Publications by authors named "Takao Iwawaki"

104 Publications

The unfolded protein response transducer IRE1α promotes reticulophagy in podocytes.

Biochim Biophys Acta Mol Basis Dis 2022 06 15;1868(6):166391. Epub 2022 Mar 15.

Department of Medicine, McGill University Health Centre, McGill University, Montreal, Canada. Electronic address:

Glomerular diseases involving podocyte/glomerular epithelial cell (GEC) injury feature protein misfolding and endoplasmic reticulum (ER) stress. Inositol-requiring enzyme 1α (IRE1α) mediates chaperone production and autophagy during ER stress. We examined the role of IRE1α in selective autophagy of the ER (reticulophagy). Control and IRE1α knockout (KO) GECs were incubated with tunicamycin to induce ER stress and subjected to proteomic analysis. This showed IRE1α-dependent upregulation of secretory pathway mediators, including the coat protein complex II component Sec23B. Tunicamycin enhanced expression of Sec23B and the reticulophagy adaptor reticulon-3-long (RTN3L) in control, but not IRE1α KO GECs. Knockdown of Sec23B reduced autophagosome formation in response to ER stress. Tunicamycin stimulated colocalization of autophagosomes with Sec23B and RTN3L in an IRE1α-dependent manner. Similarly, during ER stress, glomerular α5 collagen IV colocalized with RTN3L and autophagosomes. Degradation of RTN3L and collagen IV increased in response to tunicamycin, and the turnover was blocked by deletion of IRE1α; thus, the IRE1α pathway promotes RTN3L-mediated reticulophagy and collagen IV may be an IRE1α-dependent reticulophagy substrate. In experimental glomerulonephritis, expression of Sec23B, RTN3L, and LC3-II increased in glomeruli of control mice, but not in podocyte-specific IRE1α KO littermates. In conclusion, during ER stress, IRE1α redirects a subset of Sec23B-positive vesicles to deliver RTN3L-coated ER fragments to autophagosomes. Reticulophagy is a novel outcome of the IRE1α pathway in podocytes and may play a cytoprotective role in glomerular diseases.
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http://dx.doi.org/10.1016/j.bbadis.2022.166391DOI Listing
June 2022

Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression.

EMBO Mol Med 2022 04 22;14(4):e15344. Epub 2022 Feb 22.

Department of Cardiology, Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.
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http://dx.doi.org/10.15252/emmm.202115344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8988208PMC
April 2022

Targeting IRE1 endoribonuclease activity alleviates cardiovascular lesions in a murine model of Kawasaki disease vasculitis.

JCI Insight 2022 03 22;7(6). Epub 2022 Mar 22.

Department of Pediatrics, Division of Infectious Diseases and Immunology.

Kawasaki disease (KD) is the leading cause of noncongenital heart disease in children. Studies in mice and humans propound the NLRP3/IL-1β pathway as the principal driver of KD pathophysiology. Endoplasmic reticulum (ER) stress can activate the NLRP3 inflammasome, but the potential implication of ER stress in KD pathophysiology has not been investigated to our knowledge. We used human patient data and the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis to characterize the impact of ER stress on the development of cardiovascular lesions. KD patient transcriptomics and single-cell RNA sequencing of the abdominal aorta from LCWE-injected mice revealed changes in the expression of ER stress genes. Alleviating ER stress genetically, by conditional deletion of inositol-requiring enzyme 1 (IRE1) in myeloid cells, or pharmacologically, by inhibition of IRE1 endoribonuclease (RNase) activity, led to significant reduction of LCWE-induced cardiovascular lesion formation as well as reduced caspase-1 activity and IL-1β secretion. These results demonstrate the causal relationship of ER stress to KD pathogenesis and highlight IRE1 RNase activity as a potential new therapeutic target.
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http://dx.doi.org/10.1172/jci.insight.157203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8986066PMC
March 2022

Mechanisms of liver injury in high fat sugar diet fed mice that lack hepatocyte X-box binding protein 1.

PLoS One 2022 14;17(1):e0261789. Epub 2022 Jan 14.

Division of Gastroenterology and Hepatology, Department of Medicine, Northwestern University, Chicago, Illinois, United States of America.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of liver diseases in the United States and can progress to cirrhosis, end-stage liver disease and need for liver transplantation. There are limited therapies for NAFLD, in part, due to incomplete understanding of the disease pathogenesis, which involves different cell populations in the liver. Endoplasmic reticulum stress and its adaptative unfolded protein response (UPR) signaling pathway have been implicated in the progression from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH). We have previously shown that mice lacking the UPR protein X-box binding protein 1 (XBP1) in the liver demonstrated enhanced liver injury and fibrosis in a high fat sugar (HFS) dietary model of NAFLD. In this study, to better understand the role of liver XBP1 in the pathobiology of NAFLD, we fed hepatocyte XBP1 deficient mice a HFS diet or chow and investigated UPR and other cell signaling pathways in hepatocytes, hepatic stellate cells and immune cells. We demonstrate that loss of XBP1 in hepatocytes increased inflammatory pathway expression and altered expression of the UPR signaling in hepatocytes and was associated with enhanced hepatic stellate cell activation after HFS feeding. We believe that a better understanding of liver cell-specific signaling in the pathogenesis of NASH may allow us to identify new therapeutic targets.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0261789PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8759640PMC
February 2022

The IRE1/XBP1 signaling axis promotes skeletal muscle regeneration through a cell non-autonomous mechanism.

