Immunopathology of terminal complement activation and complement C5 blockade creating a pro-survival and organ-protective phenotype in trauma

Abstract

Background and purpose: Traumatic haemorrhage (TH) is the leading cause of potentially preventable deaths that occur during the prehospital phase of care. No effective pharmacological therapeutics are available for critical TH patients yet. Here, we identify terminal complement activation (TCA) as a therapeutic target in combat casualties and evaluate the efficacy of a TCA inhibitor (nomacopan) on organ damage and survival in vivo.

Experimental approach: Complement activation products and cytokines were analysed in plasma from 54 combat casualties. The correlations between activated complement pathway(s) and the clinical outcomes in trauma patients were assessed. Nomacopan was administered to rats subjected to lethal TH (blast injury and haemorrhagic shock). Effects of nomacopan on TH were determined using survival rate, organ damage, physiological parameters, and laboratory profiles.

Key results: Early TCA was associated with systemic inflammatory responses and clinical outcomes in this trauma cohort. Lethal TH in the untreated rats induced early TCA that correlated with the severity of tissue damage and mortality. The addition of nomacopan to a damage-control resuscitation (DCR) protocol significantly inhibited TCA, decreased local and systemic inflammatory responses, improved haemodynamics and metabolism, attenuated tissue and organ damage, and increased survival.

Conclusion and implications: Previous findings of our and other groups revealed that early TCA represents a rational therapeutic target for trauma patients. Nomacopan as a pro-survival and organ-protective drug, could emerge as a promising adjunct to DCR that may significantly reduce the morbidity and mortality in severe TH patients while awaiting transport to critical care facilities.

Keywords: complement; mortality; organ failure; prehospital care; traumatic haemorrhage.

FIGURE 1

Workflow of translational study design. Blood plasma from 54 casualties on admission, 8 and 24 h after admission to a hospital and 10 civilian volunteers was used for the analysis of the complement activation. On the basis of the products of complement activation in the casualties' plasma, complement component C5 was identified as a reasonable therapeutic target. Prophylactic and therapeutic effects of nomacopan, an inhibitor of C5, were tested in rat models of blast (B) and haemorrhage (H) injury. Abbreviation: NOM, nomacopan

FIGURE 2

Early activation of complement terminal and alternative pathways after trauma in military casualties. Plasma levels of C5a (a), sC5b‐9 (b), Bb (c), and C4d (d) were measured to determine activation of terminal complement (C5a and sC5b9), alternative pathway (Bb), and classical and lectin pathways (C4d) by ELISA in healthy donors, and trauma patients on admission to hospital, 8 and 24 h after admission. The data are expressed as μg·mg⁻¹ plasma protein, except for C5a and C5b‐9, which are shown as ng·mg⁻¹ plasma protein. Data shown are means ± SEM, from the group sizes shown (n). *P < 0.05, significantly different from values in healthy subjects (Ctrl); Mann–Whitney U test). (e–h) Correlation of Bb and C4d with either C5a or sC5b‐9 in the injured patients at admission. Correlation analysis between complement factors bb or C4d and C5a or sC5b‐9 were performed using Spearman's rank correlation. Data are shown as individual values with the correlation coefficient (r_s). Significant correlations (P < 0.05) are indicated by boldface type.

FIGURE 3

Activation of complement terminal and alternative pathways is related to systemic inflammatory response after trauma in battlefield casualties. Inflammatory factors and cytokines were measured by ELISA and by Bio‐Plex kits, respectively. Positive correlation between plasma concentrations of C5a in the trauma patients and IL‐6 (a), MCP‐1 (b), and MPO (c) in the blood plasma of the patients on admission. Positive correlation of plasma levels of C5b‐9 on admission with IL‐6 (d), MCP‐1 (e), and MPO (f) in the injured patients on admission. Plasma concentration of bb on admission positively correlated with IL‐6 (g), MCP‐1 (h), and MPO (i), whereas the plasma levels of C4d on admission inversely correlated with IL‐1β (j), IL‐17 (k), and TNF‐α (l). Correlation analysis between complement factors (C5a, C5b‐9, bb and C4d) and inflammatory factors/cytokines were performed by using Spearman's rank correlation. Data are shown as individual values with the correlation coefficient (r_s). Significant correlations (P < 0.05) are indicated by boldface type.

