Tyk2-deficient Mice Research Articles

Augmentative effects of leukemia inhibitory factor reveal a critical role for TYK2 signaling in vascular calcification

Medial vascular calcification in chronic kidney disease (CKD) involves pro-inflammatory pathways induced by hyperphosphatemia. Several interleukin 6 family members have been associated with pro-calcific effects in vascular smooth muscle cells (VSMCs) and are considered as therapeutic targets. Therefore, we investigated the role of leukemia inhibitory factor (LIF) during VSMC calcification. LIF expression was found to be increased following phosphate exposure of VSMCs. LIF supplementation aggravated, while silencing of endogenous LIF or LIF receptor (LIFR) ameliorated the pro-calcific effects of phosphate in VSMCs. The soluble LIFR mediated antagonistic effects towards LIF and reduced VSMC calcification. Mechanistically, LIF induced phosphorylation of the non-receptor tyrosine-protein kinase 2 (TYK2) and signal transducer and activator of transcription-3 (STAT3) in VSMCs. TYK2 inhibition by deucravacitinib, a selective, allosteric oral immunosuppressant used in psoriasis treatment, not only blunted the effects of LIF, but also interfered with the pro-calcific effects induced by phosphate. Conversely, TYK2 overexpression aggravated VSMC calcification. Ex vivo calcification of mouse aortic rings was ameliorated by Tyk2 pharmacological inhibition and genetic deficiency. Cholecalciferol-induced vascular calcification in mice was improved by Tyk2 inhibition and in the Tyk2-deficient mice. Similarly, calcification was ameliorated in Abcc6/Tyk2-deficient mice after adenine/high phosphorus-induced CKD. Thus, our observations indicate a role for LIF in CKD-associated vascular calcification. Hence, the effects of LIF identify a central pro-calcific role of TYK2 signaling, which may be a future target to reduce the burden of vascular calcification in CKD.

Tyrosine kinase 2 modulates splenic B cells through type I IFN and TLR7 signaling

Tyrosine kinase 2 (TYK2) is involved in type I interferon (IFN-I) signaling through IFN receptor 1 (IFNAR1). This signaling pathway is crucial in the early antiviral response and remains incompletely understood on B cells. Therefore, to understand the role of TYK2 in B cells, we studied these cells under homeostatic conditions and following in vitro activation using Tyk2-deficient (Tyk2−/−) mice. Splenic B cell subpopulations were altered in Tyk2−/− compared to wild type (WT) mice. Marginal zone (MZ) cells were decreased and aged B cells (ABC) were increased, whereas follicular (FO) cells remained unchanged. Likewise, there was an imbalance in transitional B cells in juvenile Tyk2−/− mice. RNA sequencing analysis of adult MZ and FO cells isolated from Tyk2−/− and WT mice in homeostasis revealed altered expression of IFN-I and Toll-like receptor 7 (TLR7) signaling pathway genes. Flow cytometry assays corroborated a lower expression of TLR7 in MZ B cells from Tyk2−/− mice. Splenic B cell cultures showed reduced proliferation and differentiation responses after activation with TLR7 ligands in Tyk2−/− compared to WT mice, with a similar response to lipopolysaccharide (LPS) or anti-CD40 + IL-4. IgM, IgG, IL-10 and IL-6 secretion was also decreased in Tyk2−/− B cell cultures. This reduced response of the TLR7 pathway in Tyk2−/− mice was partially restored by IFNα addition. In conclusion, there is a crosstalk between TYK2 and TLR7 mediated by an IFN-I feedback loop, which contributes to the establishment of MZ B cells and to B cell proliferation and differentiation.