Elife 2021 11 23;10. Epub 2021 Nov 23.

Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, United States.

Skeletal muscle regeneration is regulated by coordinated activation of multiple signaling pathways. The unfolded protein response (UPR) is a major mechanism that detects and alleviates protein-folding stresses in the endoplasmic reticulum. However, the role of individual arms of the UPR in skeletal muscle regeneration remain less understood. In the present study, we demonstrate that IRE1α (also known as ERN1) and its downstream target, XBP1, are activated in skeletal muscle of mice upon injury. Myofiber-specific ablation of IRE1α or XBP1 in mice diminishes skeletal muscle regeneration that is accompanied with reduced number of satellite cells. Ex vivo cultures of myofiber explants demonstrate that ablation of IRE1α reduces the proliferative capacity of myofiber-associated satellite cells. Myofiber-specific ablation of IRE1α dampens Notch signaling and canonical NF-κB pathway in skeletal muscle of adult mice. Finally, targeted ablation of IRE1α also reduces Notch signaling, abundance of satellite cells, and skeletal muscle regeneration in the mdx mice, a model of Duchenne muscular dystrophy. Collectively, our experiments suggest that the IRE1α-mediated signaling promotes muscle regeneration through augmenting the proliferation of satellite cells in a cell non-autonomous manner.
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http://dx.doi.org/10.7554/eLife.73215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8635982PMC
November 2021

Inhibition of skin fibrosis in systemic sclerosis by botulinum toxin B via the suppression of oxidative stress.

J Dermatol 2021 Jul 11;48(7):1052-1061. Epub 2021 Apr 11.

Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Japan.

Oxidative stress has been reported to play an important role in the pathogenesis of skin fibrosis in systemic sclerosis (SSc). We previously identified that botulinum toxin (BTX) injection suppresses pressure ulcer formation in a cutaneous ischemia-reperfusion injury mouse model by regulation of oxidative stress. However, the therapeutic possibility of BTX administration for preventing skin fibrosis in SSc is unclear. The objective of this study was to investigate the effect of BTX-B on skin fibrosis in a murine model of SSc and determine the underlying mechanism. We found that BTX-B injection significantly reduced dermal thickness and inflammatory cell infiltration in bleomycin-induced skin fibrosis lesion in mice. We also identified that the oxidative stress signal detected through bioluminescence in OKD48 mice after bleomycin injection in the skin was significantly decreased by BTX-B. Additionally, mRNA levels of oxidative stress associated factors (NOX2, HO-1, Trx2) were significantly decreased by BTX-B. Apoptotic cells in the lesional skin of bleomycin-treated mice were significantly reduced by BTX-B. Oxidant-induced intracellular accumulation of reactive oxygen species in SSc fibroblasts was also inhibited by BTX-B. In conclusion, BTX-B might improve bleomycin-induced skin fibrosis via the suppression of oxidative stress and inflammatory cells in the skin. BTX-B injection may have a therapeutic effect on skin fibrosis in SSc.
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http://dx.doi.org/10.1111/1346-8138.15888DOI Listing
July 2021

Spatiotemporal analysis of the UPR transition induced by methylmercury in the mouse brain.

Arch Toxicol 2021 04 16;95(4):1241-1250. Epub 2021 Jan 16.

Department of Medicinal Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, 700-8530, Japan.

Methylmercury (MeHg), an environmental toxicant, induces neuronal cell death and injures a specific area of the brain. MeHg-mediated neurotoxicity is believed to be caused by oxidative stress and endoplasmic reticulum (ER) stress but the mechanism by which those stresses lead to neuronal loss is unclear. Here, by utilizing the ER stress-activated indicator (ERAI) system, we investigated the signaling alterations in the unfolded protein response (UPR) prior to neuronal apoptosis in the mouse brain. In ERAI transgenic mice exposed to MeHg (25 mg/kg, S.C.), the ERAI signal, which indicates activation of the cytoprotective pathway of the UPR, was detected in the brain. Interestingly, detailed ex vivo analysis showed that the ERAI signal was localized predominantly in neurons. Time course analysis of MeHg exposure (30 ppm in drinking water) showed that whereas the ERAI signal was gradually attenuated at the late phase after increasing at the early phase, activation of the apoptotic pathway of the UPR was enhanced in proportion to the exposure time. These results suggest that MeHg induces not only ER stress but also neuronal cell death via a UPR shift. UPR modulation could be a therapeutic target for treating neuropathy caused by electrophiles similar to MeHg.
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http://dx.doi.org/10.1007/s00204-021-02982-9DOI Listing
April 2021

Role of IRE1α in podocyte proteostasis and mitochondrial health.

Cell Death Discov 2020 Nov 19;6(1):128. Epub 2020 Nov 19.

Department of Medicine, McGill University Health Centre Research Institute, McGill University, Montreal, QC, Canada.

