MAdCAM-1 Co-stimulation Combined with Retinoic Acid and TGF-ß Induces Blood CD8+ T cells to Adopt a Gut CD101+ TRM Phenotype.

Resident memory T cells (TRMs) help control local immune homeostasis and contribute to tissue protective immune responses. The local cues that guide their differentiation and localization are poorly defined. We demonstrate that MAdCAM-1, a ligand for the gut homing receptor α4ß7 integrin, in the presence of retinoic acid and TGF-ß provide a costimulatory signal that induces blood CD8+ T cells to adopt a TRM -like phenotype. These cells express CD103 (integrin αE) and CD69, the two major TRM cell surface markers, along with CD101. They also express CCR5, CCR9 and α4ß7, three receptors associated with gut homing. A subset also express E-cadherin, a ligand for αEß7. Fluorescent lifetime imaging indicated an αEß7 and E-cadherin cis interaction on the plasma membrane. This report advances our understanding of the signals that drive the differentiation of CD8+ T cells into TRMs and provides a means to expand these cells in vitro, thereby affording an avenue to generate more effective tissue specific immunotherapies.

Murine alveolar macrophages rapidly accumulate intranasally administered SARS-CoV-2 Spike protein leading to neutrophil recruitment and damage.

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study, we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques, we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Widespread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α (tumor necrosis factor-α) and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

Regulator of G-protein signaling 1 critically supports CD8+ TRM cell-mediated intestinal immunity.

Members of the Regulator of G-protein signaling (Rgs) family regulate the extent and timing of G protein signaling by increasing the GTPase activity of Gα protein subunits. The Rgs family member Rgs1 is one of the most up-regulated genes in tissue-resident memory (TRM) T cells when compared to their circulating T cell counterparts. Functionally, Rgs1 preferentially deactivates Gαq, and Gαi protein subunits and can therefore also attenuate chemokine receptor-mediated immune cell trafficking. The impact of Rgs1 expression on tissue-resident T cell generation, their maintenance, and the immunosurveillance of barrier tissues, however, is only incompletely understood. Here we report that Rgs1 expression is readily induced in naïve OT-I T cells in vivo following intestinal infection with Listeria monocytogenes-OVA. In bone marrow chimeras, Rgs1 -/- and Rgs1 +/+ T cells were generally present in comparable frequencies in distinct T cell subsets of the intestinal mucosa, mesenteric lymph nodes, and spleen. After intestinal infection with Listeria monocytogenes-OVA, however, OT-I Rgs1 +/+ T cells outnumbered the co-transferred OT-I Rgs1- /- T cells in the small intestinal mucosa already early after infection. The underrepresentation of the OT-I Rgs1 -/- T cells persisted to become even more pronounced during the memory phase (d30 post-infection). Remarkably, upon intestinal reinfection, mice with intestinal OT-I Rgs1 +/+ TRM cells were able to prevent the systemic dissemination of the pathogen more efficiently than those with OT-I Rgs1 -/- TRM cells. While the underlying mechanisms are not fully elucidated yet, these data thus identify Rgs1 as a critical regulator for the generation and maintenance of tissue-resident CD8+ T cells as a prerequisite for efficient local immunosurveillance in barrier tissues in case of reinfections with potential pathogens.

Murine Alveolar Macrophages Rapidly Accumulate Intranasally Administered SARS-CoV-2 Spike Protein leading to Neutrophil Recruitment and Damage.

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Wide-spread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

A CRISPR screen targeting PI3K effectors identifies RASA3 as a negative regulator of LFA-1-mediated adhesion in T cells.

