Patients with hypothermic sepsis have a unique gene expression profile compared to patients with fever and sepsis

Abstract The pathophysiology of hypothermia during sepsis is unclear. Using genomic profiling of blood leukocytes, we aimed to determine if hypothermia is associated with a different gene expression profile compared to fever during sepsis. Patients with sepsis and either hypothermia or fever within 24 hours after ICU admission were included in the study (n = 168). Hypothermia was defined as body temperature below 36 °C. Fever was defined as body temperature equal to or above 38.3°C. We compared blood gene expression (whole‐genome transcriptome in leukocytes) in hypothermic septic compared to febrile septic patients in an unmatched analysis and matched for APACHE IV score and the presence of shock. In total, 67 septic patients were hypothermic and 101 patients were febrile. Hypothermia was associated with a distinct gene expression profile in both unmatched and matched analyses. There were significant differences related to the up‐ and downregulation of canonical signalling pathways. In the matched analysis, the top upregulated gene was cold‐inducible mRNA binding protein (CIRBP) which plays a role in cold‐induced suppression of cell proliferation. In addition, we found three signalling pathways significantly upregulated in hypothermic patients compared to febrile patients; tryptophan degradation X, phenylalanine degradation IV and putrescine degradation III. In conclusion, there are distinct signalling pathways and genes associated with hypothermia, including tryptophan degradation and CIRBP expression, providing a possible link to the modulation of body temperature and early immunosuppression. Future studies may focus on the canonical signalling pathways presented in this paper to further investigate spontaneous hypothermia in sepsis.


| INTRODUC TI ON
Body temperature changes are common in sepsis. 1 Patients who present with spontaneous hypothermia suffer from substantially increased morbidity and mortality compared to their normothermic or febrile counterparts. 2,3 It is unclear whether hypothermia simply represents a symptom of severe inflammation or that hypothermia itself drives mortality through a yet unknown mechanism. 4,5 Animal studies even indicate that hypothermia may be an adaptive response to severe inflammation in order to limit metabolism and prevent hypoxia. 6 To determine the aetiology of the hypothermic response during sepsis, studies have mainly focused on the ability to generate an adequate host immune response, often with levels of pro-inflammatory cytokines as a read-out. However, studies have not confirmed a defective immune host response, as septic patients with hypothermia had similar, 7,8 or even increased proinflammatory cytokine levels 9 in comparison with normothermic or febrile patients. Patients with hypothermic sepsis do develop persistent lymphopenia, a marker of immunosuppression. 10 Alternatively, the cardiovascular system may play a role. We previously showed that markers of endothelial injury are increased in hypothermic sepsis compared to nonhypothermic controls. 7 Taken together, the pathophysiology of hypothermic response in sepsis remains ill defined. Whole blood transcriptome analysis has provided valuable insights in the complex pathophysiology of the sepsis syndrome. 11 In this study, we aimed to determine if hypothermia in sepsis patients is associated with a different blood leukocyte gene expression profile compared to fever. We hypothesized that the blood transcriptomes of hypothermic sepsis patients differed from those obtained in febrile sepsis patients, which in turn reflect on variations in the host response.

| Study design, setting and patient identification
This study was performed within the Molecular Diagnosis and Risk Stratification of Sepsis (MARS) project, a prospective observational cohort study in mixed ICUs of two tertiary teaching hospitals (Academic Medical Center in Amsterdam and University Medical Center in Utrecht) in the Netherlands (ClinicalTrials.gov identifier NCT01905033). 12,13 Between January 2011 and July 2012, patients older than 18 years of age with an expected length of stay longer than 24 h were included via an opt-out consent method approved by ethical committees of both hospitals (IRB no. 10-056). During this study, demographic, clinical, microbiology and interventional data were collected daily by trained research physicians. The plausibility of an infection was assessed using a four-point scale (none, possible, probable or definite) using Centers for Disease Control and Prevention and International Sepsis Forum consensus definitions 14,15 as described previously. 12 We included patients diagnosed with sepsis and having blood microarray data obtained within the first 24 h of ICU admission. Sepsis was defined on ICU admission as having definite or probable infection, 12 combined with at least one parameter of inflammatory dysfunction, hemodynamic dysfunction, organ dysfunction or deranged tissue perfusion. 16 To limit the occurrence of iatrogenic hypothermia, patients admitted from the operating room (OR), ICU readmissions, patients undergoing active cooling, patients transferred from another ICU and patients with immunosuppression were excluded from this study. 7 Shock was defined as hypotension requiring treatment with vasopressors at a dose of 0.1 mcg/kg/min during at least 50% of the day. Clinical severity was assessed by Acute Physiology and Chronic Health Evaluation (APACHE) IV and Sequential Organ Failure Assessment (SOFA) scores. Temperature was removed from the APACHE IV scores which are presented in the tables and subsequent matched analyses.
Body temperatures were prospectively validated every hour by the treating ICU nurse and subsequently the researcher. To control for any body temperatures that may have been inadvertently entered in the database (i.e. a rectal sensor that has been displaced and is exposed to ambient temperature), patients with unreliably low measurements of temperature (below 33°C) were excluded.
Also, patients with only one registered temperature measurement during the first 24 h were not included. Temperature was measured using a rectal, nasal, inguinal or tympanic temperature probes.
Core temperatures were used in preference to inguinal or tympanic measurements.
Hypothermia was defined as a minimum body temperature <36.0°C and a maximum body temperature <38.3°C in the first 24 h of ICU admission. Conversely, fever was defined as a maximum body temperature ≥38.3°C and a minimum body temperature ≥36.0°C as defined in previous studies. 10,17 Normothermia was defined a minimum and maximum body temperature between ≥36.0°C and <38.3°C. Patients were defined as "both" if they had both hypothermia and fever in the first 24 h of ICU admission.

