Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions

TL;DR

Heme released during hemolysis worsens Gram-negative sepsis not by feeding bacteria iron but by disrupting phagocyte actin dynamics, phagocytosis, and migration. Heme acts through DOCK8-dependent Cdc42 activation, and quinine can block this effect, restore phagocytosis, reduce bacterial burden, and improve survival in mice.

Key findings

• In mice infected i.p. with *E. coli* PMV-1 (1×10⁴ CFU), plasma heme correlated with bacteremia (Pearson r=0.5572, p=0.0013), and heme pretreatment (25 µmol/kg i.p.) increased plasma heme, bacterial burden in blood/liver, and impaired survival versus PBS.
• Heme increased bacterial burden independently of heme-iron acquisition: an isogenic *E. coli* ΔdppC::KanR mutant unable to use heme-iron still showed increased blood/liver CFU in heme-treated mice, and equimolar free ferric iron did not reproduce the heme effect.
• Heme inhibited phagocytosis of *E. coli*, *P. aeruginosa*, *S. pneumoniae*, and *L. monocytogenes* by mouse and human phagocytes, including human whole blood neutrophils/monocytes at 5–50 µM; washing heme restored phagocytosis, and heme induced actin remodeling, filopodia, and Cdc42 activation via DOCK8.
• *Dock8−/−*>WT bone marrow chimeras were protected from heme during sepsis, with a 10-fold reduction in bacterial numbers in liver, blood, and peritoneal cavity; quinine restored phagocytosis in vitro and in vivo, reduced bacterial counts, and improved survival in heme-treated septic mice.

Methods

• Murine sepsis/peritonitis models used *E. coli* PMV-1 or isogenic mutants, *L. monocytogenes*, LPS, heme (25 µmol/kg i.p.), iron, quinine (60 mg/kg), and LysM-Cre+/−Hmox1fl/fl or Dock8−/− bone marrow chimeras.
• In vitro assays measured phagocytosis, efferocytosis, bacterial killing, cell shape/cytoskeleton by microscopy, Cdc42 activation by G-LISA, and heme-binding proteins by heme-agarose affinity purification with LC-MS/MS, SAINT/CRAPome scoring, and STRING network analysis.
• A chemical screen tested 389 compounds by automated microscopy for restoration of phagocytosis in heme-treated RAW264.7 macrophages; statistics used t-tests, Mann-Whitney, ANOVA with Tukey, Pearson correlation, and Mantel-Cox survival analysis.

Limitations

• The paper states that DOCK8 deletion improved bacterial clearance only incompletely, likely reflecting a tradeoff with immune defects caused by DOCK8 deficiency, and that how heme gains access to the phagocyte cytoplasm remains unclear.
• The paper states quinine’s mechanism of action is not fully understood, though it may interfere with heme-DOCK8 association; animal studies were performed without prior randomization or blinding.
• Evident from the text: the main evidence is from mouse sepsis models and in vitro human cells, not human clinical trials; quinine also improved survival in PBS-treated mice, suggesting effects beyond heme blockade.