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A5813 - Epithelial Cell Injury Causes Unjamming of the Cell Layer
Author Block: M. J. O'Sullivan, J. Mitchel, C. Rhodes, Z. Nagel, J. Park; Dept of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, United States.
Rationale: Airway epithelial cells play an important role in the development of asthma, a disease for which there is no cure. In asthmatic airways, the epithelium is known to be dysfunctional, lacking proper repair processes and contributing to both structural remodeling and inflammation. In our previous report (Park et al., Nat Mater. 2015) using well-differentiated primary human bronchial epithelial cells (HBECs), we found that HBECs exhibit distinct biophysical phenotypes depending on the maturation and injury status. When cells are mature, HBECs are jammed, in which the cells are caged by their neighbors and immobile. When cells are mechanically compressed to mimic the effect of asthmatic bronchoconstriction, these cells become unjammed, in which the cells freely rearrange with their neighbors and are mobile. This unjamming behavior is associated with asthma. However, mechanisms underlying the unjamming transition is unknown. Here, we explore the mechanistic link between epithelial cell injury and unjamming.
Methods: HBECs were cultured at an air-liquid interface (ALI). On ALI days 7, 10, and 14, HBECs were exposed to ionizing x-rays (Rad Source) at 0.05, 0.5 and 5 Gy to induce injury. HBECs were pretreated with a TGFβR1 inhibitor, SB 431542, for one hour before each radiation exposure. Unjamming was determined by the analysis of live-cell imaging following the last radiation exposure. HBEC injury was evaluated by immunofluorescent staining and by western blotting for γH2A.X, a marker of nuclear damage. To determine the impact of HBEC injury on the degree of differentiation, fixed cells were examined by immunofluorescent staining for p63α (basal cells), MUC5AC (goblet cells), and FOXJ1 (ciliated cells). Trans-epithelial electrical resistance (TEER) measurements were performed to assess barrier function.
Results: Repeated radiation exposure induced unjamming of HBECs in a dose dependent manner. Pre-treatment of HBECs with 10μM SB 431542 partially reduced the unjamming caused by irradiation. Repeated radiation exposure increased γH2A.X protein. Furthermore, immunofluorescence staining demonstrated increased and condensed staining of γH2A.X in the nuclei. However, repeated radiation exposure had no effect on the differentiation of HBECs nor did it alter the barrier function as measured by TEER.
Conclusion: Our data demonstrate that ionizing radiation exposure causes epithelial cell unjamming accompanied by cell injury which depends partially on TGFβ signaling. Our findings support the idea that injured epithelium exhibits a distinct physical phenotype, which is known as unjamming, and this should be investigated further for a comprehensive understanding of injured epithelium and its role in chronic lung disease.