Heterotrimeric G Protein Signaling In Allergic Inflammation

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Mast cells (MCs), basophils, and lymphocytes are integral to the development of an allergic response. Degranulation of MCs and granulocytes, and cytokine production by T cells and antibody production in B cells is induced primarily by cross-linking of the receptor for antigen. However, allergic inflammation may also be generated through activation of receptors coupled to heterotrimeric G proteins (GPCRs). The purpose of this study is to understand mechanisms of G protein-mediated signal transduction in immune cells and subsequent pathways to inflammation. GPCRs activate a core pathway of heterotrimeric G proteins, which bind guanosine triphosphate (GTP) in exchange for guanosine diphosphate (GDP). The GTP-bound form of the G protein alpha subunit induces downstream signaling cascades, including intracellular calcium flux responsible for MC/basophil degranulation. This project focuses on a family of regulators of G protein signaling (RGS proteins), which inhibit the function of G alpha-i and G alpha-q, but not G alpha-s, proteins by increasing their GTPase activity. G alpha subunits oscillate between GDP- (inactive) and GTP- (active) bound forms based on ligand occupancy of the associated receptor. The GTPase accelerating (GAP) activity of RGS proteins limits the time of interaction of active G-alpha and its effectors, resulting in desensitization of GCPR signaling. Despite a growing body of knowledge concerning the biochemical mechanisms of RGS action, relatively little is known about the physiological role of these proteins in allergic inflammation. A major area of investigation is the recruitment of inflammatory cells to sites of inflammation. Chemokines are a major class of compounds acting on leukocyte GPCRs, which orchestrate immune cell trafficking, and RGS proteins including RGS13 and RGS16 inhibit chemokine signaling by desensitizing GPCR signals. CXCL12 and CXCL13 shape the formation of germinal centers (GCs) in lymphoid organs such as spleen and lymph nodes during a high affinity antibody response. GCs provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. We showed that IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies in autoimmune BXD2 mice. The majority of CXCR5+ TFH cells from BXD2-Il17ra-/- mice were mis-localized in the GC light zone (LZ). Acute interruption of IL-17 signaling disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of RGS16 to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16-/- mice. The results suggests that IL-17 induces a chemokine stop signal (RGS16) that acts on postdifferentiated IL-17RA+ TFH to enable its interaction with responder B cells in the LZ. The mechanisms by which RGS13 promotes the generation of pathogenic autoantibodies in GCs were determined using BXD2-Rgs13-/- mice. In spleens of BXD2 mice, RGS13 was mainly expressed by GC B cells and was stimulated by IL-17 but not IL-21. IL-17 upregulated Rgs13 in A20 GC but not 70Z/3 non-GC B cells. BXD2-Rgs13-/- mice had smaller GCs and lower adenosine induced deaminase (AID) levels, suggesting lower somatic hypermutation and affinity maturation during the antibody response. There were, however, increased IgMbright plasmablasts, upregulation of plasma program genes Irf4, Blimp1, Xbp1 and pCREB target genes Fosb and Obf1, with downregulation of GC program genes Aicda, Pax5 and Bach2 in GC B cells of BXD2-Rgs13-/- mice. BXD2-Rgs13-/- mice showed lower titers of IgG autoantibodies and IgG deposits in the kidney glomeruli, suggesting reduced autoantibody pathogenicity. These results indicate that RGS13 deficiency is associated with reduction in GC program genes and exit of less pathogenic IgM plasmablasts in BXD2 mice. A prolonged GC program, mediated by upregulation of RGS13, enhanced AID expression and enabled generation of pathogenic autoantibodies in autoreactive GCs. A second research area is the mechanisms controlling mast cell and basophil trafficking in allergic responses. Many allergens contain intrinsic proteolytic activity and bind protease activated GPCRs. Although sensitization to protease allergens, such as papain, helminth infection, chronic allergic skin inflammation, and nasal rhinitis are associated with basophil recruitment to inflamed tissue or to draining lymph nodes (LNs), the precise role of basophils and mechanisms involved in their recruitment is incompletely understood. Basophils have the capacity to present antigen to naive T cells and promote TH2 differentiation directly or indirectly through IL-4 production. We are currently examining the chemotaxis of basophils to various chemoattractants in vitro with the goal of determining which are responsible for basophil recruitment to tissues during the aforementioned allergic processes. We are generating mouse strains containing mast cells or basophils hyper- or hyporesponsive to chemokines in order to study the contribution of these cells to various allergic responses.