Inhibition of lipopolysaccharide-induced inflammatory responses by 1'-acetoxychavicol acetate.

Lipopolysaccharide on gram negative bacteria can be detected by Toll-like receptor 4 (TLR4) to elicit a series of innate immune responses, leading to inflammation to eliminate the targeted pathogen. However, dysregulation in the responses results in excessive inflammation. The 1'-acetoxychavicol acetate (ACA) is a bioactive compound originated from Alpinia species known to have anti-inflammatory and apoptosis-inducing properties. Here, we found that ACA inhibits lipopolysaccharide-induced expression and production of proinflammatory cytokines such as interleukin 6 and TNFα by macrophages. ACA suppresses the activation of NF-κB and MAP kinases in TLR4 signaling. Moreover, ACA also inhibits TLR4-mediated induction of type I interferon by suppressing IRF3 activation. In lipopolysaccharide-challenged mice, ACA treatment successfully increased the survival of mice and alleviated inflammation in the lung. Thus, ACA is a potential anti-inflammatory agent to regulate excessive inflammation.

1'-Acetoxychavicol acetate inhibits NLRP3-dependent inflammasome activation via mitochondrial ROS suppression.

The nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing (NLRP) 3 inflammasome is a multiprotein complex that triggers Caspase-1-mediated IL-1ß production and pyroptosis, and its dysregulation is associated with the pathogenesis of inflammatory diseases. 1'-Acetoxychavicol acetate (ACA) is a natural compound in the rhizome of tropical ginger Alpinia species with anti-microbial, anti-allergic and anti-cancer properties. In this study, we found that ACA suppressed NLRP3 inflammasome activation in mouse bone marrow-derived macrophages and human THP-1 monocytes. ACA inhibited Caspase-1 activation and IL-1ß production by NLRP3 agonists such as nigericin, monosodium urate (MSU) crystals, and ATP. Moreover, it suppressed oligomerization of the adapter molecule, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1-mediated cleavage of pyroptosis executor Gasdermin D. Mechanistically, ACA inhibited generation of mitochondrial reactive oxygen species (ROS) and prevented release of oxidized mitochondrial DNA, which trigger NLRP3 inflammasome activation. ACA also prevented NLRP3 inflammasome activation in vivo, as evidenced in the MSU crystal-induced peritonitis and dextran sodium sulfate-induced colitis mouse models accompanied by decreased Caspase-1 activation. Thus, ACA is a potent inhibitor of the NLRP3 inflammasome for prevention of NLRP3-associated inflammatory diseases.

Deletion of PIKfyve alters alveolar macrophage populations and exacerbates allergic inflammation in mice.

Alveolar macrophages (AMs) are specialized tissue-resident macrophages that orchestrate the immune responses to inhaled pathogens and maintain organ homeostasis of the lung. Dysregulation of AMs is associated with allergic inflammation and asthma. Here, we examined the role of a phosphoinositide kinase PIKfyve in AM development and function. Mice with conditionally deleted PIKfyve in macrophages have altered AM populations. PIKfyve deficiency results in a loss of AKT activation in response to GM-CSF, a cytokine critical for AM development. Upon exposure to house dust mite extract, mutant mice display severe lung inflammation and allergic asthma accompanied by infiltration of eosinophils and lymphoid cells. Moreover, they have defects in production of retinoic acid and fail to support incorporation of Foxp3+ Treg cells in the lung, resulting in exacerbation of lung inflammation. Thus, PIKfyve plays a role in preventing excessive lung inflammation through regulating AM function.

The transcription factor Jdp2 controls bone homeostasis and antibacterial immunity by regulating osteoclast and neutrophil differentiation.

Jdp2 is an AP-1 family transcription factor that regulates the epigenetic status of histones. Previous in vitro studies revealed that Jdp2 is involved in osteoclastogenesis. However, the roles of Jdp2 in vivo and its pleiotropic functions are largely unknown. Here we generated Jdp2(-/-) mice and discovered its crucial roles not only in bone metabolism but also in differentiation of neutrophils. Jdp2(-/-) mice exhibited osteopetrosis resulting from impaired osteoclastogenesis. Jdp2(-/-) neutrophils were morphologically normal but had impaired surface expression of Ly6G, bactericidal function, and apoptosis. We also found that ATF3 was an inhibitor of neutrophil differentiation and that Jdp2 directly suppresses its expression via inhibition of histone acetylation. Strikingly, Jdp2(-/-) mice were highly susceptible to Staphylococcus aureus and Candida albicans infection. Thus, Jdp2 plays pivotal roles in in vivo bone homeostasis and host defense by regulating osteoclast and neutrophil differentiation.

