Conditional ablation of TYK2 in immunity to viral infection and tumor surveillance.

Tyrosine kinase 2 (TYK2) has a pivotal role in immunity to infection and tumor surveillance. It is associated with several cytokine receptor chains including type I interferon (IFN) receptor 1 (IFNAR1), interleukin- (IL-) 12 receptor beta 1 (IL-12Rb1) and IL-10R2. We have generated a mouse with a conditional Tyk2 null allele and proved integrity of the conditional Tyk2 locus. TYK2 was successfully removed by the use of ubiquitous and tissue-specific Cre-expressing mouse strains. Myeloid TYK2 was found to critically contribute to the defense against murine cytomegalovirus. Ubiquitous TYK2 ablation severely impaired tumor immunosurveillance, while deletion in myeloid, dendritic or T cells alone showed no effect. The conditional Tyk2 mouse strain will be instrumental to further dissect TYK2 functions in infection, inflammation and cancer.

TYK2 kinase activity is required for functional type I interferon responses in vivo.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2(K923E)) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein's stability. An inhibitory function was only observed upon over-expression of TYK2(K923E)in vitro. Tyk2(K923E) mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors.

Generation of mice with a conditional Stat1 null allele.

Interferons (IFNs) are key cytokines in the innate immune response that also bridge the gap to adaptive immunity. Signaling upon stimulation by IFN type I, II and III is mediated by the Jak-Stat pathway. STAT1 is activated by all three IFN receptor complexes and absence of STAT1 from mice increases their susceptibility to pathogens. In addition, depending on the setting, STAT1 can act as tumor suppressor or oncogene. Here we report the generation and detailed functional characterization of a conditional Stat1 knockout mouse. We show the integrity of the conditional Stat1 locus and report successful in vivo deletion by means of a ubiquitous and a tissue-specific Cre recombinase. The conditional Stat1 null allele represents an important tool for identifying novel and cell-autonomous STAT1 functions in infection and cancer.

Tristetraprolin-driven regulatory circuit controls quality and timing of mRNA decay in inflammation.

For a successful yet controlled immune response, cells need to specifically destabilize inflammatory mRNAs but prevent premature removal of those still used. The regulatory circuits controlling quality and timing in the global inflammatory mRNA decay are not understood. Here, we show that the mRNA-destabilizing function of the AU-rich element-binding protein tristetraprolin (TTP) is inversely regulated by the p38 MAPK activity profile such that after inflammatory stimulus the TTP-dependent decay is initially limited to few mRNAs. With time, the TTP-dependent decay gradually spreads resulting in cumulative elimination of one third of inflammation-induced unstable mRNAs in macrophages in vitro. We confirmed this sequential decay model in vivo since LPS-treated mice with myeloid TTP ablation exhibited similar cytokine dysregulation profile as macrophages. The mice were hypersensitive to LPS but otherwise healthy with no signs of hyperinflammation seen in conventional TTP knockout mice demonstrating the requirement for myeloid TTP in re-installment but not maintenance of immune homeostasis. These findings reveal a TTP- and p38 MAPK-dominated regulatory mechanism that is vital for balancing acute inflammation by a temporally and qualitatively controlled mRNA decay.

Tyk2 and signal transducer and activator of transcription 1 contribute to intestinal I/R injury.

Previously, we have shown that the Jak-signal transducer and activator of transcription signaling constituents Tyk2 and STAT1 play a role in the development of multiple organ failure during endotoxin shock. Here, we report that Tyk2 and STAT1 contribute to death caused by intestinal I/R injury. Tyk2- and STAT1-deficient mice showed increased survival to I/R because their intestines were protected from gross histomorphological tissue destruction and neutrophil infiltration. On the molecular level, the reduced ischemia induced inflammatory response in mutant versus wild-type mice was accompanied by an impaired up-regulation of the adhesion molecules P-selectin and intercellular adhesion molecule 1 and of the matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MMP-14 in the reperfused intestine. In conclusion, this study demonstrates for the first time that Tyk2 or STAT1 promote intestinal I/R-induced shock based on a deregulated local inflammatory response and a destruction of the gut intestinal barrier.

