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.
Much of the tissue damage in multiple sclerosis (MS), rheumatoid arthritis, lupus inflammatory bowel disease and other increasingly common autoimmune disorders have been linked to a type of lymphocytes called Th17 cells that are important for clearing fungal and bacterial infections. However, in autoimmune disease, Th17 cells are further converted into `ex-Th17 cells' that produce inflammatory mediators and act as agents of tissue injury and inflammation. We recently discovered a mechanism based on the protein serpinB1 that restricts the number of Th17 cells generated and controls the survival of ex-Th17 cells; characterization of this mechanism has potential for improved therapeutics to limit the number of pathogenic ex-Th17 cells in MS and other autoimmune diseases.
| Hou, Lifei; Cooley, Jessica; Swanson, Richard et al. (2015) The protease cathepsin L regulates Th17 cell differentiation. J Autoimmun 65:56-63 |
