Neutrophil-specific expression of JAK2-V617F or CALRmut induces distinct inflammatory profiles in myeloproliferative neoplasia.

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-1ß. 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.

Cross-regulation of Listeria monocytogenes and the host ubiquitin system in listeriosis.

The facultative intracellular bacterium Listeria (L.) monocytogenes may cause severe diseases in humans and animals. The control of listeriosis/L. monocytogenes requires the concerted action of cells of the innate and adaptive immune systems. In this regard, cell-intrinsic immunity of infected cells, activated by the immune responses, is crucial for the control and elimination intracellular L. monocytogenes. Both the immune response against L. monocytogenes and cell intrinsic pathogen control are critically regulated by post-translational modifications exerted by the host ubiquitin system and ubiquitin-like modifiers (Ubls). In this review, we discuss our current understanding of the role of the ubiquitin system and Ubls in listeriosis, as well as future directions of research.

The deubiquitinating enzyme OTUD7b protects dendritic cells from TNF-induced apoptosis by stabilizing the E3 ligase TRAF2.

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.

Partial otubain 1 deficiency compromises fetal well-being in allogeneic pregnancies despite no major changes in the dendritic cell and T cell compartment.

OBJECTIVE: Pregnancy is characterized by well-defined immunological adaptions within the maternal immune cell compartment allowing the survival of a genetically disparate individual in the maternal womb. Phenotype and function of immune cells are largely determined by intracellular processing of external stimuli. Ubiquitinating and deubiquitinating enzymes are known to critically regulate immune signaling either by modulating the stability or the interaction of the signaling molecules. Accordingly, if absent, critical physiological processes may be perturbed such as fetal tolerance induction. Based on previous findings that mice hemizygous for the deubiquitinating enzyme otubain 1 (OTUB1) do not give rise to homozygous progeny, here, we investigated whether partial OTUB1 deficiency influences fetal-wellbeing in a syngeneic or an allogeneic pregnancy context accompanied by changes in the dendritic cell (DC) and T cell compartment. RESULTS: We observed increased fetal rejection rates in allogeneic pregnant OTUB1 heterozygous dams but not syngeneic pregnant OTUB1 heterozygous dams when compared to OTUB1 wildtype dams. Fetal demise in allogeneic pregnancies was not associated with major changes in maternal peripheral and local DC and T cell frequencies. Thus, our results suggest that OTUB1 confers fetal protection, however, this phenotype is independent of immune responses involving DC and T cells.

OTUB1 prevents lethal hepatocyte necroptosis through stabilization of c-IAP1 during murine liver inflammation.

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.

The deubiquitinase OTUB1 augments NF-κB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13.

Dendritic cells (DCs) are indispensable for defense against pathogens but may also contribute to immunopathology. Activation of DCs upon the sensing of pathogens by Toll-like receptors (TLRs) is largely mediated by pattern recognition receptor/nuclear factor-κB (NF-κB) signaling and depends on the appropriate ubiquitination of the respective signaling molecules. However, the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood. Here, we reveal that the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) is upregulated in DCs upon murine Toxoplasma gondii infection and lipopolysaccharide challenge. Stimulation of DCs with the TLR11/12 ligand T. gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-κB activation in OTUB1-competent cells, resulting in elevated interleukin-6 (IL-6), IL-12, and tumor necrosis factor (TNF) production, which was also observed upon the specific stimulation of TLR2, TLR3, TLR7, and TLR9. Mechanistically, OTUB1 promoted NF-κB activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13, resulting in increased K63-linked ubiquitination of IRAK1 (IL-1 receptor-associated kinase 1) and TRAF6 (TNF receptor-associated factor 6). Consequently, DC-specific deletion of OTUB1 impaired the production of cytokines, in particular IL-12, by DCs over the first 2 days of T. gondii infection, resulting in the diminished production of protective interferon-γ (IFN-γ) by natural killer cells, impaired control of parasite replication, and, finally, death from chronic T. encephalitis, all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection. In contrast, impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology. Collectively, these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.

Blood-Brain Barrier in Cerebral Malaria: Pathogenesis and Therapeutic Intervention.

