We have shown that RGS proteins modulate signaling through a variety of G-protein coupled receptors including chemokine receptors. Chemokine receptors signal predominantly by triggering Gαi nucleotide exchange. Humans and mice have three Gi isoforms although Gαi2 (encoded by Gnai2) and Gαi3 (encoded by Gnai3) predominate in lymphoid cells. We have found that Gnai2 -/- T and B cells have severe defects in chemokine-receptor signaling while Gnai2 +/- T and B cells exhibit modest defects. In vivo, the Gnai2-/- B cells fail to properly access lymph node follicles and the Gnai2-/- T cells failing to properly enter the T cell zone. We have studies the immune system with a focus on immune cell trafficking in various RGS deficient mice. We have supplemented these studies with in vitro studies using mRNA knock-downs as well as over expression studies using RGS proteins fused to a fluorescent marker. The recognition of the importance of both G-protein signaling and RGS proteins in the regulation of lymphocyte responses to chemokines has led us some of our studies to hematopoietic cell types. RGS13 is strongly expressed in both human and mouse germinal center B cells and is often found highly expressed in human B cell lymphomas of germinal center origin. RGS13 is also well expressed in mast cells and in adult T cell leukemia cells. To facilitate our studies of RGS13 and to provide a potential resource for in vivo imaging endogenous germinal center B cells we have generated mice where GFP (green fluorescent protein) has been knocked-in (KI) into the Rgs13 locus replacing the RGS13 coding sequence. Flow cytometry revealed high levels of GFP expression in germinal center centroblasts and centrocytes. Intravital microscopy documented the presence of brightly GFP positive cells in the interfollicular region and at the T-B border within a day of immunization. Later strong expression is observed in B cells located in germinal centers. Immune phenotyping of these mice revealed a minor defect in early B cell development with a normal peripheral lymphoid compartment with the exception of a 25% expansion in the number of Peyers patches. Upon immunization the Rgs13-/- mice exhibited an enhanced extra-follicular antibody response, an exaggerated germinal center response, and a disturbance of germinal center architecture. Analysis of mRNA expression in Rgs13GFP positive B cells revealed a marked enrichment in germinal center specific genes as well as genes involved in cell proliferation. These studies are aimed at understanding the role RGS13 plays in germinal center B cell function. Rgs14 is well expressed in lymphocytes and initially targeting in mice indicated that its absence resulted in embryonic lethality at the two cell stage. To analyze the role of Rgs14 in immune cells we created Rgs14LoxP mice. However, deletion of Rgs14 was not deleterious so we have analyzed mice globally lacking Rgs14 expression. Initial analysis of the immune system of these mice was unrevealing. Rgs19 is well expressed in B lymphocytes and less so in T cells. It is also well expressed in stem cells and hematopoietic progenitors. GFP has been inserted into the Rgs19 locus and appropriated targeted mice identified. Flow cytometry revealed that GFP is well expressed in mature B and T cells and in progenitors in the bone marrow. Consistently the Rgs19 GFP KI mice had an enlarged spleen and thymus compared to wild type mice. Chemotaxis experiments comparing wild type and Rgs19-/- lymphocytes indicate significant increases to a panel of chemokines with the KO B cells. Serum levels of IgM, IgG2a, and IgG2b are elevated in these mice. Current studies are aimed understanding the pathogenesis of the splenomegaly and enhanced B cell responsiveness in the Rgs19-/- mice. To better understand the functional importance of Rgs19 expression we have compared gene expression profiles in the mutant versus wild type B cells by high throughput RNA sequencing. The results of this analysis are pending. As another approach to assessing the role of RGS proteins we have begun the analysis of mice with a Gnai2 knock-in obtained from Richard Neubig (University of Michigan). This mutation renders Gαi2 resistant to the effect of RGS proteins. These KI mice possess a striking phenotype verifying the overall important of RGS proteins in G-protein regulation. They have a marked reduction in