The overall goal of this revised proposal is to identify the mechanisms by which IL-17 and IFN1 act in concert to initiate the development of B cells that produce autoantibodies in the BXD2 model of autoimmunity. We have found that both IL-17 and IFN1 are required for the formation of germinal centers (GCs) that enable the production of pathogenic autoantibodies in autoimmune BXD2 mice, i.e., a deficiency of either of these cytokines abrogates GC formation, autoantibody formation, and autoimmune disease in these mice. Analysis of the effects of IL-17 on GC formation in the BXD2 mice suggest that IL-17 facilitates the interaction of CD4 T cells and B cells thereby enhancing the formation and stabilization of spontaneous germinal centers (GCs), and that this effect is mediated by upregulation of regulators of G-protein signaling (Rgs)13 and Rgs16 in the B cells. The principal investigator's most recent results indicate that there is an increased number of plasmacytoid DCs (pDCs) in the marginal sinus in BXD2 mice and that these pDCs exhibit significantly elevated expression of IFN1. Notably, there is a robust marginal zone precursor B cell population (MZP) that exhibits an enhanced ability to transport antigen (Ag) directly into a GC light zone area. Based on our accumulated preliminary data, we have formulated a hypothetical model in which IL-17 and IFNa act in a coordinated manner to facilitate the spontaneous GC response through regulating the migration behavior of Ag-transporting MZP B cells. We propose to test this hypothetical model by addressing two of its essential components: (1) Does IFNa regulate influx and IL-17 regulate the retention of MZP in the GC LZ area? (2) Do IFNa and IL-17 regulate Ag transport by MZP B cells leading to GC development? To answer the first question, the ability of IL-17 and IFNa to regulate chemotaxis will be determined in vitro using a transwell chamber, and in vivo by analyzing homing of GFP+ MZP B cells in BXD2 mice in which IL-17R, IFNaR, or Rgs genes has been modulated. This will be achieved by using B cells obtained from BXD2-Il-17r-/-, BXD2-Ifnar-/-, BXD2-Rgs13-/- and BXD2-Rgs16-/- mice. To answer the second question, in vivo Ag capture and transportation by MZP B cells will be determined by FACS and confocal imaging analyses. The Ag transport function of MZP B cells will be studied using the hen egg lysozyme (HEL) or HEL-ovalbumin (OVA) conjugate strategies in combination with BXD2 mice that express the MD4 B-cell receptor transgene and the OVA T-cell receptor transgene.
Successful accomplishment of the project will help to identify critical migratory signals and upstream mediators that facilitate the transportation of Ag to initiate a GC response. The results will provide new insights into the mechanisms of action of currently used therapies based on modulation of IFN1 as well as the proposed targeting of Th17 cells in autoimmune disease as well as suggesting novel targets for therapeutic intervention.
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