Inflammation and pyroptosis mediate muscle expansion in an IL-1β -dependent manner Subhash Haldar 1 , Christopher Dru 1 , Diptiman Choudhury 1,2 , Rajeev Mishra 1 , Ana Fernandez 1,2 , Shea Biondi 1,2 , Zhenqiu Liu 1 , Kenichi Shimada 3 , Moshe Arditi 3 , Neil Bhowmick 1,2, * 1 Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA. 2 Greater Los Angeles Veterans Administration, Los Angeles, CA. 3 Department of Pediatrics, Cedars-Sinai Medical Center, Los Angeles, CA Running Title: DNA damage induced cell death results in adjacent muscle expansion. * Corresponding author: Neil A. Bhowmick, Ph.D., Department of Medicine, Cedars-Sinai Medical Center 8750 Beverly Blvd., Atrium 103, Los Angeles, CA 90048. Tel: (310) 423-5992, Fax: (310) 423-8543 Email: bhowmickn@cshs.org Keywords: bladder inflammation, pyroptosis, hypertrophy, hyperplasia Background: Hyperplasia is a common phenomenon in inflamed muscle. Results: IL-1β secreted associated with pyroptotic cell death mediate IGF1-dependent detrusor expansion. Conclusion: Bladder muscle hyperplasia resulting from NLRP3 inflammatory cascade can be attenuated by neutralization of IL-1β and IGF1. Significance: Antagonizing IL-1β can have therapeutic benefit for subjects with muscle hyperplasia resulting from chronic inflammatory diseases. ABSTRACT Muscle inflammation is often associated with its expansion. Bladder smooth muscle inflammation- induced cell death is accompanied by hyperplasia and hypertrophy, as the primary cause for poor bladder function. In mice, DNA damage initiated by chemotherapeutic drug cyclophosphamide, activated caspase 1 through the formation of the NLRP3 complex resulting in detrusor hyperplasia. Cyclophosphamide metabolite, acrolein, caused global DNA methylation and accumulation of DNA damage in a mouse model of bladder inflammation and in cultured bladder muscle cells. In correlation, global DNA methylation and NLRP3 expression was up regulated in human chronic bladder inflammatory tissues. The epigenetic silencing of DNA damage repair gene, Ogg1, could be reversed by the use of demethylating agents. In mice, demethylating agents reversed cyclophosphamide- induced bladder inflammation and detrusor expansion. The transgenic knockout of Ogg1 in as few as 10% of the detrusor cells tripled the proliferation of the remaining wild type counterparts in an in vitro co- culture titration experiment. Antagonizing IL-1β with Anakinra, a rheumatoid arthritis therapeutic, prevented detrusor proliferation in conditioned media experiments as well as in a mouse model of bladder inflammation. Radiation treatment validated the role of DNA damage in the NLRP3-assocaited caspase 1- mediated IL-1β secretory phenotype. A protein array analysis identified IGF1 to be downstream of IL-1β signaling. IL-1β induced detrusor proliferation and hypertrophy could be reversed with the use of Anakinra as well as an IGF1 neutralizing antibody. IL- 1β antagonists in current clinical practice can exploit the revealed mechanism for DNA damage-mediated muscular expansion. INTRODUCTION Muscle inflammation and associated expansion is well recognized in multiple tissues with significant morbidity and mortality. Bladder inflammation is a model muscular tissue subjected to inflammation-associated expansion. Fortunately bladder cystitis is normally self limiting, however it can endure in some patients (1). Conditions such as trauma, microbial, and non-microbial activators can contribute to inflammation. In the United States alone up to 7.9 million women have inflammation-associated bladder function impairment impacting the quality of life. Urothelial changes are striking in bladder inflammation, associated with denudation and ulceration (2). However, the lamina propria and detrusor muscle respond to inflammatory signals in controlling bladder compliance (3). The multi-factorial physiological response to inflammation involves epigenetic mechanisms associated DNA and histone methylation, associated with the http://www.jbc.org/cgi/doi/10.1074/jbc.M114.617886 The latest version is at JBC Papers in Press. Published on January 16, 2015 as Manuscript M114.617886 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on March 4, 2015 http://www.jbc.org/ Downloaded from