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      CYLD Enhances Severe Listeriosis by Impairing IL-6/STAT3-Dependent Fibrin Production

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          Abstract

          The facultative intracellular bacterium Listeria monocytogenes (Lm) may cause severe infection in humans and livestock. Control of acute listeriosis is primarily dependent on innate immune responses, which are strongly regulated by NF-κB, and tissue protective factors including fibrin. However, molecular pathways connecting NF-κB and fibrin production are poorly described. Here, we investigated whether the deubiquitinating enzyme CYLD, which is an inhibitor of NF-κB-dependent immune responses, regulated these protective host responses in murine listeriosis. Upon high dose systemic infection, all C57BL/6 Cyld −/− mice survived, whereas 100% of wildtype mice succumbed due to severe liver pathology with impaired pathogen control and hemorrhage within 6 days. Upon in vitro infection with Lm, CYLD reduced NF-κB-dependent production of reactive oxygen species, interleukin (IL)-6 secretion, and control of bacteria in macrophages. Furthermore, Western blot analyses showed that CYLD impaired STAT3-dependent fibrin production in cultivated hepatocytes. Immunoprecipitation experiments revealed that CYLD interacted with STAT3 in the cytoplasm and strongly reduced K63-ubiquitination of STAT3 in IL-6 stimulated hepatocytes. In addition, CYLD diminished IL-6-induced STAT3 activity by reducing nuclear accumulation of phosphorylated STAT3. In vivo, CYLD also reduced hepatic STAT3 K63-ubiquitination and activation, NF-κB activation, IL-6 and NOX2 mRNA production as well as fibrin production in murine listeriosis. In vivo neutralization of IL-6 by anti-IL-6 antibody, STAT3 by siRNA, and fibrin by warfarin treatment, respectively, demonstrated that IL-6-induced, STAT3-mediated fibrin production significantly contributed to protection in Cyld −/− mice. In addition, in vivo Cyld siRNA treatment increased STAT3 phosphorylation, fibrin production, pathogen control and survival of Lm-infected WT mice illustrating that therapeutic inhibition of CYLD augments the protective NF-κB/IL-6/STAT3 pathway and fibrin production.

          Author Summary

          Listeria monocytogenes causes high mortality in immunocompromised patients and fetuses. Murine studies have revealed that innate immune responses and fibrin, a major product of hepatocytes, are important to control Listeria. In the present study, we analysed whether the deubiquitinating enzyme CYLD impairs protective host responses in severe listeriosis and is a potential therapeutic target molecule. Using wildtype and Cyld −/− mice, we show that CYLD significantly reduced pathogen control and production of interferon (IFN)-γ, interleukin (IL)-6, and NOX2 mRNA in liver and spleen resulting in death of wildtype but not of Cyld −/− mice upon high-dose systemic infection. In vitro, CYLD impaired NF-κB-dependent pathogen control, reactive oxygen production, and IL-6 secretion in IFN-γ-stimulated, infected macrophages. We newly identified that CYLD directly removed K63-ubiquitin from STAT3, inhibited STAT3 activation and nuclear translocation resulting in reduced hepatocyte fibrin production. In Listeria-infected Cyld −/− mice, hepatic STAT3 K63-ubiquitination and activation, NF-κB activation, IL-6 production, and fibrin deposition were also increased. Neutralization experiments confirmed that the improved survival and pathogen control of Cyld −/− mice was dependent on IL-6-STAT3-mediated fibrin deposition. Finally, Cyld siRNA treatment of Listeria-infected wildtype mice significantly increased activated STAT3 and fibrin production, improved pathogen control and reduced mortality illustrating a therapeutic potential of CYLD inhibition.

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          Most cited references30

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          The JAK-STAT signaling pathway: input and output integration.

          Universal and essential to cytokine receptor signaling, the JAK-STAT pathway is one of the best understood signal transduction cascades. Almost 40 cytokine receptors signal through combinations of four JAK and seven STAT family members, suggesting commonality across the JAK-STAT signaling system. Despite intense study, there remain substantial gaps in understanding how the cascades are activated and regulated. Using the examples of the IL-6 and IL-10 receptors, I will discuss how diverse outcomes in gene expression result from regulatory events that effect the JAK1-STAT3 pathway, common to both receptors. I also consider receptor preferences by different STATs and interpretive problems in the use of STAT-deficient cells and mice. Finally, I consider how the suppressor of cytokine signaling (SOCS) proteins regulate the quality and quantity of STAT signals from cytokine receptors. New data suggests that SOCS proteins introduce additional diversity into the JAK-STAT pathway by adjusting the output of activated STATs that alters downstream gene activation.
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            Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naïve cells.

            The rules that govern memory T cell differentiation are not well understood. This study shows that after antigenic stimulation naïve CD8+ T cells become committed to dividing at least seven times and differentiating into effector and memory cells. Once the parental naïve CD8+ T cell had been activated, this developmental process could not be interrupted and the daughter cells continued to divide and differentiate in the absence of further antigenic stimulation. These data indicate that initial antigen encounter triggers an instructive developmental program that does not require further antigenic stimulation and does not cease until memory CD8+ T cell formation.
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              Cyld inhibits tumor cell proliferation by blocking Bcl-3-dependent NF-kappaB signaling.

              Mutations in the CYLD gene cause tumors of hair-follicle keratinocytes. The CYLD gene encodes a deubiquitinase that removes lysine 63-linked ubiquitin chains from TRAF2 and inhibits p65/p50 NF-kappaB activation. Here we show that mice lacking Cyld are highly susceptible to chemically induced skin tumors. Cyld-/- tumors and keratinocytes treated with 12-O-tetradecanoylphorbol-13 acetate (TPA) or UV light are hyperproliferative and have elevated cyclin D1 levels. The cyclin D1 elevation is caused not by increased p65/p50 action but rather by increased nuclear activity of Bcl-3-associated NF-kappaB p50 and p52. In Cyld+/+ keratinocytes, TPA or UV light triggers the translocation of Cyld from the cytoplasm to the perinuclear region, where Cyld binds and deubiquitinates Bcl-3, thereby preventing nuclear accumulation of Bcl-3 and p50/Bcl-3- or p52/Bcl-3-dependent proliferation. These data indicate that, depending on the external signals, Cyld can negatively regulate different NF-kappaB pathways; inactivation of TRAF2 controls survival and inflammation, while inhibition of Bcl-3 controls proliferation and tumor growth.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                June 2013
                June 2013
                27 June 2013
                : 9
                : 6
                : e1003455
                Affiliations
                [1 ]Institute of Medical Microbiology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
                [2 ]Department of Neuropathology, University Hospital Cologne, Cologne, Germany
                [3 ]Department of Laboratory Medicine, Lund University, Malmö, Sweden
                [4 ]Institute of Experimental Internal Medicine, Otto-von-Guericke University Magdeburg, Magdeburg, Germany
                [5 ]Helmholtz Centre for Infection Research, Braunschweig, Germany
                National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MN DS. Performed the experiments: GN MD KW. Analyzed the data: GN MD KW MN DS. Contributed reagents/materials/analysis tools: RM KS MN. Wrote the paper: GN DS.

                Article
                PPATHOGENS-D-12-02589
                10.1371/journal.ppat.1003455
                3695090
                23825949
                93bd4c77-abab-4e7d-8323-a9f21b5128f2
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 19 October 2012
                : 10 May 2013
                Page count
                Pages: 17
                Funding
                This study was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 854, TP5) to MN and DS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Immunology
                Immunity
                Immunity to Infections

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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