Cerebral malaria is a life-threatening complication of malaria caused by the parasite Plasmodium falciparum. The growing problem of drug resistance and the dearth of new antiparasitic drugs are a serious threat to the antimalaria treatment regimes. Studies on humans and the murine model have implicated the disruption of the blood-brain barrier (BBB) in the lethal course of the disease. Therefore, efforts to alleviate the BBB dysfunction could serve as an adjunct therapy. Here, we review the mechanisms associated with the disruption of the BBB. In addition, we discuss the current, still limited, knowledge on the contribution of different cell types, microparticles, and the kynurenine pathway in the regulation of BBB dysfunction, and how these molecules could be used as potential new therapeutic targets.

OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes.

Astrocytes are critical regulators of neuroinflammation in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Growing evidence indicates that ubiquitination of signaling molecules is an important cell-intrinsic mechanism governing astrocyte function during MS and EAE Here, we identified an upregulation of the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) in astrocytes during MS and EAE Mice with astrocyte-specific OTUB1 ablation developed more severe EAE due to increased leukocyte accumulation, proinflammatory gene transcription, and demyelination in the spinal cord as compared to control mice. OTUB1-deficient astrocytes were hyperactivated in response to IFN-γ, a fingerprint cytokine of encephalitogenic T cells, and produced more proinflammatory cytokines and chemokines than control astrocytes. Mechanistically, OTUB1 inhibited IFN-γ-induced Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling by K48 deubiquitination and stabilization of the JAK2 inhibitor suppressor of cytokine signaling 1 (SOCS1). Thus, astrocyte-specific OTUB1 is a critical inhibitor of neuroinflammation in CNS autoimmunity.

A Listeria monocytogenes ST2 clone lacking chitinase ChiB from an outbreak of non-invasive gastroenteritis.

An outbreak with a remarkable Listeria monocytogenes clone causing 163 cases of non-invasive listeriosis occurred in Germany in 2015. Core genome multi locus sequence typing grouped non-invasive outbreak isolates and isolates obtained from related food samples into a single cluster, but clearly separated genetically close isolates obtained from invasive listeriosis cases. A comparative genomic approach identified a premature stop codon in the chiB gene, encoding one of the two L. monocytogenes chitinases, which clustered with disease outcome. Correction of this premature stop codon in one representative gastroenteritis outbreak isolate restored chitinase production, but effects in infection experiments were not found. While the exact role of chitinases in virulence of L. monocytogenes is still not fully understood, our results now clearly show that ChiB-derived activity is not required to establish L. monocytogenes gastroenteritis in humans. This limits a possible role of ChiB in human listeriosis to later steps of the infection.

The Deubiquitinating Enzyme Cylindromatosis Dampens CD8+ T Cell Responses and Is a Critical Factor for Experimental Cerebral Malaria and Blood-Brain Barrier Damage.

Cerebral malaria is a severe complication of human malaria and may lead to death of Plasmodium falciparum-infected individuals. Cerebral malaria is associated with sequestration of parasitized red blood cells within the cerebral microvasculature resulting in damage of the blood-brain barrier and brain pathology. Although CD8+ T cells have been implicated in the development of murine experimental cerebral malaria (ECM), several other studies have shown that CD8+ T cells confer protection against blood-stage infections. Since the role of host deubiquitinating enzymes (DUBs) in malaria is yet unknown, we investigated how the DUB cylindromatosis (CYLD), an important inhibitor of several cellular signaling pathways, influences the outcome of ECM. Upon infection with Plasmodium berghei ANKA (PbA) sporozoites or PbA-infected red blood cells, at least 90% of Cyld-/- mice survived the infection, whereas all congenic C57BL/6 mice displayed signatures of ECM, impaired parasite control, and disruption of the blood-brain barrier integrity. Cyld deficiency prevented brain pathology, including hemorrhagic lesions, enhanced activation of astrocytes and microglia, infiltration of CD8+ T cells, and apoptosis of endothelial cells. Furthermore, PbA-specific CD8+ T cell responses were augmented in the blood of Cyld-/- mice with increased production of interferon-γ and granzyme B and elevated activation of protein kinase C-θ and nuclear factor "kappa light-chain enhancer" of activated B cells. Importantly, accumulation of CD8+ T cells in the brain of Cyld-/- mice was significantly reduced compared to C57BL/6 mice. Bone marrow chimera experiments showed that the absence of ECM signatures in infected Cyld-/- mice could be attributed to hematopoietic and radioresistant parenchymal cells, most likely endothelial cells that did not undergo apoptosis. Together, we were able to show that host deubiqutinating enzymes play an important role in ECM and that CYLD promotes ECM supporting it as a potential therapeutic target for adjunct therapy to prevent cerebral complications of severe malaria.

