Shuyu Hao1, Erica A Roesch2, Aura Perez1, Rebecca L Weiner2, Leigh C Henderson1, Linda Cummings3, Paul Consiglio1, Joseph Pajka1, Amy Eisenberg1, Lauren Yeh1, Calvin U Cotton4, Mitchell L Drumm5. 1. Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH 44106, United States of America. 2. Department of Pediatrics, Case Western Reserve University, Cleveland, OH 44106, United States of America. 3. Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, United States of America. 4. Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH 44106, United States of America; Department of Pediatrics, Case Western Reserve University, Cleveland, OH 44106, United States of America. 5. Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH 44106, United States of America; Department of Pediatrics, Case Western Reserve University, Cleveland, OH 44106, United States of America. Electronic address: mxd34@case.edu.
Abstract
BACKGROUND: Individuals with cystic fibrosis (CF) experience elevated inflammation in multiple organs, but whether this reflects an inherent feature of CF cells or is a consequence of a pro-inflammatory environment is not clear. METHOD: Using CRISPR/Cas9-mediated mutagenesis of CFTR, 17 subclonal cell lines were generated from Caco-2 cells. Clonal lines with functional CFTR (CFTR+) were compared to those without (CFTR-) to directly address the role of CFTR in inflammatory gene regulation. RESULTS: All lines maintained CFTR mRNA production and formation of tight junctions. CFTR+ lines displayed short circuit currents in response to forskolin, while the CFTR- lines did not. Baseline expression of cytokines IL6 and CXCL8 (IL8) was not different between the lines regardless of CFTR genotype. All lines responded to TNFα and IL1β by increasing IL6 and CXCL8 mRNA levels, but the CFTR- lines produced more CXCL8 mRNA than the CFTR+ lines. Transcriptomes of 6 CFTR- and 6 CFTR+ lines, before and after stimulation by TNFα, were compared for differential expression as a function of CFTR genotype. While some genes appeared to be differentially expressed simply because of CFTR's absence, others required stimulation for differences to be apparent. CONCLUSION: Together, these data suggest cells respond to CFTR's absence by modulating transcriptional networks, some of which are only apparent when cells are exposed to different environmental contexts, such as inflammation. With regards to inflammation, these data suggest a model in which CFTR's absence leads to a poised, pro-inflammatory state of cells that is only revealed by stimulation.
BACKGROUND: Individuals with cystic fibrosis (CF) experience elevated inflammation in multiple organs, but whether this reflects an inherent feature of CF cells or is a consequence of a pro-inflammatory environment is not clear. METHOD: Using CRISPR/Cas9-mediated mutagenesis of CFTR, 17 subclonal cell lines were generated from Caco-2 cells. Clonal lines with functional CFTR (CFTR+) were compared to those without (CFTR-) to directly address the role of CFTR in inflammatory gene regulation. RESULTS: All lines maintained CFTR mRNA production and formation of tight junctions. CFTR+ lines displayed short circuit currents in response to forskolin, while the CFTR- lines did not. Baseline expression of cytokines IL6 and CXCL8 (IL8) was not different between the lines regardless of CFTR genotype. All lines responded to TNFα and IL1β by increasing IL6 and CXCL8 mRNA levels, but the CFTR- lines produced more CXCL8 mRNA than the CFTR+ lines. Transcriptomes of 6 CFTR- and 6 CFTR+ lines, before and after stimulation by TNFα, were compared for differential expression as a function of CFTR genotype. While some genes appeared to be differentially expressed simply because of CFTR's absence, others required stimulation for differences to be apparent. CONCLUSION: Together, these data suggest cells respond to CFTR's absence by modulating transcriptional networks, some of which are only apparent when cells are exposed to different environmental contexts, such as inflammation. With regards to inflammation, these data suggest a model in which CFTR's absence leads to a poised, pro-inflammatory state of cells that is only revealed by stimulation.
Authors: Katherine M Antosca; Diana A Chernikova; Courtney E Price; Kathryn L Ruoff; Kewei Li; Margaret F Guill; Natalie R Sontag; Hilary G Morrison; Shuyu Hao; Mitchell L Drumm; Todd A MacKenzie; Dana B Dorman; Lynn M Feenan; Molly A Williams; John Dessaint; Irene H Yuan; Brian J Aldrich; Lisa A Moulton; Lily Ting; Ana Martinez-Del Campo; Edward J Stewart; Margaret R Karagas; George A O'Toole; Juliette C Madan Journal: J Bacteriol Date: 2019-07-24 Impact factor: 3.490
Authors: Cameron B Morrison; Kendall M Shaffer; Kenza C Araba; Matthew R Markovetz; Jason A Wykoff; Nancy L Quinney; Shuyu Hao; Martial F Delion; Alexis L Flen; Lisa C Morton; Jimmy Liao; David B Hill; Mitchell L Drumm; Wanda K O'Neal; Mehmet Kesimer; Martina Gentzsch; Camille Ehre Journal: Eur Respir J Date: 2022-02-03 Impact factor: 16.671