Publications by authors named "Zhaozhao Jiang"

26 Publications

  • Page 1 of 1

cGAS-STING Pathway Does Not Promote Autoimmunity in Murine Models of SLE.

Front Immunol 2021 29;12:605930. Epub 2021 Mar 29.

Program in Innate Immunity, Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, United States.

Detection of DNA is an important determinant of host-defense but also a driver of autoinflammatory and autoimmune diseases. Failure to degrade self-DNA in DNAseII or III(TREX1)-deficient mice results in activation of the cGAS-STING pathway. Deficiency of cGAS or STING in these models ameliorates disease manifestations. However, the contribution of the cGAS-STING pathway, relative to endosomal TLRs, in systemic lupus erythematosus (SLE) is controversial. In fact, STING deficiency failed to rescue, and actually exacerbated, disease manifestations in Fas-deficient SLE-prone mice. We have now extended these observations to a chronic model of SLE induced by the i.p. injection of TMPD (pristane). We found that both cGAS- and STING-deficiency not only failed to rescue mice from TMPD-induced SLE, but resulted in increased autoantibody production and higher proteinuria levels compared to cGAS STING sufficient mice. Further, we generated cGASFas mice on a pure MRL/Fas background using Crispr/Cas9 and found slightly exacerbated, and not attenuated, disease. We hypothesized that the cGAS-STING pathway constrains TLR activation, and thereby limits autoimmune manifestations in these two models. Consistent with this premise, mice lacking cGAS and Unc93B1 or STING and Unc93B1 developed minimal systemic autoimmunity as compared to cGAS or STING single knock out animals. Nevertheless, TMPD-driven lupus in B6 mice was abrogated upon AAV-delivery of DNAse I, implicating a DNA trigger. Overall, this study demonstrated that the cGAS-STING pathway does not promote systemic autoimmunity in murine models of SLE. These data have important implications for cGAS-STING-directed therapies being developed for the treatment of systemic autoimmunity.
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http://dx.doi.org/10.3389/fimmu.2021.605930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040952PMC
March 2021

The long non-coding RNA LUCAT1 is a negative feedback regulator of interferon responses in humans.

Nat Commun 2020 12 11;11(1):6348. Epub 2020 Dec 11.

Program in Innate Immunity, University of Massachusetts Medical School, Worcester, MA, 01605, USA.

Long non-coding RNAs are important regulators of biological processes including immune responses. The immunoregulatory functions of lncRNAs have been revealed primarily in murine models with limited understanding of lncRNAs in human immune responses. Here, we identify lncRNA LUCAT1 which is upregulated in human myeloid cells stimulated with lipopolysaccharide and other innate immune stimuli. Targeted deletion of LUCAT1 in myeloid cells increases expression of type I interferon stimulated genes in response to LPS. By contrast, increased LUCAT1 expression results in a reduction of the inducible ISG response. In activated cells, LUCAT1 is enriched in the nucleus where it associates with chromatin. Further, LUCAT1 limits transcription of interferon stimulated genes by interacting with STAT1 in the nucleus. Together, our study highlights the role of the lncRNA LUCAT1 as a post-induction feedback regulator which functions to restrain the immune response in human cells.
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http://dx.doi.org/10.1038/s41467-020-20165-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733444PMC
December 2020

Succination inactivates gasdermin D and blocks pyroptosis.

Science 2020 09 20;369(6511):1633-1637. Epub 2020 Aug 20.

Program in Innate Immunity, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Activated macrophages undergo a metabolic switch to aerobic glycolysis, accumulating Krebs' cycle intermediates that alter transcription of immune response genes. We extended these observations by defining fumarate as an inhibitor of pyroptotic cell death. We found that dimethyl fumarate (DMF) delivered to cells or endogenous fumarate reacts with gasdermin D (GSDMD) at critical cysteine residues to form S-(2-succinyl)-cysteine. GSDMD succination prevents its interaction with caspases, limiting its processing, oligomerization, and capacity to induce cell death. In mice, the administration of DMF protects against lipopolysaccharide shock and alleviates familial Mediterranean fever and experimental autoimmune encephalitis by targeting GSDMD. Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for fumarate-based therapeutics that include DMF, for the treatment of multiple sclerosis.
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http://dx.doi.org/10.1126/science.abb9818DOI Listing
September 2020

A Mitochondrial Micropeptide Is Required for Activation of the Nlrp3 Inflammasome.

J Immunol 2020 01 13;204(2):428-437. Epub 2019 Dec 13.