Role of tyrosine kinase 2 signals during progression of psoriasis

Psoriasis is a skin disease characterized by scaly erythema, parakeratosis, and epidermal hyperplasia. Application of imiquimod (IMQ), a ligand for Toll-like receptor 7, produces a mouse model for psoriasis. IMQ application induces scaling, erythema, and thickness in skin lesions, and the symptoms are milder in interleukin-23 p19 (Il23p19)-deficient and Il17a-deficient mice than in wild-type mice; this suggests that the interleukin-23 (IL-23)/T helper 17 (Th17) axis and Th17 cell-secreting cytokines play essential roles in the IMQ-induced psoriasis model. It is notable that a genome-wide association study identified the human tyrosine kinase 2 (TYK2) gene within the psoriasis susceptibility locus. After IMQ application, mice lacking Tyk2, a mouse homologue of the human TYK2 gene, exhibited significantly lower symptom scores of psoriasis and diminished inflammatory cell infiltration in the skin lesions. Tyk2-deficient mice also failed to increase CD4+IL-17+ or CD4+ interferon-γ+ (IFN-γ+) T cells in the draining lymph nodes or to produce Th17 cell-related cytokines after IMQ application. Furthermore, Tyk2 deficiency led to diminished skin inflammation induced by IL-23 and IL-22 injections. These results indicate that Tyk2-mediated signals in mice contribute to multiple steps of immune and inflammatory responses during the development of psoriasis; therefore, TYK2 targeting may be a promising strategy to treat patients with psoriasis. Recent clinical trials have shown that TYK2 inhibitors have a high overall response rate with good tolerability in the management of psoriasis. This review describes the fundamental mechanisms of Tyk2 inhibition in immune/inflammatory diseases.

Current understanding of the role of tyrosine kinase 2 signaling in immune responses.

Immune system is a complex network that clears pathogens, toxic substrates, and cancer cells. Distinguishing self-antigens from non-self-antigens is critical for the immune cell-mediated response against foreign antigens. The innate immune system elicits an early-phase response to various stimuli, whereas the adaptive immune response is tailored to previously encountered antigens. During immune responses, B cells differentiate into antibody-secreting cells, while naïve T cells differentiate into functionally specific effector cells [T helper 1 (Th1), Th2, Th17, and regulatory T cells]. However, enhanced or prolonged immune responses can result in autoimmune disorders, which are characterized by lymphocyte-mediated immune responses against self-antigens. Signal transduction of cytokines, which regulate the inflammatory cascades, is dependent on the members of the Janus family of protein kinases. Tyrosine kinase 2 (Tyk2) is associated with receptor subunits of immune-related cytokines, such as type I interferon, interleukin (IL)-6, IL-10, IL-12, and IL-23. Clinical studies on the therapeutic effects and the underlying mechanisms of Tyk2 inhibitors in autoimmune or chronic inflammatory diseases are currently ongoing. This review summarizes the findings of studies examining the role of Tyk2 in immune and/or inflammatory responses using Tyk2-deficient cells and mice.

Therapeutic Advantage of Tyk2 Inhibition for Treating Autoimmune and Chronic Inflammatory Diseases

Tyrosine kinase 2 (Tyk2) is a member of the Janus family of protein tyrosine kinases (Jaks). Tyk2 associates with interferon (IFN)-α, IFN-β, interleukin (IL)-6, IL-10, IL-12, and IL-23 receptors and mediates their downstream signaling pathways. Based on our data using Tyk2-deficient mice and cells, Tyk2 plays crucial roles in the differentiation, maintenance, and function of T helper 1 (Th1) and Th17 cells, and its dysregulation may promote autoimmune and/or inflammatory diseases. IFN-α-induced growth inhibition of B lymphocyte progenitors is dependent on Tyk2-mediated signals to regulate death-associated protein (Daxx) nuclear localization and Daxx-promyelocytic leukemia protein interactions. Tyk2-deficient mice show impaired constitutive production of type I IFNs by macrophages under steady-state conditions. When heat-killed Cutibacterium acnes is injected intraperitoneally, Tyk2-deficient mice show less granuloma formation through enhanced prostaglandin E2 and protein kinase A activities, leading to high IL-10 production by macrophages. Thus, Tyk2 is widely involved in the immune and inflammatory response at multiple events; therefore, Tyk2 is likely to be a suitable target for treating patients with autoimmune and/or chronic inflammatory diseases. Clinical trials of Tyk2 inhibitors have shown higher response rates and improved tolerability in the treatment of patients with psoriasis and inflammatory bowel diseases. Taken together, Tyk2 inhibition has great potential for clinical application in the management of a variety of diseases.