peripheral T cells;an increase in mature T cells in the thymus;a thymocyte egress defect;an absence of many peripheral lymph nodes;disorganized lymphoid organ architecture;splenomegaly;abnormal lymphocyte responses to chemokines;and reduced serum levels of IgG3, but increased levels of Ig2b. Neutrophils from these mice accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Intravital imaging revealed a failure of the mutant neutrophils to accumulate at and stabilize sites of sterile inflammation. Furthermore, these mice could not control a non-lethal Staphylococcus aureus infection. Neutrophil RGS proteins establish a threshold for Gαi activation helping to coordinate desensitization mechanisms. Their loss renders neutrophils functionally incompetent. Further analysis of B cells from these mice revealed have they markedly elevated basal calcium levels, but poor chemokine induced increases;enhanced non-specific migration, but extremely poor chemotaxis. In striking contrast, the same B cells exhibited enhanced sensitivity to Sphingosine 1-Phosphate (S1P). Mice with the Gαi2 mutation displayed excessive numbers of distorted germinal centers;abnormal serum immunoglobulin profiles;and aberrant B lymphocyte trafficking. These findings established an essential role for RGS proteins in B cell chemoattractant signaling and for the proper position of B lymphocytes in lymphoid organs. It has been increasing evident that G-proteins and G-protein signaling have role in cells beyond their known roles as receptor transducers. We have begun a study of the role of G-proteins in macrophage phagocytosis. Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors whether such signaling pathways affect phagocytosis is contentious. We have shown that Gαi nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin and Gαi proteins to cell protrusions, phagocytic cups, and early phagosomes. Inhibiting Gαi nucleotide exchange or pharmacologically interrupting Gβγsignaling impairs phagocytosis while favoring the duration that Gαi remains GTP bound promotes it. Macrophages lacking Gαi2 or Gαi3, or with lowered expression of the Gαi nucleotide exchange factor Ric-8A have reduced phagocytic capacities. These studied revealed an important for Gαi signaling in coordinating actin-driven cell protrusions and the cell remodeling needed to phagocytize large particles.

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Hwang, Il-Young; Boularan, Cedric; Harrison, Kathleen et al. (2018) G?i Signaling Promotes Marginal Zone B Cell Development by Enabling Transitional B Cell ADAM10 Expression. Front Immunol 9:687
Lee, Hong Kyung; Kim, Hyung Sook; Kim, Ji Sung et al. (2017) CCL2 deficient mesenchymal stem cells fail to establish long-lasting contact with T cells and no longer ameliorate lupus symptoms. Sci Rep 7:41258
Hwang, Il-Young; Park, Chung; Harrison, Kathleen et al. (2017) Normal Thymocyte Egress, T Cell Trafficking, and CD4+ T Cell Homeostasis Require Interactions between RGS Proteins and G?i2. J Immunol 198:2721-2734
Hwang, Il-Young; Harrison, Kathleen; Park, Chung et al. (2017) Loss of G?i proteins impairs thymocyte development, disrupts T-cell trafficking, and leads to an expanded population of splenic CD4(+)PD-1(+)CXCR5(+/-) T-cells. Sci Rep 7:4156
Kehrl, John H (2016) The impact of RGS and other G-protein regulatory proteins on G?i-mediated signaling in immunity. Biochem Pharmacol 114:40-52
Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C et al. (2016) Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection. J Immunol 196:846-56
Park, Chung; Hwang, Il-Young; Kehrl, John H (2016) Intravital Two-Photon Imaging of Lymphocytes Crossing High Endothelial Venules and Cortical Lymphatics in the Inguinal Lymph Node. Methods Mol Biol 1407:195-206
Boularan, Cedric; Hwang, Il-Young; Kamenyeva, Olena et al. (2015) B Lymphocyte-Specific Loss of Ric-8A Results in a G? Protein Deficit and Severe Humoral Immunodeficiency. J Immunol 195:2090-102
Hwang, Il-Young; Park, Chung; Harrison, Kathleen et al. (2015) An essential role for RGS protein/G?i2 interactions in B lymphocyte-directed cell migration and trafficking. J Immunol 194:2128-39
Branham-O'Connor, Melissa; Robichaux 3rd, William G; Zhang, Xian-Kui et al. (2014) Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3). J Biol Chem 289:10738-47

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