Role of heme in lung bacterial infection after trauma hemorrhage and stored red blood cell transfusion: A preclinical experimental study

Abstract

BackgroundTrauma is the leading cause of death and disability in patients aged 1–46 y. Severely injured patients experience considerable blood loss and hemorrhagic shock requiring treatment with massive transfusion of red blood cells (RBCs). Preclinical and retrospective human studies in trauma patients have suggested that poorer therapeutic efficacy, increased severity of organ injury, and increased bacterial infection are associated with transfusion of large volumes of stored RBCs, although the mechanisms are not fully understood.Methods and findingsWe developed a murine model of trauma hemorrhage (TH) followed by resuscitation with plasma and leukoreduced RBCs (in a 1:1 ratio) that were banked for 0 (fresh) or 14 (stored) days. Two days later, lungs were infected with Pseudomonas aeruginosa K-strain (PAK). Resuscitation with stored RBCs significantly increased the severity of lung injury caused by P. aeruginosa, as demonstrated by higher mortality (median survival 35 h for fresh RBC group and 8 h for stored RBC group; p < 0.001), increased pulmonary edema (mean [95% CI] 106.4 μl [88.5–124.3] for fresh RBCs and 192.5 μl [140.9–244.0] for stored RBCs; p = 0.003), and higher bacterial numbers in the lung (mean [95% CI] 1.2 × 107 [−1.0 × 107 to 2.5 × 107] for fresh RBCs and 3.6 × 107 [2.5 × 107 to 4.7 × 107] for stored RBCs; p = 0.014). The mechanism underlying this increased infection susceptibility and severity was free-heme-dependent, as recombinant hemopexin or pharmacological inhibition or genetic deletion of toll-like receptor 4 (TLR4) during TH and resuscitation completely prevented P. aeruginosa–induced mortality after stored RBC transfusion (p < 0.001 for all groups relative to stored RBC group). Evidence from studies transfusing fresh and stored RBCs mixed with stored and fresh RBC supernatants, respectively, indicated that heme arising both during storage and from RBC hemolysis post-resuscitation plays a role in increased mortality after PAK (p < 0.001). Heme also increased endothelial permeability and inhibited macrophage-dependent phagocytosis in cultured cells. Stored RBCs also increased circulating high mobility group box 1 (HMGB1; mean [95% CI] 15.4 ng/ml [6.7–24.0] for fresh RBCs and 50.3 ng/ml [12.3–88.2] for stored RBCs), and anti-HMGB1 blocking antibody protected against PAK-induced mortality in vivo (p = 0.001) and restored macrophage-dependent phagocytosis of P. aeruginosa in vitro. Finally, we showed that TH patients, admitted to the University of Alabama at Birmingham ER between 1 January 2015 and 30 April 2016 (n = 50), received high micromolar–millimolar levels of heme proportional to the number of units transfused, sufficient to overwhelm endogenous hemopexin levels early after TH and resuscitation. Limitations of the study include lack of assessment of temporal changes in different products of hemolysis after resuscitation and the small sample size precluding testing of associations between heme levels and adverse outcomes in resuscitated TH patients.ConclusionsWe provide evidence that ...

Total plasma heme concentration increases after red blood cell transfusion and predicts mortality in critically ill medical patients

“…Our results also suggest that clinically relevant storage duration may not be the major determinant of transfusion toxicity, since RBC unit age did not predict posttransfusion total plasma heme or hospital mortality, consistent with previous studies . The mean storage time of RBC units transfused in the United States is 18 days, and in our study the median storage time was 24 days with a minimum of 5 days.…”

“…These mechanisms for heme accumulation are accentuated in septic models . Our results suggest that these concerted relationships track more precisely with the severity of critical illness than the presence of sepsis per se.…”

“…We observed an independent association between posttransfusion total plasma heme and mortality. The toxicity of cell‐free heme is supported by in vitro studies, animal models, and human subjects . The adverse effects include nitric oxide scavenging and vasoconstriction, increased vascular permeability, oxidative stress, inflammation, and immune dysregulation with increased risk of infection .…”

“…We found that posttransfusion total plasma heme levels were similar in those patients with longer time intervals between transfusion and plasma sampling, even though the correlation between RBC unit total cell‐free heme and posttransfusion total plasma heme declined as the time interval lengthened. There are at least three interrelated explanations for transfusion‐related total plasma heme accumulation, apart from the quantity directly transfused: 1) posttransfusion hemolysis of storage‐damaged RBCs, 2) transfusion‐associated damage and disintegration of host RBCs, and 3) impairments in the critically ill host's capacity to clear heme …”

“…The resulting cell‐free hemoglobin (Hb; free in solution and contained in RBC microparticles) can undergo oxidation and release cell‐free heme . These mediators may cause toxicity when transfused because cell‐free Hb promotes circulatory dysfunction by scavenging nitric oxide and cell‐free heme promotes inflammation, immunosuppression, and infection . The risks of transfusion may be particularly harmful during acute illnesses that may aggravate hemolysis and impair clearance of circulating cell‐free Hb and heme …”