Tyk2-mediated homeostatic control by regulating the PGE<sub>2</sub>-PKA-IL-10 axis

<abstract> Tyrosine kinase 2 (Tyk2), which associates with the receptors for type I interferon (IFN) and interleukins (IL)-6, IL-10, IL-12, and IL-23, is critical to mediate cytokine-induced signals. Tyk2 plays an essential role in the constitutive production of small amount of type I IFNs and in the promotion of differentiation from naïve T cells into Th1 or Th17 effector cells via IL-12- and IL-23-induced signals. Additionally, Tyk2-mediated signaling suppresses the <italic>in vivo</italic> production of IL-10, which is a strong anti-inflammatory cytokine. The elevated IL-10 production in the peritoneal cells of Tyk2-deficient mice are alleviated by treatment with either diclofenac, a cyclooxygenase inhibitor, or H-89, a protein kinase A inhibitor. Notably, significantly higher basal prostaglandin E₂ (PGE₂) production is observed in peritoneal cavity of Tyk2-deficient mice than that of wild-type mice. Phosphorylation of cAMP response element-binding protein, induced by <italic>P. acnes</italic> and PGE₂ addition, is upregulated in Tyk2-deficient macrophages. This indicates that higher IL-10 production in Tyk2-deficient mice is likely a result of the enhanced PGE₂-protein kinase A pathway. Thus, Tyk2-mediated signaling regulates multiple events during immune and/or inflammatory responses. </abstract>

IκB-ζ Expression Requires Both TYK2/STAT3 Activity and IL-17-Regulated mRNA Stabilization.

Cytokine IL-17A (IL-17) acts on various cell types, including epidermal keratinocytes, and induces antimicrobial peptide and chemokine production to elicit antibacterial and antifungal defense responses. Excess IL-17 leads to inflammatory skin diseases such as psoriasis. The IκB family protein IκB-ζ mediates IL-17-induced responses. However, the mechanism controlling IκB-ζ expression in IL-17-stimulated cells remains elusive. In this study, we showed that JAK kinase TYK2 positively regulates IL-17-induced IκB-ζ expression. TYK2-deficient mice showed reduced inflammation and concomitant reduction of IκB-ζ mRNA compared with wild-type mice in imiquimod-induced skin inflammation. The analysis of the IκB-ζ promoter activity using human cell lines (HaCaT and HeLa) revealed that catalytic activity of TYK2 and its substrate transcription factor STAT3, but not IL-17, is required for IκB-ζ promoter activity. In contrast, IL-17-induced signaling, which did not activate STAT3, posttranscriptionally stabilized IκB-ζ mRNA via its 3'-untranslated region. IL-17 signaling protein ACT1 was required to counteract constitutive IκB-ζ mRNA degradation by RNase Regnase-1. These results suggested that transcriptional activation by TYK2-STAT3 pathway and mRNA stabilization by IL-17-mediated signals act separately from each other but complementarily to achieve IκB-ζ induction. Therefore, JAK/TYK2 inhibition might be of significance in regulation of IL-17-induced inflammatory reactions.

NK Cells Require Cell-Extrinsic and -Intrinsic TYK2 for Full Functionality in Tumor Surveillance and Antibacterial Immunity.

Tyrosine kinase 2 (TYK2) is a widely expressed receptor-associated kinase that is involved in signaling by a variety of cytokines with important immune regulatory activities. Absence of TYK2 in mice results in impaired NK cell maturation and antitumor activity, although underlying mechanisms are largely unknown. Using conditional ablation of TYK2 in NK cells we show that TYK2 is required for IFN-γ production by NK cells in response to IL-12 and for an efficient immune defense against Listeria monocytogenes Deletion of TYK2 in NK cells did not impact NK cell maturation and IFN-γ production upon NK cell activating receptor (actR) stimulation. Similarly, NK cell-mediated tumor surveillance was unimpaired upon deletion of TYK2 in NK cells only. In line with the previously reported maturation-associated Ifng promoter demethylation, the less mature phenotype of Tyk2-/- NK cells correlated with an increased CpG methylation at the Ifng locus. Treatment with the DNA hypomethylating agent 5-aza-2-deoxycytidine restored the ability of Tyk2-/- NK cells to produce IFN-γ upon actR but not upon IL-12 stimulation. NK cell maturation was dependent on the presence of TYK2 in dendritic cells and could be rescued in Tyk2-deficient mice by treatment with exogenous IL-15/IL-15Rα complexes. IL-15 treatment also rescued the in vitro cytotoxicity defect and the impaired actR-induced IFN-γ production of Tyk2-/- NK cells. Collectively, our findings provide the first evidence, to our knowledge, for a key role of TYK2 in the host environment in promoting NK cell maturation and antitumor activity.

