To better understand the role of MAP4K2 and MAP4K5 in vivo, the murine map4k2 and map4k5 genes have been isolated. Both map4k2-/- and map4k5-/- mice have been created and backcrossed on to a C57Bl/6 background used to generate double knock-out (KO) mice. We have 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, elevated serum antibodies, 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. With the recognition that MAP4Ks can participate in the Hippo pathway, we have acquired mice lacking LATS1 and mice with a floxed allele of LATS2. These mice are being analyzed to assess the role of LATS kinases in the phenotype found in the map4k2/5 double KO mice. Leucine-rich repeat kinase 2 (LRRK2) is a large, 2527 amino acid protein with multiple functional and protein interaction domains. It is well expressed in B lymphocytes and macrophages. Autosomal dominant mutations in LRRK2 are the most common genetic cause of Parkinsons disease and genetic linkage studies have identified its potential involvement in Crohn's disease and leprosy. Given that patients with LRRK2 mutations show changes in autophagy, lysosomes, and in immune activation, we investigated whether LRRK2 regulated Transcription Factor EB (TFEB),a basic helix-loop-helix protein considered the master transcriptional regulator of autophagy and lysosomal biogenesis. Our studies show that TFEB is an immune response gene in macrophages and B cells. In dissecting the molecular mechanisms, we found that LRRK2 increases CREB activity, which controls TFEB transcription. In addition, LRRK2 phosphorylates TFEB on its C-terminus, which stabilizes the protein. We also showed that Lrrk2 mediated TFEB nuclear translocation is dependent on the lysosomal calcium channel TPC2 and calcineurin. Besides it role in Lrrk2 signaling we have also identified TFEB as a negative regulator of the Wnt signaling pathway. To confirm this result we developed a HEK293T cell line that lacks TFEB expression by disrupting the gene using CRISPR. 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. 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. Preliminary studies have also examined the localization and delivery of HIV-1 viral like particles (VLPs). Using GPF labeled VLPs we used intravital 2-photon microscopy and thick lymph node confocal microscopy to study the uptake and delivery of HIV VLPs to the follicular dendritic cell network and eventually to B cells. In a collaboration with Paolo Lusso laboratory we found that HIV-1 VLPs expressing alpha4beta7 target to Peyer's patch high endothelium via an interaction with MAdCAM. Severe acute respiratory syndrome (SARS) is a recently recognized viral infectious disease. 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). In more recent studies we have found that ORF8b rapidly aggregates in cells triggering the activation of NLRP3 inflammasomes. This leads to a rapid inflammatory cell death. When transiently expressed ORF8b co-localizes in cytosolic dot-like structures with NLRP3 and ASC. Studies of ORF-3a indicate that the expression of ORF-3a causes lysosomal damage triggering the translocation of 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. We found 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.

Project Start
Project End
Budget Start
Budget End
Support Year
22
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
Zip Code
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
Harris, James; Lang, Tali; Thomas, Jacinta P W et al. (2017) Autophagy and inflammasomes. Mol Immunol 86:10-15
Nabar, Neel R; Kehrl, John H (2017) The Transcription Factor EB Links Cellular Stress to the Immune Response??. Yale J Biol Med 90:301-315
Klionsky, Daniel J (see original citation for additional authors) (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12:1-222
Shi, Chong-Shan; Kehrl, John H (2016) Cytochrome c Negatively Regulates NLRP3 Inflammasomes. PLoS One 11:e0167636
Hu, Guowu; McQuiston, Travis; Bernard, Amélie et al. (2016) Tor-dependent post-transcriptional regulation of autophagy: Implications for cancer therapeutics. Mol Cell Oncol 3:e1078923
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
Chae, Jae Jin; Park, Yong Hwan; Park, Chung et al. (2015) Connecting two pathways through Ca 2+ signaling: NLRP3 inflammasome activation induced by a hypermorphic PLCG2 mutation. Arthritis Rheumatol 67:563-7
Hu, Guowu; McQuiston, Travis; Bernard, Amélie et al. (2015) TOR-dependent post-transcriptional regulation of autophagy. Autophagy 11:2390-2
Hu, Guowu; McQuiston, Travis; Bernard, Amélie et al. (2015) A conserved mechanism of TOR-dependent RCK-mediated mRNA degradation regulates autophagy. Nat Cell Biol 17:930-942

Showing the most recent 10 out of 33 publications