The Function of IL-12 and IL-23 in Central Nervous System Autoimmune Disease

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2009.When an infection takes place in an otherwise healthy individual, CD4+ T-helper (Th) cells specific for bacterial or viral epitopes receive instructions from antigen presenting cells, often in the form of cytokine signals, allowing Th cells to respond appropriately to the pathogen. Through mechanisms that are not fully understood, Th cells specific for self-antigens, such as myelin, are sometimes improperly activated by cytokines, leading to inflammation and autoimmunity. Adoptive transfer experiments have shown that CD4+ T cells reactive against myelin proteins initiate destructive inflammation in experimental autoimmune encephalomyelitis (EAE), a widely used animal model of multiple sclerosis (MS). The observation that an MHC class II allele is the genetic locus most strongly associated with MS susceptibility, implicates CD4+ T cells in the human disease as well. A comprehensive understanding of the cytokine signals that lead to CD4+ T cell-driven autoimmunity is critical for the design of therapeutic agents that target specific inflammatory mediators. Interleukin-12 (IL-12) and interleukin-23 (IL-23) constitute the p40 family of cytokines and are potent factors for the polarization and stabilization of CD4+ T cells. Administration of anti-IL-12 p40 neutralizing antibodies suppresses EAE, and these results were initially interpreted as demonstrating a critical role for IL-12 p40 and by extension, IFN-producing Th1 cells, in disease induction. However, the p40 subunit is shared by IL-23, which is composed of the p40 and p19 subunits and is important for stabilization and maintenance of IL-17 producing Th17 cells in vivo. Subsequent studies revealed that mice deficient in IL-23 p19 are resistant to EAE by active immunization, while IL-12 p35 deficient mice are susceptible, indicating that IL-23 rather than IL-12 is the critical cytokine for EAE induction. Nevertheless, experiments in cytokine deficient mice come with many caveats, due to the potential of those mice to acquire compensatory pathways during development. In fact, some of the EAE data generated with cytokine deficient mice are discrepant with studies examining the role of cytokines in wild-type mice. For example, while IL-12p35 deficient mice are susceptible to EAE, incubation of wild-type myelin-specific CD4+ T cells with recombinant IL-12 enhances their encephalogenicity in several experimental systems. Furthermore, unlike IL-23p19 deficient mice, mice lacking IL-17 remain susceptible to EAE. Hence, much remains to be elucidated regarding the mechanism of action of IL-12 and IL-23 in disease pathogenesis. In order to further explore the roles of IL-12/23 and Th1/Th17 cells in EAE, we carefully measured myelin-specific cytokine responses during relapsing remitting disease. These studies revealed that both IFN+ and IL-17+ cells accumulate in the lymphoid tissues and CNS in association with disease activity. Th1 and Th17 responses remained focused on the immunizing epitope and did not spread to secondary epitopes throughout the course of multiple relapses. We found that both IL-12- and IL-23-modulated cells were encephalitogenic in two different mouse models of adoptively transferred EAE, and that each cell type caused disease with distinct characteristics. In SJL mice, IL-12-modulated T cells induced EAE characterized by macrophage-rich CNS lesions and up-regulation of CXCL9-11 mRNA. On the other hand, IL-23-modulated cells induced an influx of neutrophils into the spinal cord, concomitant with up-regulation of CXCL1 and CXCL2. While IL-23-modulated EAE was ameliorated by treatment with neutralizing antibodies to IL-17 and GM-CSF, IL-12-modulated EAE was unaffected by these therapies. We questioned whether myelin-specific T cells modulated with IL-12 could induce disease independent of IFN production or IL-17 signaling. First we transferred IL-12-polarized, myelin-specific CD4+ T cells into either wild-type or IL-17RA deficient mice. We found that the encephalitogenicity of Th1 cells is not compromised in the complete absence of IL-17A/F signaling. Next we compared the encephalitogenic properties of IL-12-modulated wild-type and IFN deficient (IFN -/-) T cells. Consistent with studies from other laboratories, IFN-/- T cells were encephalitogenic but induced an atypical form of EAE characterized by ataxia and neutrophil-rich brainstem infiltrates. Interestingly, the transfer of IL-12-modulated IFN -/- CD4+ T cells to IL-17RA deficient mice resulted in conventional EAE, demonstrating an absolute requirement for IL-17A/F signaling in atypical EAE. Conventional EAE in IL-17RA deficient mice receiving IFN -/- “Th1” cells was completely abrogated upon neutralization of GM-CSF, indicating that GM-CSF can mediate the effector phase of EAE in the absence of IL-17A/F and myelin-specific T cell-derived IFN. These studies demonstrate that there are numerous independent, but related, immunological pathways capable of causing inflammatory autoimmune disease within the central nervous system. A fuller understanding of these pathways will greatly enhance our ability to identify critical cytokines in distinct subclasses of patients, allowing for more specific therapies with increased efficacy