Regulation of Th17 immune responses by serpinB1

Boston Children'S Hospital, Boston MA

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Abstract

? DESCRIPTION (provided by applicant): At mucosal surfaces, Th17 cells and their products IL-17A, IL-17F and IL-22 protect the host from fungal and bacterial infections. However, in autoinflammatory and autoimmune settings, the initially generated Th17 cells are converted into ex-Th17 cells that produce IFN? and GM-CSF and are agents of pathogenesis of rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis (MS), inflammatory bowel disease and other increasingly common chronic inflammatory autoimmune diseases. We recently discovered a regulatory module based on serpinB1 that controls Th17 cells at two key events: (1) differentiation of naive CD4 T cells to Th17 cells and (2) homeostasis of ex-Th17 cells. We present new evidence that serpinB1 KO (sb1-/-) mice immunized with KLH or MOG generate increased antigen-specific splenic Th17 cells that produce more IL-17 than WT (Hou et al, manuscript submitted). In the EAE model, we found that sB1 regulates pathogenicity as predicted; however the findings are opposite from predicted and paradoxical. Despite increased splenic MOG- specific Th17 cells, immunized sb1-/- mice are resistant to EAE. Preliminary findings suggest that the critical event affected by sB1 in EAE is the paucity, likely the survival of GM-CSF+ and IFN+ ex-Th17 cells in the CNS. The proposed project is focused on solidifying the evidence that (i) sB1 of CD4 T cells is a serious force controlling pathogenicity in EAE, (ii) outlining the underlying mechanism and (iii) identifying the cathepsin protease that partners with sB1 in the regulatory module. In aim 1 we will cross sb1flox/flox mice with CD4Cre to create sb1?CD4 mice with which we will demonstrate/ verify that attenuation of pathogenicity and paucity of sb1- /- ex-Th17 cells are due to CD4-cell intrinsic sB1. We will determine in BrdU `pulse-chase' studies whether the paucity of sB1neg ex-Th17 cells is due to inadequate proliferation or inadequate survival. We anticipate finding decreased survival (accelerated cell death). We will verify accelerated cell death of sB1neg ex-Th17 cells by transcriptome analysis of ex-Th17 cells generated by MOG immunization of Rag1-/ recipients bearing mixed adoptively transferred CD45.1 sb1flox/flox and CD45.2 sb1?CD4 CD4 T cells. Because protease inhibition is sB1's only known biochemical function, we postulated that regulation of Th17 responses by sB1 requires the intermediacy of a protease. For Th17 differentiation, we identified the co-regulator as the cysteine protease cathepsin L (catL). Aim 2 will identify the protease that co-regulates (by counteracting sB1) survival of ex-Th17 cells and pathogenicity of EAE. We will determine the effect of pharmacological cathepsin inhibitors on density of ex-Th17 cells in MOG immunized mice. In aim 2.2, we will determine whether deleting the catL gene (ctsl) in sb1-/- mice rescues the attenuation of encephalomyelitis and the paucity of ex-Th17 cells. For technical reasons (details in aim 2) the mice that will be compared will be MOG-immunized Rag-/ chimeric mice bearing WT or sb1-/- or ctsl-/-sb1-/- CD4 T cells. Increased understanding of this central regulatory mechanism in murine EAE pathogenicity is relevant for improved therapy of MS and other Th17 dominated autoimmune diseases.