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.
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.