Program in Innate Immunity, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605; and

Functional peptides encoded by short open reading frames are emerging as important mediators of fundamental biological processes. In this study, we identified a micropeptide produced from a putative long noncoding RNA (lncRNAs) that is important in controlling innate immunity. By studying lncRNAs in mice macrophages, we identified lncRNA 1810058I24Rik, which was downregulated in both human and murine myeloid cells exposed to LPS as well as other TLR ligands and inflammatory cytokines. Analysis of lncRNA 1810058I24Rik subcellular localization revealed that this transcript was localized in the cytosol, prompting us to evaluate its coding potential. In vitro translation with S-labeled methionine resulted in translation of a 47 aa micropeptide. Microscopy and subcellular fractionation studies in macrophages demonstrated endogenous expression of this peptide on the mitochondrion. We thus named this gene mitochondrial micropeptide-47 (Mm47). Crispr-Cas9-mediated deletion of , as well as small interfering RNA studies in mice primary macrophages, showed that the transcriptional response downstream of TLR4 was intact in cells lacking Mm47. In contrast, -deficient or knockdown cells were compromised for Nlrp3 inflammasome responses. Activation of Nlrc4 or Aim2 inflammasomes were intact in cells lacking Mm47. This study therefore identifies, to our knowledge, a novel mitochondrial micropeptide Mm47 that is required for the activation of the Nlrp3 inflammasome. This work further highlights the functional activity of short open reading frame-encoded peptides and underscores their importance in innate immunity.
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http://dx.doi.org/10.4049/jimmunol.1900791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370245PMC
January 2020

Control of antiviral innate immune response by protein geranylgeranylation.

Sci Adv 2019 05 29;5(5):eaav7999. Epub 2019 May 29.

Division of Rheumatology and Immunology, Department of Medicine, Duke University School of Medicine, 207 Research Drive, Durham, NC 27710, USA.

The mitochondrial antiviral signaling protein (MAVS) orchestrates host antiviral innate immune response to RNA virus infection. However, how MAVS signaling is controlled to eradicate virus while preventing self-destructive inflammation remains obscure. Here, we show that protein geranylgeranylation, a posttranslational lipid modification of proteins, limits MAVS-mediated immune signaling by targeting Rho family small guanosine triphosphatase Rac1 into the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) at the mitochondria-ER junction. Protein geranylgeranylation and subsequent palmitoylation promote Rac1 translocation into MAMs upon viral infection. MAM-localized Rac1 limits MAVS' interaction with E3 ligase Trim31 and hence inhibits MAVS ubiquitination, aggregation, and activation. Rac1 also facilitates the recruitment of caspase-8 and cFLIP to the MAVS signalosome and the subsequent cleavage of Ripk1 that terminates MAVS signaling. Consistently, mice with myeloid deficiency of protein geranylgeranylation showed improved survival upon influenza A virus infection. Our work revealed a critical role of protein geranylgeranylation in regulating antiviral innate immune response.
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http://dx.doi.org/10.1126/sciadv.aav7999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541464PMC
May 2019

HiChIRP reveals RNA-associated chromosome conformation.

Nat Methods 2019 06 27;16(6):489-492. Epub 2019 May 27.

Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA, USA.

Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS).
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http://dx.doi.org/10.1038/s41592-019-0407-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638558PMC
June 2019

CNBP controls IL-12 gene transcription and Th1 immunity.

J Exp Med 2018 12 15;215(12):3136-3150. Epub 2018 Nov 15.

Program in Innate Immunity, Department of Medicine, University of Massachusetts Medical School, Worcester, MA

An inducible program of inflammatory gene expression is a hallmark of antimicrobial defenses. Recently, cellular nucleic acid-binding protein (CNBP) was identified as a regulator of nuclear factor-kappaB (NF-κB)-dependent proinflammatory cytokine gene expression. Here, we generated mice lacking CNBP and found that CNBP regulates a very restricted gene signature that includes IL-12β. CNBP resides in the cytosol of macrophages and translocates to the nucleus in response to diverse microbial pathogens and pathogen-derived products. -deficient macrophages induced canonical NF-κB/Rel signaling normally but were impaired in their ability to control the activation of c-Rel, a key driver of IL-12β gene transcription. The nuclear translocation and DNA-binding activity of c-Rel required CNBP. Lastly, -deficient mice were more susceptible to acute toxoplasmosis associated with reduced production of IL-12β, as well as a reduced T helper type 1 (Th1) cell IFN-γ response essential to controlling parasite replication. Collectively, these findings identify CNBP as important regulator of c-Rel-dependent IL-12β gene transcription and Th1 immunity.
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http://dx.doi.org/10.1084/jem.20181031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279399PMC
December 2018

Genetic Models Reveal cis and trans Immune-Regulatory Activities for lincRNA-Cox2.

