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 cells from these mice have a shortened lifespan and exhibit an exaggerated activation of caspase 3 following in vitro activation. 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. Recently, we have also begun to examine how various antigens are delivered to B and T cells in the lymph node by intravital microscopy. The envelope protein of the human immunodeficiency virus gp120 is being tested as a vaccine candidate. However little is known about the cellular transit mechanism in lymph nodes by which gp120 is delivered to B and T cells. Using intravital microscopy to follow the transit of gp120 we uncovered a novel mechanism by which gp120 is captured and delivered to lymphocytes and dendritic cells. Our findings elucidate the major target cells for gp120 in the lymph node and demonstrate an antigen delivery mechanism for high molecular weight protein antigens in lymph nodes. 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. Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. We investigated the neutrophil influx following the local injection of Staphylococcus aureus bioparticles adjacent to the inguinal lymph node and determined their impact on the lymph node humoral response. Using intravital microscopy we show that local immunization recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells and their depletion augments IgM and IgG production. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF-beta1 dependent manner. Our data reveals that initial wave of neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampens the early humoral response in the lymph node. Severe acute respiratory syndrome (SARS) is a recently recognized viral infectious disease. A 2002-3 international outbreak occurred where patients suffered a severe respiratory illness with close to 10% mortality. We identified three mechanisms by which open reading frame-9b (ORF-9b) of the SARS coronavirus (SARS-CoV) contributes to disease pathogenesis. ORF-9b localizes to mitochondria causing mitochondrial elongation by ubiquitination and proteasomal degradation of DRP1, a host protein involved in mitochondrial fission; it inhibits antiviral immune responses by targeting the mitochondrial-associated adaptor molecule MAVS signalosome for PCBP2/AIP4 dependent destruction; and it triggers ATG5-dependent autophagy. We have also shown that ORF8b activates NLRP3 inflammasomes in a macrophages It likely does so by directly targeting the Leucine Rich Repeat domain of NLRP3. Expression of ORF8b triggered NLRP3, ASC, and caspase-1 dependent IL-1β secretion without need for Toll-like receptor (TLR) priming. When transiently expressed ORF8b co-localizes in cytosolic dot-like structures with NLRP3 and ASC, and caused THP-1 cell death. We conclude that SARS-CoV expresses ORF8b to target NLRP3 inflammasomes. In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs Galpha i3, Activator of G-protein Signaling-3 (AGS3/GPSM1), and Regulator of G-protein Signaling 19 (RGS19). As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and formation of LC3 puncta in bone marrow derived macrophages from wild type, Gnai3-/-, Gpsm1-/-, or Rgs19-/- mice following amino acid starvation or nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3-/-, and Gpsm1-/- macrophages. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked Galpha i3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while Galpha i signaling may impact autophagy in some cell types it does not in macrophages. The omega-3 (ω3) fatty acid docosahexaenoic acid (DHA) can suppress inflammation, specifically IL-1β production through poorly understood molecular mechanisms. We have shown that DHA reduces macrophage IL-1β production by limiting inflammasome activation. Exposure to DHA reduced IL-1β production by ligands that stimulate the NLRP3, AIM2, and NAIP5/NLRC4 inflammasomes. The inhibition required Free Fatty Acid Receptor (FFAR) 4 (also known as GPR120), a G-protein coupled receptor (GPR) known to bind DHA. The exposure of cells to DHA recruited the adapter protein β-arrestin1/2 to FFAR4, but not to a related lipid receptor. DHA treatment reduced the initial inflammasome priming step by suppressing the nuclear translocation of NF-κB. DHA also reduced IL-1β levels by enhancing autophagy in the cells. As a consequence macrophages derived from mice lacking the essential autophagy protein ATG7 were partially resistant to suppressive effects of DHA. Thus, DHA suppresses inflammasome activation by two distinct mechanisms, inhibiting the initial priming step and by augmenting autophagy, which limits inflammasome activity.
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