OP25NLRC4-inflammasome mediated Caspase-1 activity is a novel driver of fibrogenesis in stricturing Crohn’s disease

Abstract Background During Crohn’s disease (CD), an impaired gut barrier provides an escape route for microbe-associated molecular patterns (MAMPs), like flagellin (FLA), leading to selective activation of human intestinal myofibroblasts (HIMF) producing a fibrotic extracellular matrix (ECM) 1. This leads to increased exposure of FLA with intestinal immune and non-immune cells via NLRC4 inflammasome complex, leading to intestinal inflammation. However, inflammasome expression in primary HIMFs has not been previously reported. Here, we explored the implications of the NLRC4 inflammasome and its effector, Caspase-1 (Casp-1), in intestinal fibrosis. Methods Strictured CD (CDs) specimens exhibited elevated expression of inflammasome-related genes compared with non-involved CD and NL tissues. ECM deposition assays for fibronectin (FN), collagen (Col) I and III were performed in HIMFs stimulated with NLRC4-dependent (FLA/Needle-Tox) and -independent (TLR2/6/4, NOD1) ligands. NLRC4-inflammasome formation and actin filament arrangements were assessed via immunofluorescence (IF). Casp-1 activity assay was performed with NLRC4 agonists. Casp-1 small molecule inhibitor (YVAD) and knockdown (KD) were performed in HIMFs, followed by treatment with S. typhimurium FLAWT or FLAmutant(ΔfljB/fliC) to test the profibrotic effects. Bulk RNA-seq of YVAD and Needle-tox exposed NL HIMFs was performed. The Casp-1 inhibitor YVAD was tested in the DSS-induced murine fibrosis model. Results Elevated inflammasome-related gene expression signatures, including NLRC4, AIM2, PYRIN, TLR5, and CASP1, were observed in freshly resected strictured CD specimens compared to non-involved CD and NL. scRNA-seq identified increased NLRC4 expression in HIMFs from CDs patients, confirmed by IHC and IB. Activation of NLRC4 by bacterial FLA or Needle-Tox induced intracellular NLRC4–ASC–Caspase-1 complex assembly, enhanced Casp-1 (cleaved p20) activity, increased fibronectin and collagen I/III deposition, and promoted actin filament rearrangement. RNAseq data in HIMFs revealed Casp-1–dependent activation of profibrotic pathways, implicating actin cytoskeleton and vesicle trafficking as a downstream mechanism. Pharmacologic or genetic inhibition of Casp-1, as well as blockade of actin cytoskeleton remodeling, attenuated NLRC4-dependent ECM deposition in HIMFs. In colitis mouse models, YVAD1-mediated inhibition of Casp-1 prevented inflammation and alleviated fibrosis scores in experimental fibrosis in vivo. Conclusion Bacterial flagellin (FLA/Needle-tox)–mediated NLRC4 inflammasome activation drives intestinal fibrosis through Caspase-1–dependent pathways. Targeting Caspase-1 alleviates intestinal fibrosis in vivo, highlighting a novel therapeutic avenue for stricturing Crohn’s disease. Reference: 1.Selective deletion of MyD88 signaling in α-SMA positive cells ameliorates experimental intestinal fibrosis via post-transcriptional regulation. Zhao, Shuai et al. Mucosal Immunology, Volume 13, Issue 4, 665-678 Conflict of interest: Chauhan, Gaurav: No conflict of interest Mukherjee, Pranab: No conflict of interest Tam Nguyen, Quang: No conflict of interest Zhao, Shuai: No conflict of interest Czarnecki, Doug: No conflict of interest Veisman, Ido: No conflict of interest Massey, William J: No conflict of interest Wang, Yan: No conflict of interest Chandra, Jyotsna: No conflict of interest Liu, Weiwei: No conflict of interest Banerjee, Suhanti: No conflict of interest Prasad, Ankita: No conflict of interest Qazi, Taha: No conflict of interest Abushamma, Suha: No conflict of interest West, Gail: No conflict of interest Braga Neto, Manuel: No conflict of interest Ahern, Philip: No conflict of interest Zafar, Atif: No conflict of interest Ivanov, Andrei: No conflict of interest Garantziotis, Stavros: No conflict of interest Rieder, Florian: Grant: This work is supported by the Crohn’s and Colitis Foundation grant, USA. Florian Rieder is consultant to Adiso, Adnovate, Agomab, Allergan, AbbVie, Arena, AstraZeneca, Bausch & Lomb, Boehringer-Ingelheim, Celgene/BMS, Celltrion, CDISC, Celsius, Cowen, Eugit, Ferring, Galapagos, Galmed, Genentech, Gilead, Gossamer, Granite, Guidepoint, Helmsley, Horizon Therapeutics, Image Analysis Limited, Index Pharma, Landos, Jannsen, Koutif, Mestag, Metacrine, Mirum, Mopac, Morphic, Myka Labs, Organovo, Origo, Palisade, Pfizer, Pliant, Prometheus Biosciences, Receptos, RedX, Roche, Samsung, Sanofi, Surmodics, Surrozen, Takeda, Techlab, Teva, Theravance, Thetis, Trix Bio, UCB, Ysios, and 89Bio.