The integrin lymphocyte function-associated antigen 1 (LFA-1) helps to coordinate the migration, adhesion, and activation of T cells through interactions with intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. LFA-1 is activated during the engagement of chemokine receptors and the T cell receptor (TCR) through inside-out signaling, a process that is partially mediated by phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3). To evaluate potential roles of PI3K in LFA-1 activation, we designed a library of CRISPR/single guide RNAs targeting known and potential PIP3-binding proteins and screened for effects on the ability of primary mouse T cells to bind to ICAM-1. We identified multiple proteins that regulated the binding of LFA-1 to ICAM-1, including the Rap1 and Ras GTPase-activating protein RASA3. We found that RASA3 suppressed LFA-1 activation in T cells, that its expression was rapidly reduced upon T cell activation, and that its activity was inhibited by PI3K. Loss of RASA3 in T cells led to increased Rap1 activation, defective lymph node entry and egress, and impaired responses to T-dependent immunization in mice. Our results reveal a critical role for RASA3 in T cell migration, homeostasis, and function.

A B-cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation.

B-cell activation and immune synapse (IS) formation with membrane-bound antigens are actin-dependent processes that scale positively with the strength of antigen-induced signals. Importantly, ligating the B-cell integrin, LFA-1, with ICAM-1 promotes IS formation when antigen is limiting. Whether the actin cytoskeleton plays a specific role in integrin-dependent IS formation is unknown. Here, we show using super-resolution imaging of mouse primary B cells that LFA-1:ICAM-1 interactions promote the formation of an actomyosin network that dominates the B-cell IS. This network is created by the formin mDia1, organized into concentric, contractile arcs by myosin 2A, and flows inward at the same rate as B-cell receptor (BCR):antigen clusters. Consistently, individual BCR microclusters are swept inward by individual actomyosin arcs. Under conditions where integrin is required for synapse formation, inhibiting myosin impairs synapse formation, as evidenced by reduced antigen centralization, diminished BCR signaling, and defective signaling protein distribution at the synapse. Together, these results argue that a contractile actomyosin arc network plays a key role in the mechanism by which LFA-1 co-stimulation promotes B-cell activation and IS formation.

The immune system has the ability to recognize a vast array of infections and trigger rapid responses. This defense mechanism is mediated in part by B cells which make antibodies that can neutralize or destroy specific disease-causing agents. When pathogens (such as bacteria or viruses) invade the body, a specialized immune cell called an 'antigen presenting cell' holds it in place and presents it to the B cell to examine. Receptors on the surface of the B cell then bind to the infectious agent and launch the B cell into action, triggering the antibody response needed to remove the pathogen. This process relies on B cells and antigen presenting cells making a close connection called an immune synapse, which has a bulls-eye pattern with the receptor in the middle surrounded by sticky proteins called adhesion molecules. A network of actin filaments coating the inside of the B cell are responsible for arranging the proteins into this bulls-eye shape. Once fully formed, the synapse initiates the production of antibodies and helps B cells to make stronger versions of these defensive proteins. So far, most studies have focused on the role the receptor plays in B cell activation. However, when there are only small amounts of the pathogen available, these receptors bind to the antigen presenting cell very weakly. When this happens, adhesion molecules have been shown to step in and promote the formation of the mature synapse needed for B cell activation. But it is not fully understood how adhesion molecules do this. To investigate, Wang et al. looked at mouse B cells using super resolution microscopes. This revealed that when B cells receive signals through both their receptors and their adhesion molecules, they rearrange their actin into a circular structure composed of arc shapes. Motors on the actin arcs then contract the structure inwards, pushing the B cell receptors into the classic bullseye pattern. This only happened when adhesion molecules were present and signals through the B cell receptors were weak. These findings suggest that adhesion molecules help form immune synapses and activate B cells by modifying the actin network so it can drive the re-patterning of receptor proteins. B cells are responsible for the long-term immunity provided by vaccines. Thus, it is possible that the findings of Wang et al. could be harnessed to create vaccines that trigger a stronger antibody response.

Interactions of Autophagy and the Immune System in Health and Diseases.

