The paradigm of SIRS-to-CARS transition implies that hyperinflammation triggers acute sepsis mortality, while hypoinflammation (release of anti-inflammatory cytokines) in late sepsis induces chronic deaths. However, the exact humoral inflammatory mechanisms attributable to sepsis outcomes remain elusive. In part I of the study, we characterized the systemic dynamics of the chronic inflammation in dying (DIE) and surviving (SUR) mice suffering from CLP sepsis (days 6-28). In part II, we combined the current chronic and previous acute/chronic sepsis data to compare the outcome-dependent inflammatory signatures between these two phases. To compare global inflammatory responses, a composite cytokine score (CCS) was calculated. Mice were never sacrificed but sampled daily (20μl) for blood. Part I: parameters from chronic DIE mice were clustered into the 72h, 48h and 24h prior-to-death time-points and compared to SUR of the same post-CLP day. Cytokine increases were mixed and never preceded chronic deaths earlier than 48h (3 to 180-fold increase). CCS demonstrated simultaneous and similar upregulation of pro-and anti-inflammatory compartments at 24h prior to chronic death (DIE 80 and 50-fold higher vs. SUR). Part II: cytokine ratios across sepsis phases/outcomes indicated steady pro-vs. anti-inflammatory balance. CCS showed the inflammatory response in chronic DIE was 5-fold lower versus acute DIE mice, yet identical to acute SUR. Concluding, the systemic MARS-like pattern (concurrent release of pro-and anti-inflammatory cytokines) occurs irrespectively of the sepsis phase, response magnitude and/or outcome. Although different in magnitude, neither acute nor chronic septic mortality is associated with a predominating pro-and/or anti-inflammatory signature in the blood.

Age/gender may likely influence the course of septic complications after trauma. We aimed to characterize the influence of age/gender on the response of circulating cytokines, cells and organ function in post-traumatic sepsis. We additionally tested whether post-traumatic responses alone can accurately predict outcomes in subsequent post-traumatic sepsis. A mouse 2-hit model of trauma/hemorrhage (TH, 1st hit) and cecal ligation and puncture (CLP, 2nd hit) was employed. 3, 15 and 20 month (m) old female (♀) and male (♂) CD-1 mice underwent sublethal TH followed by CLP 2 days later. Blood was sampled daily until day 6 post-TH and survival was followed for 16 days. To compare general response patterns among groups, we calculated two scores: the inflammatory response (including KC, MIP-1α, TNFα, MCP-1, IFNγ, IL-1β,-5,-6,-10) and the organ dysfunction score (Urea, ALT, AST and LDH). Moreover, mice were retrospectively divided into survivors (SUR) and dying (DIE) based on post-CLP outcome. In general, females survived better than males and their survival did not correspond to any specific estrus cycle phase. Pre-CLP phase: the post-TH inflammatory score was weakest in 3 m♂ but there were no changes among remaining groups (similar lack of differences in the organ dysfunction score). TH induced a 40% increase of IFNγ, MIP-1α and IL-5 in 15 m♂ SUR (vs. DIE) but predictive accuracy for post-CLP outcomes was moderate. Post-CLP phase: while stable in males, inflammatory response score in 15 m and 20 m females decreased with age at day 1 and 2 post-CLP. SUR vs. DIE differences in inflammatory and organ dysfunction score were evident but their magnitude was comparable across age/gender. Nearly identical activation of the humoral inflammatory and organ function compartments, both across groups and according to sepsis severity, suggests that they are not directly responsible for the age/gender-dependent disparity in TH-CLP survival in the studied young-to-mature population.

