Measurement of circulating biomarkers constitutes the most practical method to rapidly describe the immuno-inflammatory status of a septic ER/ICU patient. To characterize the chronic pre-lethal inflammatory response in the first part of the study (-), we incorporated three vital experimental elements: 1) a model of moderate severity sepsis (displaying frequent late mortality) by polymicrobial, CLP-induced peritonitis, 2) a study design where mice were never sacrificed to allow a natural progression of the disease to survival/death, and 3) small-volume daily sampling during the chronic phase of sepsis (days 6-28). The above design was dictated by the clinical reality, where a large fraction of septic patients die in the chronic disease stage, the onset of sepsis is typically unknown (compared to pre-clinical studies), and patients are monitored frequently to provide optimal management and avoid another and ultimate reference point - death.
Given that plethora of pro- and anti-inflammatory cytokines were shown to correlate, both clinically (47
) and experimentally (8
) with sepsis severity/poor prognosis, a clinical focus has been put on selecting subjects with the highest risk of death to produce relatively homogenous cohorts more suitable for personalized treatments. Comparison of the Composite Cytokine Scores in the SUR and DIE groups at 24h prior to death () shows that similar to acute sepsis (8
) late deaths can also be preceded by a marked release of circulating cytokines. In line with our initial findings (35
), the current data re-confirm that individual cytokine/subject responses in chronic sepsis are highly mixed, regarding both the magnitude of the increase and the selection of released mediators. By longitudinally plotting the data from the chronic phase of sepsis in the current study, we demonstrate an aspect of the chronic inflammatory response never shown before. Specifically, virtually no cytokine increases were observed earlier than 48h pre-death, regardless of whether the mediator was in the pro-or anti-inflammatory panel. This closely mirrors the cytokine response in acute CLP (8
) where (SUR vs. DIE) differences did not appear earlier than the 48h prior-to-death. In the CLP setting, therefore, septic deaths (regardless whether acute or chronic) cannot be predicted earlier than approx. 48h prior to the event. In the current chronic sepsis study, none of the biomarkers exceeded AUC > 0.7 at 48h before late deaths (except AUC=0.77 for TNF srI, data not shown). Although many, seemingly promising, clinical studies investigated the viability of cytokine-prediction for long-term (typically 28 days) septic outcomes (51
), none of the biomarkers/biomarker sets has ever translated into a routine and clinically employable tool (5
). Current observations in the chronic sepsis part of the study may, at least partially, explain this deficit. We show that compared to the relatively steady (hyperinflammatory) signature of acute sepsis (8
), the feasibility of predicting outcome in chronic sepsis is much weaker. This weakness is primarily related to the transient and erratic appearance of cytokines in the bloodstream, limiting their use as predictors, regardless whether as a single marker or in combination. Another aspect is the diverse strength of the signal, since cytokine values are much higher in the acute phase. In other words, although chronic phase spikes in selected circulating cytokines (e.g. MIP-2) are predictive for late deaths, the magnitude of these increases is negligible when compared to their massive release in the acute phase. Interestingly, with the expanded range of biomarkers measured we observed that the inflammatory response in mice dying in chronic sepsis displays an “all or nothing” character: i.e. whenever an increase (or lack of response) in a given cytokine was recorded, a similar increasing (or no-responsiveness) pattern was also true for virtually all other (or majority of) remaining biomarkers.
The two most valuable findings, however, come from a combined, across-the-board comparison of outcome-dependent cytokine responses in the acute and chronic phases of sepsis. First, based on the comparison of pro/anti-inflammatory ratios from this (chronic sepsis) and our two previous (acute and chronic sepsis) experiments (8
), we prove that the proposed “Sepsis: Always in MARS” paradigm (54
) can be uniformly applied regardless of outcome and the phase of disease. Specifically, whenever sepsis provokes a given systemic inflammatory response, the associated release of anti-inflammatory cytokines closely matches the speed and/or robustness of the pro-inflammatory mediator secretion (and vice versa). Consequently, neither acute nor chronic sepsis mortality appear to be associated with a distinct predominant pro-and/or anti-inflammatory signature in the blood (despite differences in the relative response magnitude – see below). The most recent clinical studies corroborate the above observations (10
). The clinical translation of the above argued concept is simple: in the blood, the true cytokine make-up of SIRS/CARS is a one of MARS, and these definitions should be redefined accordingly.
Second, we demonstrate that due to the 5-fold difference in the magnitude of the response between acute DIE and chronic DIE, an effective identification of late CLP mortality in the milieu of an acute cytokine response is next to impossible. In other words, a 5ng/ml of circulating IL-6 measured in a CLP mouse may herald either its impeding death (hence a need for an immediate therapeutic intervention) in the chronic phase of sepsis, or conversely, indicate that the host mounted a successful struggle against the disease in its early, acute stage (discouraging an aggressive anti-inflammatory treatment). The above can be also directly extrapolated to the hospital ICU. The cytokine response profiles represented by Normalized Cytokine Scores indicate that the current precept of selecting septic subjects with the highest risk of death (based on the secreted cytokines) is heavily biased toward the hyper-inflammatory (MARS-like) signature, i.e., it tends to include only those who display a relatively strong cytokine response, while leaving both “non- and “low-responders” behind. As a result, chronic septic subjects with a typically weaker pre-lethal cytokine response are unable to rise above a given diagnostic threshold calibrated on the magnitude of the early inflammatory response. Hence, these subjects will be neither identified nor treated (with the latter omission paradoxically beneficial in case of aggressive immunosuppressive/anti-inflammatory therapies). The above might have been partially reflected in the MONARCS trial (15
) that relied on IL-6 to direct an anti-TNF treatment in septic patients: the trial improved the overall survival by approx. 6%, yet the target group of IL-6 test positive patients was relatively small (n= 998) despite the size of the entire (n=2634) enrolled population. The hyper-inflammatory bias may be further exacerbated depending on the type/source of sepsis since innate/adaptive inflammatory signature of abdominal sepsis differs from, for example, pulmonary sepsis developing in ventilated ICU patients (55
). Finally, it needs to be stressed out that plasma cytokines may not mirror the functional state of the immune cells in tissues/organs. Cavaillion et al., described this disparity as “compartmentalization” of the septic inflammatory response (56
). Indeed, a robust systemic release of inflammatory cytokines (in sepsis) was shown to coincide with evident signs of cellular immunossupression in various tissue compartments, both in clinical (25
) and CLP studies (59
The above data do not deny a role for circulating biomarkers as a valuable tool for guiding specific immunomodulatory therapies in septic patients. In contrary, such a scenario still appears feasible, at least to some degree. Given the aforementioned differences in the magnitude of humoral cytokine response, it needs to be carefully established under what circumstances and for which patient cohorts the most clinical benefit is to be produced. Perhaps, a superior solution for parallel identification of some high-risk-of-death patients (and their immuno-inflammatory status) is to combine the most accurate existing scoring systems (e.g. Pitt bacteremia score, APACHE II and MEDS scores) (62
) with a relatively uncomplicated (and rapid) profiling of an array of suitable circulating cytokines and/or cells. As accuracy of the latter element(s) is heavily dependent on the phase of sepsis, it should not be ignored that the absence/low levels of biomarker(s) may/should be as alerting as their overwhelming presence.