| Blood gene expression microarrays and bioinformatics
Whole blood was collected in PAXgene tubes (Becton-Dickinson) within 24 h after ICU admission and total RNA was isolated using the PAXgene blood mRNA kit (Qiagen, Venlo, the Netherlands) in combination with QIAcube automated system (Qiagen), as previously described. 13,18,19 The RNA isolation excluded material from erythrocytes. Microarray data (Affymetrix Human Genome U219 96-array plates) are accessible to the public via the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) accession GSE65682. Briefly, raw scans were pre-processed by means of the robust multi-average (RMA) method, normalized (quantile), summarized by median polish and log2 transformed using the affy method. 20 Nonexperimental chip effects were assessed and corrected by means of the combat method in the surrogate variable analysis R package. 21 Comparisons between groups were done using multi-variate linear models, including age and gender as covariates, implemented in the limma method. 22 Benjamini-Hochberg (BH)adjusted p-values <0.05 defined genome-wide significance. To assess the association with canonical signalling pathways, we used Ingenuity Pathway Analysis software (Qiagen Bioinformatics). Fisher exact test BH-adjusted p-values <0.05 demarcated significance.
Human species and Ingenuity gene knowledgebase were specified.
All other parameters were default.

| Statistical analysis
All data were analysed using R studio (version 3.2.2, R Core Team 2013, Vienna, Austria). In this study, we performed two analyses.
Firstly, we compared genomic profiles between septic hypothermic and febrile patients without correcting for disease severity.
Subsequently, in order to determine whether hypothermia was associated with a specific genomic profile irrespective of severity of disease, hypothermic patients were 1:1 matched to fever patients using their APACHE IV scores and presence of shock. Data are presented as numbers (percentages), parametric data as mean ± SD and nonparametric data as median and 25th-75th percentages; Q1-Q3.

Data distribution was assessed by the Kolmogorov-Smirnov test.
Mann-Whitney U or a Kruskal-Wallis test was used to analyse continuous nonparametric data, whereas continuous parametric data were analysed using Student's t-test or analysis of variance (twosided analysis of variance). All categorical data were analysed using a chi-square or Fisher exact test. A p-value less than 0.05 was considered to be of statistical significance for clinical data. Matching was done using "optimal matching" with R-package "MatchIt" (calliper 0.35 standard deviations of the logit), which locates matched samples with the smallest average absolute distance across all matched pairs.

| Patients
The selection of study patients can be seen in Figure 1. Out of a total of 579 sepsis admissions, 168 patients were included in the microarray analysis. Table 1 shows the baseline characteristics of these patients arranged by temperature group (baseline characteristics of patients that were normothermic (n = 75) or both hypothermic and febrile (n = 18) are shown in Table S1 for the purpose of interpretation, but these were not included in the microarray analysis).
Of the 168 included patients, 67 patients were hypothermic and 101 patients were febrile. Minimum temperature in the hypothermic group was lower compared to the febrile group (35.0°C ± 0.9 vs.
37.2°C ± 0.7) as was the maximum temperature (37.1°C ± 0.9 vs. 39.3°C ± 0.9). Hypothermic patients were also older compared to febrile patients, but BMI and gender distribution were similar between groups. There was also a similar distribution of site of infection between hypothermic and febrile patients.
Patients in the hypothermic group compared to febrile patients had higher APACHE IV scores and SOFA scores, increased incidence of shock and higher rates of mortality at 30 days post-ICU admission (29 (43%) vs. 22 (22%)).

