment, important molecules that are part of the purinergic signaling as adenosine triphosphate (ATP) and adenosine have essential features in the immune and inflammatory responses.ATP acts as a damageassociated molecular pattern (DAMP) through P2 receptors exerting pro-inflammatory effects to promote chemotaxis, phagocytosis, and cytokine release by immune cells following an infectious insult or sterile inflammation [10].Adenosine operates predominantly anti-inflammatory effects via P1 receptor sensitization on immune cells, contrasting and equilibrating the ATP pro-inflammatory activities [11].An apparatus of extracellular nucleotide converting enzymes to the respective nucleoside, combined with specialized membrane nucleoside transporters, assures tight control of pericellular purine concentrations.The ectoenzyme CD39 (ectonucleoside triphosphate diphosphohydrolase; E-NTPDase/ CD39) hydrolyzes ATP to adenosine diphosphate (ADP), a molecule with signaling functions in inflammation and platelet adhesion.The same enzyme converts ADP to adenosine monophosphate (AMP), the substrate for extracellular adenosine production through CD73 (ecto-5'nucleotidase/5'NT).Finally, adenosine deaminase (ADA) terminates adenosine signaling by deaminating this nucleoside into inosine [12].
Pre-clinical research proposes a role for purinergic signaling in neutrophil chemotaxis, phagocytosis, and inflammasome NLRP3 activation on macrophages, among other features of sepsis immune modulation, pointing this signaling system as a potential therapeutic target for regulating inflammation in sepsis [13].In this study, we sought to carry out a comprehensive exploration of purinergic signaling in septic patients.However, investigations on purinergic signaling in human sepsis pathophysiology are scarce.We conducted a case-control study of septic patients and their controls (the control group was splitted into two subgroups: healthy adults, and a second control group composed by ward, non-septic patients with chronic conditions that could hypothetically modulate purinergic signaling).We determined the serum levels of purinergic agonists, the serum activity of enzymes involved in agonists metabolism, and the expression of selected purinergic receptors in circulating neutrophils and monocytes.We hypothesized that as a reflection of deregulation on purinergic signaling during sepsis, these patients have differential measurements of serum adenine purines, activity of nucleotidases and ADA, and purinergic receptors gene expression in neutrophils and monocytes when compared to controls.Additionally, we further investigated the value of serum purine nucleotide/ nucleoside levels and/or nucleotidase activities as diagnostic markers in human sepsis.

Ethical and funding considerations
The local Research Ethics Committee approved the study protocol (registration numbers 49959315.5.0000.5530and 49959315.5.3001.5345 in Brazilian national ethical committee-CONEP).Every subject or proxy provided written informed consent before inclusion.We declare that funding agencies had no role in the study design, execution, and results reporting.

Settings
The study venues are a 59-bed general intensive care unit (ICU) and the wards of an 800-bed, public, tertiary-care hospital in Brazil.Academic laboratories from two Brazilian universities performed the biochemical analysis.

Study design
We conducted a case-control study of septic patients and their controls.The dependent variable is sepsis diagnosis and the independent variables are measurements of several elements of purinergic signaling.We determined the serum levels of purinergic agonists, the serum activity of enzymes involved in nucleotide/nucleoside metabolism, and the expression of selected purinergic receptors in circulating neutrophils and monocytes at the mRNA level.Laboratory personnel was not blinded.We planned to enroll cases and controls at a 1:2 ratio.Due to concerns that the typical control group comprised of healthy and young subjects are inaccurate controls for the average ICU patient, and that some chronic conditions are associated with mild inflammation, we split the control group into two subgroups: ward, non-septic patients with chronic conditions that could hypothetically modulate purinergic signaling, and a second control group comprised only by young, healthy adults.We described concurrent diseases in the first control subgroup but did not attempt to match for the same concurrent diseases observed in the sepsis group.