Autophagy is a highly conserved process that utilizes lysosomes to selectively degrade a variety of intracellular cargo, thus providing quality control over cellular components and maintaining cellular regulatory functions. Autophagy is triggered by multiple stimuli ranging from nutrient starvation to microbial infection. Autophagy extensively shapes and modulates the inflammatory response, the concerted action of immune cells, and secreted mediators aimed to eradicate a microbial infection or to heal sterile tissue damage. Here, we first review how autophagy affects innate immune signaling, cell-autonomous immune defense, and adaptive immunity. Then, we discuss the role of non-canonical autophagy in microbial infections and inflammation. Finally, we review how crosstalk between autophagy and inflammation influences infectious, metabolic, and autoimmune disorders.

LRRK2 is required for CD38-mediated NAADP-Ca2+ signaling and the downstream activation of TFEB (transcription factor EB) in immune cells.

CD38 is a cell surface receptor capable of generating calcium-mobilizing second messengers. It has been implicated in host defense and cancer biology, but signaling mechanisms downstream of CD38 remain unclear. Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common genetic cause of Parkinson disease; it is also a risk factor for Crohn disease, leprosy, and certain types of cancers. The pathogenesis of these diseases involves inflammation and macroautophagy/autophagy, processes both CD38 and LRRK2 are implicated in. Here, we mechanistically and functionally link CD38 and LRRK2 as upstream activators of TFEB (transcription factor EB), a host defense transcription factor and the master transcriptional regulator of the autophagy/lysosome machinery. In B-lymphocytes and macrophages, we show that CD38 and LRRK2 exist in a complex on the plasma membrane. Ligation of CD38 with the monoclonal antibody clone 90 results in internalization of the CD38-LRRK2 complex and its targeting to the endolysosomal system. This generates an NAADP-dependent calcium signal, which requires LRRK2 kinase activity, and results in the downstream activation of TFEB. lrrk2 KO macrophages accordingly have TFEB activation defects following CD38 or LPS stimulation and fail to switch to glycolytic metabolism after LPS treatment. In overexpression models, the pathogenic LRRK2G2019S mutant promotes hyperactivation of TFEB even in the absence of CD38, both by stabilizing TFEB and promoting its nuclear translocation via aberrant calcium signaling. In sum, we have identified a physiological CD38-LRRK2-TFEB signaling axis in immune cells. The common pathogenic mutant, LRRK2G2019S, appears to hijack this pathway.Abbreviations:ADPR: ADP-ribose; AMPK: AMP-activated protein kinase; BMDM: bone marrow-derived macrophage; cADPR: cyclic-ADP-ribose; COR: C-terminal of ROC; CTSD: cathepsin D; ECAR: extracellular acidification rate; EDTA: ethylenediaminetetraacetic acid; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GPN: Gly-Phe ß-naphthylamide; GSK3B/GSK3ß: glycogen synthase kinase 3 beta; GTP: guanosine triphosphate; KD: knockdown; LAMP1: lysosomal-associated membrane protein 1; LRR: leucine rich repeat; LRRK2: leucine rich repeat kinase 2; mAb: monoclonal antibody; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAPK/ERK: mitogen-activated protein kinase; MCOLN1: mucolipin 1; MFI: mean fluorescence intensity; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; NAADP: nicotinic acid adenine dinucleotide phosphate; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; PD: Parkinson disease; PPP3CB: protein phosphatase 3, catalytic subunit, beta isoform; q-RT-PCR: quantitative reverse transcription polymerase chain reaction; ROC: Ras of complex; siRNA: small interfering RNA; SQSTM1/p62: sequestome 1; TFEB: transcription factor EB; TPCN: two pore channel; TRPM2: transient receptor potential cation channel, subfamily M, member 2; ZKSCAN3: zinc finger with KRAB and SCAN domains 3.

An optimized confocal intravital microscopy protocol for long-term live imaging of murine F-actin organization during naïve lymphocyte migration.

Actin plays a crucial role during cell motility, but the organization of F-actin filaments during lymphocyte migration has not been visualized in vivo. Here, we present a 4D imaging platform using high-resolution confocal intravital microscopy to precisely determine the F-actin filament profile during lymphocyte transendothelial migration and interstitial migration. This protocol allows prolonged live imaging by laser scanning microscopy with advanced spatial resolution compared with the traditional multi-photon intravital microscopy techniques. For complete details on the use and execution of this protocol, please refer to Yan et al. (2019).