In vivo tumor surveillance by NK cells requires TYK2 but not TYK2 kinase activity

Tyrosine kinase 2 (TYK2) is a Janus kinase (JAK) that is crucially involved in inflammation, carcinogenesis and defense against infection. The cytotoxic activity of natural killer (NK) cells in TYK2-deficient (Tyk2 −/−) mice is severely reduced, although the underlying mechanisms are largely unknown. Using Tyk2 −/− mice and mice expressing a kinase-inactive version of TYK2 (Tyk2 K923E ), we show that NK cell function is partly independent of the enzymatic activity of TYK2. Tyk2 −/− and Tyk2 K923E NK cells develop normally in the bone marrow, but the maturation of splenic Tyk2 −/− NK cells (and to a lesser extent of Tyk2 K923E NK cells) is impaired. In contrast, the production of interferon γ (IFNγ) in response to interleukin 12 (IL-12) or to stimulation through NK cell-activating receptors strictly depends on the presence of enzymatically active TYK2. The cytotoxic activity of Tyk2 K923E NK cells against a range of target cells in vitro is higher than that of Tyk2 −/− NK cells. Consistently, Tyk2 K923E mice control the growth of NK cell-targeted tumors significantly better than TYK2-deficient mice, showing the physiological relevance of the finding. Inhibitors of TYK2's kinase activity are being developed for the treatment of inflammatory diseases and cancers, but their effects on tumor immune surveillance have not been investigated. Our finding that TYK2 has kinase-independent functions in vivo suggests that such inhibitors will leave NK cell mediated tumor surveillance largely intact and that they will be suitable for use in cancer therapy.

Role of Tyk-2 in Th9 and Th17 cells in allergic asthma.

In a murine model of allergic asthma, we found that Tyk-2(−/−) asthmatic mice have induced peribronchial collagen deposition, mucosal type mast cells in the lung, IRF4 and hyperproliferative lung Th2 CD4+ effector T cells over-expressing IL-3, IL-4, IL-5, IL-10 and IL-13. We also observed increased Th9 cells expressing IL-9 and IL-10 as well as T helper cells expressing IL-6, IL-10 and IL-21 with a defect in IL-17A and IL-17F production. This T helper phenotype was accompanied by increased SOCS3 in the lung of Tyk-2 deficient asthmatic mice. Finally, in vivo treatment with rIL-17A inhibited local CD4+CD25+Foxp3+ T regulatory cells as well as Th2 cytokines without affecting IL-9 in the lung. These results suggest a role of Tyk-2 in different subsets of T helper cells mediated by SOCS3 regulation that is relevant for the treatment of asthma, cancer and autoimmune diseases.

Tyk2-dependent bystander activation of conventional and nonconventional Th1 cell subsets contributes to innate host defense against Listeria monocytogenes infection.

IL-12, which is produced in response to intracellular bacteria, such as Listeria monocytogenes, promotes the development of pathogen-specific Th1 cells that play an important role in host defense. However, it has also been known that CD44(high) memory-phenotype CD4 T cells with Th1 functions naturally occur in naive mice, and that lymphopenia-induced proliferation of naive CD4 T cells generates memory-phenotype CD4 T cells with Th1 functions, although their differentiation mechanism and contribution to host defense are unclear. In this study, we analyzed the development and the functions of the different subsets of Th1 cells by using mice lacking tyrosine kinase 2 (Tyk2), a member of the Janus kinase family critically involved in IL-12 signaling. In contrast with the case of conventional Ag-specific Th1 cells, the development of naturally occurring Th1 cells was not impaired in Tyk2-deficient mice. In addition, Th1 cells were normally generated from Tyk2-deficient naive CD4 T cells via lymphopenia-induced proliferation. Nevertheless, all these Th1 subsets, including conventional Ag-induced Th1 cells, produced IFN-γ in response to IL-12 in a Tyk2-dependent manner. Importantly, such Tyk2-dependent bystander IFN-γ production of any Th1 subsets conferred early protection against L. monocytogenes infection. Thus, Tyk2-mediated IL-12 signaling is differentially required for the development of different Th1 cell subsets but similarly induces their bystander IFN-γ production, which contributes to innate host defense against infection with intracellular bacteria.