BackgroundThe mechanisms of sepsis mortality remain undefined. While there is some evidence of organ damage, it is not clear whether this damage alone is sufficient to cause death. Therefore, we aimed to examine contribution of organ injury/dysfunction to early deaths in the mouse abdominal sepsis.MethodsFemale OF-1 mice underwent either medium-severity cecal ligation and puncture (CLP-Only) or non-lethal CLP-ODam (CLP with cisplatin/carbontetrachloride to induce survivable hepatotoxicity and nephrotoxicity). In the first experiment, blood was collected daily from survivors (SUR; CLP-Only and CLP-ODam groups) or until early death (DIED; CLP-Only). In the second experiment (CLP-Only), early outcome was prospectively predicted based on body temperature (BT) and pairs of mice predicted to survive (P-SUR) and die (P-DIE) were sacrificed post-CLP. The overall magnitude of organ injury/dysfunction was compared in retrospectively and prospectively stratified mice.ResultsAt day 7 post-CLP, survival in CLP-Only was 48%, while CLP-ODam was non-lethal. In CLP-Only mice within 24 h of death, urea increased to 78 (versus 40 mg/dl in SUR), ALT to 166 (vs. 108 U/l), LDH to 739 (vs. 438 U/l) and glucose declined to 43 (vs. 62 mg/dl). In CLP-ODam, hypoglycemia was exacerbated (by 1.5-fold) and ALT and LDH were 20- and 8-fold higher versus DIED (CLP-Only) mice. In CLP-Only, predicted deaths (P-DIE) were preceded by a significant rise only in cystatin C (268 vs. 170 ng/ml in P-SUR) but not in creatinine and troponin I. Respiratory function of mitochondria in the liver and kidney of P-SUR and P-DIE CLP-Only mice was not impaired (vs. controls) and ATP level in organs remained similar among all groups. Histologic injury scores in the liver, kidney, heart and lung showed no major disparities among dying, surviving and control mice.ConclusionsIn CLP-Only mice, although the deregulation of parameters indicative of organ injury/dysfunction was greater in dying versus surviving mice, it never exceeded the changes in surviving CLP-ODam animals, and it was not followed by histopathological damage and/or mitochondrial dysfunction. This shows that interpretation of the contribution of the organ injury/dysfunction to early deaths in the CLP model is not straightforward and depends on the pathophysiological origin of the profiled disturbances.Electronic supplementary materialThe online version of this article (doi:10.1186/s40635-015-0048-z) contains supplementary material, which is available to authorized users.

Blood-based monitoring of immunoinflammatory and organ function fluctuations is essential in models of critical illness. This is challenging in diseased mice as repetitive blood collection may be harmful and/or affect end points. We studied the influence of daily sampling in acutely septic (days 1-5) mice upon survival and selected hematologic and organ function parameters. In addition, we tested the reliabilty of complete blood cell (CBC) count using resuspended blood cells. Female OF-1 and CD-1 mice underwent cecal ligation and puncture (CLP) and were subdivided into Daily and Day 5 groups. Blood was collected daily for 5 days in the Daily group and only on day 5 post-CLP in the Day 5 group. We tested 20 μL (both strains) and 35 μL (OF-1 mice) sampling volumes. The 35-μL volume simultaneously served to test the CBC reliabilty in resuspended versus unprocessed blood. Daily sampling did not affect the 14-day CLP mortality. Compared with the Day 5 group, daily 35-μL sampling in OF-1 mice decreased the red blood cell count and hemoglobin concentration by 22% and 23% (P < 0.05). In neither strain did daily 20-μL sampling affect the red blood cell count, whereas there was a 9% hemoglobin decrease (P < 0.05) in OF-1 mice. Although alanine aminotransferase, lactate dehydrogenase, and glucose levels were comparable, urea significantly increased by 24% in the Daily group (20-μL volume, OF-1 mice). Interleukin 6, platelets, and white blood cell counts remained unaffected. There was an excellent correlation between regular and resuspended CBC for all cell types (r ≥ 0.9; slope, ≥0.9), except lymphocytes (r > 0.5; slope, >0.5). This method provides a feasible and safe translation of clinically relevant daily immunomonitoring to the mouse sepsis model.

