Extracellular histones promote pulmonary fibrosis by multicellular interactions

Abstract

Pulmonary fibrosis is characterized by a dysregulated accumulation of extracellular matrix components. This chronic and irreversible disease has a poor prognosis because of limited treatment options. In the presented work, the cellular processes and cytokine networks initiated by extracellular histones during bleomycin-induced pulmonary fibrosis were studied in mice. As the cellular sources of extracellular histones, neutrophils and non-hematopoietic cells were identified by using chimeric H2B-eGFP reporter mice. In addition, citrullinated extracellular histones derived from neutrophil extracellular traps (NETs) were reduced after depletion of neutrophils in Ly6GCre/iDTR mice. This transient depletion of Ly6G+ neutrophils during the early phase of bleomycin-induced pulmonary fibrosis protected from extracellular matrix accumulation (collagen I,V) and the infiltration of pro-fibrotic Th17 cells into the lungs. Furthermore, blockade of extracellular histones (anti-histone H4 antibodies) protected from collagen I,V accumulation as well as epithelial/endothelial to mesenchymal transition. The blocked extracellular histones also led to reduced stress-induced gene expression levels in T regulatory cells. Histones activated platelets, followed by release of TGFβ1 during ex vivo studies. Lower concentrations of TGFβ1 and less collagen I,V accumulation were noted in PF4Cre/TGFβ1flox/flox mice. TGFβ1 inhibited IL-27 production in macrophages in a TGFbRI and TGFbRII dependent manner. The influence of TGFbRII on the release of IL-27 during bleomycin-induced pulmonary fibrosis was shown in LysMCre/TbRIIflox/flox mice. IL-27 apparently exerted anti-fibrotic activities, since IL-27RA-/- mice were more prone to fibrosis. The molecular inhibition mechanism of TGFβ1 was further analyzed by several in vitro studies: TGFβ1 engaged multiple downstream proteins such as p38 MAPK, Tristetraprolin and IL-10. A central signaling molecule of TGFβ1, Smad3, was shown to bind directly to the il-27p28 and ebi3 promoter regions. This work identifies extracellular histones as central key players during experimental pulmonary fibrosis. The extracellular histones appear to skew the reciprocal balance of IL-27 and TGFβ1 by triggering the release of platelet-derived TGFβ1. These data set extracellular histones in the focus of the search for future treatment options for pulmonary fibrosis.