Patients
Enrollment started in November 2015 and finished in December 2018.We enrolled patients and controls in convenience samples on the days that the laboratory could receive fresh blood specimens for the neutrophil isolation procedure.We searched for and enrolled septic patients on their first morning after ICU admission.Inclusion criteria were the presence of acute infection and the concurrent development of acute organic failure.The attending ICU staff defined the acute infection diagnosis.At patient triage, we defined acute organic failure by the incidence, in the last 48 h before ICU admission, of at least one of the following [14].All patients included in the study fulfilled the Sepsis-3 criteria.
(1) Hypotension: Systolic arterial pressure < 90 mmHg or mean arterial pressure < 70 mmHg; (2) Arterial lactate > 2 mEq/L; (3) Urine output < 0.5 mL/kg/h for 2 consecutive hours, in the absence of hypovolemia; (4) PaO 2 / FiO 2 ratio < 200, if pneumonia, or < 250 in the absence of pneumonia; (5)  The first control subgroup was composed of healthy volunteers.We confirmed the healthy state in the absence of chronic diseases and new symptoms in the last 48 h.The second control subgroup included ward patients with chronic diseases but not septic.The non-septic status required the absence of active infection, as defined by the attending team, and also the lack of signs of clinical deterioration in the last 48 h.

Clinical variables
For each septic patient, we recorded a set of 11 numeric, categorical, or binary variables.Numeric variables: age (years), SOFA score (Sequential Organ Failure Assessment; points), and ICU length-of-stay (days).Categorical variable: infection site.Binary variables: female gender, arterial hypertension, manifested atherosclerotic disease, cancer, chronic pulmonary disease, diabetes, and inhospital death during the index admission.The variable "manifested atherosclerotic disease" includes chronic renal failure, coronary disease, or cerebrovascular disease.The variables describing co-morbid states were selected for being prevalent among Brazilian ICU patients [15].The binary variable "fulfillment of sepsis-3 qSOFA criteria" required the presence of two out of the three Sepsis-3 qSOFA criteria [1].This variable is a post-hoc addition after the issuance of the Sepsis-3 consensus.For each patient of the control groups, we registered the numeric variable age and the binary variable female gender.We recorded the variables describing the presence of concurrent diseases in the first control group.We collected the clinical variables at the bedside and from the hospital's electronic medical records and consolidated data in a deidentified fashion in an electronic sheet.

Neutrophil and monocyte isolation
Circulating human neutrophils and monocytes were isolated from 5 mL of total blood collected with heparin using Polymorphprep™ gradient (density 1.113 g/mL; Accurate Chemical, NY, USA), as previously described [16].Isolated neutrophils and monocytes were immediately harvested in TRIzol Reagent (Invitrogen Co., Carlsbad, CA, USA) and stored at -80 °C freezer for further RNA isolation and realtime PCR analyses.

Expression assessment by quantitative PCR (qPCR)
Total RNA was isolated from neutrophil and monocyte samples following the instructions of the fabricant (TRIzol Reagent, Invitrogen Co., Carlsbad, CA, USA) and samples were DNase-treated with a DNA-free kit (Promega, Madison, WI, USA) following the manufacturer's instructions.We performed first-strand cDNA synthesis with 1 μg of RNA using the Reverse Transcription kit (Promega) according to the manufacturer's instructions.The qPCR reactions ran on an Applied-Biosystem Step One Plus real-time PCR system using GoTaq PCR Master Mix (Promega) using specific primers for human P2Y 2 , P2X7, and A2a purinergic receptors (Supplementary Table 1).We analyzed all data by the 2 −Δ/ΔCT method, as described previously [17].TBP expression was the internal control gene for all relative expression calculations.

Serum nucleotides and nucleosides measurements
Human blood samples were drawn into vacutainer tubes containing a coagulation activator and processed up to 15 min after collection.Briefly, the samples were centrifuged at 1,300 × g at 4 °C for 10 min.The supernatants were transferred into a new microcentrifuge tube, rapidly frozen and stored at − 80 °C for further measurement of purine levels and ectonucleotidase activity.
For HPLC analysis, the serum (150 µL) was deproteinized using perchloric acid (100 µL PCA, 0.6N), followed by centrifugation at 16,000 × g at 4 °C for 20 min.The supernatants were transferred into a new microcentrifuge tube, neutralized by adding KOH (4 M), and centrifuged once more at 16,000 × g at 4 °C for 20 min.The supernatant was collected and immediately used for purine level quantification.ATP, ADP, AMP, adenosine, and inosine levels were analyzed by HPLC Shimadzu Prominence (Shimadzu, Kyoto, Japan) according to a previously described procedure [18].Standard solutions and sample serum (50 µL) were injected at a flow rate of 1.00 mL.min −1 and separated on a Shimadzu column Shim-pack CLC-ODS(M)® C18 (150 × 4.6 mm × 5 μm).The mobile phase was constituted by solvent A, a buffer solution prepared with 60 mM KH 2 PO 4 , 5.0 mM C 16 H 36 ClN (pH 5.0); and solvent B, with 60 mM KH 2 PO 4 , 5.0 mM C 16 H 36 ClN, 30% methanol (pH 5.0) (Sigma, St Louis, MO, USA).We set up a linear gradient program with 0-25 min 100% A, 25-26 min 100% B and 26-45 min 100% A. Data were acquired and processed using the LC Solution Software (Shimadzu) and the amounts of nucleotides/nucleosides were measured by absorption at 254 nm and compared with their respective standard solution (25 µM concentration).