FIGURE 4

Complement activation correlated with clinical outcomes in trauma patients on admission. (a–d) Correlation of C5a plasma levels with clinical scores, infused fluid and INR, a standard coagulation test; (e–h) correlation of C5b‐9 with clinical scores, infused fluid and INR; (i–l) correlation of fragment bb with clinical scores, infused fluid and INR; (m, n) the receiver‐operator characteristic curve (ROC) analysis tested diagnostic ability of C5a and fragment bb in identifying traumatic brain injury (TBI); the cut‐off value, specificity, and sensitivity are shown (q); (o) patients on mechanical ventilation had significantly higher plasma levels of C5a in comparison to those not‐ventilated; (p) ROC analysis showed that the C4d plasma levels had a strong predictive value for the survival; the cut‐off value, specificity and sensitivity are presented (q). Data are shown as individual values with the correlation coefficient (r_s). Significant correlations (P < 0.05) are indicated by boldface type.

FIGURE 5

Effects of nomacopan treatment early after blast injury on MAP, CH50, and blood chemistry changes in a clinically relevant rat model of traumatic haemorrhagic shock. (a) Experimental design; (b) changes of MAP were monitored via the carotid artery using the BIOPAC system. During the shock and resuscitation period, the MAP was recorded every 5 min. Data are presented as mean ± SEM; (c) haemolytic TCA of sera was measured by CH50 and normalized to baseline level, which was a pre‐blast injury; the percentages of baseline are shown; (d) PaO₂/FiO₂ ratio (PFR) following injuries. The PFR was calculated at each time point based on the artery i‐STAT data. A PFR of less than 300 (dashed line) suggests acute respiratory distress syndrome (ARDS). E‐G, the blood chemistry of lactate, BE/BD, and K⁺ in injured and treated animals were measured by I‐stat and presented. Data shown are means ± SEM from groups of rats, sizes (n) as indicated. *P < 0.05, significant effect of NOM; unpaired t‐test with Welch's correction; †P < 0.05, significantly different as indicated; two‐way ANOVA. Abbreviations: BOP = blast overpressure; B + H = blast + haemorrhagic shock; NOM = nomacopan given to injured rats; MAP = mean arterial pressure; EOS = end of the study

FIGURE 6

Effect of nomacopan treatment early after injury on systemic and local inflammatory response in rats after blast injury and haemorrhage. (a, b) Inflammatory mediators HMGB1and MPO, were measured by ELISA. Data shown are means ± SEM from groups of rats, sizes (n) as indicated. *P < 0.05, significantly different from B+H at this time point; unpaired t‐test with Welch's correction; †P < 0.05, group data significantly difference in group data: two‐way ANOVA. (c–f) Cytokines/chemokines were measured in the lung homogenates. Data shown are means ± SEM from groups of rats, sizes (n) as indicated. †P < 0.05, significantly different from sham; Mann–Whitney U test. (g) Lung tissue at necropsy or at the end of study were collected, fixed by PFA, and stained by IHC. The representative images of immunostaining of MPO (red) and ICAM1 (green) are shown; (h) antibodies for detecting C5b‐9 (red) and C3 (green) were used to detect complement deposition in the lung tissue. Representative images (g, h) are shown (original magnification, 200×), and semi‐quantitative analysis (i) of the positively stained cells to total cells are presented. Data are shown as individual values with means +SEM; n = 3‐4 animals per group. Scale bar = 50 μm. Abbreviations: B + H = blast + haemorrhagic shock; NOM = nomacopan treated rats; MPO = myeloperoxidase

FIGURE 7

Effect of nomacopan treatment on histological changes in rats after blast injury and haemorrhage. (a, b) Representative H&E photomicrographs of organs collected at necropsy (a), and organ injury scored based on the criteria described in Section 2 (b). The data are presented as means ± SEM. †P < 0.05, significantly different from sham; *P < 0.05, significantly different from B + H; Mann–Whitney U test).

FIGURE 8

Effect of nomacopan on injury‐induced mortality. All rats were subjected to blast and severe haemorrhage (B + H), treated with vehicle control (saline) or nomacopan (NOM), and monitored for survival up to 25 h after blast injury. Survival distribution of these two groups was determined by using the log‐rank Mantel‐Cox test. *P < 0.05, significantly different from B + H.