Unrestrained Gαi2 Signaling Disrupts Neutrophil Trafficking, Aging, and Clearance.

Neutrophil trafficking, homeostatic and pathogen elicited, depends upon chemoattractant receptors triggering heterotrimeric G-protein Gαißγ signaling, whose magnitude and kinetics are governed by RGS protein/Gαi interactions. RGS proteins typically limit Gαi signaling by reducing the duration that Gαi subunits remain GTP bound and able to activate downstream effectors. Yet how in totality RGS proteins shape neutrophil chemoattractant receptor activated responses remains unclear. Here, we show that C57Bl/6 mouse neutrophils containing a genomic knock-in of a mutation that disables all RGS protein-Gαi2 interactions (G184S) cannot properly balance chemoattractant receptor signaling, nor appropriately respond to inflammatory insults. Mutant neutrophils accumulate in mouse bone marrow, spleen, lung, and liver; despite neutropenia and an intrinsic inability to properly mobilize from the bone marrow. In vitro they rapidly adhere to ICAM-1 coated plates, but in vivo they poorly adhere to blood vessel endothelium. Those few neutrophils that cross blood vessels and enter tissues migrate haphazardly. Following Concanavalin-A administration fragmented G184S neutrophils accumulate in liver sinusoids leading to thrombo-inflammation and perivasculitis. Thus, neutrophil Gαi2/RGS protein interactions both limit and facilitate Gαi2 signaling thereby promoting normal neutrophil trafficking, aging, and clearance.

AKT Regulates NLRP3 Inflammasome Activation by Phosphorylating NLRP3 Serine 5.

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 that contains the adapter proteins ASC and caspase-1, whose activation triggers the maturation and release of the proinflammatory cytokines IL-1ß and IL-18. S5 phosphorylation of NLRP3 prevents its oligomerization and activation, whereas dephosphorylation of this residue by the phosphatase PP2A allows NLRP3 activation. However, the protein kinase that mediates NLRP3 S5 phosphorylation is unknown. In this study, we show 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-1ß production. Inhibition of AKT reduced IL-1ß production following the i.p. injection of LPS into mice. We propose that AKT, Trim31, and PP2A together modulate NLRP3 protein levels and the tendency to oligomerize, thereby setting a tightly regulated threshold for NLRP3 activation.

Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD 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. Here, we identify a BH3-like domain in both GSDMD and MLKL that mediates an interaction with B-cell lymphoma 2 (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.

An integrin/MFG-E8 shuttle loads HIV-1 viral-like particles onto follicular dendritic cells in mouse lymph node.

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. Here, we identify a subset of lymphoid organ sinus lining macrophage (SMs) that provide a cell-cell contact portal, which facilitates the uptake of HIV-1 viral-like particles (VLPs) by FDCs and B cells in mouse lymph node. Central for portal function is the bridging glycoprotein MFG-E8. Using a phosphatidylserine binding domain and an RGD motif, MFG-E8 helps target HIV-1 VLPs to αv integrin bearing SMs. Lack of MFG-E8 or integrin blockade severely limits HIV-1 VLP spread onto FDC networks. Direct SM-FDC virion transfer also depends upon short-lived FDC network abutment, likely triggered by SCSM antigen uptake. This provides a mechanism for rapid FDC loading broadening the opportunity for rare, antigen reactive follicular B cells to acquire antigen, and a means for HIV virions to accumulate on the FDC network.

Biased S1PR1 Signaling in B Cells Subverts Responses to Homeostatic Chemokines, Severely Disorganizing Lymphoid Organ Architecture.