PtdIns3P phosphatases MTMR3 and MTMR4 negatively regulate innate immune responses to DNA through modulating STING trafficking.

The innate immune system plays an essential role in initial recognition of pathogen infection by producing inflammatory cytokines and type I interferons. cGAS is a cytoplasmic sensor for DNA derived from DNA viruses. cGAS binding with DNA induces the production of cGAMP, a second messenger that associates with STING in endoplasmic reticulum (ER). STING changes its cellular distribution from ER to perinuclear Golgi, where it activates the protein kinase TBK1 that catalyzes the phosphorylation of IRF3. Here we found that STING trafficking is regulated by myotubularin-related protein (MTMR) 3 and MTMR4, members of protein tyrosine phosphatases that dephosphorylate 3' position in phosphatidylinositol (PtdIns) and generate PtdIns5P from PtdIns3,5P2 and PtdIns from PtdIns3P. We established MTMR3 and MTMR4 double knockout (DKO) RAW264.7 macrophage cells and found that they exhibited increased type I interferon production after interferon-stimulatory DNA (ISD) stimulation and herpes simplex virus 1 infection concomitant with enhanced IRF3 phosphorylation. In DKO cells, STING rapidly trafficked from ER to Golgi after ISD stimulation. Notably, DKO cells exhibited enlarged cytosolic puncta positive for PtdIns3P and STING was aberrantly accumulated in this puncta. Taken together, these results suggest that MTMR3 and MTMR4 regulate the production of PtdIns3P, which plays a critical role in suppressing DNA-mediated innate immune responses via modulating STING trafficking.

Identification of nucleoporin 93 (Nup93) that mediates antiviral innate immune responses.

RIG-I-like receptors (RLRs) are cytoplasmic sensors for viral RNA that elicit antiviral innate immune responses. RLR signaling culminates in the activation of the protein kinase TBK1, which mediates phosphorylation and nuclear translocation of IRF3 that regulates expression of type I interferon genes. Here, we found that Nucleoporin 93 (Nup93), components of nuclear pore complex (NPC), plays an important role in RLR-mediated antiviral responses. Nup93-deficient RAW264.7 macrophage cells exhibited decreased expression of Ifnb1 and Cxcl10 genes after treatment with a synthetic RLR agonist stimulation as well as Newcastle Disease Virus infection. Silencing Nup93 in murine primary macrophages and embryonic fibroblasts also resulted in reduced expression of these genes. IRF3 nuclear translocation during RLR signaling was impaired in Nup93-deficient RAW264.7 cells. Notably, the activation of TBK1 during RLR signaling was also decreased in Nup93-deficient cells. We found that Nup93 formed a complex with TBK1, and Nup93 overexpression enhanced TBK1-mediated IFNß promoter activation. Taken together, our findings suggest that Nup93 regulates antiviral innate immunity by enhancing TBK1 activity and IRF3 nuclear translocation.

The Ca2+-dependent pathway contributes to changes in the subcellular localization and extracellular release of interleukin-33.

Interleukin-33 (IL-33) is a member of the IL-1 cytokine family and plays critical roles in facilitating type-2 immune responses. IL-33 is localized in the nucleus and released to the extracellular milieu during cell death, although the precise mechanisms underlying IL-33 mobilization remain unclear. Here, we found that nigericin, a toxin derived from Streptomyces hygroscopicus, promoted IL-33 translocation from the nucleus to the cytosol before extracellular release. This translocation was inhibited by chelating Ca2+ with EGTA or membrane protection by glycine treatment. Ca2+ ionophore A23187 stimulation caused IL-33 translocation to the cytoplasm but was not sufficient for extracellular release. However, IL-33 release was induced by detergent treatment, which indicates that membrane rupture is required for IL-33 release. The pore-forming pyroptosis executor gasdermin D was cleaved following nigericin stimulation, and overexpression of the cleaved gasdermin D-N-terminal fragment that forms the membrane pore sufficiently induced IL-33 release, which was blocked by EGTA and glycine. Together, these findings suggest that Ca2+-dependent signals and gasdermin D pore formation are required for robust IL-33 production.

