The deubiquitinase CYLD controls protective immunity against helminth infection by regulation of Treg cell plasticity.

BACKGROUND: Type 2 immunity can be modulated by regulatory T (Treg) cell activity. It has been suggested that the deubiquitinase cylindromatosis (CYLD) plays a role in the development or function of Treg cells, implying that it could be important for normal protective immunity, where type 2 responses are prevalent. OBJECTIVE: We sought to investigate the role of CYLD in Treg cell function and TH2 cell immune responses under steady-state conditions and during helminth infection. METHODS: Foxp3-restricted CYLD conditional knockout (KO) mice were examined in mouse models of allergen-induced airway inflammation and Nippostrongylus brasiliensis infection. We performed multiplex magnetic bead assays, flow cytometry, and quantitative PCR to understand how a lack of CYLD affected cytokine production, homing, and suppression in Treg cells. Target genes regulated by CYLD were identified and validated by microarray analysis, coimmunoprecipitation, short hairpin RNA knockdown, and transfection assays. RESULTS: Treg cell-specific CYLD KO mice showed severe spontaneous pulmonary inflammation with increased migration of Treg cells into the lung. CYLD-deficient Treg cells furthermore produced high levels of IL-4 and failed to suppress allergen-induced lung inflammation. Supporting this, the conditional KO mice displayed enhanced protection against N brasiliensis infection by contributing to type 2 immunity. Treg cell conversion into IL-4-producing cells was due to augmented mitogen-activated protein kinase and nuclear factor κB signaling. Moreover, Scinderin, a member of the actin-binding gelsolin family, was highly upregulated in CYLD-deficient Treg cells, and controlled IL-4 production through forming complexes with mitogen-activated protein kinase kinase/extracellular receptor kinase. Correspondingly, both excessive IL-4 production in vivo and the protective role of CYLD-deficient Treg cells against N brasiliensis were reversed by Scinderin ablation. CONCLUSIONS: Our findings indicate that CYLD controls type 2 immune responses by regulating Treg cell conversion into TH2 cell-like effector cells, which potentiates parasite resistance.

Molecular cloning and functional analysis of deubiquitinase CYLD in rainbow trout, Oncorhynchus mykiss.

Deubiquitinase cylindromatosis (CYLD) inhibits MAPK and NF-κB activation pathways by deubiquitinating upstream regulatory factors. Although CYLD has been identified and actively studied in mammals, nothing is known about its putative function in fish. In this study, we identified the gene encoding CYLD (OmCYLD) from rainbow trout, Oncorhynchus mykiss, and examined its role during pathogenic infections. The deduced amino acid sequence of OmCYLD contains conserved CAP-Gly and USP domains. In RTH-149 cells, the expression of OmCYLD was increased by stimulation with Edwardsiella tarda and Streptococcus iniae. Gain-of-function and loss-of-function experiments showed that OmCYLD down-regulates the activation of MAPK and NF-κB and the expression of pro-inflammatory cytokines in E. tarda-stimulated RTH-149 cells. These findings suggest that OmCYLD might function like those of mammals to negatively regulate bacteria-triggered signaling pathway in fish.

Prion protein is required for tumor necrosis factor α (TNFα)-triggered nuclear factor κB (NF-κB) signaling and cytokine production.

The expression of normal cellular prion protein (PrP) is required for the pathogenesis of prion diseases. However, the physiological functions of PrP remain ambiguous. Here, we identified PrP as being critical for tumor necrosis factor (TNF) α-triggered signaling in a human melanoma cell line, M2, and a pancreatic ductal cell adenocarcinoma cell line, BxPC-3. In M2 cells, TNFα up-regulates the expression of p-IκB-kinase α/ß (p-IKKα/ß), p-p65, and p-JNK, but down-regulates the IκBα protein, all of which are downstream signaling intermediates in the TNF receptor signaling cascade. When PRNP is deleted in M2 cells, the effects of TNFα are no longer detectable. More importantly, p-p65 and p-JNK responses are restored when PRNP is reintroduced into the PRNP null cells. TNFα also activates NF-κB and increases TNFα production in wild-type M2 cells, but not in PrP-null M2 cells. Similar results are obtained in the BxPC-3 cells. Moreover, TNFα activation of NF-κB requires ubiquitination of receptor-interacting serine/threonine kinase 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). TNFα treatment increases the binding between PrP and the deubiquitinase tumor suppressor cylindromatosis (CYLD), in these treated cells, binding of CYLD to RIP1 and TRAF2 is reduced. We conclude that PrP traps CYLD, preventing it from binding and deubiquitinating RIP1 and TRAF2. Our findings reveal that PrP enhances the responses to TNFα, promoting proinflammatory cytokine production, which may contribute to inflammation and tumorigenesis.

TNF phase III signalling in tolerant cells is tightly controlled by A20 and CYLD.

Following the acute phase of an inflammatory reaction, a strictly controlled resolution of inflammation is necessary. A dysregulation of this process leads to hyperinflammation, chronic inflammatory disease, or immune paralysis. Different mechanisms participate in the coordinated termination of the inflammatory process, e.g. the expression of antiinflammatory molecules and different forms of tolerance. To better understand the processes which mediate resolution of TNF-dependent inflammation and induce tolerance, it is necessary to characterize the signal transduction quality during TNF long-term (pre)incubation. Within a time frame from 12 to 48h, designated as phase III of the TNF response, we measured an ongoing, constitutive activation of TNFR1/NF-κB-dependent pathways in monocytic cells. Phase III signalling which was also named "constitutive signaling in TNF tolerant cells" induces the expression of low- and high-sensitive target genes including A20 which is differentially regulated by transcriptional and proteolytic events. A20 strictly controls TNF long-term constitutive signalling in an IκB kinase complex- and partially RIP-dependent manner supported by adjuvant ABIN1. In addition, CYLD proteins participate in the regulation of this late-phase signal transduction, whereas downstream molecules such as Bcl3 and p50 are not involved. A20 and CYLD are expressed with different mRNA kinetics resulting in a strong or only a modest increase in protein levels, respectively. The identification of mechanisms which contribute to the termination of inflammation will provide additional diagnostic and therapeutic aspects to specifically diagnose certain aspects of inflammation and specifically modulate them.