Glomerular epithelial cell (GEC)/podocyte proteostasis is dysregulated in glomerular diseases. The unfolded protein response (UPR) is an adaptive pathway in the endoplasmic reticulum (ER) that upregulates proteostasis resources. This study characterizes mechanisms by which inositol requiring enzyme-1α (IRE1α), a UPR transducer, regulates proteostasis in GECs. Mice with podocyte-specific deletion of IRE1α (IRE1α KO) were produced and nephrosis was induced with adriamycin. Compared with control, IRE1α KO mice had greater albuminuria. Adriamycin increased glomerular ER chaperones in control mice, but this upregulation was impaired in IRE1α KO mice. Likewise, autophagy was blunted in adriamycin-treated IRE1α KO animals, evidenced by reduced LC3-II and increased p62. Mitochondrial ultrastructure was markedly disrupted in podocytes of adriamycin-treated IRE1α KO mice. To pursue mechanistic studies, GECs were cultured from glomeruli of IRE1α flox/flox mice and IRE1α was deleted by Cre-lox recombination. In GECs incubated with tunicamycin, deletion of IRE1α attenuated upregulation of ER chaperones, LC3 lipidation, and LC3 transcription, compared with control GECs. Deletion of IRE1α decreased maximal and ATP-linked oxygen consumption, as well as mitochondrial membrane potential. In summary, stress-induced chaperone production, autophagy, and mitochondrial health are compromised by deletion of IRE1α. The IRE1α pathway is cytoprotective in glomerular disease associated with podocyte injury and ER stress.
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http://dx.doi.org/10.1038/s41420-020-00361-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677398PMC
November 2020

Hepatocyte-specific deletion of XBP1 sensitizes mice to liver injury through hyperactivation of IRE1α.

Cell Death Differ 2021 05 20;28(5):1455-1465. Epub 2020 Nov 20.

Department of Medicine, University of California San Francisco, San Francisco, CA, USA.

X-box binding protein-1 (XBP1) is a transcription factor that plays a central role in controlling cellular responses to endoplasmic reticulum (ER) stress. Under stress conditions, the transcriptionally active form of XBP1 is generated via splicing of Xbp1 mRNA by the ER-resident protein inositol-requiring enzyme-1 (IRE1α). Genetic deletion of XBP1 has multiple consequences: some resulting from the loss of the transcription factor per se, and others related to compensatory activation of IRE1α. The objective of the current study was to investigate the effects of XBP1 deletion in adult mouse liver and determine to what extent they are direct or indirect. XBP1 was deleted from hepatocytes in adult Xbp1 mice using AAV8-Transthyretin-Cre (Xbp1). Xbp1 mice exhibited no liver disease at baseline, but developed acute biochemical and histologic liver injury in response to a dietary challenge with fructose for 4 weeks. Fructose-mediated liver injury in Xbp1 mice coincided with heightened IRE1α activity, as demonstrated by Xbp1 mRNA splicing, JNK activation, and regulated IRE1α-dependent RNA decay (RIDD). Activation of eIF2α was also evident, with associated up-regulation of the pro-apoptotic molecules CHOP, BIM, and PUMA. To determine whether the adverse consequences of liver-specific XBP1 deletion were due to XBP1 loss or heightened IRE1α activity, we repeated a fructose challenge in mice with liver-specific deletion of both XBP1 and IRE1α (Xbp1;Ire1a). Xbp1;Ire1a mice were protected from fructose-mediated liver injury and failed to exhibit any of the signs of ER stress seen in mice lacking XBP1 alone. The protective effect of IRE1α deletion persisted even with long-term exposure to fructose. Xbp1 mice developed liver fibrosis at 16 weeks, but Xbp1;Ire1a mice did not. Overall, the results indicate that the deleterious effects of hepatocyte-specific XBP1 deletion are due primarily to hyperactivation of IRE1α. They support further exploration of IRE1α as a contributor to acute and chronic liver diseases.
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http://dx.doi.org/10.1038/s41418-020-00671-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8166833PMC
May 2021

Protein quality control through endoplasmic reticulum-associated degradation maintains haematopoietic stem cell identity and niche interactions.

Nat Cell Biol 2020 10 21;22(10):1162-1169. Epub 2020 Sep 21.

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.

Stem cells need to be protected from genotoxic and proteotoxic stress to maintain a healthy pool throughout life. Little is known about the proteostasis mechanism that safeguards stem cells. Here we report endoplasmic reticulum-associated degradation (ERAD) as a protein quality checkpoint that controls the haematopoietic stem cell (HSC)-niche interaction and determines the fate of HSCs. The SEL1L-HRD1 complex, the most conserved branch of ERAD, is highly expressed in HSCs. Deletion of Sel1l led to niche displacement of HSCs and a complete loss of HSC identity, and allowed highly efficient donor-HSC engraftment without irradiation. Mechanistic studies identified MPL, the master regulator of HSC identity, as a bona fide ERAD substrate that became aggregated in the endoplasmic reticulum following ERAD deficiency. Restoration of MPL signalling with an agonist partially rescued the number and reconstitution capacity of Sel1l-deficient HSCs. Our study defines ERAD as an essential proteostasis mechanism to safeguard a healthy stem cell pool by regulating the stem cell-niche interaction.
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http://dx.doi.org/10.1038/s41556-020-00581-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888538PMC
October 2020

IRE1α regulates macrophage polarization, PD-L1 expression, and tumor survival.

PLoS Biol 2020 06 10;18(6):e3000687. Epub 2020 Jun 10.

The Laboratory of Immunology, Department of Medicine and Moores Cancer Center, University of California, San Diego, La Jolla, California, United States of America.

