Multiple Sclerosis (MS) is a disease characterized by the formation of perivascular inflammatory lesions that result from the influx of leukocytes across the BBB, a process regulated by the cross-communication between astrocytes and BBB-ECs. In this regard, I have uncovered a novel and critical role of the Hedgehog (Hh) pathway on supporting BBB properties under homeostatic and inflammatory conditions (Alvarez et al., Science 2011, Alvarez et al., Brain 2015). However, the mechanims by which CNS intrinsic defense mechanisms regulate neuroinflammation remain poorly characterized. In this proposal, I aim to investigate the influence of the Hh pathway on neuroinflammatory responses. I have preliminary data demonstrating that Sonic Hh (Shh) is expressed by human astrocytes whereas the receptors and transcription factors of the Hh signaling pathway are express by T cells and microglia. In addition, blocking the Hh pathway during the course of Experimental Autoimmune Encephalomyelitis (EAE), the mouse model of MS, exacerbates disease severity. Thus, I hypothesize that astrocyte-secreted Shh tampers the inflammatory phenotype of peripheral (T cells) and endogenous (microglia) immune elements. To address this hypothesis, I propose the following 2 specific aims:
Aim 1 : To assess the influence of the Hh pathway on T cell function. In this aim we will determine the effect of Hh activation in the skewing of inflammatory T helper cells (Th1/Th17) derived from control and MS patients. We will also study the impact of Shh on the adhesion and migration of these cells across human BBB- ECs. In vivo validation will be performed using the EAE model by passive transfer of Hh-conditioned encephalithogenic CD4 T cells into nave mice. We will also actively induce EAE in mice lacking the Hh signaling specifically on CD4 T cells (CD4-Cre; smoc/c). Aside from determining multiple pathological and immunological parameters of disease, we will also monitor T cell behavior within the CNS by intravital imaging.
Aim 2 : To determine the role of the Hh pathway in regulating the inflammatory properties of microglia. We will first determine the expression of the Hh components in vitro/in situ in microglia during health and disease (MS/EAE). Then, we will establish the in vitro effect of Hh stimulation/abrogation on microglia. In these cells we will measure multiple immunological parameters (M1 vs M2 phenotype, antigen presentation, proliferation and T cell skewing). In addition, we will include assays using co-cultures of microglia with resting and inflamed astrocytes. These studies will be complemented in vivo by inducing EAE in mice lacking Hh signaling in microglia (cxcr3-Cre; smoc/c). In these mice, we will determine the clinical, pathological and immunological parameters associated with disease. Long-term goals: These studies will provide the framework to establish my independent scientific career Given the mentoring team helping me in this endeavor and our interaction with other scientists and clinicians, I aim to build the foundations to develop a successful research program within the field of neuroimmunology.
In the last 20 years, most research investigating neuroinflammatory diseases like multiple sclerosis (MS) has focused on the individual contributions of the immune system and central nervous system (CNS) to pathology. However, given the close interaction between the two systems, I want to explore: How do CNS-intrinsic defense mechanisms regulate neuroinflammation. These studies will provide a better understanding of the pathogenesis behind neuroinflammatory disorders and will open new avenues in the design of therapeutics to control leukocyte migration into the CNS, to enhance CNS protective mechanisms or conversely, to improve delivery of chemotherapeutic agents in the CNS compartment.
