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A7683 - Extracellular Histone H3.3 Damages Lung Tissue and Induce a Systemic Inflammatory Response
Author Block: C. A. Barrero1, M. C. Rico1, B. Kosmider2, O. M. Perez-Lea1, C. Fecchio1, V. Olusajo1, N. Vij3, Y. Thanavala4, K. Bahmed2, M. F. Barbe5, G. J. Criner2; 1Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA, United States, 2Thoracic Medicine and Surgery, Temple University Lewis Katz School of Medicine, Philadelphia, PA, United States, 3Pediatrics, Johns Hopkins University, Mount Pleasant, MI, United States, 4Immunology, Roswell Park Cancer Institute, Buffalo, NY, United States, 5Anatomy and Cell Biology, Temple University Lewis Katz School of Medicine, Philadelphia, PA, United States.
Background: Abundant extracellular levels of histones are considered detrimental to cellular function and survival. We characterized the harmful effects of extracellular histone H3.3 and its acetylated form (AcH3.3) in chronic obstructive pulmonary disease (COPD). In this work, we demonstrated that AcH3.3 accumulates in response to cigarette smoke (CS) in vitro and in vivo. Furthermore, we developed an animal model using C57/BL6J mice to study the in vivo AcH3.3 effects on lung tissue and the mechanisms of its toxicity.
Methods: The effect of CS on the AcH33 accumulation was evaluated in vitro and in vivo. Lung cells and genetically modified cells expressing H33-RFP were incubated with CS condensate. AcH3.3 was detected by Western blot and high-content image analysis. C57/BL6J mice were exposed to CS for 1, 2 and 4 months. Histone lung toxicity was tested in vivo. Mice were intratracheally instilled with recombinant H3.3 (rH3.3) and its hyperacetylated form (rAcH3.3); histone 2B (rH2B) and vehicle (PBS) were used as controls. After 48 hours mice lung’s and blood were collected for structure and cell viability evaluation by histopathology, micro-CT, immunohistochemistry, proteomics and cytokine profile. Results: We observed AcH3.3 accumulation in both cells treated with CS condensate and in mice exposed to CS in a dose-dependent manner. Mice intratracheal instillation with rH3.3 and rAcH3.3 induce lung tissue damage with disruption of alveolar cell walls and immune cells infiltration. rAcH3.3 produced a significant decrease in the ratio of lung tissue volume as compared to the total lung volume by micro-CT; and an increase in apoptotic cells at the lung parenchyma. Extracellular histone exposure induced acute inflammation and high plasma concentrations of pro-inflammatory mediators. Proteome changes induced by AcH3.3 in the lung tissue evidence an increase in the proteins of specific acute response inflammatory pathways, implicated in the mechanism of the lung tissue damage. Conclusion: AcH3.3 accumulate in lung cells in response to CS. The effects in lung pathology, pro-inflammatory proteins, and cytokine levels were more pronounced in animals instilled with rAcH3.3, suggesting that the acetylation of H3.3 induces more cytotoxicity and robust immune response. These results demonstrate a crucial role of extracellular H3.3 and its acetylated form inducing lung cell death and acute inflammatory response. The activation of specific pathways by AcH3.3 contribute to the destruction of lung structure observed in the mouse model and resemble lung features of COPD pathology.