In the tumor microenvironment, local immune dysregulation is driven in part by macrophages and dendritic cells that are polarized to a mixed proinflammatory/immune-suppressive phenotype. The unfolded protein response (UPR) is emerging as the possible origin of these events. Here we report that the inositol-requiring enzyme 1 (IRE1α) branch of the UPR is directly involved in the polarization of macrophages in vitro and in vivo, including the up-regulation of interleukin 6 (IL-6), IL-23, Arginase1, as well as surface expression of CD86 and programmed death ligand 1 (PD-L1). Macrophages in which the IRE1α/X-box binding protein 1 (Xbp1) axis is blocked pharmacologically or deleted genetically have significantly reduced polarization and CD86 and PD-L1 expression, which was induced independent of IFNγ signaling, suggesting a novel mechanism in PD-L1 regulation in macrophages. Mice with IRE1α- but not Xbp1-deficient macrophages showed greater survival than controls when implanted with B16.F10 melanoma cells. Remarkably, we found a significant association between the IRE1α gene signature and CD274 gene expression in tumor-infiltrating macrophages in humans. RNA sequencing (RNASeq) analysis showed that bone marrow-derived macrophages with IRE1α deletion lose the integrity of the gene connectivity characteristic of regulated IRE1α-dependent decay (RIDD) and the ability to activate CD274 gene expression. Thus, the IRE1α/Xbp1 axis drives the polarization of macrophages in the tumor microenvironment initiating a complex immune dysregulation leading to failure of local immune surveillance.
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http://dx.doi.org/10.1371/journal.pbio.3000687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307794PMC
June 2020

Protective effect of dimethyl fumarate for the development of pressure ulcers after cutaneous ischemia-reperfusion injury.

Wound Repair Regen 2020 09 12;28(5):600-608. Epub 2020 May 12.

Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Japan.

Ischemia-reperfusion (I/R) is associated with various pathogenic conditions, and there has been increasing evidence that cutaneous I/R injury is associated with the pathogenesis of pressure ulcers (PUs), especially at the early stage presenting as non-blanchable erythema. Several studies demonstrated that oxidative stress is a key player in I/R injury, and the inhibition of oxidative stress may be capable of protecting tissue damage after I/R injury in various organs including skin. Dimethyl fumarate (DMF) approved by the Food and Drug Administration is Nrf2 activator, and recent studies revealed the antioxidative and anti-inflammatory effects of DMF on I/R injury in animal models. Our objective was to assess the effects of oral administration of DMF on the development of PUs after cutaneous I/R injury in mice. We found that DMF administration significantly decreased the size of PUs after cutaneous I/R. Cutaneous I/R-induced oxidative stress was also significantly inhibited by DMF in OKD48 mice, in which oxidative stress can be visually assessed. In addition, DMF treatment decreased hypoxic area, the numbers of apoptotic cells, and vascular loss in I/R area. DMF treatment suppressed the infiltration of MPO neutrophils and the production of proinflammatory cytokines in I/R site after cutaneous I/R injury. in vitro experiments, DMF treatment suppressed the production of reactive oxygen species in pericyte-like cells. These results suggest that DMF treatment might prevent the formation of PUs induced by cutaneous I/R injury via suppressing oxidative stress and subsequent inflammation. DMF treatment during the early phase of decubitus ulcers might protect against further progression.
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http://dx.doi.org/10.1111/wrr.12824DOI Listing
September 2020

AAV-mediated ERdj5 overexpression protects against P23H rhodopsin toxicity.

Hum Mol Genet 2020 05;29(8):1310-1318

UCL Institute of Ophthalmology, London EC1V 9EL, UK.

Rhodopsin misfolding caused by the P23H mutation is a major cause of autosomal dominant retinitis pigmentosa (adRP). To date, there are no effective treatments for adRP. The BiP co-chaperone and reductase ERdj5 (DNAJC10) is part of the endoplasmic reticulum (ER) quality control machinery, and previous studies have shown that overexpression of ERdj5 in vitro enhanced the degradation of P23H rhodopsin, whereas knockdown of ERdj5 increased P23H rhodopsin ER retention and aggregation. Here, we investigated the role of ERdj5 in photoreceptor homeostasis in vivo by using an Erdj5 knockout mouse crossed with the P23H knock-in mouse and by adeno-associated viral (AAV) vector-mediated gene augmentation of ERdj5 in P23H-3 rats. Electroretinogram (ERG) and optical coherence tomography of Erdj5-/- and P23H+/-:Erdj5-/- mice showed no effect of ERdj5 ablation on retinal function or photoreceptor survival. Rhodopsin levels and localization were similar to those of control animals at a range of time points. By contrast, when AAV2/8-ERdj5-HA was subretinally injected into P23H-3 rats, analysis of the full-field ERG suggested that overexpression of ERdj5 reduced visual function loss 10 weeks post-injection (PI). This correlated with a significant preservation of photoreceptor cells at 4 and 10 weeks PI. Assessment of the outer nuclear layer (ONL) morphology showed preserved ONL thickness and reduced rhodopsin retention in the ONL in the injected superior retina. Overall, these data suggest that manipulation of the ER quality control and ER-associated degradation factors to promote mutant protein degradation could be beneficial for the treatment of adRP caused by mutant rhodopsin.
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http://dx.doi.org/10.1093/hmg/ddaa049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7254845PMC
May 2020

IRE1α Promotes Zika Virus Infection via XBP1.

