Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that is widely believed to be mediated by myelin-specific CD4+ T cells. Th17 cells, a recently defined subset of CD4+ T cells that secrete the cytokine IL-17 upon antigenic activation, have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), widely used as an animal model of MS. Upon commitment to the Th17 lineage, myelin-specific T cells acquire potent pathogenic properties and are readily recruited across the blood-brain-barrier following injection into naive syngeneic hosts. Furthermore, IL-17 transcripts have been detected in MS lesions but not normal appearing white matter.
In Aim 1 of this proposal we will expand upon preliminary data demonstrating that myelin-specific Th17 cells express a distinctive panel of chemokine receptors (ie, CXCR4, CCR6 and CXCR5) and adhesion molecules (such as α4β7 integrin) that could potentially facilitate their homing to the CNS. Specifically, we will polarize both murine and human myelin-specific T cells lines (the latter derived from patients with MS in addition to age and sex matched healthy controls) to either a Th17, Th1 or Th2 lineage and compare them among themselves, as well as to uncommitted memory cells, for expression of candidate homing molecules by real time RT-PCR and flow cytometric analyses. Based on the results of these experiments, we will determine which cell surface molecules are critical for the infiltration of the CNS by murine Th17 cells in the context of the EAE model. To do so, we will park CFSE-labeled myelin-specific Th17 cells into CD45.1 congenic, myelin-sensitized hosts and track their activation, expansion, and migration patterns in the presence or absence of antibodies that block selected chemokine or adhesion pathways.
In Aim 2 we will test our hypothesis that progressive CNS damage in MS (as documented by magnetic resonance/ diffusion tensor imaging) is associated with a cytokine milieu that favors the differentiation and expansion of Th17 cells. We have been monitoring chronic MS patients, off immunomodulatory medications, for evidence of acute inflammatory activity (by MRI with triple dose gadolinium) and for incremental tissue loss/ demyelination in the corpus callosum (via diffusion tensor imaging) on a bimonthly basis over the course of one year. We propose to measure levels of Th17 polarizing factors in peripheral blood mononuclear cells, collected from these same subjects over the same time frame, by real time RT-PCR and Elispot analyses. We predict that IL-6, TGF-β and IL-1α, which collectively act as a potent Th17 inducing stimulus, will be upregulated during, or immediately prior to, the formation of gadolinium enhancing lesions. Furthermore, we predict that those patients who develop enhancing lesions and exhibit cytokine dysregulation will be more likely to experience ongoing tissue loss/damage throughout the observation period.
We are hopeful that the data generated by the proposed studies will help identify novel radiological and cellular surrogate markers that are predictive of, or coincident with, MS disease activity, and provide insights into novel therapies designed to inhibit the development and/or CNS homing of encephalitogenic Th17 cells in patients with MS.