F I G U R E 1
Flowchart showing the selection of study patients. (Some patients were excluded for multiple reasons and were counted multiple times for reason of exclusion).

| Alterations in microarray gene expression in hypothermia compared to fever
A total of 1930 transcripts were significantly altered in hypothermic patients compared to febrile patients, of which 1425 were reduced and 505 transcripts were elevated (Figures S1 and   S2A). Figure S2B shows the significant canonical pathways associated with these genes. Subsequently, we matched patients for APACHE IV scores and presence of shock. In total, 55 patients in each group remained for further analysis (characteristics of whom are shown in Table S2). After matching, there was no difference between groups in terms of APACHE IV scores and SOFA scores were elevated (Figure 2A)  Table S3. Pathway analysis of significantly altered, high expression genes resulted in significant associations with tryptophan degradation X, phenylalanine degradation IV and putrescine degradation III canonical signalling pathways ( Figure 2B). These signalling pathways relate to immunometabolic reactions that function in the degradation of amino acids and (poly)amines.

| DISCUSS ION
In this study of canonical pathways in blood leukocytes in septic patients, we found that hypothermic septic patients have a unique gene expression profile compared to sepsis patient presenting with fever.
After correcting for disease severity, hypothermic septic patients showed a surprisingly similar gene expression profile compared patients with febrile sepsis. However, there were distinct upregulated signalling pathways related to degradation of amino acids and (poly) amines were strongly associated with hypothermia, both in uncorrected analyses and analyses corrected for disease severity.
Patients with hypothermic sepsis show significant alterations in genomic pathways compared to febrile patients. These included downregulated pathways related to protein catabolism and translation, cell growth proliferation and mobility, cardiovascular signalling, pattern recognition receptor and cytokine signalling and lymphocyte pathways. In addition, pathways relating to amino acid and (poly) amine degradation were significantly upregulated. In a different sepsis cohort, similar defects in metabolic and immunologic signalling pathways have been associated with underlying immunoparalysis encountered in the acute phase of an infection. 23 In line with this finding, a recent retrospective analysis of hypothermic septic patients found increased incidence of lymphopenia associated with hypothermia compared to nonhypothermic septic patients. 10 However, other studies focusing on the host response have revealed remarkably few differences between hypothermic and nonhypothermic or febrile patients regarding typical pro-and anti-inflammatory cytokine responses and thereby signal that hypothermic patients are initially able to mount an adequate host response. 7,8 It is important to note that on the basis of blood transcriptomic data alone, the presence of generalized sepsis immunosuppression is In the matched analysis, three canonical pathways associated with amino acid and polyamine degradation were significantly upregulated in hypothermic septic patients compared to febrile septic patients. First, the tryptophan degradation X pathway was upregulated. Tryptophan. 33 Metabolites regulate anti-inflammatory effects of the immune response. 33,34 In sepsis, increased degradation from tryptophan to kynurenine is associated with decreased lymphocyte counts. 35 In vitro and in vivo evidence shows that tryptophan metabolites are fatal for T-cell survival. 36 Tryptophan is also degraded to serotonin. Interestingly, serotonin deficient mice are extremely susceptible to temperature variations and show a profound hypothermic response when placed in a cold environment. 37 Also, serotonin receptor antagonists has improved survival in experimental settings, but has not been evaluated in humans. 38 Secondly, the putrescine degradation III pathway was upregulated. Putrescine has shown the potential to modulate the innate immune response. 39 In a retrospective study in patients with community acquired pneumonia, putrescine levels were associated with disease severity and mortality. 40 In addition, putrescine is essential to the survival of important pathogens such as Streptococcus pneumoniae 41,42 and Escherischia coli. 43 Phenylalanine degradation IV pathway was the third pathway which was significantly upregulated; phenylalanine is a precursor to L-dopa and catecholamines. Increased serum levels of phenylalanine have been shown to be increased in patients after trauma, burns and sepsis 44,45 and predict mortality in patients with a severe infection. 46 Increased serum levels of phenylalanine are related to insufficient tissue perfusion and impaired cellular energy production. Impaired phenylalanine metabolism can interfere with the production of catecholamine and augment shock. 45 In general, the aetiology and pathophysiology of the hypothermic response remains unclear. Our study suggests that patients with hypothermia have distinct differences in gene expression profiles.
Several pathways were altered in the unmatched analysis but not in the matched analysis, suggesting that these pathways are not specific to hypothermia. However, by matching for disease severity, important pathways relating hypothermia and sepsis may be overlooked as hypothermia may represent a symptom of disease severity. 47 How the genes and pathways found in this study relate to the hypothermic response and increased mortality remains to be seen. In conclusion, hypothermic patients were characterized by largely similar, but also significant changes in leukocyte transcriptomes. Genes were associated with distinct cellular biological pathways, including tryptophan metabolism and serotonin-signalling.
Cold-inducible mRNA binding protein (CIRBP) expression was particularly elevated in sepsis patients with hypothermia. These signalling pathways provide a possible link to the modulation of body temperature and early immunosuppression. Future functional studies on the canonical signalling pathways and specific genes presented in this paper are warranted.

CO N FLI C T O F I NTE R E S T
The authors of this study declare no conflict of interest.