Computational evidence for an early, amplified systemic inflammation program in polytrauma patients with severe extremity injuries

Abstract

Extremity and soft tissue injuries contribute significantly to inflammation and adverse in-hospital outcomes for trauma survivors; accordingly, we examined the complex association between clinical outcomes inflammatory responses in this setting using in silico tools. Two stringently propensity-matched, moderately/severely injured (Injury Severity Score > 16) patient sub-cohorts of ~30 patients each were derived retrospectively from a cohort of 472 blunt trauma survivors and segregated based on their degree of extremity injury severity (above or below 3 on the Abbreviated Injury Scale). Serial blood samples were analyzed for 31 plasma inflammatory mediators. In addition to standard statistical analyses, Dynamic Network Analysis (DyNA) and Principal Component Analysis (PCA) were used to model systemic inflammation following trauma. Patients in the severe extremity injury sub-cohort experienced longer intensive care unit length of stay (LOS), total LOS, and days on a mechanical ventilator, with higher Marshall Multiple Organ Dysfunction (MOD) Scores over the first 7 days post-injury as compared to the mild/moderate extremity injury sub-cohort. The higher severity cohort had statistically significant elevated lactate, base deficit, and creatine phosphokinase on first blood draw, along with significant changes in multiple circulating inflammatory mediators. DyNA pointed to a sustained role for type 17 immunity in both sub-cohorts, along with IFN-γ in the severe extremity injury group. DyNA network complexity increased over 7 days post-injury in the severe injury group, while generally decreasing over this same time period in the mild/moderate injury group. PCA suggested a more robust activation of multiple pathways in the severe extremity injury group as compared to the mild/moderate injury group. These studies thus point to the possibility of self-sustaining inflammation following severe extremity injury vs. resolving inflammation following less severe extremity injury.

Fig 1. Stringently matched sub-cohorts differ only in the extremities component of the Abbreviated Injury Scale.

Trauma patients were recruited following IRB approval and informed consent. The Abbreviated Injury Scale (AIS) score was statistically significantly higher in the extremities regions in the severe extremity injury sub-cohort when compared to a stringently matched mild/moderate extremity injury sub-cohort (*P<0.05 vs. mild injury analyzed by One-Way ANOVA).

Fig 2. Clinical biochemistry parameters are significantly altered as a function of extremity/soft tissue injury severity in stringently matched sub-cohorts.

(A) Plasma lactate levels were statistically significant higher in the severe extremity injury sub-cohort when compared to a stringently matched mild/moderate extremity injury sub-cohort over the first 24 h post injury. (B) pH was statistically significant lower in the severe extremity injury sub-cohort when compared to a stringently matched mild/moderate extremity injury sub-cohort over the first 24 h post injury. (C) Base deficit (BD) was statistically significantly higher in the severe extremity injury sub-cohort when compared to a stringently matched mild/moderate extremity injury sub-cohort over the first 24 h post-injury. (D) Creatine Phosphokinase (CPK) was statistically significantly higher in the severe extremity injury sub-cohort when compared to a stringently matched mild/moderate extremity injury sub-cohort over the first 24 h post-injury.

Fig 3. Greater severity of multiple organ dysfunction as a function of extremity/soft tissue injury severity over 7 days.

Daily Marshall MODScore analysis of organ failure suggests that the severe extremity injury sub-cohort had higher degree of MOD from day 2 up to day 7 post-injury when compared to a stringently matched mild/moderate extremity injury sub-cohort. *P<0.05 by Two-Way ANOVA.

Fig 4. Time course analysis of inflammation biomarkers in the mild/moderate, and severe extremity injury sub-cohorts from time of injury up to 7 days.