Nucleotidase activity
Briefly, the reaction mixture containing ATP (3 mM), ADP (3 mM) or AMP (6 mM) as substrate (Sigma, St Louis, MO, USA), 112.5 mM Tris-HCl buffer (pH 8.0) was incubated with approximately 1.0 mg of serum protein at 37 °C for 40 min in a final volume of 200 μL.The reaction was stopped by adding 200 μL 10% trichloroacetic acid.The amount of inorganic phosphate (Pi) liberated was measured using KH 2 PO 4 as standard at 630 nm following the malachite green method [19].Protein was determined by the Coomassie Blue method, using bovine serum albumin as the standard [20].Enzyme activities were expressed as nmol of Pi released per minute per milligram of protein.

ADA activity
ADA activity assay was started by the addition of the substrate (adenosine; Sigma, St Louis, MO, USA) to a final concentration of 21 mM; the incubation was carried out for 1 h at 37 °C and pH 6.5.We then stopped the reaction by adding 106 mM/0.16 mM phenol-nitroprusside/mL solution.We immediately mixed the reaction mixtures to 125 mM/11 mM alkaline hypochlorite (sodium hypochlorite) and vortexes.Ammonium sulfate 75 μM was used as the ammonium standard.Ammonia concentration is directly proportional to the absorption of indophenol at 620 nm.The specific activity report unity is U/L.One unit (1 U) of ADA represents the amount of enzyme required to release one mMol of ammonia per minute from adenosine at standard assay conditions [21].

In silico transcriptome analysis
We analyzed the expression of A2a, P2Y 2 , P2X7, CD39, CD73, and ADA in two human whole blood transcriptome datasets of septic patients and non-septic controls.To allow comparison with our data, we analyzed samples drawn in the first ICU day following a sepsis diagnosis in septic patients (datasets number GSE137340 and GSE54514 available at the NCBI GEO data repository).The characteristics and the overall design of these two Gene Expression Omnibus (GEO) datasets analyzed are shown in Supplementary Table 2.

Statistical analysis
We used Stata/IC 15.1 software (StataCorp, College Station, TX, USA) for statistical analysis and GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA) for graphic presentation of data.We report categorical variables as proportions and continuous variables as means with standard deviation (SD) if normally distributed or as medians with interquartile range (IQR) if not.Kruskal-Wallis test followed by Dunn's correction for multiple comparisons was used to compare the p-values among the groups.We tested the normality of continuous variables using the Shapiro-Wilk test.The comparison between the groups whose variables had normal distribution was evaluated by One-Way ANOVA with post-hoc Tukey's test.The significance threshold was set at a two-sided p-value < 0.05.

Outliers
According to previous findings, we expected an overall concentration of serum purines spanning from the low nanomolar to the low micromolar range, combined with marked higher measurements in some patients, so we kept the observed values in micromolar scale without transformation and did not discard near-zero values as outliers as others [22,23].In contrast, we regarded extremely high measurements as outliers.

Hypothesis test
We tested the hypothesis of unequal measurements of purinergic agonists, nucleotidase activity, and receptor expression among cases and control groups by mean comparison tests in normally distributed variables or by ranksum tests otherwise.We evaluated differences in categorical variables using Fisher exact test.

Diagnostic inferences
All patients were included in this analysis.In order to assess the association of sepsis diagnosis with the serum levels of purines and/or serum enzyme activities we performed univariate logistic regression analysis with sepsis diagnosis as the dependent variable and each candidate biomarker as the independent variable.Association was measured as odds ratios.Discrimination was estimated by both areas under the curve for the receiver operator characteristics curve (AUC-ROC) and the precision-recall curve (AUC-PRC) [24].

