Kiefmann, M.; Hammerschmidt, S.; Dietrich, A.; Kiefmann, R.

Severe infection with Streptococcus (S.) pneumoniae is a leading cause of acute lung injury, multiorgan failure, and death despite appropriate antibiotic therapy. A major contributor to tissue damage is pneumolysin (PLY), a cholesterol-dependent pore-forming toxin that is released in large amounts during bacterial lysis. Because antibiotic treatment enhances PLY release, understanding the mechanisms that control PLY-induced host injury is critical for the development of strategies that limit tissue damage without impairing antibacterial defense. Using isolated perfused lungs and in situ two-photon imaging, we show that S. pneumoniae induces platelet and leukocyte recruitment, pulmonary edema, perfusion failure, and intravascular bacterial trapping within pulmonary arterioles. These responses required PLY and were associated with endothelial Ca2+ influx and plasma membrane permeabilization. The oxysterol 25-hydroxycholesterol (25-HC) prevented PLY-dependent Ca2+ entry, vascular leakage, and perfusion failure by reducing accessible membrane cholesterol through activation of acyl-CoA:cholesterol acyltransferase (ACAT). Unexpectedly, pneumococcal infection suppressed expression of cholesterol-25-hydroxylase (CH25H), the enzyme responsible for 25-HC synthesis, although induction of this pathway would be predicted to protect against toxin-mediated injury. We show that this downregulation results from inhibition of platelet-activating factor (PAF) receptor (PAFR) signaling by pneumococcal phosphocholine-containing cell wall components. Remarkably, both PAF itself and the anti-phosphocholine antibody TEPC directly inhibited PLY pore formation independently of PAFR, while preserving intravascular coagulation and bacterial trapping. Thus, although 25-HC protects against toxin-mediated vascular injury, its suppression may allow coagulation-dependent bacterial containment, with PAF acting as a compensatory antagonist of PLY. These findings identify a lipid-based host defense system in which 25-HC and PAF differentially regulate PLY activity to balance tissue protection with bacterial clearance and suggest new therapeutic approaches to limit the harmful effects of antibiotic-induced PLY release during pneumococcal infection.