Ligand-engaged chemoattractant receptors trigger Gαi subunit nucleotide exchange, stimulating the activation of downstream effector molecules. Activated chemoattractant receptors also dock G protein-coupled receptor kinases (GRKs) that help mediate receptor desensitization. In this study, we show that the B cell-specific loss of GRK2 severely disrupts B cell trafficking and immune cell homeostasis. The GRK2 deficiency in developing murine B cells leads to a severe immune phenotype, including a major reduction of bone marrow IgD+ cells, splenomegaly with a loss of white pulp and grossly expanded red pulp, a deficit of Peyer patches, and small lymph nodes with marked reductions in B cell numbers. The major phenotypes in these mice arise from excessive S1PR1 signaling combined with inadequate homeostatic chemokine receptor signaling. CXCL13 signaling is the most severely compromised. In B cells, our data also indicate that S1PR1 signals constitutively, as blocking S1PR1 signaling with an S1PR1 antagonist enhanced CXCL13-triggered wild-type B cell migration. Furthermore, blocking S1PR1 signaling in the GRK2-deficient B cells partially corrected their poor response to chemokines. Treating mice lacking GRK2 expression in their B cells with an S1PR1 antagonist partially normalized B cell trafficking into lymph node and splenic follicles. These findings reveal the critical interdependence of Gαi-linked signaling pathways in controlling B lymphocyte trafficking.

In Vivo F-Actin Filament Organization during Lymphocyte Transendothelial and Interstitial Migration Revealed by Intravital Microscopy.

Actin is essential for many cellular processes including cell motility. Yet the organization of F-actin filaments during lymphocyte transendothelial migration (TEM) and interstitial migration have not been visualized. Here we report a high-resolution confocal intravital imaging technique with LifeAct-GFP bone marrow reconstituted mice, which allowed visualization of lymphocyte F-actin in vivo. We find that naive lymphocytes preferentially cross high endothelial venules (HEVs) using paracellular rather than the transcellular route. During both modes of transmigration F-actin levels rise at the lymphocyte leading edge as the cell engages the TEM site. Once the lymphocytes breach the endothelium, they briefly reside in HEV pockets before crossing into the parenchyma. During interstitial migration dynamic actin-based protrusions rapidly form and collapse to help drive motility. Using a panel of inhibitors, we established roles for actin regulators and myosin II in lymphocyte TEM. This study provides further insights into lymphocyte TEM and interstitial migration in vivo.

E-protein-regulated expression of CXCR4 adheres preselection thymocytes to the thymic cortex.

Preselection thymocytes are normally retained in the thymic cortex, but the mechanisms responsible remain incompletely understood. We now report that deletion of genes encoding the E-protein transcription factors E2A and HEB disorders chemokine receptor expression on developing thymocytes to allow escape of preselection TCR-CD8+ thymocytes into the periphery. We document that CXCR4 expression normally anchors preselection thymocytes to the thymic cortex via interaction with its ligand CXCL12 on cortical thymic epithelial cells, and that disruption of CXCR4-CXCL12 engagements release preselection thymocytes from the thymic cortex. We further document that CXCR4 expression must be extinguished by TCR-mediated positive selection signals to allow migration of TCR-signaled thymocytes out of the thymic cortex into the medulla. Thus, E-protein transcription factors regulate the ordered expression pattern of chemokine receptors on developing thymocytes, and the interaction of the chemokine receptor CXCR4 with its ligand adheres TCR-unsignaled preselection thymocytes to the thymic cortex.

SARS-Coronavirus Open Reading Frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes.

The SARS (severe acute respiratory syndrome) outbreak was caused by a coronavirus (CoV) named the SARS-CoV. SARS pathology is propagated both by direct cytotoxic effects of the virus and aberrant activation of the innate immune response. Here, we identify several mechanisms by which a SARS-CoV open reading frame (ORF) activates intracellular stress pathways and targets the innate immune response. We show that ORF8b forms insoluble intracellular aggregates dependent on a valine at residue 77. Aggregated ORF8b induces endoplasmic reticulum (ER) stress, lysosomal damage, and subsequent activation of the master regulator of the autophagy and lysosome machinery, Transcription factor EB (TFEB). ORF8b causes cell death in epithelial cells, which is partially rescued by reducing its ability to aggregate. In macrophages, ORF8b robustly activates the NLRP3 inflammasome by providing a potent signal 2 required for activation. Mechanistically, ORF8b interacts directly with the Leucine Rich Repeat domain of NLRP3 and localizes with NLRP3 and ASC in cytosolic dot-like structures. ORF8b triggers cell death consistent with pyroptotic cell death in macrophages. While in those cells lacking NLRP3 accumulating ORF8b cytosolic aggregates cause ER stress, mitochondrial dysfunction, and caspase-independent cell death.