Patients
A total of 74 patients were divided into three groups.Twenty subjects composed the first (healthy) control subgroup.The septic group (test) and the second (ward, nonseptic patients) control subgroup have 27 patients each.Figure 1 displays subject enrollment.The age median was 62 (52-71) years in the septic group, 26 (21.75-30.75) in the first control subgroup and 69 (63-76) in the second control subgroup.Female gender accounted for 44.4% of patients in the septic group and 65% and 55.5% in the first and second control group, respectively.The differences in prevalence of chronic pulmonary disease (22.2% vs 18.5%), cancer (18.5% vs 11.1%), atherosclerotic disease (25.0%vs 44.4%), and arterial hypertension (51.8% vs 74.0%) between the septic group and the second control subgroup did not reach statistical significance.That does not apply to the prevalence of type 2 diabetes (22.2% vs 51.8%, p = 0.047).Septic patients had a mean SOFA score of 6.1 at inclusion.Respiratory (44%) and intra-abdominal (44%) accounted for 88% of infection sites.Hospital mortality among septic patients was 59%.All septic patients satisfied SEPSIS-3 diagnosis criteria.Table 1 summarizes demographic data.

Human circulating neutrophils and monocytes from septic subjects overexpress A2a adenosine purinoceptor
Samples of 51 subjects from the three study groups were included in the analysis of P2Y 2 , P2X7 and A2a purinergic receptors gene expression in circulating human neutrophils and monocytes by qPCR, as described in the materials and methods section.Results are displayed in Fig. 2 and in supplementary Table 3, whereas no statistical difference was found in the P2Y 2 and P2X7 mRNA levels among groups (Fig. 2A and B), the A2a expression was ~ 6.5 and 9 times higher in neutrophils from septic patients compared to control 1 (healthy) and control 2 (ward, non-septic patients), respectively (p < 0.0034; Fig. 2C and Table 3S).Similarly, the A2a expression was also increased by ~ 4 and 3.4 times in circulating monocytes compared to control 1 and 2 groups, respectively (p = 0.0034; Fig. 2D and Table 3S).This finding is supported by the presence of significantly higher A2a expression in whole blood samples of septic patients from two independent transcriptome datasets (Fig. 1S).Interestingly, the expression of P2Y 2 and P2X7 purinoceptors was divergent between the datasets analyzed, and just the GSE54514 dataset for these two receptors was similar to the present study.The difference of the transcriptomic profile observed in the datasets may be related to the intrinsic biological variation as well as the number of participants enrolled in those studies.Besides that A2a receptor expression increase in septic patients is consistent in both in silico and ex-vivo experimental approaches applied in the current study.These data suggest the importance of A2a purinoceptor on sepsis-associated innate immune response disruption.

Fig. 1 Organizational chart of subjects of investigation and experimental design
Table 1 Demographics data of septic patients and the control groups 1 Binary and categorical variables presented as percentages.

Septic subjects exhibit higher ATP and ADP serum levels compared to controls
We next determined the circulating ATP levels and its products of metabolism (ADP to inosine) in serum samples from septic patients and control groups by HPLC.We measured the serum purine levels in 58 subjects, 20 from the sepsis group, 22 wards, non-septic patients, and 16 healthy controls.Results are expressed as median levels (μM) with IQR (Fig. 3; Table 4S).We found significantly higher ATP (~ 6 times; p = 0.033; Fig. 3A) and ADP levels (~ 18 times; p = 0.001; Fig. 3B) in septic patients when compared to control 1 (healthy) and control 2 (ward, non-septic) groups, respectively.However, albeit a tendency of increase in serum adenosine levels in the septic patients, no statistical difference in AMP, adenosine and inosine levels was detected among the investigated groups (Fig. 3C, D, E).

Septic patients show differential serum ATP/ADPase activities compared to control groups
The enzyme activity assays in serum samples enrolled 60 subjects, 18 septic patients, 22 ward non-septic patients, and 20 healthy controls (Fig. 4, Table 5S).Interestingly, median ATPase and ADPase activities were significantly higher in septic patients (~ 2.3 and 1.6 times, p = 0.0067 and 0.002, respectively) compared to control groups (Fig. 4A and B).On the other hand, AMPase and ADA activities were ~ 1.6 and ~ 1.5/2 times higher, respectively, in both non-septic and septic groups when compared to control 1 (healthy subjects), suggesting that these alterations were not specific to sepsis condition, but associated with a general inflammatory condition of the patients (Fig. 4C and D; Table 5S).Accordingly, we found higher ENTPD1 gene expression among septic patients in both transcriptome datasets analyzed, while no significative alterations were detected in NT5E and ADA gene expression (Fig. 1S).

