Naturally occurring short splice variant of CYLD positively regulates dendritic cell function

Abstract

Deubiquitination of NF-B members by IntroductionCYLD is a tumor suppressor gene that is mutated in familial cylindromatosis, an autosomal dominant predisposition to tumors of skin appendages. 1 CYLD removes lysine-63 polyubiquitin chains from distinct members of the NF-B pathway 2-4 and mutations in CYLD dysregulate NF-B activity.Complete deletion of CYLD in mice (CYLD ko ) renders them susceptible to skin tumors, 5 but the CYLD ko mice do not display alterations of the immune system, as we could show for B-6 and T-cell development (A.W., N.H., S. Reissig, manuscript in preparation). Other knockout mouse models of CYLD point to a role for flCYLD in immunity, including hyperinduction of IFN␣ in virus-infected dendritic cells (DCs) 7 as well as protection from infections in its absence. 8 However, mice exclusively expressing the naturally occurring short splice variant of CYLD, sCYLD, are characterized by lymphomegaly, splenomegaly, and dramatic increases in B-cell numbers 6 caused by aberrant NF-B signaling. Because this pathway is important for the function of DCs, 9 which orchestrate innate and adaptive immune responses, we investigated the effect of sCYLD overexpression on DC function. We found CYLD ex7/8 DCs to be hyperresponsive to LPS, capable of inducing superior T-cell expansion on DC immunization in vivo, and able to suppress tolerance induced by ␣-DEC-205:OVA administration. As a molecular basis for this phenotype, we identified increased nuclear Bcl-3, p50, and p65 in resting and stimulated CYLD ex7/8 bone marrowderived DCs (BMDCs). Methods Mice and BMDC generationC57BL/6 wild-type (WT), CYLD ko , 5 and CYLD ex7/8 6 mice 6 to 8 weeks old were used as recipients and for BMDC generation as previously described. 10 St42 TCR Tg mice have been previously described. 11 OT-I mice 12 were obtained from Christian Kurts (Institute for Molecular Medicine & Experimental Immunology, Bonn, Germany). Approval for these studies was obtained from the review board of the Federal State of Rhineland-Palatinate, Germany. All animal experiments were in accordance with the guidelines of the Central Animal Facility Institution of the University of Mainz. Flow cytometryFACSCanto cytometer and FlowJo software (Tree Star, Ashland, OR) using ␣-CD8 and ␣-CD90.1/CD45.1 (eBioscience, San Diego, CA) was used. ␣-IL-6, IL-10, and TNF-␣ antibodies from Cytometric Bead Array Flex system (Becton Dickinson, Franklin Lakes, NJ) were used and analyzed with FCAP Array Software (BD Biosciences, Mountain View, CA). Immune toleranceRecipient mice were given 10 6 adoptively transferred OT-I cells, immunized with 20 g DEC-205:OVA, and boosted with 50 g OVA protein (Sigma-Aldrich, St Louis, MO) as previously described. 13Western blot analysis and electrophoretic mobility shift assay Western blot analysis was performed as previously described. 8 Nuclear and cytoplasmic fractions were prepared according to standard procedures. 14,15 NF-B Consensus Oligonucleotide was purchased from For personal use only. on May 7, 2018. by guest www.bloodjournal.org ...

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells

“…A splicing variant of CYLD which lacks the TRAF2 and IKKγ binding sites increases basal rather than anti-IgM-induced NF-κB activity and enhances LPS-induced MAPK p38 activation in B cells [49]. Moreover, this shorter CYLD (sCYLD) enhances NF-κB activity accompanied by an increased nuclear translocation of the IκB molecule Bcl-3, along with nuclear p50 and p65 leading to a pro-inflammatory phenotype in dendritic cells [50]. Considering the similarities between these previous finding and ours, such as the increased basal rather induced NF-κB activity or the enhanced p38 activity in both Cyld deficient VSMCs and Cyld deficient with sCyld overexpressing B or dendritic cells, it is possible that sCYLD is responsible for pro-inflammatory activation of VSMCs whereas full-length CYLD is a negative regulator of inflammatory responses in diverse organ systems including vasculature.…”

The Tumor Suppressor CYLD Controls the Function of Murine Regulatory T Cells

“…Indeed, Treg-specific CTLA-4 deficiency impairs the Treg-mediated downregulation of CD80 and CD86 expression on dendritic cells, resulting in spontaneous development of systemic lymphoproliferation and fatal T cell-mediated autoimmune disease (23). Of note, also in CYLD ex7/8 mice, dendritic cells displayed a hyperactivated phenotype accompanied by high expression of CD80 and CD86 (22), pointing to the suppressive dysfunction of CYLD ex7/8 Tregs. In summary, our results indicate that sCYLD promotes via NFkB activation thymic-Treg lineage differentiation.…”

“…The NF-kB molecule Bcl-3 has previously been shown to interact with both FL-CYLD and sCYLD and thereby modulate NFkB activity (21,22,34). Hence, we examined the expression of Bcl-3 in CYLD ex7/8 T cells and detected increased levels of Bcl-3 in thymocytes, CD4…”

“…CYLD ex7/8 mice display enlarged secondary lymphoid organs due to the accumulation of mature B cells as a result of enhanced survival and activation of NF-kB signaling. Moreover, bone marrowderived dendritic cells generated from CYLD ex7/8 mice show a hyperactive phenotype in vitro and in vivo, correlating with increased nuclear translocation of Bcl-3 (22).…”

Tumor Suppressor Cylindromatosis (CYLD) Controls HIV Transcription in an NF-κB-Dependent Manner

“…Since CYLD is a lysine 63-linked deubiquitinase, it induces and suppresses different signaling pathways by specifically removing lysine 63-linked ubiquitin moieties (8,14). Many of these pathways regulate the development (e.g., natural killer cells) and homeostasis (e.g., T cells and dendritic cells [DCs]) of the immune system as well as the quality of the immune responses mounted to different pathogens (15)(16)(17)(18)(19). For example, CYLD inhibits type I interferon signaling by deubiquitinating the pattern recognition receptor RIG-I and downregulating Toll-like receptor 2 (TLR2) signaling (20)(21)(22).…”