Organ-specific and differential requirement of TYK2 and IFNAR1 for LPS-induced iNOS expression in vivo.

Lipopolysaccharide (LPS) is an integral structural component of the outer membrane of Gram-negative bacteria and the principal active agent in the pathogenesis of endotoxin shock. LPS is a potent inducer of a variety of cytokines and inflammatory agents that lead to a profound alteration of gene expression patterns in cells and organs. The gene coding for the inducible nitric oxide synthase (iNOS) is highly responsive to LPS in vitro and in vivo and accounts for the production of nitric oxide (NO). The Janus kinase (JAK) family member tyrosine kinase 2 (TYK2) is a constituent of the interferon (IFN) type I response pathway and an important effector in the progression of endotoxin shock. Macrophages deficient for IFNalphabeta receptor chain 1 (IFNAR1) or TYK2 were shown to have an impaired LPS-induced iNOS expression. Here we determined the contribution of IFNAR1 and TYK2 to iNOS expression in vivo in a lethal LPS challenge model. TYK2 and IFNAR1 were found to be crucial for the LPS-induced iNOS mRNA and protein expression in spleen and lung that could be attributed to the Mac3-positive population. In liver LPS-induced iNOS mRNA expression was only partially impaired in TYK2-deficient mice and was unimpaired in IFNAR1-deficient mice, indicating organ specificity. TYK2(-/-) and IFNAR1(-/-) mice also differ with respect to IFNgamma production upon LPS challenge in that TYK2(-/-) mice show a defect while IFNAR1(-/-) mice do not. Our data suggest that iNOS is induced through IFNAR1 and TYK2 in Mac3-positive cells which are the main source of iNOS in spleen and lung. The LPS-induced iNOS expression in liver is independent of IFNAR1 and partially dependent on TYK2, which is most likely due to the lack of IFNgamma production in the absence of TYK2.

A time- and dose-dependent STAT1 expression system.

BACKGROUND: The signal transducer and activator of transcription (STAT) family of transcription factors mediates a variety of cytokine dependent gene regulations. STAT1 has been mainly characterized by its role in interferon (IFN) type I and II signaling and STAT1 deficiency leads to high susceptibility to several pathogens. For fine-tuned analysis of STAT1 function we established a dimerizer-inducible system for STAT1 expression in vitro and in vivo. RESULTS: The functionality of the dimerizer-induced STAT1 system is demonstrated in vitro in mouse embryonic fibroblasts and embryonic stem cells. We show that this two-vector based system is highly inducible and does not show any STAT1 expression in the absence of the inducer. Reconstitution of STAT1 deficient cells with inducible STAT1 restores IFNgamma-mediated gene induction, antiviral responses and STAT1 activation remains dependent on cytokine stimulation. STAT1 expression is induced rapidly upon addition of dimerizer and expression levels can be regulated in a dose-dependent manner. Furthermore we show that in transgenic mice STAT1 can be induced upon stimulation with the dimerizer, although only at low levels. CONCLUSION: These results prove that the dimerizer-induced system is a powerful tool for STAT1 analysis in vitro and provide evidence that the system is suitable for the use in transgenic mice. To our knowledge this is the first report for inducible STAT1 expression in a time- and dose-dependent manner.

Novel functions of tyrosine kinase 2 in the antiviral defense against murine cytomegalovirus.

We have recently reported that tyrosine kinase 2 (Tyk2)-deficient mice have a selective defect in the in vivo defense against certain viruses. In our current study we show that Tyk2 is essential for the defense against murine CMV (MCMV). In vivo challenges with MCMV revealed impaired clearance of virus from organs and decreased survival of mice in the absence of Tyk2. Our in vitro studies demonstrate that MCMV replicates to dramatically higher titers in Tyk2-deficient macrophages compared with wild-type cells. We show an essential role of type I IFN (IFN-alphabeta) in the control of MCMV replication, with a prominent role of IFN-beta. MCMV infection leads to the activation of STAT1 and STAT2 in an IFN-alphabeta receptor 1-dependent manner. Consistent with the role of Tyk2 in IFN-alphabeta signaling, activation of STAT1 and STAT2 is reduced in Tyk2-deficient cells. However, lack of Tyk2 results in impaired MCMV-mediated gene induction of only a subset of MCMV-induced IFN-alphabeta-responsive genes. Taken together, our data demonstrate a requirement for Tyk2 in the in vitro and in vivo antiviral defense against MCMV infection. In addition to the established role of Tyk2 as an amplifier of Jak/Stat signaling upon IFN-alphabeta stimulation, we provide evidence for a novel role of Tyk2 as a modifier of host responses.

