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. 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 (HIV) gp120 is being tested as a vaccine candidate. However little is known about how gp120 is delivered to B cells in lymph nodes. 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. Preliminary studies have also examined the localization and delivery of HIV particles. Using GPF labeled virus we used intravital 2-photon microscopy and thick lymph node confocal microscopy to study the uptake and delivery of HIV particles to the follicular dendritic cell network and eventually to B cells. 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 have studied the impact of three open reading frames from the SARS virus, ORF-9b, ORF-8b and ORF-3A. The results of our studies of ORF-9b have been published (J Immunol 193:3080-9, 2014) and our studies of ORF-8b submitted for publication. In the later studies we found that ORF8b activates NLRP3 inflammasomes. It likely did so by directly targeting the Leucine Rich Repeat domain of NLRP3. Expression of ORF8b triggered NLRP3, ASC, and caspase-1 dependent IL-1 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 cell death. We conclude that SARS-CoV expresses ORF8b to target NLRP3 inflammasomes. Studies of ORF-3a are still in progress. Preliminary results indicate that the expression of ORF-3a causes lysosomal damage triggering the translocation of a master regulator of lysosomal function (transcription factor EB, TFEB) to the nucleus. Expression of SARS-3a also causes a RIP3 dependent, but MLKL independent cell death. The release of cytochrome c from the inner mitochondrial membrane, where it is anchored by caridolipin, triggers the formation of the Apaf-1 apoptosome. Cardiolipin also interacts with NLRP3 recruiting NLRP3 to mitochondria and facilitating inflammasome assembly. We have investigated whether cytosolic cytochrome c impacts NLRP3 inflammasome activation in macrophages (manuscript submitted for publication). We report that cytochrome c binds to the LRR domain of NLRP3 and that cytochrome c reduces the interactions between NLRP3 and cardiolipin and between NLRP3 and NEK7, a recently recognized component of the NLRP3 inflammasome needed for NLRP3 oligomerization. Protein transduction of cytochrome c impairs NLRP3 inflammasome activation, while partially silencing cytochrome c expression enhances it. The addition of cytochrome c to an in vitro inflammasome assay severely limited caspase-1 activation. Apoptosis, pyroptosis and necroptosis all feature the activation of effector proteins that causes cellular death. Bcl-2 inhibits the pro-apoptotic proteins by binding to the BH3 domain present in those proteins. Necroptosis uses the effector protein, MLKL, and pyroptosis the effector protein gasdermin D. We have found that both MLKL and gasdermin D contain BH3-like domains that are capable of binding Bcl-2. Expression of high levels of Bcl-2 inhibit signals that trigger necroptosis and pyroptosis. Conversely, reduction in Bcl-2 levels sensitizes cells to both necroptosis and pyroptosis.
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