In atopic dermatitis (AD), lesional skin is frequently colonized by Staphylococcus aureus, which promotes clinical symptoms of the disease. The inflammatory milieu in the skin is characterized by a Th2 response, including M2 macrophages, which cannot eradicate S. aureus. Therefore, repolarization of macrophages toward the M1 phenotype may foster control of S. aureus. Our data show that the deubiquitinating enzyme cylindromatosis (CYLD) is strongly expressed in macrophages of AD patients and prevents the clearance of S. aureus. Mechanistically, CYLD impaired M1 macrophage polarization by K63-specific deubiquitination of STAT1 and activation of the NF-κB pathway via its interaction with TRAF6, NEMO, and RIPK2. Inhibition of STAT1 and NF-κB, independently, abolished the differences between S. aureus-infected CYLD-deficient and CYLD-competent M1 macrophages. Infection of Cyld-deficient and wild-type mice with S. aureus confirmed the protective CYLD function. Collectively, our study shows that CYLD impairs the control of S. aureus in macrophages of AD patients, identifying CYLD as a potential therapeutic target.

Abstract Background Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. Methods Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1β. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. Results Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. Conclusions Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

In atopic dermatitis (AD), lesional skin is frequently colonized by Staphylococcus (S.) aureus, contributing to the severity and clinical symptoms of the disease. The inflammatory milieu in the skin is characterized by a type 2 inflammatory signature, including M2 macrophages, which cannot eradicate S. aureus. Since S. aureus is effectively controlled in macrophages activated by pattern recognition receptor (PRR)-induced NF-κB and interferon (IFN)-γ-induced STAT1 stimulation, we hypothesized that pre-treatment with LPS as a PRR/TLR4-activating agent and IFN-γ would induce effective control of S. aureus in monocyte-derived macrophages (MDMs) of AD patients. Our data show that the deubiquitinating enzyme CYLD is strongly expressed in skin macrophages and MDMs from AD patients compared to healthy controls and impairs the anti-staphylococcal activity of PRR-activated and IFN-γ-treated MDMs. Functionally, CYLD impaired M1 macrophage polarization, as evidenced by reduced expression of CD80, TNF, and IL-6 upon LPS- and IFN-γ treatment in CYLD-deficient as compared to wild-type (WT) MDM/THP1 macrophages. Mechanistically, CYLD inhibited IFN-γ-induced STAT1 phosphorylation and activation by binding to STAT1 and inducing its K63 deubiquitination. CYLD also interacted with TRAF6 and NEMO/IKKγ in the MYD88 signaling pathway and with RIPK2 in the NOD2 pathway, leading to impaired activation of NF-κB. Inhibition of both STAT1 by siRNA and NF-κB by IKK inhibitor treatment, respectively, independently abolished the control of S. aureus in both CYLD-deficient and WT THP1 macrophages, which harbored identical high numbers of the pathogen. The in vivo inhibitory function of CYLD on the control of S. aureus was also observed upon infection of Cyld-deficient and WT mice. Collectively, these data illustrate that the increased expression of CYLD in macrophages of AD patients is a factor contributing to the ineffective control of S. aureus, diminishes M1 macrophage polarization, and that CYLD deletion unleashes the break on effective STAT1 and NF-κB-dependent control of S. aureus. ### Competing Interest Statement The authors have declared no competing interest.

The cytokine tumor necrosis factor (TNF) critically regulates the intertwined cell death and pro-inflammatory signaling pathways of dendritic cells (DCs) via ubiquitin modification of central effector molecules, but the intrinsic molecular switches deciding on either pathway are incompletely defined. Here, we uncover that the ovarian tumor deubiquitinating enzyme 7b (OTUD7b) prevents TNF-induced apoptosis of DCs in infection, resulting in efficient priming of pathogen-specific CD8 + T cells. Mechanistically, OTUD7b stabilizes the E3 ligase TNF-receptor-associated factor 2 (TRAF2) in human and murine DCs by counteracting its K48-ubiquitination and proteasomal degradation. TRAF2 in turn facilitates K63-linked polyubiquitination of RIPK1, which mediates activation of NF-κB and MAP kinases, IL-12 production, and expression of anti-apoptotic cFLIP and Bcl-xL. We show that mice with DC-specific OTUD7b-deficiency displayed DC apoptosis and a failure to induce CD8 + T cell-mediated brain pathology, experimental cerebral malaria, in a murine malaria infection model. Together, our data identify the deubiquitinating enzyme OTUD7b as a central molecular switch deciding on survival of human and murine DCs and provides a rationale to manipulate DC responses by targeting their ubiquitin network downstream of the TNF receptor pathway.

In bacterial and sterile inflammation of the liver, hepatocyte apoptosis is, in contrast to necroptosis, a common feature. The molecular mechanisms preventing hepatocyte necroptosis and the potential consequences of hepatocyte necroptosis are largely unknown. Apoptosis and necroptosis are critically regulated by the ubiquitination of signaling molecules but especially the regulatory function of deubiquitinating enzymes (DUBs) is imperfectly defined. Here, we addressed the role of the DUB OTU domain aldehyde binding-1 (OTUB1) in hepatocyte cell death upon both infection with the hepatocyte-infecting bacterium Listeria monocytogenes (Lm) and D-Galactosamine (DGal)/Tumor necrosis factor (TNF)-induced sterile inflammation. Combined in vivo and in vitro experiments comprising mice lacking OTUB1 specifically in liver parenchymal cells (OTUB1LPC-KO) and human OTUB1-deficient HepG2 cells revealed that OTUB1 prevented hepatocyte necroptosis but not apoptosis upon infection with Lm and DGal/TNF challenge. Lm-induced necroptosis in OTUB1LPC-KO mice resulted in increased alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release and rapid lethality. Treatment with the receptor-interacting serine/threonine-protein kinase (RIPK) 1 inhibitor necrostatin-1s and deletion of the pseudokinase mixed lineage kinase domain-like protein (MLKL) prevented liver damage and death of infected OTUB1LPC-KO mice. Mechanistically, OTUB1 reduced K48-linked polyubiquitination of the cellular inhibitor of apoptosis 1 (c-IAP1), thereby diminishing its degradation. In the absence of OTUB1, c-IAP1 degradation resulted in reduced K63-linked polyubiquitination and increased phosphorylation of RIPK1, RIPK1/RIPK3 necrosome formation, MLKL-phosphorylation and hepatocyte death. Additionally, OTUB1-deficiency induced RIPK1-dependent extracellular-signal-regulated kinase (ERK) activation and TNF production in Lm-infected hepatocytes. Collectively, these findings identify OTUB1 as a novel regulator of hepatocyte-intrinsic necroptosis and a critical factor for survival of bacterial hepatitis and TNF challenge.