Viruses 2020 03 3;12(3). Epub 2020 Mar 3.

Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA.

Zika virus (ZIKV) is an emergent member of the family which causes severe congenital defects and other major sequelae, but the cellular processes that support ZIKV replication are incompletely understood. Related flaviviruses use the endoplasmic reticulum (ER) as a membranous platform for viral replication and induce ER stress during infection. Our data suggest that ZIKV activates IRE1α, a component of the cellular response to ER stress. IRE1α is an ER-resident transmembrane protein that possesses a cytosolic RNase domain. Upon activation, IRE1α initiates nonconventional cytoplasmic splicing of mRNA. Spliced encodes a transcription factor, which upregulates ER-related targets. We find that ZIKV infection induces mRNA splicing and induction of XBP1 target genes. Small molecule inhibitors of IRE1α, including those specific for the nuclease function, prevent ZIKV-induced cytotoxicity, as does genetic disruption of IRE1α. Optimal ZIKV RNA replication requires both IRE1α and XBP1. Spliced XBP1 has been described to cause ER expansion and remodeling and we find that ER redistribution during ZIKV infection requires IRE1α nuclease activity. Finally, we demonstrate that inducible genetic disruption of IRE1α and XBP1 impairs ZIKV replication in a mouse model of infection. Together, our data indicate that the ER stress response component IRE1α promotes ZIKV infection via XBP1 and may represent a potential therapeutic target.
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http://dx.doi.org/10.3390/v12030278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150863PMC
March 2020

Transgenic mouse model exhibiting weak red fluorescence before and strong green fluorescenceafter Cre/loxP-mediated recombination.

Exp Anim 2020 Aug 1;69(3):306-318. Epub 2020 Mar 1.

Division of Cell Medicine, Department of Life Science, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 920-0293, Japan.

The Cre/loxP system is an indispensable tool for temporal and spatial control of gene function in mice. Many mice that express Cre and carry loxP sites in their genomes have been bred for functional analysis of various genes in vivo. Also, several reporter mice have been generated for monitoring of recombination by the Cre/loxP system. We have developed a Cre reporter gene with DsRed1 and Venus that exhibits a strong red fluorescence before and a strong green fluorescence after Cre/loxP-mediated recombination in experiments using NIH3T3 cells. However, a transgenic mouse introduced with the same reporter gene exhibits a weak red fluorescence before and a strong green fluorescence after Cre/loxP-mediated recombination. This property manifested ubiquitously in this mouse model and was maintained stably in mouse-derived fibroblasts. Use of the mouse model exhibiting the stronger red fluorescence might result in confusion of the Cre-dependent signal with false signals, because the Venus signal includes some fluorescence in the red region of the spectrum and the DsRed1 signal includes some fluorescence in the green region. However, we fortuitously obtained reporter mice that exhibit a weaker red fluorescence before Cre/loxP-mediated recombination. The use of this mouse model would decrease concern regarding errors in the identification of signals and should increase certainty in the detection of Cre activity in vivo.
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http://dx.doi.org/10.1538/expanim.19-0085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445058PMC
August 2020

Apelin/APJ signaling suppresses the pressure ulcer formation in cutaneous ischemia-reperfusion injury mouse model.

Sci Rep 2020 Jan 28;10(1):1349. Epub 2020 Jan 28.

Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Japan.

Several studies have demonstrated potential roles for apelin/APJ signaling in the regulation of oxidative stress associated with ischemia-reperfusion (I/R) injury in several organs. Objective was to assess the role of apelin/APJ signaling in the development of pressure ulcers (PUs) formation after cutaneous I/R injury in mice. We identified that cutaneous I/R injury increased the expression of apelin in the skin at I/R site. Administration of apelin significantly inhibited the formation of PUs. The reductions of blood vessels, hypoxic area and apoptosis in I/R site were inhibited by apelin injection. Oxidative stress signals in OKD48 mice and the expressions of oxidative stress related genes in the skin were suppressed by apelin injection. HO-induced intracellular ROS and apoptosis in endothelial cells and fibroblasts were suppressed by apelin in vitro. Furthermore, MM07, biased agonist of APJ, also significantly suppressed the development of PUs after cutaneous I/R, and the inhibitory effect of MM07 on PUs formation was higher than that in apelin. We conclude that apelin/APJ signaling may inhibit cutaneous I/R injury-induced PUs formation by protecting the reduction of vascularity and tissue damage via suppression of oxidative stress. Exogenous application of apelin or MM07 might have therapeutic potentials against the development of PUs.
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http://dx.doi.org/10.1038/s41598-020-58452-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6987197PMC
January 2020

Danger-associated extracellular ATP counters MDSC therapeutic efficacy in acute GVHD.

Blood 2019 11;134(19):1670-1682

Division of Blood and Marrow Transplantation, Department of Pediatrics.

Myeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD), donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer, resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen-induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival, indicating in vivo biologic effects, no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1β (IL-1β), we expected that IL-1β KO donor MDSCs would be superior in subverting GVHD, but such MDSCs proved inferior relative to WT. IL-1β release and IL-1 receptor expression was required for optimal MDSC function, and exogenous IL-1β added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1β could diminish survival in GVHD. However, loss of inflammasome activation and IL-1β release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1β signaling, improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum, the ATP release site, synergized with WT MDSCs, as did regulatory T-cell infusion, which we showed reduced but did not eliminate MDSC inflammasome activation, as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments.
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http://dx.doi.org/10.1182/blood.2019001950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6871306PMC
November 2019

Inhibitory effect of kaempferol on skin fibrosis in systemic sclerosis by the suppression of oxidative stress.

J Dermatol Sci 2019 Oct 16;96(1):8-17. Epub 2019 Aug 16.

Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Japan.

Background: There is growing evidence that vasculopathy-induced hypoxia and oxidative stress enhance the process of fibrosis in systemic sclerosis (SSc). Kaempferol is a natural flavonoid widely found in various vegetables and fruits, and has been reported to have excellent antioxidant activity.

Objective: Objective was to elucidate the effect of kaempferol on skin fibrosis and the mechanism of the inhibitory regulation of fibrosis by kaempferol.

Methods: We assessed the effect of intraperitoneally administered kaempferol on bleomycin-induced dermal fibrosis in mice. The effect of kaempferol on oxidative stress in bleomycin-treated mice and SSc fibroblasts was assessed in vivo and in vitro.

Results: We identified that kaempferol injection significantly inhibited bleomycin-induced dermal fibrosis in mice. The number of αSMA myofibroblasts, CD3 T-cells, and CD68 macrophages in lesional skin was significantly decreased by kaempferol injections. Kaempferol administration also significantly suppressed the bleomycin-induced oxidative stress signal in OKD48 mice. Additionally, mRNA levels of oxidative stress-associated factors, such as HO-1 and NOX2, as well as inflammatory and pro-fibrotic cytokines, including IL-6, TGF-β and TNFα in sclerotic skin were significantly decreased by kaempferol. Kaempferol also reduced bleomycin-induced TUNEL apoptotic cells in the lesional skin of bleomycin-treated mice. Furthermore, the oxidant-induced intracellular accumulation of reactive oxygen species (ROS) in SSc fibroblasts was inhibited by kaempferol treatment. In addition, the oxidant-induced apoptosis of SSc fibroblasts was decreased by kaempferol in vitro.

Conclusion: Kaempferol might improve bleomycin-induced fibrosis by reducing oxidative stress, inflammation, and oxidative cellular damage. Administration of kaempferol might be an alternative treatment for skin fibrosis in SSc.
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http://dx.doi.org/10.1016/j.jdermsci.2019.08.004DOI Listing
October 2019

MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites.

EMBO J 2019 08 13;38(15):e100999. Epub 2019 Jun 13.

Laboratory of Molecular Biochemistry, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan.

Unresolved endoplasmic reticulum (ER) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). MITOL promotes K63-linked chain ubiquitination of IRE1α at lysine 481 (K481), thereby preventing hyper-oligomerization of IRE1α and regulated IRE1α-dependent decay (RIDD). Therefore, under ER stress, MITOL depletion or the IRE1α mutant (K481R) allows for IRE1α hyper-oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL-deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE1α ubiquitylation by MITOL at the MAM.
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http://dx.doi.org/10.15252/embj.2018100999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669929PMC
August 2019

Zinc deficiency exacerbates pressure ulcers by increasing oxidative stress and ATP in the skin.

J Dermatol Sci 2019 Aug 12;95(2):62-69. Epub 2019 Jul 12.

Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Japan. Electronic address:

Background: Zinc deficiency is believed to be a predisposing factor for the development and intractable healing of pressure ulcers (PUs); however, the mechanisms of this association have not been elucidated.

Objective: Objective was to elucidate the mechanisms of the formation of severe and prolonged PUs under the zinc deficiency condition.

Methods: We assessed PUs formation after cutaneous ischemia-reperfusion (I/R) injury in mice fed with a zinc-adequate (ZA) or a zinc-deficient (ZD) diet from 2 weeks before I/R injury. Wound size, vascular damage, apoptotic cells, adenosine triphosphate (ATP) amount, and the number of Langerhans cells (LCs) in I/R area were analyzed. We evaluated the extent of oxidative stress in I/R area in OKD48 mice through bioluminescence detection.

Results: We found that dietary zinc deficiency caused the formation of severe and prolonged PUs in mice. Zinc deficiency increased the vascular disorder, oxidative stress, and apoptosis induced by cutaneous I/R injury. I/R injury-induced oxidative stress signals were significantly higher in ZD OKD48 mice than in ZA OKD48 mice. Additionally, zinc deficiency reduced the number of LCs and increased the amount of ATP in cutaneous I/R-injured skin. Oral supplementation of zinc improved zinc deficiency-associated PUs.

Conclusion: Zinc deficiency might increase cutaneous I/R injury-induced vascular damages, oxidative stress, and apoptosis, as well as ATP amount in I/R area due to the loss of LCs. These mechanisms might partly account for zinc deficiency-induced formation of severe and prolonged PUs. Oral supplementation of zinc might be a reasonable therapeutic choice for patients with PUs and zinc deficiency.
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http://dx.doi.org/10.1016/j.jdermsci.2019.07.004DOI Listing
August 2019

IRE1α-XBP1 signaling in leukocytes controls prostaglandin biosynthesis and pain.

Science 2019 07;365(6450)

Weill Cornell Graduate School of Medical Sciences, Cornell University. New York, NY 10065, USA.