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Sepsis and sepsis-induced organ dysfunction remain lethal and common conditions among intensive care patients. Accumulating evidence suggests that the matricellular Cyr61/CCN1 (cysteine-rich, angiogenic-inducer, 61) protein is involved in the regulation of inflammatory responses and possesses organ-protective capabilities in diseases of an inflammatory etiology. However, its regulation in sepsis remains largely unexplored. The present study provides a comprehensive description of CCN1 regulation in the circulation and vital organs during experimentally induced sepsis with developing organ dysfunction. Female CD-1 mice served as baseline controls or were subjected to cecal ligation and puncture (CLP) for 18 to 96 h, and CCN1 regulation was analyzed in selected organs and in the circulation. A 5-, 5-, and 3-fold increases in circulating CCN1 protein were observed at 18, 48, and 96 h after CLP, respectively. Hepatic and pulmonary CCN1 mRNA expression was down-regulated by 80%, 60%, and 55% and 85%, 80%, and 65% at 18, 48, and 96 h after CLP and undetectable in circulating white blood cells. To identify a potential source for the circulating protein, mouse and human platelets were explored and revealed to contain CCN1. Human platelets were stimulated by thrombin and a specific PAR1 agonist (SFLLRN) in vitro. Both agonists induced an instant CCN1 release, and the effect of SFLLRN was blocked by the specific antagonist RWJ56110. The current study demonstrates that experimental sepsis is associated with a robust increase in circulating CCN1 protein levels and a paradoxical downregulation of CCN1 mRNA expression in vital organs. It provides evidence that CCN1 is released from activated platelets, suggesting that platelets constitute a novel source for CCN1 release to the circulation during sepsis.

IntroductionPlasminogen activator inhibitor 1 (PAI-1) is a key factor in trauma- and sepsis-induced coagulopathy. We examined how trauma-hemorrhage (TH) modulates PAI-1 responses in subsequent cecal ligation and puncture (CLP)-induced sepsis, and the association of PAI-1 with septic outcomes.MethodsMice underwent TH and CLP 48 h later in three separate experiments. In experiment 1, mice were sacrificed pre- and post-CLP to characterize the trajectory of PAI-1 in plasma (protein) and tissues (mRNA). Post-CLP dynamics in TH-CLP (this study) and CLP-Only mice (prior study) were then compared for modulatory effects of TH. In experiment 2, to relate PAI-1 changes to outcome, mice underwent TH-CLP and were sampled daily and followed for 14 days to compare non-survivors (DEAD) and survivors (SUR). In experiment 3, plasma and tissue PAI-1 expression were compared between mice predicted to die (P-DIE) and to live (P-LIVE).ResultsIn experiment 1, an early post-TH rise of circulating PAI-1 was contrasted by a delayed (post-TH) decrease of PAI-1 mRNA in organs. In the post-CLP phase, profiles of circulating PAI-1 were similar between TH-CLP and CLP-Only mice. Conversely, PAI-1 mRNA declined in the liver and heart of TH-CLP mice versus CLP-Only. In experiment 2, there were no DEAD/SUR differences in circulating PAI-1 prior to CLP. Post-CLP, circulating PAI-1 in DEAD was 2–4-fold higher than in SUR. PAI-1 increase heralded septic deaths up to 48 h prior but DEAD/SUR thrombomodulin (endothelial injury marker) levels were identical. In experiment 3, levels of circulating PAI-1 and its hepatic gene expression were higher in P-DIE versus P-LIVE mice and those increases closely correlated with liver dysfunction.ConclusionsTrauma modulated septic PAI-1 responses in a compartment-specific fashion. Only post-CLP increases in circulating PAI-1 predicted septic outcomes. In posttraumatic sepsis, pre-lethal release of PAI-1 was mostly of hepatic origin and was independent of endothelial injury.

To simulate and monitor the evolution of posttraumatic sepsis in mice, we combined a two-hit model of trauma/hemorrhage (TH) followed by polymicrobial sepsis with repetitive blood sampling. Anesthetized mice underwent femur fracture/sublethal hemorrhage and cecal ligation and puncture (CLP) 48 h later. To monitor outcome-dependent changes in circulating cells/biomarkers, mice were sampled daily (facial vein) for 7 days and retrospectively divided into either dead (DIE) or surviving (SUR) by post-CLP day 7. Prior to CLP, AST was 3-fold higher in DIE, while all other post-TH changes were similar between groups. There was a significant post-CLP intergroup separation. In SUR, RBC and Hb were lower, platelets and neutrophils higher, and lymphocytes mixed compared to DIE. In DIE, all organ function markers except glucose (decrease) were few folds higher compared to SUR. In summary, the combination of daily monitoring with an adequate two-hit model simulates the ICU setting, allows insight into outcome-based responses, and can identify biomarkers indicative of death in the acute posttraumatic sepsis in mice.