A20 Curtails Primary but Augments Secondary CD8+ T Cell Responses in Intracellular Bacterial Infection.

The ubiquitin-modifying enzyme A20, an important negative feedback regulator of NF-κB, impairs the expansion of tumor-specific CD8+ T cells but augments the proliferation of autoimmune CD4+ T cells. To study the T cell-specific function of A20 in bacterial infection, we infected T cell-specific A20 knockout (CD4-Cre A20fl/fl) and control mice with Listeria monocytogenes. A20-deficient pathogen-specific CD8+ T cells expanded stronger resulting in improved pathogen control at day 7 p.i. Imaging flow cytometry revealed that A20-deficient Listeria-specific CD8+ T cells underwent increased apoptosis and necroptosis resulting in reduced numbers of memory CD8+ T cells. In contrast, the primary CD4+ T cell response was A20-independent. Upon secondary infection, the increase and function of pathogen-specific CD8+ T cells, as well as pathogen control were significantly impaired in CD4-Cre A20fl/fl mice. In vitro, apoptosis and necroptosis of Listeria-specific A20-deficient CD8+ T cells were strongly induced as demonstrated by increased caspase-3/7 activity, RIPK1/RIPK3 complex formation and more morphologically apoptotic and necroptotic CD8+ T cells. In vitro, A20 limited CD95L and TNF-induced caspase3/7 activation. In conclusion, T cell-specific A20 limited the expansion but reduced apoptosis and necroptosis of Listeria-specific CD8+ T cells, resulting in an impaired pathogen control in primary but improved clearance in secondary infection.

Listeriosis downregulates hepatic cytochrome P450 enzymes in sublethal murine infection.

PURPOSE: Listeria monocytogenes (Lm) can cross the intestinal barrier in humans and then disseminates into different organs. Invasion of the liver occurs even in sublethal infections, however, knowledge of affected physiological processes is scarce. This study employed a sublethal murine infection model to investigate liver responses systematically by proteomics. EXPERIMENTAL DESIGN: Liver samples from three stages of the sublethal infection covering the initial invasion, the peak of infection, and the clearance phase (1, 3, 9 days postinoculation) were analyzed in comparison to samples from noninfected mice. Apart from flow cytometry and RT-PCRs for immune status control, liver responses were analyzed by quantitative peptide sequencing (HPLC-Orbitrap Fusion) using 4-plex iTRAQ-labeling. RESULTS: Accurate MS characterized about 3600 proteins and statistics revealed 15% of the hepatic proteome as regulated. Immunological data as well as protein regulation dynamics strongly indicate stage-specific hepatic responses in sublethal infections. Most notably, this study detected a comprehensive deregulation of drug metabolizing enzymes at all stages, including 25 components of the cytochrome P450 system. CONCLUSIONS AND CLINICAL RELEVANCE: Sublethal Lm infection deregulates hepatic drug metabolizing pathways. This finding indicates the need to monitor drug administration along Lm infections, especially in all patients needing constant medication.

Protective dendritic cell responses against listeriosis induced by the short form of the deubiquitinating enzyme CYLD are inhibited by full-length CYLD.

The deubiquitinating enzyme CYLD is an important tumor suppressor and inhibitor of immune responses. In contrast to full-length CYLD, the immunological function of the naturally occurring short splice variant of CYLD (sCYLD) is insufficiently described. Previously, we showed that DCs, which lack full-length CYLD but express sCYLD, exhibit augmented NF-κB and DC activation. To explore the function of sCYLD in infection, we investigated whether DC-specific sCYLD regulates the pathogenesis of listeriosis. Upon Listeria monocytogenes infection of CD11c-Cre Cyld(ex7/8 fl/fl) mice, infection of CD8α(+) DCs, which are crucial for the establishment of listeriosis in the spleen, was not affected. However, NF-κB activity of CD11c-Cre Cyld(ex7/8 fl/fl) DCs was increased, while activation of ERK and p38 was normal. In addition, CD11c-Cre Cyld(ex7/8 fl/fl) DCs produced more TNF, IL-10, and IL-12 upon infection, which led to enhanced stimulation of IFN-γ-producing NK cells. In addition CD11c-Cre Cyld(ex7/8 fl/fl) DCs presented Listeria Ag more efficiently to CD8(+) T cells resulting in a stronger pathogen-specific CD8(+) T-cell proliferation and more IFN-γ production. Collectively, the improved innate and adaptive immunity and survival during listeriosis identify the DC-specific FL-CYLD/sCYLD balance as a potential target to modulate NK-cell and Ag-specific CD8(+) T-cell responses.