Hu Antigen R Regulates Antiviral Innate Immune Responses through the Stabilization of mRNA for Polo-like Kinase 2.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), RIG-I, and melanoma differentiation-associated gene 5 (MDA5) play a critical role in inducing antiviral innate immune responses by activating IFN regulatory factor 3 (IRF3) and NF-κB, which regulates the transcription of type I IFN and inflammatory cytokines. Antiviral innate immune responses are also regulated by posttranscriptional and translational mechanisms. In this study, we identified an RNA-binding protein HuR as a regulator for RLR signaling. Overexpression of HuR, but not of other Hu members, increased IFN-ß promoter activity. HuR-deficient macrophage cells exhibited decreased Ifnb1 expression after RLR stimulation, whereas they showed normal induction after stimulation with bacterial LPS or immunostimulatory DNA. Moreover, HuR-deficient cells displayed impaired nuclear translocation of IRF3 after RLR stimulation. In HuR-deficient cells, the mRNA expression of Polo-like kinase (PLK) 2 was markedly reduced. We found that HuR bound to the 3' untranslated region of Plk2 mRNA and increased its stabilization. PLK2-deficient cells also showed reduced IRF3 nuclear translocation and Ifnb mRNA expression during RLR signaling. Together, these findings suggest that HuR bolsters RLR-mediated IRF3 nuclear translocation by controlling the stability of Plk2 mRNA.

Intravesicular Acidification Regulates Lipopolysaccharide Inflammation and Tolerance through TLR4 Trafficking.

TLRs recognize pathogen components and drive innate immune responses. They localize at either the plasma membrane or intracellular vesicles such as endosomes and lysosomes, and proper cellular localization is important for their ligand recognition and initiation of signaling. In this study, we disrupted ATP6V0D2, a component of vacuolar-type H+ adenosine triphosphatase (V-ATPase) that plays a central role in acidification of intracellular vesicles, in a macrophage cell line. ATP6V0D2-deficient cells exhibited reduced cytokine production in response to endosome-localized, nucleic acid-sensing TLR3, TLR7, and TLR9, but enhanced inflammatory cytokine production and NF-κB activation following stimulation with LPS, a TLR4 agonist. Moreover, they had defects in internalization of cell surface TLR4 and exhibited enhanced inflammatory cytokine production after repeated LPS stimulation, thereby failing to induce LPS tolerance. A component of the V-ATPase complex interacted with ARF6, the small GTPase known to regulate TLR4 internalization, and ARF6 deficiency resulted in prolonged TLR4 expression on the cell surface. Taken together, these findings suggest that ATP6V0D2-dependent intravesicular acidification is required for TLR4 internalization, which is associated with prevention from excessive LPS-triggered inflammation and induction of tolerance.

DNA-Containing Exosomes Derived from Cancer Cells Treated with Topotecan Activate a STING-Dependent Pathway and Reinforce Antitumor Immunity.

Danger-associated molecular patterns derived from damaged or dying cells elicit inflammation and potentiate antitumor immune responses. In this article, we show that treatment of breast cancer cells with the antitumor agent topotecan (TPT), an inhibitor of topoisomerase I, induces danger-associated molecular pattern secretion that triggers dendritic cell (DC) activation and cytokine production. TPT administration inhibits tumor growth in tumor-bearing mice, which is accompanied by infiltration of activated DCs and CD8+ T cells. These effects are abrogated in mice lacking STING, an essential molecule in cytosolic DNA-mediated innate immune responses. Furthermore, TPT-treated cancer cells release exosomes that contain DNA that activate DCs via STING signaling. These findings suggest that a STING-dependent pathway drives antitumor immunity by responding to tumor cell-derived DNA.

Bone-protective Functions of Netrin 1 Protein.

Netrin 1 was initially identified as an axon guidance factor, and recent studies indicate that it inhibits chemokine-directed monocyte migration. Despite its importance as a neuroimmune guidance cue, the role of netrin 1 in osteoclasts is largely unknown. Here we detected high netrin 1 levels in the synovial fluid of rheumatoid arthritis patients. Netrin 1 is potently expressed in osteoblasts and synovial fibroblasts, and IL-17 robustly enhances netrin 1 expression in these cells. The binding of netrin 1 to its receptor UNC5b on osteoclasts resulted in activation of SHP1, which inhibited VAV3 phosphorylation and RAC1 activation. This significantly impaired the actin polymerization and fusion, but not the differentiation of osteoclast. Strikingly, netrin 1 treatment prevented bone erosion in an autoimmune arthritis model and age-related bone destruction. Therefore, the netrin 1-UNC5b axis is a novel therapeutic target for bone-destructive diseases.