Tyk2 and Stat3 Regulate Brown Adipose Tissue Differentiation and Obesity

Mice lacking the Jak tyrosine kinase member Tyk2 become progressively obese due to aberrant development of Myf5+ brown adipose tissue (BAT). Tyk2 RNA levels in BAT and skeletal muscle, which shares a common progenitor with BAT, are dramatically decreased in mice placed on a high-fat diet and in obese humans. Expression of Tyk2 or the constitutively active form of the transcription factor Stat3 (CAStat3) restores differentiation in Tyk2(-/-) brown preadipocytes. Furthermore, Tyk2(-/-) mice expressing CAStat3 transgene in BAT also show improved BAT development, normal levels of insulin, and significantly lower body weights. Stat3 binds to PRDM16, a master regulator of BAT differentiation, and enhances the stability of PRDM16 protein. These results define Tyk2 and Stat3 as critical determinants of brown fat lineage and suggest that altered levels of Tyk2 are associated with obesity in both rodents and humans.

STAT1-Deficient Mice Are Resistant to Cecal Ligation and Puncture–Induced Septic Shock

STAT1 (signal transducer and activator of transcription 1) is a member of the JAK-STAT signaling family and plays a key role in facilitating gene transcription in response to activation of the types I and II interferon (IFN) receptors. TYK2 is essential for type I, but not type II, IFN-induced STAT1 activation. Previous studies show that STAT1-deficient mice are resistant to endotoxin-induced shock. The goal of the present study was to assess the response of STAT1- and TYK2-deficient mice to septic shock caused by cecal ligation and puncture (CLP). End points included survival, core temperature, organ injury, systemic cytokine production, and bacterial clearance. Results showed that survival rates were significantly higher in STAT1 knockout (STAT1KO) mice compared with wild-type controls (80% vs. 10%). The improved survival of STAT1KO mice was associated with less hypothermia, metabolic acidosis, hypoglycemia, and hepatocellular injury. Plasma interleukin 6, MIP-2, CXCL10, and IFN-α concentrations were significantly lower in STAT1KO mice than in wild-type mice. In the absence of antibiotic treatment, blood and lung bacterial counts were significantly lower in STAT1KO mice than in controls. However, treatment with antibiotics ablated that difference. A survival advantage was not observed in TYK2-deficient mice compared with control. However, CLP-induced hypothermia and systemic interleukin 6 and CXCL10 production were significantly attenuated in TYK2-deficient mice. These results indicate that STAT1 activation is an important factor in the pathogenesis of CLP-induced septic shock and is associated with the development of systemic inflammation and organ injury. TYK2 activation also appears to contribute to CLP-induced inflammation, but to a lesser extent than STAT1.

Tyk2 signaling pathway and Mycobacterium tuberculosis (Mtb) infection (174.19)

Abstract IL-12 is required for the optimal IFNg T-cell response, which is required for control of Mtb growth. IL-12R signaling is mediated by Tyrosine kinase 2 (Tyk2) which belongs to the janus kinase (JAK) family. Tyk2 phosphorylates STAT4 and STAT3, which subsequently induce transcription of target genes. We wanted to define the role of Tyk2 in IL-12R signaling during control of Mtb infection. In order to do this, we infected Tyk2 deficient mice via the aerosol route with Mtb and followed progression of the disease. We found that absence of Tyk2 resulted in a modest increase in bacterial burden associated with slightly reduced cytokine secretion (IL-17, IFNg) and T cell maturation. These data suggest Tyk2 plays a minor role in the expression of the protective response during Mtb infection. It is likely therefore that the other components of IL-12R signaling (JAK1,2 and 3) are the principal mediators of IL-12 activity in this model. These data may also reflect a role of Tyk2 in other signaling pathways such as those initiated by IL-10 and type I IFNs.