A20 expression in dendritic cells protects mice from LPS-induced mortality.

DCs contribute to immune homeostasis under physiological conditions and regulate the immune activation during infection. The deubiquitinase A20 inhibits the activation of NF-κB-dependent immune reactions, and prevents the hyperactivation of DCs under steady-state conditions. However, the role of DC-specific A20 under pathological conditions is unknown. Here, we demonstrate that upon injection of low-dose LPS, mice with DC-specific A20 deletion (CD11c-Cre A20(fl/fl) ) died within 6 h, whereas A20(fl/fl) controls survived. LPS-induced mortality in CD11c-Cre A20(fl/fl) mice was characterized by increased serum levels of IL-2, IL-10, IL-12, IFN-γ, and TNF. Upon LPS stimulation, the activation of NF-κB and ERK-NFATc3 pathways were enhanced in A20-deficient DCs, resulting in an increased production of IL-2, IL-12, and TNF both in vitro and in vivo. Targeted inhibition of ERK in A20-deficient DCs abolished the increased production of IL-2. A20-deficient DCs failed to induce LPS tolerance, which was independent of T cells and the intestinal flora, since T-cell depletion and decolonization of CD11c-Cre A20(fl/fl) mice could not prevent death of LPS-challenged CD11c-Cre A20(fl/fl) mice. In conclusion, these findings show that DC-specific A20 preserves immune homeostasis in steady-state conditions and is also required for LPS tolerance.

Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages.

Upon infection with intracellular bacteria, nucleotide oligomerization domain protein 2 recognizes bacterial muramyl dipeptide and binds, subsequently, to receptor-interacting serine/threonine kinase 2 (RIPK2), which activates immune responses via the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinase (ERK) pathways. Activation of RIPK2 depends on its K63 ubiquitination by E3 ligases, whereas the deubiquitinating enzyme A20 counter regulates RIPK2 activity by cleaving K63-polyubiquitin chains from RIPK2. Here, we newly identify the deubiquitinating enzyme CYLD as a new inhibitor of RIPK2. We show that CYLD binds to and removes K63-polyubiquitin chains from RIPK2 in Listeria monocytogenes (Lm) infected murine bone marrow-derived macrophages. CYLD-mediated K63 deubiquitination of RIPK2 resulted in an impaired activation of both NF-κB and ERK1/2 pathways, reduced production of proinflammatory cytokines interleukin-6 (IL-6), IL-12, anti-listerial reactive oxygen species (ROS) and nitric oxide (NO), and, finally, impaired pathogen control. In turn, RIPK2 inhibition by siRNA prevented activation of NF-κB and ERK1/2 and completely abolished the protective effect of CYLD deficiency with respect to the production of IL-6, NO, ROS, and pathogen control. Noteworthy, CYLD also inhibited autophagy of Listeria in a RIPK2-ERK1/2-dependent manner. The protective function of CYLD deficiency was dependent on interferon gamma (IFN-γ) prestimulation of infected macrophages. Interestingly, the reduced NF-κB activation in CYLD-expressing macrophages limited the protective effect of IFN-γ by reducing NF-κB-dependent signal transducers and activators of transcription-1 (STAT1) activation. Taken together, our study identifies CYLD as an important inhibitor of RIPK2-dependent antibacterial immune responses in macrophages.

IFN-γ regulates CD8+ memory T cell differentiation and survival in response to weak, but not strong, TCR signals.