Inositol-requiring enzyme 1[α] (IRE1[α])-X-box binding protein spliced (XBP1) signaling maintains endoplasmic reticulum (ER) homeostasis while controlling immunometabolic processes. Yet, the physiological consequences of IRE1α-XBP1 activation in leukocytes remain unexplored. We found that induction of prostaglandin-endoperoxide synthase 2 (/Cox-2) and prostaglandin E synthase (/mPGES-1) was compromised in IRE1α-deficient myeloid cells undergoing ER stress or stimulated through pattern recognition receptors. Inducible biosynthesis of prostaglandins, including the pro-algesic mediator prostaglandin E2 (PGE), was decreased in myeloid cells that lack IRE1α or XBP1 but not other ER stress sensors. Functional XBP1 transactivated the human and genes to enable optimal PGE production. Mice that lack IRE1α-XBP1 in leukocytes, or that were treated with IRE1α inhibitors, demonstrated reduced pain behaviors in PGE-dependent models of pain. Thus, IRE1α-XBP1 is a mediator of prostaglandin biosynthesis and a potential target to control pain.
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http://dx.doi.org/10.1126/science.aau6499DOI Listing
July 2019

Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1.

Cell Rep 2019 07;28(3):746-758.e4

Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; Tohoku Medical Megabank Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8573, Japan. Electronic address:

The Keap1-Nrf2 system plays a central role in the oxidative stress response; however, the identity of the reactive oxygen species sensor within Keap1 remains poorly understood. Here, we show that a Keap1 mutant lacking 11 cysteine residues retains the ability to target Nrf2 for degradation, but it is unable to respond to cysteine-reactive Nrf2 inducers. Of the 11 mutated cysteine residues, we find that 4 (Cys226/613/622/624) are important for sensing hydrogen peroxide. Our analyses of multiple mutant mice lines, complemented by MEFs expressing a series of Keap1 mutants, reveal that Keap1 uses the cysteine residues redundantly to set up an elaborate fail-safe mechanism in which specific combinations of these four cysteine residues can form a disulfide bond to sense hydrogen peroxide. This sensing mechanism is distinct from that used for electrophilic Nrf2 inducers, demonstrating that Keap1 is equipped with multiple cysteine-based sensors to detect various endogenous and exogenous stresses.
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http://dx.doi.org/10.1016/j.celrep.2019.06.047DOI Listing
July 2019

ERdj5 in Innate Immune Cells Is a Crucial Factor for the Mucosal Adjuvanticity of Cholera Toxin.

Front Immunol 2019 4;10:1249. Epub 2019 Jun 4.

College of Pharmacy, and Research Institute of Pharmaceutical Science and Technology (RIPST), Ajou University, Suwon-si, South Korea.

Cholera toxin (CT) is one of most strong mucosal adjuvants, but it cannot be clinically used owing to its toxicity. The cytosolic A1 subunit of CT (CTA1) is the molecule responsible for its immunostimulatory activity, which increases the concentration of cyclic AMP and causes the induction of pro-inflammatory cytokines in innate immune cells. However, the importance of endoplasmic reticulum (ER) molecules involved in CTA1 retro-translocation to induce immune responses remained to be investigated. ERdj5 is an ER protein which is expected to transfer CTA1 to the Hrd1 complex for the retro-translocation of CTA1. In this study, we investigated the physiological relevance of ERdj5 in immune stimulation by CT. ERdj5-knockout (ERdj5 KO) mice had decreased production of antigen-specific IgG in the serum and IgA in the mucosal secretion after intranasal immunization with Ag and CT. Especially, IgG2c isotypes were specifically reduced in the absence of ERdj5. ERdj5 KO dendritic cells (DCs) failed to full activation with decreased expression of costimulatory molecules, such as MHC class II, CD80, and CD 86. In ERdj5 KO DCs, secretion of pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, was reduced. The cytokine signatures of several helper T cells were reduced in ERdj5 KO mice following intranasal CT immunization. The absence of ERdj5 affects the immunostimulatory properties of CT but does not affect the response to the CTB pentamer, the response to alum, total antibody production, or cytokine release from DCs exposed to CpG. Interestingly, CT enhanced the expression of ER stress proteins in ERdj5 KO innate immune cells. These results suggested that ERdj5 contributed as a decisive factor to the immunostimulatory capacity of CT via CTA1 retro-translocation.
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http://dx.doi.org/10.3389/fimmu.2019.01249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593289PMC
October 2020

Ablation of the Chaperone Protein ERdj5 Results in a Sjögren's Syndrome-Like Phenotype in Mice, Consistent With an Upregulated Unfolded Protein Response in Human Patients.

Front Immunol 2019 22;10:506. Epub 2019 Mar 22.

Division of Microbiology and Molecular Medicine, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.

