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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.

The omega-3 (ω3) fatty acid docosahexaenoic acid (DHA) can suppress inflammation, specifically IL-1β production through poorly understood molecular mechanisms. Here, we show that DHA reduces macrophage IL-1β production by limiting inflammasome activation. Exposure to DHA reduced IL-1β production by ligands that stimulate the NLRP3, AIM2, and NAIP5/NLRC4 inflammasomes. The inhibition required Free Fatty Acid Receptor (FFAR) 4 (also known as GPR120), a G-protein coupled receptor (GPR) known to bind DHA. The exposure of cells to DHA recruited the adapter protein β-arrestin1/2 to FFAR4, but not to a related lipid receptor. DHA treatment reduced the initial inflammasome priming step by suppressing the nuclear translocation of NF-κB. DHA also reduced IL-1β levels by enhancing autophagy in the cells. As a consequence macrophages derived from mice lacking the essential autophagy protein ATG7 were partially resistant to suppressive effects of DHA. Thus, DHA suppresses inflammasome activation by two distinct mechanisms, inhibiting the initial priming step and by augmenting autophagy, which limits inflammasome activity.

Autophagosomes deliver cytoplasmic constituents to lysosomes for degradation. Kehrl and colleagues show that inflammasome activation triggers autophagosome formation, which in turn eliminates active inflammasomes. Autophagosomes delivers cytoplasmic constituents to lysosomes for degradation, whereas inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1β (IL-1β) and IL-18. Here we show that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered activation of the G protein RalB and autophagosome formation. The induction of autophagy did not depend on the adaptor ASC or capase-1 but was dependent on the presence of the inflammasome sensor. Blocking autophagy potentiated inflammasome activity, whereas stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes.

The omega-3 ([omega]3) fatty acid docosahexaenoic acid (DHA) can suppress inflammation, specifically IL-1[beta] production through poorly understood molecular mechanisms. Here, we show that DHA reduces macrophage IL-1[beta] production by limiting inflammasome activation. Exposure to DHA reduced IL-1[beta] production by ligands that stimulate the NLRP3, AIM2, and NAIP5/NLRC4 inflammasomes. The inhibition required Free Fatty Acid Receptor (FFAR) 4 (also known as GPR120), a G-protein coupled receptor (GPR) known to bind DHA. The exposure of cells to DHA recruited the adapter protein [beta]-arrestin1/2 to FFAR4, but not to a related lipid receptor. DHA treatment reduced the initial inflammasome priming step by suppressing the nuclear translocation of NF-[kappa]B. DHA also reduced IL-1[beta] levels by enhancing autophagy in the cells. As a consequence macrophages derived from mice lacking the essential autophagy protein ATG7 were partially resistant to suppressive effects of DHA. Thus, DHA suppresses inflammasome activation by two distinct mechanisms, inhibiting the initial priming step and by augmenting autophagy, which limits inflammasome activity.

Autophagosomes delivers cytoplasmic constituents to lysosomes for degradation, whereas inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1[beta] (IL-1[beta]) and IL-18. Here we show that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered activation of the G protein RalB and autophagosome formation. The induction of autophagy did not depend on the adaptor ASC or capase-1 but was dependent on the presence of the inflammasome sensor. Blocking autophagy potentiated inflammasome activity, whereas stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes.

Ligand bound chemoattractant receptors activate the heterotrimeric G-protein G i to stimulate downstream signaling pathways to properly position lymphocytes in lymphoid organs. Here, we show how variations in the expression of a chemokine receptor and in two components in the signaling pathway, Gα i 2 and RGS1, affect the output fidelity of the signaling pathway. Examination of B cells from mice with varying numbers of intact alleles of Ccr7, Rgs1, Gnai2, and Gnai3 provided the basis for these results. Loss of a single allele of either Gnai2 or Rgs1 affected CCL19 triggered chemotaxis, whereas the loss of a single allele of Ccr7, which encodes the cognate CCL19 receptor, had little effect. Emphasizing the importance of Gnai2, B cells lacking Gnai3 expression responded to chemokines better than did wild-type B cells. At an organismal level, variations in Rgs1 and Gnai2 expression affected marginal zone B-cell development, splenic architecture, lymphoid follicle size, and germinal center morphology. Gnai2 expression was also needed for the proper alignment of MOMA-1 + macrophages and MAdCAM-1 + endothelial cells along marginal zone sinuses in the spleen. These data indicate that chemoattractant receptors, heterotrimeric G-proteins, and RGS protein expression levels have a complex interrelationship that affects the responses to chemoattractant exposure.

Alpha kinase 1 (ALPK1) is a cytosolic sensor of microbial sugar metabolites that activates NF-κB signaling through phosphorylation of the adaptor protein TIFA. Although canonically linked to NF-κB, individuals with gain-of-function ALPK1 mutations also show features of interferon-driven inflammation. Here, we show that ALPK1 activation enhances multiple outputs of the stimulator of interferon genes (STING) pathway, including both canonical and noncanonical responses such as STING proton channel-dependent LC3B lipidation and NLRP3 inflammasome activation. Furthermore, ALPK1 signaling activates eIF2α, an effector of the integrated stress response. Conversely, STING activation increases ALPK1 protein expression and triggers TIFA-Threonine 9 phosphorylation. Clinically, individuals with ALPK1-mediated disease exhibit premature intracranial mineralization and elevated cerebrospinal fluid neopterin, both associated with dysregulated interferon signaling. These findings support a model of bidirectional signaling between ALPK1 and STING, in which microbial and nucleic acid sensing pathways can amplify one another. This crosstalk provides a mechanistic framework for understanding innate immune signaling relevant to both homeostasis and disease.