Diagnostic inferences-ATP and ADP metabolism as candidate biomarkers of sepsis
The examination of purinergic signaling potential in sepsis diagnosis included all patients.We included all products of ATP metabolism until adenosine and the serum ectonucleotidase and ADA activities as candidate biomarkers (Table 2).Serum ATP and ADP have a significant association with sepsis diagnosis.The odds ratio for serum ATP level is 1.27, with a p-value equal to 0.007, AUC-PRC 0.65 (baseline 0.34), and AUC-ROC 0.70.The odds ratio for serum ADP levels is 1.64, with a p-value of 0.004, AUC-PRC 0.59 (baseline 0.26), and AUC-ROC equal to 0.81.The activity of ATP and ADP hydrolysis is also significantly higher in the septic patient group.

Discussion
Purines and their metabolites, most well described ATP and adenosine, are essential components controlling intracellular energy homoeostasis and nucleotide synthesis.Extracellularly, they act as chemical messengers throughout tissues and have special importance in the immunological response during systemic inflammation and sepsis [25][26][27][28][29][30].In the present study, we investigated how purinergic signaling modulation would be associated with sepsis pathophysiology in humans.It was a transversal, observational, case-control study in which septic patients were recruited from the ICU and control group was splitted in two subgroups to discard that the findings would be related to general inflammation and not sepsis itself.The first control subgroup was composed of healthy volunteers and the second control subgroup was composed of ward patients with chronic diseases presenting mild inflammation.It was observed that serum ATP and ADP levels were higher in the septic group when compared to the control groups.In agreement with this finding, the ATPase and ADPase activities were higher in septic group.The A2a mRNA expression was found increased in circulating neutrophils and also in monocytes from septic patients.Interestingly, described for the first time in clinical human sepsis, at least to our knowledge, higher ATP/ ADP levels and ATPase/ADPase activities were found to be associated with sepsis diagnosis.We have recently reported higher CD39 expression in neutrophils harvested from septic patients [7].Together, these findings are corroborated by the increased expression of A2a receptor and CD39 we found in the transcriptome analysis.The pro-inflammatory effects of extracellular ATP and its role as DAMP is well described in the literature [10].This nucleotide is released from tissues in necrosis, hypoxia, apoptotic cells, and immune cells during infection, causing an up-regulation of the inflammatory response [5].For example, the NLRP3 inflammasome activation in monocytes from septic patients is compromised, where P2X7 expression is associated with mitochondrial dysfunction, and the increased NLRP3 disfunction is connected to sepsis-related death of patients.Authors suggest that therapies aiming to decrease extracellular ATP or to block the P2X7R may improve survival rates among septic patients [31].In a phase-2 clinical trial of recombinant alkaline phosphatase in sepsis-induced acute kidney injury, Pickkers et al. (2012) found evidence of improved renal function, suggesting that an increase in ATP hydrolysis exerted a protective effect and reduced inflammation in septic patients [32].Furthermore, reducing serum ATP levels with apyrase restored human neutrophil chemotaxis in an ex-vivo study of neutrophil function [33].CD39 is responsible for metabolizing ATP and ADP, and a study using the experimental model of sepsis cecal ligation and puncture (CLP) showed that peripheral injections of CD39 mimic apyrase decreased inflammation, organ damage, immune cell apoptosis, and bacterial load.
The CD39 knockout mouse model, when submitted to sepsis, had a higher mortality rate when compared to wild type group [34].
In sepsis, neutrophil activation and chemotaxis require ATP release via pannexin-1 channels that induce purinergic signaling [34].Another experimental approach used suramin and apyrase to block endogenous or systemic effects of ATP during sepsis (CLP model).The blockage of endogenous ATP reduced neutrophil activation and organ injury and increased mortality; in contrast, the blockage of systemic ATP improved neutrophil chemotaxis and host defense.These observations demonstrated the complexity of ATP function in sepsis [33].On the other hand, experimentally increasing adenosine levels with pentostatin, an ADA inhibitor, appears to curb systemic inflammation, restoring proper neutrophil-endothelium interactions [35].
Adenosine modulates inflammation and prevents associated organ injury by activation of its receptors [36].The fact that we evaluated the patients on the first day when enrolled in the ICU can explain why we did not observe any difference in adenosine or its precursor AMP in the septic group.Subjects that received E. coli endotoxin (lipopolysaccharide) intravenously similar to an infection event had increased A2a and A2b receptor mRNA expression [36], in line with what was found in the present study.Another recent study demonstrated the relevance of purinergic signaling using both pre-clinical and clinical experimental approaches.In alignment with the thesis that purinergic signaling is involved in the pathophysiology of sepsis, Nascimento and collaborators have shown that sepsis induces expansion of an immunesuppressive CD39-expressing B cell population that produces adenosine which impairs macrophage bacterial killing [37].
If a dysregulated inflammatory response driven by high ATP levels is present, the higher serum nucleotidase activity we observed in sepsis may be an adaptive countermeasure to reduce ATPergic signaling through ATP hydrolysis and adenosine generation.Therefore, our findings suggest that in early human sepsis there is an orchestrated response aiming to control ATPergic-mediated proinflammatory/cell damage processes and to promote adenosinergic signaling by both increasing the A2a expression in neutrophils and monocytes and by stimulating the ATP hydrolysis toward adenosine.It was not possible in the present study to observe increased adenosine levels in the serum of patients in the early stage of sepsis.However, the potentiation of adenosinergic signaling through sepsis progression may promote adenosine accumulation in the late stages of sepsis, which has been associated with the long-term immunosuppression in sepsis survivors [37].Future studies should be conducted collecting samples at 3-5 days or even at longer time-points from septic patients from the ICU and correlate both ATP and adenosine levels with clinical outcomes.
Hypothetically, a physician at the bedside could infer the current state of purinergic signaling by analyzing serum purine levels and serum nucleotidase activity.Our analysis shows promising results for serum ATP levels and serum ATPase/ADPase activities as biomarkers of severe acute inflammation.If the inflammation cause is an infection, these could be biomarkers of sepsis.In the corollary, a patient exhibiting the features of severe inflammation associated with high serum ATP and ADP levels may benefit from therapeutic control of these levels.
All together, the present data are an important addition to the present literature on sepsis, but limitations may apply and should be taken in consideration when interpreting the present findings.First, we did not examine the correlation of gene expression with the protein content of purinergic receptors, especially the one we found difference (A2a).Second, we held to sepsis diagnosis as a clinical entity.The diagnosis of sepsis is a clinical construct that lacks a formal description concerning inflammatory hallmarks.Therefore, we did not describe our cases or controls in terms of any inflammatory pathway other than the purinergic.Finally, serum alkaline phosphatase activity appears to be higher in sepsis [38].Our nucleotidase assay could not discriminate between the enzyme or enzymes that promoted the release of Pi from the nucleotides.

