To better understand the role of GCK and GCKR in vivo, the murine Gck and Gckr genes have been isolated. Both Gck-/- and Gckr -/- mice have been created and backcrossed on to a C57Bl/6 background used to generate double knock-out (KO) mice. The mutations did not affect mouse development as the Gck, Gckr, and double KO mice are born with normal Mendelian frequencies. We have nearly completed the analysis of these mice and are now focused on the double KO mice as they exhibit a sharp reduction in the number of follicular B cells, an expansion of marginal zone B cells, hypergammaglobulemia, defective humoral responses to neo-antigens, and evidence of autoimmunity. B lymphocyte recirculation through lymph nodes requires crossing endothelial barriers and chemoattractant-triggered cell migration. We have shown how lymph node anatomy and chemoattractant receptor signaling organize B lymphocyte trafficking through lymph nodes. Surprisingly B and T cells differ in their entrances and initial trafficking through high endothelial venules and their entrance into lymph node follicle and T cell zone, respectively. We have also begun a study of how local immunization alters homeostatic lymphocyte trafficking through the draining lymph node. We have shown how immunization induced changes in lymph node architecture along with cell intrinsic factors facilitate and how these changes help coordinate the trafficking of B cells into and through lymph nodes following immunization. We have shown that immunization leads to a rapid recruitment of newly arrived B cells into the lymph node follicle and causes the cells to be retained within the follicle. We are currently trying to understand the underlying mechanisms that alters B lymphocyte trafficking. We have also begun to examine how various antigens are delivered to B and T cells in the lymph node by intravital microscopy. In addition we have developed methods for intravital imaging lymphoid and hematopoietic progenitor in the bone marrow. Our studies of chemokine receptor signaling have focused on the proximal elements in the signaling pathway. These receptors predominantly use the heterotrimeric G protein Gi to link to downstream signaling pathways. We have shown Gi alpha proteins regulate and co-localize with F-actin at actin-rich structures, including microspikes or filopodia, lamellipodia, adhesion sites, phagocytic cups, actin comet tails, sub-cortical ruffles and stress fibers. Consistently, reduction of Gi alpha protein expression altered cell morphology, modulated actin filaments and microtubules, reduced cell migration, and reduced macrophage phagocytosis. That Gi alpha facilitates the interplay of actin and microtubule was further supported by observations that Gi alpha depletion and PTX treatment both altered the dynamics and distribution of myosin X, Rab5, Rac, and the actin bundling protein fascin, which directs myosin X to tips of filopodia. Conversely, CXCL12 treatment induced the translocation of Gi alpha, myosin X, Rab5 and fascin to the plasma membrane, and increased their co-localization at filopodia, periphery ruffle and circular ruffle. Interestingly, in conjunction with Dr. S. Venkatesan (NIAID) we have shown that Gi alpha2 is a target of the Nef protein of the human immunodeficiency virus (HIV). Nef binds Gi alpha2 and leads to its endolysomal degradation. Since Nef can be secreted by infected cells or transferred to non-infected cells via intracellular bridges, non-infected cells will be subject to Nef-mediated Gi alpha2 degradation, thereby impacting the trafficking and homing of non-infected cells in HIV patients. This likely contributes to the early immune cell dysfunction following HIV infection. Nef has hijacked a normal mechanism that regulates Gi alpha2 stability. As a potential downstream effector in the chemokine receptor signaling pathway, we have examined the functional role of the non-muscle myosin Myo1e, which is significantly enriched in B lymphocytes. We have established Myo1e-/- mice and are studying the consequences of the loss of this protein on B lymphocyte function. We have also crossed the Myo1e-/- mice with mice lacking Myo1f, another non-muscle myosin that is enriched in both B and T lymphocytes. The study of the double knock-out mice is being done in conjunction with Dr. Steve Shaws laboratory (NCI). Also in collaboration with Steve Shaw's laboratory we have used intravital microscopy to investigate the behavior of T cells from mice transgenic for ezrin (T567D). This mutation confers a phosphominetic phenotype upon erzin. Chemokine receptor signaling normally leads to ezrin T567 dephosphorylation. T cells from these mice adhere normally to high endothelial venules, but compared to wild type cells they have impaired transendothelial migration. In addition, these T cells have a reduced velocity in the lymph node cortex and impaired lymph node egress. This study implicates Ezin dephosphorylation as important step for T lymphocyte transmigration and in normal motility. We have also assisted Dr. Richard Proias laboratory in the analysis of S1pr4 deficient mice. Autophagy delivers cytoplasmic constituents to autophagolysosomes and is linked to both innate andadaptive immunity. Toll-like receptor 4 (TLR4) signaling induces autophagy and recruits Beclin-1, the mammalian homolog of yeast Atg6, to the receptor complex. We found that tumor necrosis factor receptor (TNFR)associated factor 6 (TRAF6)mediated, Lys63 (K63)linked ubiquitination of Beclin-1 is critical for TLR4-triggered autophagy in macrophages. Inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1 beta and IL-18. Suggesting interplay between these pathways, lipopolysaccharide induces inflammasome activation in macrophages genetically deficient in autophagy proteins, but not in wild type macrophages. We have shown that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered RalB activation and autophagosome formation. The induction of autophagy did not depend upon ASC or capase-1, but did depend upon the inflammasome sensor. Blocking autophagy potentiated inflammasome activity while stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes. We have recently initiated a project of understand the role of G-protein signaling in the regulation of inflammasome activity. Preliminary result indicate that GPCR signaling alters inflammasome activity. Treatment of macrophages with omega-3 fatty acids leads to a reduction of inflammasome activity that depends upon the expression of the GPCR GPR120, a known receptor for omega 3 fatty acids.
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