DNA sensor-associated type I interferon signaling is increased in ulcerative colitis and induces JAK-dependent inflammatory cell death in colonic organoids

Abstract

DNA sensor pathways can initiate inflammasome, cell death and type I interferon (IFN) signalling in immune-mediated inflammatory diseases (IMIDs); including type I interferonopathies. We investigated the involvement of these pathways in the pathogenesis of ulcerative colitis (UC); by analysing expression of DNA sensor, inflammasome, and type I IFN biomarker genes in colonic mucosal biopsy tissue from control (n=31), inactive UC (n=31), active UC (n=33) and a UC single cell RNA-Seq dataset. The effects of type I IFN (IFN-β), IFN-γ and TNF-α on gene expression, cytokine production and cell death were investigated in human colonic organoids. In organoids treated with cytokines alone, or in combination with NLRP3, caspase or JAK inhibitors, cell death was measured, and supernatants were assayed for IL-1β/IL-18/CXCL10. The expression of DNA sensor pathway genes - PYHIN family members (AIM2, IFI16, MNDA, PYHIN1), as well as ZBP1, cGAS and DDX41 were increased in active UC and expressed in a cell type restricted pattern. Inflammasome genes (CASP1, IL1B, IL18), type I IFN inducers (STING, TBK1, IRF3), IFNB1 and type I IFN biomarker genes (OAS2, IFIT2, MX2) were also increased in active UC. Co-treatment of organoids with IFN-β or IFN-γ and TNFα increased expression of IFI16, ZBP1, CASP1, cGAS and STING, induced cell death and IL-1β/IL-18 secretion. This inflammatory cell death was blocked by the JAK inhibitor tofacitinib but not by inflammasome or caspase inhibitors. Increased type I IFN activity may drive elevated expression of DNA sensor genes and JAK-dependent but inflammasome-independent inflammatory cell death of colonic epithelial cells in UC.

Impact of Micro‐ and Nano‐Plastics on Human Intestinal Organoid‐Derived Epithelium

“…Furthermore, the viability of organoids post‐passaging relies on stem cells (LGR5+ positive cells), which are sensitive to dissociation‐induced death, along with differentiated cells that cannot form into new organoids. Improving reproducibility may involve the use of Rho‐kinase inhibitors to mitigate dissociation‐induced cell death and promoting an enriched stem cell state before seeding and differentiation (Flood et al., 2022; Pleguezuelos‐Manzano et al., 2020).…”

Human intestinal organoids: Modeling gastrointestinal physiology and immunopathology — current applications and limitations

“…(D) Organoid secretory function can be assessed by immunoassays. The spatial distribution and number of secretory cell types can be determined using lineage markers, and secretion of mucins, antimicrobial peptides, and chemokines/cytokines can also be measured [3,63,65,69]. (E) Organoid regeneration assays measure the ability of single intestinal SCs to regenerate whole organoids following dissociation [83].…”

“…Two bioactive plant-derived compounds, andrographolide, and isoliquiritigenin, could synergistically increase gene expression of the AMP human β-defensin 3 in healthy colonoids [70]. Similarly, the JAK inhibitor tofacitinib prevents type I and II IFN-induced secretion of inflammatory cytokines and chemokines from non-IBD colonoids [69].…”

“…[16,23]; D-R assays: cell death dyes Annexin V (apoptosis), propidium iodide (PI) or SYTOX nucleic acid stain (necrosis); organoid regeneration assay; proliferationbased assays, such as EdU or Ki67; cell viability (metabolic) assays, such as CellTitre-Glo (ATP), or MTT (3-(4,5-dimethylthiazol-2-yl)-2-5diphenyltetrazolium bromide)[44,61,69,74,78,79]; ROS dyes to assess oxidative stress[79].…”

HP1γ Prevents Activation of the cGAS/STING Pathway by Preserving Nuclear Envelope and Genomic Integrity in Colon Adenocarcinoma Cells

“…Importantly, studies have shown increased expression of DNA sensors in the colon mucosa biopsy tissues of UC patients, with single-cell RNA sequencing data pointing to an increase in cGAS expression in both active and non-active UC colon epithelia [9,10]. These findings strongly suggest that a chronic or aberrant activation of DNA sensors contributes to the inflammation seen in IBD [11,12]. Yet, the mechanisms altering DNA homeostasis and triggering activation of DNA sensors in IBD remain unclear.…”