In response to primary Ag contact, naive mouse CD8(+) T cells undergo clonal expansion and differentiate into effector T cells. After pathogen clearance, most effector T cells die, and only a small number of memory T cell precursors (TMPs) survive to form a pool of long-lived memory T cells (TMs). Although high- and low-affinity CD8(+) T cell clones are recruited into the primary response, the TM pool consists mainly of high-affinity clones. It remains unclear whether the more efficient expansion of high-affinity clones and/or cell-intrinsic processes exclude low-affinity T cells from the TM pool. In this article, we show that the lack of IFN-γR signaling in CD8(+) T cells promotes TM formation in response to weak, but not strong, TCR agonists. The IFN-γ-sensitive accumulation of TMs correlates with reduced mammalian target of rapamycin activation and the accumulation of long-lived CD62L(hi)Bcl-2(hi)Eomes(hi) TMPs. Reconstitution of mammalian target of rapamycin or IFN-γR signaling is sufficient to block this process. Hence, our data suggest that IFN-γR signaling actively blocks the formation of TMPs responding to weak TCR agonists, thereby promoting the accumulation of high-affinity T cells finally dominating the TM pool.

CTLA-4 (CD152) enhances the Tc17 differentiation program.

Although CD8(+) T cells that produce IL-17 (Tc17 cells) have been linked to host defense, Tc17 cells show reduced cytotoxic activity, which is the characteristic function of CD8(+) T cells. Here, we show that CTLA-4 enhances the frequency of IL-17 in CD8(+) T cells, indicating that CTLA-4 (CD152) specifically promotes Tc17 differentiation. Simultaneous stimulation of CTLA-4(+/+) and CTLA-4(-/-) T cells in cocultures and agonistic CTLA-4 stimulation unambiguously revealed a cell-intrinsic mechanism for IL-17 control by CTLA-4. The quality of CTLA-4-induced Tc17 cells was tested in vivo, utilizing infection with the facultative intracellular bacterium Listeria monocytogenes (LM). Unlike CTLA-4(+/+) Tc17 cells, CTLA-4(-/-) were nearly as efficient as Tc1 CTLA-4(+/+) cells in LM clearance. Additionally, adoptively transferred CTLA-4(-/-) Tc17 cells expressed granzyme B after rechallenge, and produced Tc1 cytokines such as IFN-γ and TNF-α, which strongly correlate with bacterial clearance. CTLA-4(+/+) Tc17 cells demonstrated a high-quality Tc17 differentiation program ex vivo, which was also evident in isolated IL-17-secreting Tc17 cells, with CTLA-4-mediated enhanced upregulation of Tc17-related molecules such as IL-17A, RORγt, and IRF-4. Our results show that CTLA-4 promotes Tc17 differentiation that results in robust Tc17 responses. Its inactivation might therefore represent a central therapeutic target to enhance clearance of infection.

Astrocytic A20 ameliorates experimental autoimmune encephalomyelitis by inhibiting NF-κB- and STAT1-dependent chemokine production in astrocytes.

Single-nucleotide polymorphisms in the tumor necrosis factor, alpha-induced protein 3 gene, which encodes the ubiquitin-modifying protein A20, are linked to susceptibility to multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system (CNS). Since it is unresolved how A20 regulates MS pathogenesis, we examined its function in a murine model of MS, namely experimental autoimmune encephalomyelitis (EAE). Deletion of A20 in neuroectodermal cells (astrocytes, neurons, and oligodendrocytes; Nestin-Cre A20fl/fl mice) or selectively in astrocytes (GFAP-Cre A20fl/fl mice) resulted in more severe EAE as compared to control animals. In Nestin-Cre A20fl/fl and GFAP-Cre A20fl/fl mice demyelination and recruitment of inflammatory leukocytes were increased as compared to A20fl/fl control mice. Importantly, numbers of encephalitogenic CD4+ T cells producing interferon (IFN)-γ, interleukin (IL)-17, and granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively, as well as mRNA production of IFN-γ, IL-17, tumor necrosis factor (TNF), GM-CSF, IL-6, CXCL1, CCL2, and CXCL10 were significantly increased in spinal cords of Nestin-Cre A20fl/fl and GFAP-Cre A20fl/fl mice, respectively. Compared to A20-sufficient astrocytes, A20-deficient astrocytes displayed stronger activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) in response to TNF, IL-17, and GM-CSF, and of signal transducer and activator of transcription 1 (STAT1) upon IFN-γ stimulation. Due to NF-κB and STAT1 hyperactivation, A20-deficient astrocytes produced significantly more chemokines in response to these key encephalitogenic cytokines of autoimmune CD4+ T cells resulting in an amplification of CD4+ T cell recruitment to the CNS. Thus, astrocytic A20 is an important inhibitor of autoimmune-mediated demyelination in the CNS.

CYLD enhances severe listeriosis by impairing IL-6/STAT3-dependent fibrin production.

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.