Extracellular Histones in Lung Injury and Fibrosis
Final Report Abstract
Nuclear histones are released to the extracellular spaces during acute lung injury and other lung diseases. The cellular sources of extracellular histones are neutrophils and dying non-immune cells. Neutrophils generate extracellular histones in the form of neutrophil extracellular traps (NETs). The process of NET formation requires decondensation of chromatin by histone citrullination catalyzed by the enzyme, peptidyl arginine deiminase, type IV (PAD4). NETs and extracellular histones serve innate immune defenses by trapping and killing microorganisms during infections. On the other hand, extracellular histones are highly cytotoxic for resident cells in lungs and induce strong inflammatory responses. Before this project the activities of NETs/extracellular histones for the transition of acute tissue injury into an abnormal wound healing response with tissue remodeling and pulmonary fibrosis have only received little attention. In this project, we have investigated the cellular sources and functional activities of extracellular histones during experimental bleomycin-induced lung injury and fibrosis. We have employed chimeric mice with histone H2B-eGFP fluorescent reporter activity to determine the relative contributions of NETs versus non-myeloid cell derived extracellular histones in injured lungs. The functional relevance of histone citrullination was studied in cytotoxicity assays of mouse lung epithelial cell lines. Transgenic mouse strains for selective depletion of neutrophils and PAD4 were evaluated bleomycin-induced lung fibrosis. Monoclonal antibodies directed against histone H4 were successfully used to antagonize the effects of endogenous extracellular histones during lung fibrosis. We found that externalized histones were significantly increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis as compared to healthy controls. The pulmonary sources of externalized histones were Ly6G+CD11b+ neutrophils and nonhematopoietic cells after bleomycin in mice. Histones activated platelets to release TGFβ1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM+ cells to antagonize macrophage-derived IL-27 production. TGFβ1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA- deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. The endpoints of these studies included measurements of extracellular matrix accumulation, cytokine/chemokine patterns as well as intra-pulmonal and central T cell subsets. This project has provided us with better insights into the cellular and molecular pathways triggered by extracellular histones in the course of lung injury and lung fibrosis. In future, our experimental findings may be advantageous for the evaluation of extracellular histones as potential targets for therapeutic intervention in pulmonary fibrosis.
Publications
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Neutrophil extracellular traps impair fungal clearance in a mouse model of invasive pulmonary aspergillosis. Immunobiology, 225(1), 151867.
Alflen, Astrid; Aranda Lopez, Pamela; Hartmann, Ann-Kathrin; Maxeiner, Joachim; Bosmann, Markus; Sharma, Arjun; Platten, Johannes; Ries, Frederic; Beckert, Hendrik; Ruf, Wolfram & Radsak, Markus P.
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Neutrophil Extracellular Traps as an Exacerbating Factor in Bacterial Pneumonia. Infection and Immunity, 90(3).
Sanders, Nathan L.; Martin, Ian M. C.; Sharma, Arjun; Jones, Matthew R.; Quinton, Lee J.; Bosmann, Markus & Mizgerd, Joseph P.
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Externalized histones fuel pulmonary fibrosis via a platelet-macrophage circuit of TGFβ1 and IL-27. Proceedings of the National Academy of Sciences, 120(40).
Riehl, Dennis R.; Sharma, Arjun; Roewe, Julian; Murke, Florian; Ruppert, Clemens; Eming, Sabine A.; Bopp, Tobias; Kleinert, Hartmut; Radsak, Markus P.; Colucci, Giuseppe; Subramaniam, Saravanan; Reinhardt, Christoph; Giebel, Bernd; Prinz, Immo; Guenther, Andreas; Strand, Dennis; Gunzer, Matthias; Waisman, Ari; Ward, Peter A.; ... & Bosmann, Markus
