|
11. Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna:
Experimental autoimmune myocarditis (EAM) appears after infectious heart disease, the most common cause of dilated cardiomyopathy in humans. Here we report that mice lacking T-bet, a T-box transcription factor required for T helper (Th)1 cell differentiation and interferon (IFN)-gamma production, develop severe autoimmune heart disease compared to T-bet+/+ control mice. Experiments in T-bet-/- IL-4-/- and T-bet-/- IL-4Ralpha-/- mice, as well as transfer of heart-specific Th1 and Th2 cell lines, showed that autoimmune heart disease develops independently of Th1 or Th2 polarization. Analysis of T-bet-/- IL-12Rbeta1-/- and T-bet-/- IL-12p35-/- mice then identified interleukin (IL)-23 as critical for EAM pathogenesis. In addition, T-bet-/- mice showed a marked increase in production of the IL-23-dependent cytokine IL-17 by heart-infiltrating lymphocytes, and in vivo IL-17 depletion markedly reduced EAM severity in T-bet-/- mice. Heart-infiltrating T-bet-/- CD8+ but not CD8- T cells secrete IFN-gamma, which inhibits IL-17 production and protects against severe EAM. In contrast, T-bet-/- CD8+ lymphocytes completely lost their capacity to release IFN-gamma within the heart. Collectively, these data show that severe IL-17-mediated EAM can develop in the absence of T-bet, and that T-bet can regulate autoimmunity via the control of nonspecific CD8+ T cell bystander functions in the inflamed target organ.
The role of Cbl-b and dendritic cells in arthritis
1. Objectives
Besides genetic susceptibility, environmental triggers and infectious agents have been implicated in the pathogenesis of multiple autoimmune diseases, as well as in asthma. However, in most autoimmune diseases the causative environmental agents and the endogenous molecules that control immunotolerance and susceptibility to autoimmunity are not known. We have recently identified the E3 ubiquitin ligase Cbl-b as a critical and essential regulator of immunotolerance in vivo (Immunity, in press 2004). Moroever, we have developed a novel approach to trigger autoimmune heart inflammation with self antigens loaded onto dendritic cells (Nature Med. 2003). The experiments are designed to understand the role of Cbl-family proteins and Cbl-associated molecules in arthritis and to develop a novel animal model for arthritis research using dendritic cell immunizations.
Project 1: The role of the E3 ubiquitin ligase Cbl-b in arthritis
Efficient immunity is critically dependent on the activation of T cells and the generation of cytokines such as interleukin 2 (IL-2). T cell activation and cytokine production require two signals: 1) A signal via the peptide-specific TCR and 2) a signal via a costimulatory receptor. A principal costimulatory receptor in T cells is CD28 that allows long-term proliferation and T cell survival in vivo. Failure to activate the second signal results in T cell unresponsiveness. Lack of immunity to many tumors or chronic infections can be ascribed to the fact that these cells do not provide a second signal for T cell activation
The three mammalian Cbl-family proteins, c-Cbl, Cbl-b, and Cbl-3, are RING-finger containing E3 ubiquitin ligases that control degradation, localization, receptor recycling, or protein-protein interaction of multiple signaling molecules. Genetic inactivation of c-Cbl results in altered thymocyte selection whereas mutation of Cbl-b in mice leads to spontaneous autoimmunity and renders disease-resistant mice susceptible to experimental autoimmunity. Thus, our experiments in Cbl-b knock-out mice (Bachmaier et al. & Penninger, Nature 2000) provided the first molecular link between protein ubiquitination and autoimmunity. Moreover, Cbl-b mutant mice develop spontaneous diabetes in a transgenic system which is in line with a recent report that a Cbl-b mutation is the cause of type I diabetes in a rat model (Nature Genetics 2002). Mechanistically, loss of Cbl-b leads to TCR-mediated T cell activation in the absence of any CD28 costimulation. The Penninger laboratory has both c-Cbl and Cbl-b mutant mice available and has recently generated knock-out mice that carry a mutation in the third mammalian Cbl-family protein, Cbl-3 (Griffiths & Penninger, unpublished).
To extend our tolerance studies to in vivo autoimmunity, we will generate Cbl-b-/- DBA/1 mice. The mice will be immunized with collagen emulsified in mycobacteria-containing complete Freund's adjuvant (CFA) and/or incomplete Freund's adjuvant (IFA).
Besides disease incidence, histopathological features of the joints such as synovial hyperplasia and erosion of cartilage and bone, will be analyzed in in collagen-IFA primed Cbl-b+/+ and Cbl-b-/- DBA/1 mice. The production of anti-collagen antibodies is characteristic for the inflammatory responses in the joints. To examine whether Cbl-b ablation affects the pathogenic antibody production, sera from collagen-IFA immunized mice will be collected and the concentration of collagen-specific IgG2a was determined.
The molecular mechanism of Cbl-b regulated immunotolerance and autoimmunity will be anlayzed with a specific focus on proximal TCR signaling molecules such as PLCg1, PKCtheta, or p85-PI3K. These experiments are designed to privide the first genetic and experimental evidence that Cbl-b is a critical negative regulator of antigen-induced arthritis induction in vivo. These experiments will allow us to establish whether Cbl-b has a critical role in tissue specific autoimmunity and to establish a novel autoimmune animal models for arthritis. In collaboration with other members of the network, we plan to extend these mouse studies to human arthritis patients.
Project 2: Dendritic cells and arthritis: establishment of a new arthritis animal model
We have recently establish the first model that makes it possible to trigger a autoimmune heart disease using DCs. A single injection of as few as 100,000 DCs into completely naïve Blab/c mice induces massive, CD4+ T cell mediated heart inflammation followed by post-inflammatory heart failure. This system allows us to genetically dissect signaling pathways in DCs that determine whether DCs induce tolerance or induce autoimmunity and to establish new models of autoimmunity.
We will load DCs in our novel culture system with previously established autantigens for arthritis and immunize "naïve" DBA/1 (and other mouse strains) mice with antigen loaded DCs. Arhtiris will be analyzed as above. Using this system it might be also able to identify novel arthritis autoantigens. Since Cbl-b and its sibling c-Cbl are expressed in DCs, we will use this system to determine whether genetic inactivation of Cbl-b or c-Cbl alters the function of DCs. The Cbl-b and c-Cbl regulated signaling pathways in DCs will be determined: receptor recycling, activation of common downstream pathways, does Cbl-b or c-Cbl regulate raft and receptor clustering in DCs, cytokine production. One critical signal for DC-induced autoimmunity is the activation of Toll-like receptors indicating that autoimmunity can only occur in the presence of tissue injury and infections. We will therefore study the role of infectious particles and Toll-like receptors in autoimmunity.
|
|