TYK2 is a key regulator of the surveillance of B lymphoid tumors.

Aberrant activation of the JAK-STAT pathway has been implicated in tumor formation; for example, constitutive activation of JAK2 kinase or the enforced expression of STAT5 induces leukemia in mice. We show here that the Janus kinase TYK2 serves an opposite function. Mice deficient in TYK2 developed Abelson-induced B lymphoid leukemia/lymphoma as well as TEL-JAK2-induced T lymphoid leukemia with a higher incidence and shortened latency compared with WT controls. The cell-autonomous properties of Abelson murine leukemia virus-transformed (A-MuLV-transformed) TYK2(-/-) cells were unaltered, but the high susceptibility of TYK2(-/-) mice resulted from an impaired tumor surveillance, and accordingly, TYK2(-/-) A-MuLV-induced lymphomas were easily rejected after transplantation into WT hosts. The increased rate of leukemia/lymphoma formation was linked to a decreased in vitro cytotoxic capacity of TYK2(-/-) NK and NKT cells toward tumor-derived cells. RAG2/TYK2 double-knockout mice succumbed to A-MuLV-induced leukemia/lymphoma faster than RAG2(-/-)TYK2(+/-) mice. This defines NK cells as key players in tumor surveillance in Abelson-induced malignancies. Our observations provide compelling evidence that TYK2 is an important regulator of lymphoid tumor surveillance.

Control of Leishmania major in the absence of Tyk2 kinase.

IL-12 is indispensable for the control of many intracellular pathogens, but the components of the signaling pathway that are essential for its function in vivo are incompletely understood. Here, we investigated in the Leishmania major mouse model whether Tyk2 kinase is required for the generation of a protective immune response. Unlike C57BL/6 controls, Tyk2(-/-)mice developed severe skin lesions after infection that frequently ulcerated, but ultimately healed. NK cell cytotoxicity was absent in infected Tyk2(-/-) mice, even after IL-12 pretreatment, which correlated with a STAT4 activation defect. IFN-alpha / beta, which was still able to activate STAT1 in Tyk2(-/-) NK cells, reconstituted their cytotoxic activity, but not their IL-12 responsiveness. The IL-12-induced production of IFN-gamma by NK cells and CD8(+) T cells was strongly suppressed in Tyk2(-/-) mice at day 1 of infection, but partly regained during the late phase of infection. Tyk2(-/-) CD4(+) T cells developed into Th1 cells (although in a delayed fashion) and infected Tyk2(-/-) mice expressed normals levels of inducible NO synthase. Thus, Tyk2 is required for the IL-12 response of NK cells and CD8(+) T cells in L. major-infected mice, but not for the generation of Th1 cells and the ultimate control of the disease.

Phosphorylation of the Stat1 transactivation domain is required for full-fledged IFN-gamma-dependent innate immunity.

Stat1 is phosphorylated on serine 727 within its transactivating domain (TAD) in response to interferons or other immunological signals. We generated gene-targeted mutant mice expressing a serine727-alanine mutant of Stat1. These animals showed increased mortality upon infection with Listeria monocytogenes and impaired clearance of the bacteria from spleen and liver. The Stat1S727A mice were more resistant to the LPS-induced septic shock syndrome, suggesting that Stat1 serine phosphorylation promotes inflammatory responses. Expression of IFN-gamma-induced genes was strongly reduced in macrophages expressing Stat1(S727A). While mutation of Stat1 at S727 did not reduce its binding to chromatin, association with the coactivator CBP and histone acetylation at the interferon-responsive GBP promoter was strongly reduced, suggesting defective recruitment of histone acetylases as the mechanism underlying IFN-gamma hyporesponsiveness. Our data demonstrate that the increase in transcription factor activity caused by Stat serine phosphorylation contributes to macrophage activation and to IFN-gamma-dependent immune responses in vivo.