Sjögren's syndrome (SS) is a chronic autoimmune disorder that affects mainly the exocrine glands. Endoplasmic reticulum (ER) stress proteins have been suggested to participate in autoimmune and inflammatory responses, either acting as autoantigens, or by modulating factors of inflammation. The chaperone protein ERdj5 is an ER-resident disulfide reductase, required for the translocation of misfolded proteins during ER-associated protein degradation. In this study we investigated the role of ERdj5 in the salivary glands (SGs), in association with inflammation and autoimmunity. expression of ERdj5 and XBP1 activation were studied immunohistochemically in minor SG tissues from primary SS patients and non-SS sicca-complaining controls. We used the mouse model of ERdj5 ablation and characterized its features: Histopathological, serological (antinuclear antibodies and cytokine levels), and functional (saliva flow rate). ERdj5 was highly expressed in the minor SGs of SS patients, with stain intensity correlated to inflammatory lesion severity and anti-SSA/Ro positivity. Moreover, SS patients demonstrated higher XBP1 activation within the SGs. Remarkably, ablation of ERdj5 in mice conveyed many of the cardinal features of SS, like spontaneous inflammation in SGs with infiltrating T and B lymphocytes, distinct cytokine signature, excessive cell death, reduced saliva flow, and production of anti-SSA/Ro and anti-SSB/La autoantibodies. Notably, these features were more pronounced in female mice. Our findings suggest a critical connection between the function of the ER chaperone protein ERdj5 and autoimmune inflammatory responses in the SGs and provide evidence for a new, potent animal model of SS.
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http://dx.doi.org/10.3389/fimmu.2019.00506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438897PMC
September 2020

Adaptive endoplasmic reticulum stress signalling via IRE1α-XBP1 preserves self-renewal of haematopoietic and pre-leukaemic stem cells.

Nat Cell Biol 2019 03 18;21(3):328-337. Epub 2019 Feb 18.

Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.

Over their lifetime, long-term haematopoietic stem cells (HSC) are exposed to a variety of stress conditions that they must endure. Many stresses, such as infection/inflammation, reactive oxygen species, nutritional deprivation and hypoxia, activate unfolded protein response signalling, which induces either adaptive changes to resolve the stress or apoptosis to clear the damaged cell. Whether unfolded-protein-response signalling plays any role in HSC regulation remains to be established. Here, we report that the adaptive signalling of the unfolded protein response, IRE1α-XBP1, protects HSCs from endoplasmic reticulum stress-induced apoptosis. IRE1α knockout leads to reduced reconstitution of HSCs. Furthermore, we show that oncogenic N-Ras activates IRE1α-XBP1, through MEK-GSK3β, to promote HSC survival under endoplasmic reticulum stress. Inhibiting IRE1α-XBP1 abolished N-Ras-mediated survival under endoplasmic reticulum stress and diminished the competitive advantage of Nras HSCs in transplant recipients. Our studies illuminate how the adaptive endoplasmic reticulum stress response is advantageous in sustaining self-renewal of HSCs and promoting pre-leukaemic clonal dominance.
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http://dx.doi.org/10.1038/s41556-019-0285-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745703PMC
March 2019

IRE1α Activation in Bone Marrow-Derived Dendritic Cells Modulates Innate Recognition of Melanoma Cells and Favors CD8 T Cell Priming.

Front Immunol 2018 4;9:3050. Epub 2019 Jan 4.

Program of Immunology, Laboratory of Immunology and Cellular Stress, Faculty of Medicine, Institute of Biomedical Sciences, Universidad de Chile, Santiago, Chile.

The IRE1α/XBP1s signaling pathway is an arm of the unfolded protein response (UPR) that safeguards the fidelity of the cellular proteome during endoplasmic reticulum (ER) stress, and that has also emerged as a key regulator of dendritic cell (DC) homeostasis. However, in the context of DC activation, the regulation of the IRE1α/XBP1s axis is not fully understood. In this work, we report that cell lysates generated from melanoma cell lines markedly induce XBP1s and certain members of the UPR such as the chaperone BiP in bone marrow derived DCs (BMDCs). Activation of IRE1α endonuclease upon innate recognition of melanoma cell lysates was required for amplification of proinflammatory cytokine production and was necessary for efficient cross-presentation of melanoma-associated antigens without modulating the MHC-II antigen presentation machinery. Altogether, this work provides evidence indicating that activation of the IRE1α/XBP1 pathway in BMDCs enhances CD8 T cell specific responses against tumor antigens.
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http://dx.doi.org/10.3389/fimmu.2018.03050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338037PMC
October 2019

Stabilization of cytokine mRNAs in iNKT cells requires the serine-threonine kinase IRE1alpha.

Nat Commun 2018 12 17;9(1):5340. Epub 2018 Dec 17.

Unit for Molecular Immunology and Inflammation, VIB Center for Inflammation Research, Technologiepark 927, 9052, Zwijnaarde (Ghent), Belgium.

Activated invariant natural killer T (iNKT) cells rapidly produce large amounts of cytokines, but how cytokine mRNAs are induced, stabilized and mobilized following iNKT activation is still unclear. Here we show that an endoplasmic reticulum stress sensor, inositol-requiring enzyme 1α (IRE1α), links key cellular processes required for iNKT cell effector functions in specific iNKT subsets, in which TCR-dependent activation of IRE1α is associated with downstream activation of p38 MAPK and the stabilization of preformed cytokine mRNAs. Importantly, genetic deletion of IRE1α in iNKT cells reduces cytokine production and protects mice from oxazolone colitis. We therefore propose that an IRE1α-dependent signaling cascade couples constitutive cytokine mRNA expression to the rapid induction of cytokine secretion and effector functions in activated iNKT cells.
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http://dx.doi.org/10.1038/s41467-018-07758-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297233PMC
December 2018
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