Sepsis chronically in MARS: systemic cytokine responses are always mixed regardless of the outcome, magnitude, or phase of sepsis

Abstract

The paradigm of systemic inflammatory response syndrome-to-compensatory anti-inflammatory response syndrome transition implies that hyperinflammation triggers acute sepsis mortality, whereas 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 the first part of this study, we characterized the systemic dynamics of the chronic inflammation in dying (DIE) and surviving (SUR) mice suffering from cecal ligation and puncture sepsis (days 6-28). In the second part, we combined the current chronic and previous acute/chronic sepsis data to compare the outcome-dependent inflammatory signatures between these two phases. A composite cytokine score (CCS) was calculated to compare global inflammatory responses. Mice were never sacrificed but were sampled daily (20 μl) for blood. In the first part of the study, parameters from chronic DIE mice were clustered into the 72, 48, and 24 h before death time points and compared with SUR of the same post-cecal ligation and puncture day. Cytokine increases were mixed and never preceded chronic deaths earlier than 48 h (3- to 180-fold increase). CCS demonstrated simultaneous and similar upregulation of proinflammatory and anti-inflammatory compartments at 24 h before chronic death (DIE 80- and 50-fold higher versus SUR). In the second part of the study, cytokine ratios across sepsis phases/outcomes indicated steady proinflammatory versus anti-inflammatory balance. CCS showed the inflammatory response in chronic DIE was 5-fold lower than acute DIE mice, but identical to acute SUR. The systemic mixed anti-inflammatory response syndrome-like pattern (concurrent release of proinflammatory and anti-inflammatory cytokines) occurs irrespective of the sepsis phase, response magnitude, and/or outcome. Although different in magnitude, neither acute nor chronic septic mortality is associated with a predominating proinflammatory and/or anti-inflammatory signature in the blood.

Figure 1. 28-day survival curve after CLP-induced sepsis

CLP was performed (total n=97) with an 18-gauge needle to produce ~30% mortality during the end of chronic phase of sepsis. Using a subjective cut-off, 28-day follow up was separated into the acute (days 1-5) and chronic (days 6-28) phase. A total of 21 deaths (21 out of 71) occurred in chronic sepsis. Animals included in the analysis (15 mice) underwent daily 20μl blood sampling between days 6-28. Solid line indicates the end of the acute sepsis phase, whereas dotted lines indicate the day of death of mice included in the analysis.

Figure 2. Protracted profiles of WBC, lymphocyte and neutrophil counts based on outcome during chronic sepsis

Daily blood samples were collected between days 6-28 post-CLP. The day of death (any day between 6-28 days) served as the reference point. DIE values were retrospectively plotted in the 72h, 48h and 24h prior-to-death trajectory and were time-matched with SUR values from the same post-CLP day (see study design). 2:1 SUR to DIE ratio at each time-point for each parameter: n= 20/10 (SUR/DIE) at 72h; n=28/14at 48h and 24h. Data (box and whiskers) presented as mean + SD. Dotted lines indicate normal range.* p < 0.05.

Figure 3. Protracted profiles of TNFα, IL-6, MIP-2 and MCP-1 based on outcome during chronic sepsis

Daily blood samples were collected between days 6-28 post-CLP. The day of death (any day between 6-28 days) served as the reference point. DIE values were retrospectively plotted in the 72h, 48h and 24h prior-to-death trajectory and were time-matched with SUR values from the same post-CLP day (see study design). 2:1 SUR to DIE ratio at each time-point for each parameter: n= 18/9 (SUR/DIE) at 72h; n= 26/13 at 48h; n=30/15 at 24h. Data presented as scatter dot plot: each dot represents an individual mouse, horizontal line represents mean.* p < 0.01.

Figure 4. Protracted profiles of IL-1ra, IL-10, TNF srI and TNF srII based on outcome during chronic sepsis

Daily blood samples were collected between days 6-28 post-CLP. The day of death (any day between 6-28 days) served as the reference point. DIE values were retrospectively plotted in the 72h, 48h and 24h prior-to-death trajectory and were time-matched with SUR values from the same post-CLP day (see study design). 2:1 SUR to DIE ratio at each time-point for each parameter: n= 18/9 (SUR/DIE) at 72h; n= 26/13 at 48h; n=29/15 at 24h. Data presented as scatter dot plot: each dot represents an individual mouse, horizontal line represents mean.* p < 0.01.

Figure 5. Composite Cytokine Scores for pro-and anti-inflammatory compartments based on outcome in chronic sepsis

All cytokine values were normalized (each mediator individually) to the median DIE value of that specific cytokine. The individual activation scores for each tested cytokine were then combined into the pro-inflammatory (i.e. IL-1β, IL-2, IL-5, IL-6, IL-12, IL-17, TNFα, IFNγ, ICAM-1, MIP-1α, MIP-2, MCP-1, Eotaxin, Eotaxin-2) and anti-inflammatory (IL-1ra, IL-4, IL-10, IL-13, TNF srI, TNF srII) panels, and the average response scores were compared based on outcome. DIE (any 6-28 post-CLP day) n=15 SUR (alive by 28 post-CLP day) n=30. Chronic DIE bar represents pooled cytokine values collected within 24h of death. Cytokine values collected from SUR animals were sampled on the same post-CLP day as the DIE mice (see study design). Data presented as mean + SEM. * p < 0.01.

Figure 6. Comparison of pro/anti-inflammatory ratios in acute and chronic sepsis

To enable comparison between septic phases, cytokine values from the current and two previous studies (25,32) were combined for this analysis. Each dot represents a ratio for a given cytokine set in an individual mouse. DIE group (left) represents ratios calculated from cytokine values collected within 24h of death. SUR group (right) represents ratios calculated from cytokine values collected on the same post-CLP day as the DIE mice (see study design). n=35 in acute DIE, n=29 in chronic DIE; n=30 in both SUR groups. Data presented as scatter plot on log N scale, horizontal bars represent mean ± 95% confidence intervals.

Figure 7. Normalized Cytokine Score based on outcome in the acute and chronic sepsis

To enable comparison between septic phases, cytokine values from the current and two previous studies (25,32) were combined for this analysis. All cytokine values were normalized and cytokine scores for individual cytokines were calculated (see statistical section). Those normalized individual scores were then combined into an overall score for each group and compared between DIE and SUR and across acute and chronic sepsis. DIE bars represent pooled cytokine values collected within 24h of death in the respective phase of sepsis. Only the same cytokines that were measured in all three studies were analyzed (n = 10/each group; IL-1β, IL-6, TNFα, MIP-2, MCP-1, Eotaxin and IL-1ra, IL-10, TNF srI, TNF srII). For simplicity, pro-and anti-inflammatory cytokines were pooled in each group. Data presented as mean + SEM. * p < 0.01 and ^# p < 0.001 compared to all remaining groups.