A natural mutation in the Tyk2 pseudokinase domain underlies altered susceptibility of B10.Q/J mice to infection and autoimmunity.

The B10.Q/J strain of mice was serendipitously discovered to be highly susceptible to infection by the intracellular protozoan parasite, Toxoplasma gondii but markedly resistant to induction of autoimmune arthritis. We have previously shown that the B10.Q/J phenotype is controlled by a single recessive locus and is associated with lymphocyte hyporesponsiveness to IL-12. Using genetic approaches, we have now localized the B10.Q/J locus to chromosome 9 and established its identity as Tyk2, a Janus kinase essential for IL-12 and IFN-alpha/beta cytokine signaling. The B10.Q/J Tyk2 gene contained a single missense mutation resulting in a nonconservative amino acid substitution (E775K) in an invariant motif of the pseudokinase (Janus kinase homology 2) domain. This mutation appeared to result in the absence of the B10.Q/J-encoded Tyk2 protein, despite presence of Tyk2-specific transcripts. Phenotypically, B10.Q/J cells were indistinguishable from Tyk2-deficient cells, showing impaired signaling and biologic responses to IL-12, IL-23, and type I IFNs. The analogous E782K mutant of human Tyk2 failed to restore IFN-alpha responsiveness in Tyk2 null 11,1 cells. Our results indicate a crucial role for Tyk2 in T helper 1-mediated protective and pathogenic immune responses. An additional implication of our findings is that naturally occurring mutations in the Tyk2 gene may underlie altered susceptibilities to infectious or autoimmune diseases in human and animal populations.

Central role for type I interferons and Tyk2 in lipopolysaccharide-induced endotoxin shock.

Toll-like receptor-4 activation by lipopolysaccharide (LPS) induces the expression of interferon-beta (IFN-beta) in a MyD88-independent manner. Here we report that mice devoid of the JAK protein tyrosine kinase family member, Tyk2, were resistant to shock induced by high doses of LPS. Basal and LPS-induced expression of IFN-beta and IFN-alpha4 mRNA in Tyk2-null macrophages were diminished. However, Tyk2-null mice showed normal systemic production of nitric oxide and proinflammatory cytokines and the in vivo response to tumor necrosis factor (TNF) was unperturbed. IFN-beta-null but not STAT1-null mice were also resistant to high dose LPS treatment. Together, these data suggest that Tyk2 and IFN-beta are essential effectors in LPS induced lethality.

Species-specific regulation of Toll-like receptor 3 genes in men and mice.

Toll-like receptor 3 (TLR3) belongs to a family of evolutionary conserved innate immune recognition molecules and recognizes double-stranded RNA, a molecular pattern associated with viral infections. Earlier studies suggested a differential expression pattern in men and mice; the molecular basis for this observation, however, was unknown. Here we demonstrate that species-specific differences in tissue expression and responses to lipopolysaccaride (LPS) coincide with the presence of different, evolutionary non-conserved promoter sequences in both species. Despite the overall unrelatedness of TLR3 promoter sequences, mRNA expression of both TLR3 orthologues was induced by interferons, particularly by interferon (IFN)-beta. The basal and IFN-beta-induced activation of promoters from both species largely depended on similar interferon regulatory factor (IRF) elements, which constitutively bound IRF-2 and recruited IRF-1 after stimulation. In murine macrophages, IFN-beta-induced TLR3 up-regulation required IFNAR1, STAT1, and in part IRF-1, but not the Janus kinase (Jak) family member Tyk2. We also show that LPS specifically up-regulates TLR3 expression in murine cells through the induction of autocrine/paracrine IFN-beta. In humans, however, IFN-beta-induced up-regulation of TLR3 was blocked by pretreatment with LPS, despite the efficient induction of IRF-1. Our findings reveal a mechanistic basis for the observed differences as well as similarities in TLR3 expression in men and mice. The IFN-beta-TLR3 link further suggests a role of TLR3 in innate and adaptive immune responses to viral infections. It will be interesting and important to clarify whether the observed differences in the transcriptional regulation of TLR3 influence innate immune responses in a species-specific manner.