Conclusion
In conclusion, our results advanced the translation of purinergic signaling from pre-clinical models into the clinical setting.We found that septic patients in the first morning after ICU admission have a higher serum level of ATP/ADP accompanied by a higher serum ATPase/ADPase activities.These increased biomarkers levels were found to be promising candidates for systemic inflammation at the early onset of sepsis.These findings indicate that purinergic signaling is active in early human sepsis and offers an opportunity for diagnostic and therapeutic innovation.

Fig. 3
Fig. 3 Serum purine levels in septic patients and in control groups.A ATP, B ADP, C AMP, D adenosine (ADO), and E inosine (INO) circulating levels were determined in blood serum of healthy (Control 1), ward/non-septic (Control 2), and septic subjects (Test) by HPLC.Data were expressed as median (IQR) of purine levels (μM)

Fig. 4
Fig.4 Serum purine metabolism in septic patients and in control groups.A ATPase, B ADPase, C AMPase, D ADA (adenosine deaminase) activities were determined in blood serum of healthy (Control 1), ward/ non-septic (Control 2), and septic subjects (Test).ATPase and AMPase activities were analyzed by Kruskal-Wallis, while ADPase and ADA activities were analyzed by ANOVA followed by Tukey's post-hoc.* different from healthy subject (p ≤ 0.033); # different from nonseptic patients (p ≤ 0.001)

Table 2
Diagnostic inferences regarding serum purine levels and its metabolism in sepsis casesData were submitted to logistic regression analysis, pointing sepsis diagnosis as the dependent variable and reported the odds ratios of each candidate biomarker, as indicated.