Addition of adenosine 5 $\\sp\\prime$ -triphosphate (ATP) to quiescent cells increased intracellular pH $\\sb{\\rm i}$ , Na $\\sp+$ /K $\\sp+$pump activity, glucose and uridine uptake. DNA synthesis and cell proliferation were greatly stimulated by ATP in synergistic combination with other growth factors. ATP was a competence factor. The mitogenic effect of ATP was not due to its hydrolysis to adenosine. ATP-dependent DNA synthesis in 3T3 and 3T6 cells occurred primarily via the arachidonic acid (AA) pathway. This involves the release of AA, its oxidation to prostaglandin E $\\sb2$ , followed by elevation in cyclic AMP levels. A similar picture was obtained for A431 cells except that activation of protein kinase C played a role, and the AA pathway accounted for only a part of the ATP-dependent mitogenesis. That ATP receptors are coupled to phospholipase A $\\sb2$by a pertussis toxin (PTX) sensitive G protein is evidenced by the following: PTX inhibited AA release and PGE $\\sb2$production by ATP; exogenous AA or PGE $\\sb2$was able to overcome the inhibitory effect of PTX on ATP-induced DNA synthesis; PTX-induced in vivo ADP-ribosylation of a 41K protein followed the dose of inhibition on DNA synthesis; Guanosine 5 $\\sp\\prime$ -o-thiotriphosphate stimulated and guanosine 5 $\\sp\\prime$ -o-2-thiodiphosphate inhibited the AA release by ATP in permeabilized cells. DNA synthesis, AA metabolism and uridine uptake were mediated by ATP, ADP, adenosine 5 $\\sp\\prime$ - ( $\\beta$ , $\\gamma$ -imido) triphosphate (AMP-PNP), and 3 $\\sp\\prime$ -o-(4-benzoyl)benzoyl adenosine 5 $\\sp\\prime$ -triphosphate (BzATP) indicating a P $\\sb{\\rm 2Y}$purinoceptor. These events were desensitized by prior incubation of quiescent cultures with ATP, ADP or AMP-PNP. Stimulation of AA release and cyclic AMP accumulation by photoincorporated BzATP was diminished in both ATP and BzATP desensitized cells. Recovery from desensitization required protein synthesis. These studies provide further evidence for a P $\\sb{\\rm 2Y}$receptor mediated mitogenesis. Finally, the possible physiological role for ATP (and ADP) as mitogens in the nervous system, in wound healing, in aged cells as well as in smooth muscle cells has been explored.

3'-O-(4-Benzoyl)benzoyl-ATP (BzATP), a photoaffinity analog of ATP, was used as a ligand for a P2Ypurinoceptor (adenine nucleotide receptor) present in intact Swiss 3T3 and 3T6 cells and A-431 epidermoid carcinoma cells. Photolysis of serum-starved cells in the presence of 10-50 μM BzATP, followed by extensive washing to remove unicorporated BzATP, induced the release of arachidonic acid. A trace (<0.01%) of photoincorporated BzATP was as effective as when 50 μM BzATP or ATP was contained in the incubation medium during the assay. Photoincorporated BzATP also stimulated the production of prostaglandin E2and the accumulation of cyclic AMP. In previous studies, we demonstrated that these three events are obligatory early steps in a pathway leading to DNA synthesis in the above cell lines. The evidence indicated that the purinoceptor activated by extracellular ATP or BzATP was linked to a pertussis toxin-sensitive GTP-binding protein. Consistent with these observations, we now find that pertussis toxin inhibits the effect of photoincorporated BzATP on arachidonic acid release. These results indicate that BzATP is an effective agonist for the P2Ypurinoceptor concerned with stimulation of DNA synthesis in 3T3, 3T6, and A-431 cells. Furthermore, after photolysis it becomes irreversibly associated with intact cells and promotes the activation of early events required for DNA synthesis.

3'-O-(4-Benzoyl)benzoyl-ATP (BzATP), a photoaffinity analog of ATP, was used as a ligand for a P sub(2Y) purinoceptor (adenine nucleotide receptor) present in intact Swiss 3T3 and 3T6 cells and A-431 epidermoid carcinoma cells. Photoincorporated BzATP also stimulated the production of prostaglandin E sub(2) and the accumulation of cyclic AMP. In previous studies, we demonstrated that these three events are obligatory early steps in a pathway leading to DNA synthesis in the above cell lines. The results indicate that BzATP is an effective agonist for the P sub(2Y) purinoceptor concerned with stimulation of DNA synthesis in 3T3, 3T6, and A-431 cells. Furthermore, after photolysis it becomes irreversibly associated with intact cells and promotes the activation of early events required for DNA synthesis.