PhospholipaseCγ1/calcium-dependent membranous localization of Gsdmd-N drives endothelial pyroptosis, contributing to lipopolysaccharide-induced fatal outcome

Our study newly reveals that phospholipase Cγ1 (PLCγ1) contributes to gasdermin D (Gsdmd)-mediated endothelial pyroptosis in a calcium-dependent fashion. Cytosolic calcium signaling promotes activated NH2-terminal fragment of Gsdmd (Gsdmd-N) to translocate to the plasma membrane, enhancing endothelial pyroptosis induced by cytoplasmic LPS. Genetic or pharmacologic inhibition of endothelial PLCγ1 attenuated breakdown of endothelial barrier, reduced vascular leakage, improve perfusion disturbances, and decrease mortality of mice in endotoxemia.

Exocytosis of large-diameter lysosomes mediates interferon γ-induced relocation of MHC class II molecules toward the surface of astrocytes

Astrocytes are the key homeostatic cells in the central nervous system; initiation of reactive astrogliosis contributes to neuroinflammation. Pro-inflammatory cytokine interferon γ (IFNγ) induces the expression of the major histocompatibility complex class II (MHCII) molecules, involved in antigen presentation in reactive astrocytes. The pathway for MHCII delivery to the astrocyte plasma membrane, where MHCII present antigens, is unknown. Rat astrocytes in culture and in organotypic slices were exposed to IFNγ to induce reactive astrogliosis. Astrocytes were probed with optophysiologic tools to investigate subcellular localization of immunolabeled MHCII, and with electrophysiology to characterize interactions of single vesicles with the plasmalemma. In culture and in organotypic slices, IFNγ augmented the astrocytic expression of MHCII, which prominently co-localized with lysosomal marker LAMP1-EGFP, modestly co-localized with Rab7, and did not co-localize with endosomal markers Rab4A, EEA1, and TPC1. MHCII lysosomal localization was corroborated by treatment with the lysosomolytic agent glycyl-l-phenylalanine-β-naphthylamide, which reduced the number of MHCII-positive vesicles. The surface presence of MHCII was revealed by immunolabeling of live non-permeabilized cells. In IFNγ-treated astrocytes, an increased fraction of large-diameter exocytotic vesicles (lysosome-like vesicles) with prolonged fusion pore dwell time and larger pore conductance was recorded, whereas the rate of endocytosis was decreased. Stimulation with ATP, which triggers cytosolic calcium signaling, increased the frequency of exocytotic events, whereas the frequency of full endocytosis was further reduced. In IFNγ-treated astrocytes, MHCII-linked antigen surface presentation is mediated by increased lysosomal exocytosis, whereas surface retention of antigens is prolonged by concomitant inhibition of endocytosis.

Modification of cytosolic calcium signaling by subplasmalemmal microdomains

To investigate the hypothesis that Na+ concentration in subplasmalemmal microdomains regulates Ca2+ concentrations in cellular microdomains ([Ca]md), the cytosol ([Ca]cyt), and sarcoplasmic reticulum (SR; [Ca]sr), we modeled transport events in those compartments. Inputs to the model were obtained from published measurements in descending vasa recta pericytes and other smooth muscle cells. The model accounts for major classes of ion channels, Na+/Ca2+ exchange (NCX), and the distributions of Na+-K+-ATPase α1- and α2-isoforms in the plasma membrane. Ca2+ release from SR stores is assumed to occur via ryanodine (RyR) and inositol trisphosphate (IP3R) receptors. The model shows that the requisite existence of a significant Na+ concentration difference between the cytosol ([Na]cyt) and microdomains ([Na]md) necessitates restriction of intercompartmental diffusion. Accepting the latter, the model predicts resting ion concentrations that are compatible with experimental measurements and temporal changes in [Ca]cyt similar to those observed on NCX inhibition. An important role for NCX in the regulation of Ca2+ signaling is verified. In the resting state, NCX operates in “forward mode,” with Na+ entry and Ca2+ extrusion from the cell. Inhibition of NCX respectively raises and reduces [Ca]cyt and [Na]cyt by 40 and 30%. NCX translates variations in Na+-K+-ATPase activity into changes in [Ca]md, [Ca]sr, and [Ca]cyt. Taken together, the model simulations verify the feasibility of the central hypothesis that modulation of [Na]md can influence both the loading of Ca2+ into SR stores and [Ca2+]cyt variation.