Cell Rep 2018 11;25(6):1511-1524.e6

Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA, USA. Electronic address:

An inducible gene expression program is a hallmark of the host inflammatory response. Recently, long intergenic non-coding RNAs (lincRNAs) have been shown to regulate the magnitude, duration, and resolution of these responses. Among these is lincRNA-Cox2, a dynamically regulated gene that broadly controls immune gene expression. To evaluate the in vivo functions of this lincRNA, we characterized multiple models of lincRNA-Cox2-deficient mice. LincRNA-Cox2-deficient macrophages and murine tissues had altered expression of inflammatory genes. Transcriptomic studies from various tissues revealed that deletion of the lincRNA-Cox2 locus also strongly impaired the basal and inducible expression of the neighboring gene prostaglandin-endoperoxide synthase (Ptgs2), encoding cyclooxygenase-2, a key enzyme in the prostaglandin biosynthesis pathway. By utilizing different genetic manipulations in vitro and in vivo, we found that lincRNA-Cox2 functions through an enhancer RNA mechanism to regulate Ptgs2. More importantly, lincRNA-Cox2 also functions in trans, independently of Ptgs2, to regulate critical innate immune genes in vivo.
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http://dx.doi.org/10.1016/j.celrep.2018.10.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6291222PMC
November 2018

Fas ligand promotes an inducible TLR-dependent model of cutaneous lupus-like inflammation.

J Clin Invest 2018 07 11;128(7):2966-2978. Epub 2018 Jun 11.

Department of Medicine, University of Massachusetts School of Medicine, Worcester, Massachusetts, USA.

Toll-like receptors TLR7 and TLR9 are both implicated in the activation of autoreactive B cells and other cell types associated with systemic lupus erythematosus (SLE) pathogenesis. However, Tlr9-/- autoimmune-prone strains paradoxically develop more severe disease. We have now leveraged the negative regulatory role of TLR9 to develop an inducible rapid-onset murine model of systemic autoimmunity that depends on T cell detection of a membrane-bound OVA fusion protein expressed by MHC class II+ cells, expression of TLR7, expression of the type I IFN receptor, and loss of expression of TLR9. These mice are distinguished by a high frequency of OVA-specific Tbet+, IFN-γ+, and FasL-expressing Th1 cells as well as autoantibody-producing B cells. Unexpectedly, contrary to what occurs in most models of SLE, they also developed skin lesions that are very similar to those of human cutaneous lupus erythematosus (CLE) as far as clinical appearance, histological changes, and gene expression. FasL was a key effector mechanism in the skin, as the transfer of FasL-deficient DO11gld T cells completely failed to elicit overt skin lesions. FasL was also upregulated in human CLE biopsies. Overall, our model provides a relevant system for exploring the pathophysiology of CLE as well as the negative regulatory role of TLR9.
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http://dx.doi.org/10.1172/JCI98219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6025993PMC
July 2018

Control of the innate immune response by the mevalonate pathway.

Nat Immunol 2016 08 6;17(8):922-9. Epub 2016 Jun 6.

Division of Rheumatology and Immunology, Department of Medicine, Duke University School of Medicine, Durham, North Carolina, USA.

Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1β that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome.
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http://dx.doi.org/10.1038/ni.3487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4955724PMC
August 2016

S6K-STING interaction regulates cytosolic DNA-mediated activation of the transcription factor IRF3.

Nat Immunol 2016 May 4;17(5):514-522. Epub 2016 Apr 4.

McMaster Immunology Research Centre, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.

Cytosolic DNA-mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway.
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http://dx.doi.org/10.1038/ni.3433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917298PMC
May 2016

Cutting Edge: A Natural Antisense Transcript, AS-IL1α, Controls Inducible Transcription of the Proinflammatory Cytokine IL-1α.

J Immunol 2015 Aug 15;195(4):1359-63. Epub 2015 Jul 15.