Negative regulation of melanoma differentiation-associated gene 5 (MDA5)-dependent antiviral innate immune responses by Arf-like protein 5B.

RIG-I-like receptors (RLRs), including retinoic acid-inducible gene-I (RIG-I) and MDA5, constitute a family of cytoplasmic RNA helicases that senses viral RNA and mounts antiviral innate immunity by producing type I interferons and inflammatory cytokines. Despite their essential roles in antiviral host defense, RLR signaling is negatively regulated to protect the host from excessive inflammation and autoimmunity. Here, we identified ADP-ribosylation factor-like protein 5B (Arl5B), an Arl family small GTPase, as a regulator of RLR signaling through MDA5 but not RIG-I. Overexpression of Arl5B repressed interferon ß promoter activation by MDA5 but not RIG-I, and its knockdown enhanced MDA5-mediated responses. Furthermore, Arl5B-deficient mouse embryonic fibroblast cells exhibited increased type I interferon expression in response to MDA5 agonists such as poly(I:C) and encephalomyocarditis virus. Arl5B-mediated negative regulation of MDA5 signaling does not require its GTP binding ability but requires Arl5B binding to the C-terminal domain of MDA5, which prevents interaction between MDA5 and poly(I:C). Our results, therefore, suggest that Arl5B is a negative regulator for MDA5.

Recognition of nucleic acids by pattern-recognition receptors and its relevance in autoimmunity.

Host cells trigger signals for innate immune responses upon recognition of conserved structures in microbial pathogens. Nucleic acids, which are critical components for inheriting genetic information in all species including pathogens, are key structures sensed by the innate immune system. The corresponding receptors for foreign nucleic acids include members of Toll-like receptors, RIG-I-like receptors, and intracellular DNA sensors. While nucleic acid recognition by these receptors is required for host defense against the pathogen, there is a potential risk to the host of self-nucleic acids recognition, thus precipitating autoimmune and autoinflammatory diseases. In this review, we discuss the roles of nucleic acid-sensing receptors in guarding against pathogen invasion, discriminating between self and non-self, and contributing to autoimmunity and autoinflammatory diseases.

Store-operated Ca2+ entry through ORAI1 is critical for T cell-mediated autoimmunity and allograft rejection.

ORAI1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, which is responsible for store-operated Ca(2+) entry in lymphocytes. A role for ORAI1 in T cell function in vivo has been inferred from in vitro studies of T cells from human immunodeficient patients with mutations in ORAI1 and Orai1(-/-) mice, but a detailed analysis of T cell-mediated immune responses in vivo in mice lacking functional ORAI1 has been missing. We therefore generated Orai1 knock-in mice (Orai1(KI/KI)) expressing a nonfunctional ORAI1-R93W protein. Homozygosity for the equivalent ORAI1-R91W mutation abolishes CRAC channel function in human T cells resulting in severe immunodeficiency. Homozygous Orai1(KI/KI) mice die neonatally, but Orai1(KI/KI) fetal liver chimeric mice are viable and show normal lymphocyte development. T and B cells from Orai1(KI/KI) mice display severely impaired store-operated Ca(2+) entry and CRAC channel function resulting in a strongly reduced expression of several key cytokines including IL-2, IL-4, IL-17, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells. Cell-mediated immune responses in vivo that depend on Th1, Th2, and Th17 cell function were severely attenuated in ORAI1-deficient mice. Orai1(KI/KI) mice lacked detectable contact hypersensitivity responses and tolerated skin allografts significantly longer than wild-type mice. In addition, T cells from Orai1(KI/KI) mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease. These findings reaffirm the critical role of ORAI1 for T cell function and provide important insights into the in vivo functions of CRAC channels for T cell-mediated immunity.

Antigen Presentation in the Lung.

The lungs are constantly exposed to environmental and infectious agents such as dust, viruses, fungi, and bacteria that invade the lungs upon breathing. The lungs are equipped with an immune defense mechanism that involves a wide variety of immunological cells to eliminate these agents. Various types of dendritic cells (DCs) and macrophages (MACs) function as professional antigen-presenting cells (APCs) that engulf pathogens through endocytosis or phagocytosis and degrade proteins derived from them into peptide fragments. During this process, DCs and MACs present the peptides on their major histocompatibility complex class I (MHC-I) or MHC-II protein complex to naïve CD8+ or CD4+ T cells, respectively. In addition to these cells, recent evidence supports that antigen-specific effector and memory T cells are activated by other lung cells such as endothelial cells, epithelial cells, and monocytes through antigen presentation. In this review, we summarize the molecular mechanisms of antigen presentation by APCs in the lungs and their contribution to immune response.

