Fine-tuning the reverse genetics of Tyk2
network contributionThe Janus kinase (Jak) Tyk2 is an important determinant in host immunity both in mice and in humans. Tyk2 deficiency results in increased sensitivity to microbial infections and increased tumor development but improves clinical symptoms in several autoimmune and inflammatory diseases. Tyk2 inhibitors are in development and are considered to be promising tools for the treatment of human diseases, including psoriasis, rheumatoid arthritis and inflammatory bowel disease. However, several lines of evidence suggest that Jaks may have kinase-independent activities, e.g. receptor stabilizing and adapter functions. Our major goals are to dissect kinase-dependent and -independent functions of Tyk2 in vivo. During the previous funding periods we have generated mice that express a kinase-inactive Tyk2 protein (Tyk2K923E) and could confirm our hypothesis that the lack of Tyk2 kinase activity does not phenocopy Tyk2 deficiency. Unexpectedly, we found that kinase-inactive Tyk2 contributes to tumor surveillance and our results point towards an involvement of natural killer (NK) cells in the process. In contrast, Tyk2 kinase activity is essential for canonical type I interferon (IFNα/β) signaling and to control viral infections in vivo. The most prominent defect in Tyk2-/- mice is impaired interleukin-12 (IL-12) signaling and a consequent failure to produce IFNγ upon a wide range of immunological challenges. This defect crucially contributes to both the increased sensitivity to infections and the resistance against inflammatory diseases. It may also account, at least partially, for the defective tumor surveillance observed in Tyk2-/- mice. IL-12 signaling is similarly impaired in Tyk2-/- and Tyk2K923E lymphocytes but surprisingly we found Tyk2 kinase-independent, delayed IFNγ production after Listeria monocytogenes infection in vivo.
Within the proposed project part we aim to determine how kinase-inactive Tyk2 contributes to NK cell activity and tumor surveillance. Furthermore, we aim at further dissecting kinase-dependent and -independent functions of Tyk2 in the regulation of IFN production and during innate and adaptive immune responses to L. monocytogenes infections.
Goals and Key Hypotheses:
Our research program is based on the following hypotheses:
(1) Kinase-inactive Tyk2 contributes to NK cell- but not CD8+ T cell-mediated tumor immune surveillance.
(2) Kinase-dependent and -independent functions of Tyk2 control NK cell development/maturation and/or NK cell cytotoxicity.
(3) (a) The presence of kinase-inactive Tyk2 impairs innate IFNγ production but triggers alternative adaptive immune cell activation, or (b) kinase-inactive Tyk2 blocks negative immune regulatory pathways acting on IFNγ-producing cells.
(4) Kinase-inactive Tyk2 can contribute to the control of low-dose L. monocytogenes infection and to the development of adaptive immunity.
Our specific goals are as follows:
(1) Determine tumor control in Tyk2K923E compared to Tyk2-/- and wild-type mice upon systemic and local transplantation of the melanoma cell line B16F10.
(2) Compare the NK cell developmental and maturation state in bone marroe, spleen, lymph nodes and blood derived from Tyk2-/-, Tyk2K923E and wild-type mice.
(3) Analyse Tyk2-/-, Tyk2K923E and wild-type NK cell cytotoxicity in vitro: induction of cytolytic machinery, activation of signaling pathways and transcriptional responses.
(4) Define the IFNγ-producing cell types in L. monocytogenes infected Tyk2-/-, Tyk2K923E and wild-type mice over time and, dependent on the results, further characterize immune cell activation and cytokine responses.
(5) Determine the sensitivity of Tyk2-/-, Tyk2K923E and wild-type mice to low-dose L. monocytogenes infections (bacterial growth/clearance over time).
(6) Determine the protective effect of vaccination against L. monocytogenes-infected Tyk2-/-, Tyk2K923E and wild-type mice (survival, bacterial load).
SFB-P08MM: Fine Tuning the Reverse Genetics of Tyk2 (Mathias Müller)
Research topic of the project part is to further understand the role of Tyk2 in infection, inflammation and cancer. During the last years, contribution of Tyk2 to lipopolysaccharide and ischemia/reperfusion induced shock/lethality was further characterized. In collaboration with the T. Decker group, the role of Tyk2 during Listeria monocytogenes infection was analyzed. Involvement of Tyk2 in NK and T cell-mediated tumor surveillance was established in collaboration with SFB-P10VS. Using proteomics and transcriptomics approaches, we identified novel Tyk2 target genes and found an as yet unrecognized role of Tyk2 in translational regulation. We furthermore successfully generated the proposed genetically modified mouse models for the fine-tuned analysis of Tyk2 function, i.e. mice expressing kinase-inactive Tyk2 and conditional Tyk2 knockout mice. Preliminary data with these mice support the hypothesis of kinase-independent functions of Tyk2 in inflammation/infection and tumorigenesis. In addition, we generated further mouse models for the detailed in vivo studies of canonical and non-canonical activities of Stat1 (conditional Stat1 knockout, Stat1 isoform-specific knock in, and transgenic mice for inducible Stat1 expression).
Report of Project Part 08: Fine Tuning the Reverse Genetics of Tyk2 and Stat1 (Birgit Strobl / Mathias Müller)
Tyk2 deficiency results in an increased sensitivity to infectious diseases in mice and humans and Tyk2-/- mice are prone to develop tumors. In contrast, Tyk2 promotes inflammatory and autoimmune diseases and is therefore considered to be a promising therapeutic target. Based on the hypothesis that inhibition of Tyk2 kinase activity does not completely mimic Tyk2 deficiency, we generated mice expressing a kinase-inactive Tyk2 protein (Tyk2K923E). Tyk2K923E mice displayed similar sensitivity to viral infections as Tyk2-/- mice but intriguingly kinase-inactive Tyk2 could control tumor growth to a similar extent as wild-type Tyk2 and partially contributes to the pathogenesis of sepsis. This clearly confirms our hypothesis and underlines the importance of defining kinase-independent functions of Tyk2 before considering therapeutic interventions.
Although it is expressed ubiquitously, Tyk2 also exerts cell type-specific functions. Conditional Tyk2 knockout mice (Tyk2fl/fl) allow us to study the contribution of Tyk2 in specific cell types to host immunity. In the previous funding period we postulated and confirmed that macrophage Tyk2 is important for the early antiviral defense against Murine Cytomegalovirus. Our data further suggest an organ-specific contribution of Tyk2 in dendritic cells (DCs).
Stat1 exists as two alternatively spliced isoforms, the full length Stat1α and the C-terminally truncated Stat1β. It is currently believed that Stat1β acts as a dominant negative isoform if Stat1 is activated as a homodimer, i.e. after interferon-γ (IFNγ) treatment. To our surprise, isoform-specific knock-in mice (Stat1Δα/Δα and Stat1Δβ/Δβ) revealed that Stat1β alone can induce transcription, albeit delayed/reduced compared to Stat1α. Consistently, IFNγ-dependent antibacterial activity is reduced but not absent in mice expressing Stat1β only (Stat1Δα/Δα). These data raise the important new issue of the extent to which Stat1α and/or Stat1β contribute to the many functions attributed to total Stat1.
The unexpected and complex findings in the Tyk2 and Stat1 mouse models have prompted us to divide our future work into two independent research proposals, concentrating on Tyk2 and Stat1 separately.
Department of Biomedical Sciences, Institute of Animal Breeding and Genetics
University of Veterinary Medicine Vienna, Vetmeduni Vienna
A-1210 Vienna, Austria
phone +43(0)1 25077-5601
fax +43(0)1 25077-5690