Tyk2 deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice

Tyrosine kinase-2 (Tyk2) participates in the signaling pathways of multiple cytokines in innate and acquired immunity. In the present study, we investigated the in vivo involvement of Tyk2 in anti-type II collagen antibody-induced arthritis (CAIA) using Tyk2-deficient mice. Hind paws of wild-type mice showed massive swelling and erythema by arthritogenic antibody injection, whereas Tyk2-deficient mice did not show any signs of arthritis. Indeed, neither the infiltration of inflammatory cells nor the fibrillation of articular cartilages was observed in Tyk2-deficient mice. Tyk2 deficiency also reduced the production of T(h)1/T(h)17-related cytokines, the other proinflammatory cytokines and matrix metalloproteases, which are induced in the CAIA paw. Our results demonstrate a critical contribution of Tyk2 in the development of arthritis, and we propose that Tyk2 might be an important candidate for drug development.

Tyk2 signaling plays a critical role in the development of T cytotoxic 1 responses following chronic rBCG-OVA infections in mice

Objective To elucidate potential roles of tyrosine kinase 2 （Tyk2） in the generation and maintenance of Ag-specific CD8＋ T cells. Methods We followed the fate of OVA-specific CD8 ＋ T cells in Tyk2-deficient （ Tyk2 -/- ） mice after infection with recombinant OVA-expressing BCG （ rBCGOVA ）. Because the immunostimulatory BCG-derived peptides recognized by CD8 ＋ T cells have not been defined, and the OVA is definite peptide for specific CD8 ＋ T cells that has been accepted widely, therefore we examine the kinetics of the OVA-Ag-specific CD8 ＋ T cell response after rBCG-OVA infection in mice.Tyk2-/- and wild type（Tyk2＋/＋ ） mice were inoculated with rBCG-OVA by intra-trachea（ i. t. ）, after the examination of bacterial growth in the lung and spleen, the population of CD8 ＋ T cells were detected by FACS analysis, the epitope-specific CD8 ＋ T cells were followed with tetrameric H-2Kb molecule folding with OVA257 264 peptide, and the kinetics of Ag-specific CD8 ＋ Tc1 cells were detected by intracellular IFN-γ production in response to OVA257-264 peptide by cytokine FACS analysis. Results After rBCG-OVA challenge,the bacteria number in spleen and lung of Tyk2 -/- mice were significantly larger than those in Tyk2 ＋/＋ mice on days 14, 21 and 49. Almost as same as that in Tyk2＋/＋ mice, the size of epitope-specific CD8＋ T cella with OVA257-264/Kb-tetramer-positive and the CD8 ＋Tc1 （T eytotoxic 1 ）cells positive for intracellular IFN-γ could proliferate to its peak on day 21, then contract and maintain to the memory phase in spleen and lung of Tyk2-/- mice, but the population of CD8＋ T cells in spleen and lung of Tyk2 -/- mice were significantly smaller than those in Tyk2＋/＋ mice on days 21 and 49, the number of epitope-specific CD8＋ T cells in spleen and lung of Tyk2 -/- mice were significantly decreased and the frequency of CD8 ＋ Tc1 cells in spleen and lung of Tyk2 -/- mice significantly reduced on day 21,49 and 70 after rBCG-OVA infection. So correspond with the larger number of bacteria in Tyk2-/- mice than those in Tyk2 ＋/＋ mice, the expansion of OVA257-264-specific CD8 ＋ T cells and CD8＋ Tc1 response were attenuated in Tyk2 -/- mice following rBCG-OVA infection. Conclusion These results suggest that the lack of Tyk2 signaling impairs the proliferation and difference of effector CD8 ＋ T cell to rBCG-OVA infection and at least, is partly responsible for the susceptible to the rBCG-OVA infection. Key words: Tyrosine kinase 2 Mice rBCG-OVA CD8＋ T cell

Tyrosine kinase 2 (TYK2) in cytokine signalling and host immunity

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signal transduction pathway is essential to transmit signals from transmembrane receptors to the nucleus in order to alter gene expression programs and to respond to extracellular cues. Tyrosine kinase 2 (TYK2) was the first member of the JAK family that was identified within a screen for molecules complementing human cell lines mutant for interferon (IFN) responses. During the last decades biochemical studies and gene-targeted mice uncovered the crucial role of TYK2 in immunity. Tyk2-deficient mice are viable and fertile but display multiple immunological defects, most prominently high sensitivity to infections and defective tumour surveillance. In contrast, absence of TYK2 results in increased resistance against allergic, autoimmune and inflammatory diseases. In support of these data, the only patient with TYK2 deficiency described so far displays high serum immunoglobulin E (IgE) levels and increased sensitivity to infectious diseases. Furthermore, numerous genome-wide association studies in humans propose a link between TYK2 genetic variants and several autoimmune diseases, inflammatory diseases and tumours. Thus, TYK2 appears as an attractive target for therapeutic intervention. Future work will be required to further delineate structure-function relationships and to fully understand the involvement of TYK2 in immune regulatory networks.

The role of Tyrosine kinase 2 on Th17 development in a murine model of allergic asthma (141.7)

Abstract Mutations of the Janus Kinase Tyrosine Kinase 2 (Tyk2) in humans are associated with hypereosinophilia, Hyper-IgE and susceptibility to viral and mycobacterial infections. This syndrome was found to be the result of defective signaling of both IL 12 and IFNalpha as well as abnormal responses to IL 6, IL-10 and IL-23. Here we describe asthmatic Tyk2 deficient mice suffered from extreme airway eosinophilic inflammation and hyper IgE production compared to wild type mice. As these symptoms are associated with an increase in Th2 cytokines IL-4 and IL-5 in the airways, the major goal of the project is to investigate the role of Tyk2 in CD4+ T cell subset differentiation in the lung in a murine model of acute asthma. Th2 and early Th17 effector T cells are important in establishing the inflammation seen in the airways in this disease. In contrast, regulatory T cells and late developed Th17 T cells have a protective function on it. Under Th17 skewing conditions naïve CD4+ T cells from Tyk2 deficient mice showed an IL-6-dependent defect on Th17 cell development. Consistently, we found hyper-proliferation of Th2 cells associated with a constitutive inhibition of the Th1 and Th17 pathway in the lung of asthmatic Tyk2 deficient mice compared to wild type mice. In summary these data suggest that overexpression of Tyk2 could lead to amelioration of the asthmatic symptoms because this would suppress the pathogenic disease-specific CD4+ T effector cells in the lung.

The role of Tyrosine kinase 2 in a murine model of allergic asthma (139.9)

Abstract Tyrosine Kinase 2 is a member of the mammalian Janus kinase (JAK) family of protein tyrosine kinases which consists of three more kinases (JAK1-3). Tyk-2 mutation in humans is associated with a completely defective in signaling in response to IFNα and IL-12, abnormal responses to IL-6, IL-10 and IL-23, susceptibility to viral infection and to mycobacterial infection and Hyper IgE Syndrome. Microscopic analyses showed that asthmatic Tyk2(-/-) mice suffered from extreme airway eosinophilic inflammation as compared to wild type mice. We found that Th1 differentiation is severely impaired in Tyk2(-/-) mice as shown by decreased IFNγ production by lung CD4+ T cells. By contrast, an increase in Th2 cytokines could be measured in the supernatants of lung CD4+ T cells isolated from Tyk2(-/-) mice. Preliminary results on T regulatory cell differentiation revealed an ascent of CD4+CD25+Foxp-3+ T regulatory cells in asthmatic Tyk2(-/-) mice by FACS analysis. Consistent with this finding, quantitative Real-Time PCR also showed an increase of the T regulatory cell-specific transcription factor Foxp3 in Tyk2(-/-) mice. As both the T regulatory inducing IL-10-pathway and the T reg inhibiting IL-6-pathway are blocked in Tyk2 deficient mice only TGFβ might be regulating Foxp-3 expression in T regulatory cells in these mice. We next wish to investigate the suppressive function of T regulatory cells in the absence of Tyk-2 in allergic models of asthma. This work is supported by Graduiertenkolleg 1043 "Antigen-specific Immunotherapy".

Selective contribution of Tyk2 to cell activation by lipopolysaccharide

Tyk2 deficient mice show a markedly reduced susceptibility to lipopolysaccharide (LPS) induced shock and a partial impairment of IL-12 and interferon (IFN) signals. To examine the underlying mechanisms, we analysed the activation of peritoneal macrophages (PMΦs) and spleen cells after LPS challenge. In PMΦs and spleen cells the contribution of Tyk2 to the induction of the T-cell co-stimulatory molecules CD86, CD40 and MHC II was small or insignificant. By contrast, induced expression of the early activation marker CD69 on PMΦs and splenic cell populations required type I interferons (IFN-I) and Tyk2. The data suggest a selective contribution of Tyk2 to the activation of inflammation-relevant cell types by LPS.