Regulation of Il6 expression by single CpG methylation in downstream of Il6 transcription initiation site.

The innate immune system is an immediate defense against infectious pathogens by the production of inflammatory cytokines and other mediators. Deficiencies of epigenetic regulatory enzymes, such as Tet1 and Dnmt1, cause dysregulation of cytokine expression. However, it is unclear if DNA methylation at a single CpG dinucleotide in a specific gene locus can regulate gene expression. In this study, we demonstrated that CpG+286 and CpG+348 in exon 2 of the Il6 gene are similar in various primary mouse cells. In lipopolysaccharide-stimulated condition, hypomethylated CpG+286 promoted Il6 expression whereas deletion of CpG+348 led to a reduction in Il6 expression associated with enhanced CTCF binding to the Il6 locus. Moreover, hypomethylation at CpG+286 in alveolar macrophages from aged mice led to higher Il6 expression in response to LPS compared with young mice. Thus, DNA methylation at specific CpG dinucleotides plays an important regulatory role in Il6 expression.

Alveolar macrophages instruct CD8+ T cell expansion by antigen cross-presentation in lung.

Lung CD8+ memory T cells play central roles in protective immunity to respiratory viruses, such as influenza A virus (IAV). Here, we find that alveolar macrophages (AMs) function as antigen-presenting cells that support the expansion of lung CD8+ memory T cells. Intranasal antigen administration to mice subcutaneously immunized with antigen results in a rapid expansion of antigen-specific CD8+ T cells in the lung, which is dependent on antigen cross-presentation by AMs. AMs highly express interleukin-18 (IL-18), which mediates subsequent formation of CD103+CD8+ resident memory T (TRM) cells in the lung. In a mouse model of IAV infection, AMs are required for expansion of virus-specific CD8+ T cells and CD103+CD8+ TRM cells and inhibiting virus replication in the lungs during secondary infection. These results suggest that AMs instruct a rapid expansion of antigen-specific CD8+ T cells in lung, which protect the host from respiratory virus infection.

Protein kinase C-induced phosphorylation of Orai1 regulates the intracellular Ca2+ level via the store-operated Ca2+ channel.

Ca(2+) signals through store-operated Ca(2+) (SOC) channels, activated by the depletion of Ca(2+) from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca(2+) sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca(2+) entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCbeta both resulted in increased Ca(2+) influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.

Exploration of Pattern Recognition Receptor Agonists as Candidate Adjuvants.

Adjuvants are used to maximize the potency of vaccines by enhancing immune reactions. Components of adjuvants include pathogen-associated molecular patterns (PAMPs) and damage-associate molecular patterns (DAMPs) that are agonists for innate immune receptors. Innate immune responses are usually activated when pathogen recognition receptors (PRRs) recognize PAMPs derived from invading pathogens or DAMPs released by host cells upon tissue damage. Activation of innate immunity by PRR agonists in adjuvants activates acquired immune responses, which is crucial to enhance immune reactions against the targeted pathogen. For example, agonists for Toll-like receptors have yielded promising results as adjuvants, which target PRR as adjuvant candidates. However, a comprehensive understanding of the type of immunological reaction against agonists for PRRs is essential to ensure the safety and reliability of vaccine adjuvants. This review provides an overview of the current progress in development of PRR agonists as vaccine adjuvants, the molecular mechanisms that underlie activation of immune responses, and the enhancement of vaccine efficacy by these potential adjuvant candidates.

A regulated adaptor function of p40phox: distinct p67phox membrane targeting by p40phox and by p47phox.

In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47(phox), p67(phox), p40(phox), and Rac) translocate and associate with the membrane-spanning flavocytochrome b(558), leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40(phox) that regulate its translocation to membranes upon stimulation. GFP-p40(phox) translocates to early endosomes, whereas GFP-p47(phox) translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67(phox) does not translocate to membranes when expressed alone, but it is dependent on p40(phox) and p47(phox) for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40(phox) or GFP-p47(phox) to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67(phox) translocation to either membrane is abolished by mutations that disrupt its interaction with p40(phox) or p47(phox). Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40(phox) in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40(phox) and p47(phox) function as diverse p67(phox) "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms.