Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules

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An Integrin/MFG-E8 shuttle loads HIV-1 viral like particles onto follicular dendritic cells. During human immunodeficiency virus-1 (HIV-1) infection lymphoid organ follicular dendritic cells (FDCs) serve as a reservoir for infectious virus and an obstacle to curative therapies. In this study we identified a subset of lymphoid organ sinus lining macrophage (SMs) that provide a cell-cell contact portal, and a shuttling system, which facilitates the uptake of HIV-1 viral like particles (VLPs) by FDCs and B cells. Central for portal function was the bridging glycoprotein MFG-E8. Using a phosphatidylserine (PS) binding domain and an RGD motif, MFG-E8 helped target HIV-1 VLPs to v integrin bearing SMs. Both FDCs and HIV-1 gp120 specific B cells collected HIV-1 VLPs from MFG-E8 rich sites on SMs. Lack of MFG-E8 or integrin blockade severely limited the spread of HIV-1 VLPs onto FDC networks. Our results identify a mechanism for HIV-1 uptake by SMs that facilitates the cell to cell spread of HIV-1 to FDCs and B cells. Defining a CD38-LRRK2-TFEB pathway in B cells and macrophages. CD38 is a cell surface receptor highly expressed in B cells and macrophages responsible for generating several different second messengers. Leucine rich repeat kinase 2 (LRRK2) is a large multiple function protein expressed in B cells and macrophages, but not previously connected to CD38. LRRK2 is of clinical interest because mutations in it are linked to Parkinsons disease. We have found that CD38 ligation caused a calcium dependent nuclear translocation of transcription factor EB (TFEB) in B cells and macrophages. CD38 engagement triggers a bi-phasic calcium signal that depended upon extracellular and lysosomal calcium. CD38 and LRRK2 co-localized at the plasma membrane, and the two proteins robustly interacted by co-immunoprecipitation. Cells from LRRK2 null mice showed decreased calcium responses after CD38 stimulation, while cells from kinase overactive LRRK2 knock-in (KI) mice had the opposite phenotype. Consistent with these findings, LRRK2 null cells showed defects in TFEB activation following CD38 ligation. TFEB activation is known to help mediate the switch from oxidative phosphorylation to glycolysis in macrophages. Accordingly, LRRK2 null macrophages had decreased glycolytic activity after LPS stimulation, while LRRK2 KI macrophages had increased activity. Interestingly we also found that CD38 stimulation limits macrophage inflammasome induced IL-1 release, and this inhibition depended upon the presence of LRRK2. In sum, we have identified a previously unknown CD38-LRRK2-TFEB signaling axis with functional implications for both B cells and macrophages. Bcl-2 regulates pyroptosis and necroptosis by targeting BH-3 like domains in gasdermin D and MLKL. Apoptosis is a form of programmed cell death in multicellular organisms. Bcl-2 prevents apoptosis and promotes cellular survival by neutralizing BH3 domain containing proteins, which directly activate the pore-forming proteins BAX and BAK. However, Bcl-2 is not known to regulate other cell death effectors such as gasdermin D (GSDMD) or mixed lineage kinase domain-like (MLKL), whose activation causes pyroptosis and necroptosis, respectively. In this study, we identified a BH3-like domain in both GSDMD and MLKL that mediates an interaction with Bcl-2. The presence of Bcl-2 reduced GSDMD cleavage at D275 by caspase-1, 4 or 5, and enhanced the GSDMD cleavage at D87. The GSDMD D87 cleavage inactivates the pyroptotic execution program. The presence of Bcl-2 also limited RIP3 mediated phosphorylation of MLKL, which reduced MLKL oligomerization and tempered the induction of necroptosis. Our observations suggest that the presence of Bcl-2 limits the induction of three forms of cell death apoptosis, pyroptosis, and necroptosis. The cytosolic pattern recognition receptor NLRP3 senses host-derived danger signals and certain microbe-derived products in both humans and rodents. NLRP3 activation assembles an inflammasome complex, which contains the adapter protein ASC and caspase-1, whose activation triggers the maturation and release of the proinflammatory cytokines IL-1 and IL-18. Serine 5 (S5) phosphorylation of NLRP3 prevents its oligomerization and activation, while dephosphorylation of this residue by the phosphatase PP2A allows NLRP3 activation. However, the protein kinase that mediates NLRP3 S5 phosphorylation is unknown. We have shown that AKT associates with NLRP3 and phosphorylates it on S5, limiting NLRP3 oligomerization. This phosphorylation event also stabilizes NLRP3 by reducing its ubiquitination on lysine 496, which inhibits its proteasome mediated degradation by the E3 ligase Trim31. Pharmacologic manipulation of AKT kinase activity reciprocally modulates NLRP3 inflammasome mediated IL-1beta production. Inhibition of AKT reduced IL-1beta production following the intraperitoneal injection of LPS into mice. We propose that AKT, Trim31 and PP2A together modulate NLRP3 protein levels and tendency to oligomerize, thereby setting a tightly regulated threshold for NLRP3 activation.