Gene coexpression networks reveal a broad role for lncRNAs in inflammatory bowel disease
John L. Johnson, Davit Sargsyan, Eric M. Neiman, Amy Hart, Aleksandar Stojmirovic, Roman Kosoy, Haritz Irizar, Mayte Suárez-Fariñas, Won-Min Song, Carmen Argmann, Stefan Avey, Liraz Shmuel-Galia, Tim Vierbuchen, Gerold Bongers, Yu Sun, Leonard Edelstein, Jacqueline Perrigoue, Jennifer E. Towne, Aisling O’Hara Hall, Katherine A. Fitzgerald, Kasper Hoebe
John L. Johnson, Davit Sargsyan, Eric M. Neiman, Amy Hart, Aleksandar Stojmirovic, Roman Kosoy, Haritz Irizar, Mayte Suárez-Fariñas, Won-Min Song, Carmen Argmann, Stefan Avey, Liraz Shmuel-Galia, Tim Vierbuchen, Gerold Bongers, Yu Sun, Leonard Edelstein, Jacqueline Perrigoue, Jennifer E. Towne, Aisling O’Hara Hall, Katherine A. Fitzgerald, Kasper Hoebe
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Resource and Technical Advance Gastroenterology Immunology

Gene coexpression networks reveal a broad role for lncRNAs in inflammatory bowel disease

Abstract

The role of long noncoding RNAs (lncRNAs) in disease is incompletely understood, but their regulation of inflammation is increasingly appreciated. We addressed the extent of lncRNA involvement in inflammatory bowel disease (IBD) using biopsy-derived RNA-sequencing data from a large cohort of deeply phenotyped patients with IBD. Weighted gene correlation network analysis revealed gene modules of lncRNAs coexpressed with protein-coding genes enriched for biological pathways, correlated with epithelial and immune cell signatures, or correlated with distal colon expression. Correlation of modules with clinical features uncovered a module correlated with disease severity, with an enriched interferon response signature containing the hub lncRNA IRF1-AS1. Connecting genes to IBD-associated single nucleotide polymorphisms (SNPs) revealed an enrichment of SNP-adjacent lncRNAs in biologically relevant modules. Ulcerative colitis–specific SNPs were enriched in distal colon–related modules, suggesting that disease-specific mechanisms may result from altered lncRNA expression. The function of the IBD-associated SNP-adjacent lncRNA IRF1-AS1 was explored in human myeloid cells, and our results suggested IRF1-AS1 promoted optimal production of TNF-α, IL-6, and IL-23. A CRISPR/Cas9-mediated activation screen in THP-1 cells revealed several lncRNAs that modulated LPS-induced TNF-α responses. Overall, this study uncovered the expression patterns of lncRNAs in IBD that identify functional, disease-relevant lncRNAs.

Authors

John L. Johnson, Davit Sargsyan, Eric M. Neiman, Amy Hart, Aleksandar Stojmirovic, Roman Kosoy, Haritz Irizar, Mayte Suárez-Fariñas, Won-Min Song, Carmen Argmann, Stefan Avey, Liraz Shmuel-Galia, Tim Vierbuchen, Gerold Bongers, Yu Sun, Leonard Edelstein, Jacqueline Perrigoue, Jennifer E. Towne, Aisling O’Hara Hall, Katherine A. Fitzgerald, Kasper Hoebe

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Figure 9

IRF1-AS1 promotes inflammatory cytokine production.

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IRF1-AS1 promotes inflammatory cytokine production. (A) Expression of I...
(A) Expression of IRF1 and IRF1-AS1 in colon samples of the MSCCR data set with calculated linear regression and Pearson correlation. (B) MDMs and MoDCs were stimulated with LPS or LPS + IFN-γ for 6 hours, and the expression of IRF1-AS1 and IRF1 was determined by quantitative PCR (qPCR) and compared with unstimulated samples. Data shown are from 2 experiments (1 for MDMs and 1 for MoDCs). (C) RNA extracted from MoDCs stimulated with LPS for 6 hours was converted to cDNA and sequenced with an Oxford Nanopore device. Stringtie of resulting sequences was used to determine the transcript sequences of IRF1-AS1 (left). TPM levels were also calculated (right). (D) The transcript sequences of IRF1-AS1 from Nanopore sequencing was used to design 3 highly specific ASOs (216, 12678, and 34667) along with MALAT1 and NTC ASOs as controls. MDMs and MoDCs were differentiated from monocytes for 5 days before adding ASOs with RNAiMax for 48 hours. Cells were then stimulated for 6 hours with LPS before measuring IRF1-AS1, IRF1, and MALAT1 expression by qPCR (n = 9, n = 8, and n = 10, respectively, across 3 experiments). (E) MDMs and MoDCs were differentiated from monocytes for 5 days before adding ASOs with RNAiMax for 48 hours. Cells were then stimulated for 6 hours with LPS before collecting supernatants from the cultures and measuring cytokine concentrations using Meso Scale Discovery multiplex cytokine ELISAs. Two experiments were performed on MDMs (n = 2 and n = 3), and 2 experiments were performed with MoDCs (n = 4 and n = 3); the results were combined for a total of 12 samples. Statistical significance for cytokine concentrations was determined using a 1-way ANOVA in GraphPad Prism. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data represent mean ± SEM (B, D, and E).