cIAP1/2 antagonization by SMAC mimetic induces non‐canonical NF‐κB mediated T _H 17 cell homotypic interactions and increases their resistance to shear stress

SMAC antagonization of cIAP1/2 in TH17 cells upregulates cell adhesion and cytoskeleton genes through the NIK-RelB p52 axis. also increases homotypic interactions a non-canonical NF-κB- integrin-mediated mechanism resulting increased ability to withstand shear stress. T helper-17 (TH17) express transcription factor RORγt, produce IL-17 IL-22, are required for defense against extracellular pathogens sustain integrity epithelial barrier surfaces [1]. However, unrestrained responses have been implicated autoimmune disease pathogenesis [2, 3]. A network factors control differentiation, including RORγt [4] members NF-κB family [5]. The canonical pathway is necessary initiate activation [6], while critical balancing TH lineage decisions [7]. Activation depends on stabilization inducing Kinase (NIK), which regulated by E3 ubiquitin ligases cIAP1 cIAP2 [8]. constitutively induce degradative K48 ubiquitination NIK suppress signaling [9], however, dissociates from cIAP1/2, following receptor ligation [8, 9], turn leads nuclear translocation RelB activity stability IAP endogenously mitochondrial protein (second mitochondria-derived activator caspases) [10]. Likewise, mimetics (SM) class small chemical molecules that can either inhibit or degradation IAPs [10] be utilized modulate balance pathways activate ligand-receptor independent manner [9]. We previously reported SM-treated showed an abundance proteins associated with organization To further investigate this, we retrieved annotated reactome database as part integrin (R-HSA-354192), surface (R-HSA-216083) semaphorin interaction (R-HSA-373755) compared their expression during differentiation without SM. found induced upregulation such Vcam1, Icam, Madcam1 Itgae (Supporting Information Fig. S1A), interacting Nrp1, Plxnc1 Plxnd1 S1B) involved remodeling; Marcksl1, Fscn1, Ermn, Nebl S1C). Flow cytometry validated SM-induced some these genes; NRP1 (Nrp1), CD103 (Itgae), Vcam1 S2A B), molecule mostly stromal rather than hematopoietic cells. test if cytoskeletal NF-κB, differentiated CD4-Cre+- NIKF/F (NIK-deficient) CD4-Cre−-NIKF/F (NIK-sufficient) CD4+ toward presence absence SM, measured gene qPCR. SM upregulated Itgae, Fscn1 NIK-sufficient but not NIK-deficient S2C). This indicated was responsible differential expression. Moreover, ectopically expressed using retroviral vectors S2D) RelB, lesser extent p52, detected under conditions S2D). Furthermore, significantly expression, Marcksl1 Ermn could alone prompted us follow morphological changes formation larger clusters, DMSO controls (Fig. 1A C), suggesting enhanced interactions. Since molecules, investigated cluster In NIK, did enhance formation, indicating requirement 1D E). number Fig S1A-C) [7] were included integrins (Itgb1, Itgae), receptors (Madcam1, Icam1, Vcam1), use co-receptors (Plxnc1) often co-regulated (Fscn1, Marcksl1). assess role mediating interactions, used Arginine-Glycine-Aspartate (RGD) synthetic peptide, competes ligands binding thus inhibitor. RGD treatment inhibited clusters 1B implicating enhancement failed rescue reduction IL-17A production S4A). Treg however affect S4B-C) inhibition Foxp3 S4D). suggested might alter surrounding microenvironment. designed 3D-printed flow chambers, upon Matrigel coating, model stress 2A B; Supporting Video 1). controls, retained more chamber exposure shearing force about 2.6 dyn/cm2 2C D; S5). demonstrated may affinity adhere engage cell-to-cell tissue extravasation. It plausible switching off regulatory determines strength duration this report show induces related genes, particularly integrin-interacting molecules. provide evidence dependent p52. resulted cognate signaling. Finally, novel vitro method mimic adhesion, Th17 ECM, therefore, resisting higher thank Professor Ari Waisman (Institute Molecular Medicine, University Medical Center Johannes Gutenberg, Mainz, Germany) providing organs CD4CRE- mice. work V.B. supported Lundbeck Foundation grant R163-2013-15201. J.R. R.A. DTU PhD. scholarships. M.D. Innovation Fund Denmark (IFD) 8090-00009B Organovir 812673. authors declare no commercial financial conflict interests. peer review history article available at https://publons.com/publon/10.1002/eji.202048983 data support findings study openly ArrayExpress https://www.ebi.ac.uk/arrayexpress/, reference E-MTAB-7894. Please note: publisher content functionality any supporting information supplied authors. Any queries (other missing content) should directed corresponding author article.