Neutrophil Extracellular Traps Drive Bleomycin‐Induced Lung Fibrosis by Regulating TGFβ1‐Dependent Interactions of Platelets and Macrophages

Neutrophil extracellular traps (NETs) are extracellular histones and DNA expelled from polymorphonuclear neutrophils (PMNs). NETs are released during inflammation as an innate immune defense mechanism. NETs not only exert bactericidal activities but are toxic for host cells and thus promote tissue injury. In the past, many reports have defined the important roles of NETs during acute infections and acute inflammatory diseases. On the other hand, our current knowledge on the contribution of NETs during chronic inflammation, adaptive immunity and fibrotic diseases leaves much to be desired. In this report, we uncover the immunologic networks initiated by NETs during the natural course of bleomycin‐induced lung fibrosis in mice. First, we designed a model of inducible transient genetic depletion of Ly6G + PMNs by injection of diphtheria toxin in Ly6G‐Cre × iDTR mice. Depletion of Ly6G + PMNs simultaneously with intra‐tracheal administration of bleomycin was sufficient to decrease the build‐up of extracellular matrix, T cells and cytokines (e.g. TGFβ1) in lungs after 4 weeks. Bleomycin mediated the release of citrullinated extracellular histones in broncho‐alveolar lavage fluids in dependency of PMNs with a surge after 2 days. Neutralization of extracellular histones by monoclonal anti‐histone H4 antibodies significantly reduced the expression and accumulation of extracellular matrix proteins and TGFβ1 during lung fibrosis. Platelets represented a major source (≥70%) of plasma TGFβ1 and purified extracellular histones activated platelets to release TGFβ1. Accordingly, bleomycin‐induced lung fibrosis was ameliorated in mice with platelet‐specific genetic deletion of TGFβ1 (PF4‐Cre × TGFb1flox/flox mice). TGFβ1 signaling required TGFβ receptor II and multiple down‐stream signaling pathways in macrophages. Mice with specific genetic ablation of the TGFβ receptor II in LysM+ macrophages (LysM‐Cre × TbRIIflox/flox mice) were also protected in bleomycin‐induced fibrosis. Finally, we found that histone‐induced TGFβ1 from platelets modulated the T cell tropic cytokine production in macrophages. Our data suggest that NETs are an important factor for the pathogenesis of lung fibrosis. NETs may coordinate subsequent cytokine‐dependent interactions of platelets with innate and adaptive immune cells. In the future, pharmacologic blockade of NETs may be further evaluated as a potential treatment for chronic fibrotic diseases. Support or Funding Information This study was supported by the Federal Ministry of Education and Research (BMBF 01EO1503) and the Deutsche Forschungsgemeinschaft (BO3482/3‐1). The authors are responsible for the contents of this publication.