Inflammasome Inhibition Links IRGM to Innate Immunity.

IRGM is a risk factor for several inflammatory diseases, yet no direct link to immune regulation had been shown. In this issue of Molecular Cell, Mehto et al. (2019) report that IRGM limits NLRP3 inflammasome activation-by both direct inhibition of NLRP3/ASC oligomerization and selective autophagic destruction of NLRP3/ASC.

Gαi2 Signaling Regulates Inflammasome Priming and Cytokine Production by Biasing Macrophage Phenotype Determination.

Macrophages exist as innate immune subsets that exhibit phenotypic heterogeneity and functional plasticity. Their phenotypes are dictated by inputs from the tissue microenvironment. G-protein-coupled receptors are essential in transducing signals from the microenvironment, and heterotrimeric Gα signaling links these receptors to downstream effectors. Several Gαi-coupled G-protein-coupled receptors have been implicated in macrophage polarization. In this study, we use genetically modified mice to investigate the role of Gαi2 on inflammasome activity and macrophage polarization. We report that Gαi2 in murine bone marrow-derived macrophages (BMDMs) regulates IL-1ß release after activation of the NLRP3, AIM2, and NLRC4 inflammasomes. We show this regulation stems from the biased polarity of Gαi2 deficient (Gnai2 -/-) and RGS-insensitive Gαi2 (Gnai2 G184S/G184S) BMDMs. We determined that although Gnai2 G184S/G184S BMDMs (excess Gαi2 signaling) have a tendency toward classically activated proinflammatory (M1) phenotype, Gnai2-/- BMDMs (Gαi2 deficient) are biased toward alternatively activated anti-inflammatory (M2) phenotype. Finally, we find that Gαi2-deficient macrophages have increased Akt activation and IFN-ß production but defects in ERK1/2 and STAT3 activation after LPS stimulation. Gαi2-deficient macrophages also exhibit increased STAT6 activation after IL-4 stimulation. In summary, our data indicates that excess Gαi2 signaling promotes an M1 macrophage phenotype, whereas Gαi2 signaling deficiency promotes an M2 phenotype. Understanding Gαi2-mediated effects on macrophage polarization may bring to light insights regarding disease pathogenesis and the reprogramming of macrophages for the development of novel therapeutics.

SARS-Coronavirus Open Reading Frame-3a drives multimodal necrotic cell death.

The molecular mechanisms underlying the severe lung pathology that occurs during SARS-CoV infections remain incompletely understood. The largest of the SARS-CoV accessory protein open reading frames (SARS 3a) oligomerizes, dynamically inserting into late endosomal, lysosomal, and trans-Golgi-network membranes. While previously implicated in a non-inflammatory apoptotic cell death pathway, here we extend the range of SARS 3a pathophysiologic targets by examining its effects on necrotic cell death pathways. We show that SARS 3a interacts with Receptor Interacting Protein 3 (Rip3), which augments the oligomerization of SARS 3a helping drive necrotic cell death. In addition, by inserting into lysosomal membranes SARS 3a triggers lysosomal damage and dysfunction. Consequently, Transcription Factor EB (TFEB) translocates to the nucleus increasing the transcription of autophagy- and lysosome-related genes. Finally, SARS 3a activates caspase-1 either directly or via an enhanced potassium efflux, which triggers NLRP3 inflammasome assembly. In summary, Rip3-mediated oligomerization of SARS 3a causes necrotic cell death, lysosomal damage, and caspase-1 activation-all likely contributing to the clinical manifestations of SARS-CoV infection.