Trauma patients were recruited following IRB approval and informed consent. Plasma was obtained at multiple time points and analyzed for the presence of 27 inflammatory mediators in highly-matched sub-cohorts of patients with severe vs. mild/moderate extremity injury described in Materials and Methods. Mean circulating levels of inflammatory mediators in the mild/moderate extremity injury (n = 30), and severe extremity injury (n = 32) sub-cohorts. (A) Time course of MCP-1/CCL2. (B) Time course of MIG/CXCL9. (C) Time course of IP1-0/CXCL10. (D) Time course of MIP-1α (CCL3). (E) Time course of IL-6. (F) Time course of IL-7. (G) Time course of IL-8. (H) Time course of Eotaxin (CCL11). The indicated inflammatory mediators were assessed in serial plasma samples obtained at the indicated time points. Values are mean ± SEM. *P<0.05 by Two-Way ANOVA.

Fig 5. DyNA network complexity identifies inverse inflammation trajectories between trauma patients severe extremity injury vs. a stringently matched sub-cohort with mild/moderate extremity injury.

Trauma patients were recruited following IRB approval and informed consent. Plasma was obtained at multiple time points and analyzed for the presence of 31 inflammatory mediators in highly-matched sub-cohorts of patients with severe vs. mild/moderate extremity injury, followed by Dynamic Network Analysis (DyNA) as described in the Materials and Methods. The time-evolution of networks in severe (framed in red) vs. mild-moderate (framed in blue) extremity injury is displayed. In silico inference of inflammatory network complexity suggests a bifurcation in network progression as time progressed up to 7 days post-injury: the mild/moderate injury group reached inflammation resolution and suppression over 7 days, whereas the severe injury group multiplied in complexity over 7 days, ultimately reaching a higher network complexity than did the mild/moderate group at the onset of inflammation.

Fig 6. Differential dynamic inflammatory networks identified after 5 days in trauma patients with severe extremity injury vs. a stringently matched sub-cohort of patients with mild/moderate extremity injury.

Trauma patients were recruited following IRB approval and informed consent. Plasma was obtained at multiple time points and analyzed for the presence of 31 inflammatory mediators in highly-matched sub-cohorts of patients with severe vs. mild/moderate extremity injury, followed by Dynamic Network Analysis (DyNA) as described in the Materials and Methods. (A) Inferred dynamic networks in the mild/moderate group suggested a characteristic, lymphoid-predominant, core inflammatory network of sIL-2Rα, IL-4, IL-13, and IL-17A which contains features of a similar, lymphoid-predominant, core inflammatory network associated with survival in blunt trauma patients up to 7 days post-injury [46]. (B) The severe extremity injury group was characterized by both innate and lymphoid mediators which evolved into increasingly complex networks of 7 day, with the emergence of the pro-inflammatory TNF-α and anti-inflammatory IL-10 at Day 5–7. The prototypical Th1 cytokine IFN-γ did not appear in the inflammation networks of the mild/moderate sub-cohort until days 5–7, whereas it was increasingly connected in the inflammation networks of the severe sub-cohort over 7 days. All original DyNA outputs are included in S1 Fig.

Fig 7. Principal component analysis suggests a differential role for type 2 immune responses in the circulating inflammatory response to extremity injury.

Trauma patients were recruited following IRB approval and informed consent. Principal component analysis was carried out using the data from stringently matched sub-cohorts of patients with severe vs. mild/moderate extremity injury as described in the Materials and Methods. Both sub-cohorts shared 4 out of the 5 leading principal components: IL-1β, IL-7, IL-13, and IFN-γ, exhibiting a conserved, core inflammatory signature of extremity fracture and/or polytrauma (A) PCA of the severe injury group exhibited more components than that of the mild/moderate injury group (9 vs. 7, respectively), as well as having a slightly greater overall magnitude (~0.23 vs. 0.20, respectively). The PCA from day 0–7 identified IL-1β and its antagonist IL-1RA as leading principal mediators in the severe extremity injury group but not in the mild/moderate group. (B) The type 2 cytokine IL-4 appeared as the most characteristic principal mediator of the mild/moderate group, in addition to being identified as a key inflammatory mediators identified in the DyNA (see Fig 6A).