Program in Innate Immunity, Division of Infectious Diseases, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605;

Natural antisense transcripts (NATs) are a class of long noncoding RNAs (lncRNAs) that are complementary to other protein-coding genes. Although thousands of NATs are encoded by mammalian genomes, their functions in innate immunity are unknown. In this study, we identified and characterized a novel NAT, AS-IL1α, which is partially complementary to IL-1α. Similar to IL-1α, AS-IL1α is expressed at low levels in resting macrophages and is induced following infection with Listeria monocytogenes or stimulation with TLR ligands (Pam3CSK4, LPS, polyinosinic-polycytidylic acid). Inducible expression of IL-1α mRNA and protein were significantly reduced in macrophages expressing shRNA that target AS-IL1α. AS-IL1α is located in the nucleus and did not alter the stability of IL-1α mRNA. Instead, AS-IL1α was required for the recruitment of RNA polymerase II to the IL-1α promoter. In summary, our studies identify AS-IL1α as an important regulator of IL-1α transcription during the innate immune response.
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http://dx.doi.org/10.4049/jimmunol.1500264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4530055PMC
August 2015

3-Hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin)-induced 28-kDa interleukin-1β interferes with mature IL-1β signaling.

J Biol Chem 2014 Jun 30;289(23):16214-22. Epub 2014 Apr 30.

From the Division of Infectious Disease and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 and

Multiple clinical trials have shown that the 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors known as statins have anti-inflammatory effects. However, the underlying molecular mechanism remains unclear. The proinflammatory cytokine interleukin-1β (IL-1β) is synthesized as a non-active precursor. The 31-kDa pro-IL-1β is processed into the 17-kDa active form by caspase-1-activating inflammasomes. Here, we report a novel signaling pathway induced by statins, which leads to processing of pro-IL-1β into an intermediate 28-kDa form. This statin-induced IL-1β processing is independent of caspase-1- activating inflammasomes. The 28-kDa form of IL-1β cannot activate interleukin-1 receptor-1 (IL1R1) to signal inflammatory responses. Instead, it interferes with mature IL-1β signaling through IL-1R1 and therefore may dampen inflammatory responses initiated by mature IL-1β. These results may provide new clues to explain the anti-inflammatory effects of statins.
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http://dx.doi.org/10.1074/jbc.M114.571505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047391PMC
June 2014

The transcriptional repressor BLIMP1 curbs host defenses by suppressing expression of the chemokine CCL8.

J Immunol 2014 Mar 29;192(5):2291-304. Epub 2014 Jan 29.

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605;

The transcriptional repressor B lymphocyte-induced maturation protein 1 (BLIMP1) is a master regulator of B and T cell differentiation. To examine the role of BLIMP1 in innate immunity, we used a conditional knockout (CKO) of Blimp1 in myeloid cells and found that Blimp1 CKO mice were protected from lethal infection induced by Listeria monocytogenes. Transcriptome analysis of Blimp1 CKO macrophages identified the murine chemokine (C-C motif) ligand 8, CCL8, as a direct target of Blimp1-mediated transcriptional repression in these cells. BLIMP1-deficient macrophages expressed elevated levels of Ccl8, and consequently Blimp1 CKO mice had higher levels of circulating CCL8, resulting in increased neutrophils in the peripheral blood, promoting a more aggressive antibacterial response. Mice lacking the Ccl8 gene were more susceptible to L. monocytogenes infection than were wild-type mice. Although CCL8 failed to recruit neutrophils directly, it was chemotactic for γ/δ T cells, and CCL8-responsive γ/δ T cells were enriched for IL-17F. Finally, CCL8-mediated enhanced clearance of L. monocytogenes was dependent on γ/δ T cells. Collectively, these data reveal an important role for BLIMP1 in modulating host defenses by suppressing expression of the chemokine CCL8.
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http://dx.doi.org/10.4049/jimmunol.1301799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943885PMC
March 2014

Mouse, but not human STING, binds and signals in response to the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid.

J Immunol 2013 May 12;190(10):5216-25. Epub 2013 Apr 12.

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Vascular disrupting agents such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA) represent a novel approach for cancer treatment. DMXAA has potent antitumor activity in mice and, despite significant preclinical promise, failed human clinical trials. The antitumor activity of DMXAA has been linked to its ability to induce type I IFNs in macrophages, although the molecular mechanisms involved are poorly understood. In this study, we identify stimulator of IFN gene (STING) as a direct receptor for DMXAA leading to TANK-binding kinase 1 and IFN regulatory factor 3 signaling. Remarkably, the ability to sense DMXAA was restricted to murine STING. Human STING failed to bind to or signal in response to DMXAA. Human STING also failed to signal in response to cyclic dinucleotides, conserved bacterial second messengers known to bind and activate murine STING signaling. Collectively, these findings detail an unexpected species-specific role for STING as a receptor for an anticancer drug and uncover important insights that may explain the failure of DMXAA in clinical trials for human cancer.
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http://dx.doi.org/10.4049/jimmunol.1300097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647383PMC
May 2013

Structures of the HIN domain:DNA complexes reveal ligand binding and activation mechanisms of the AIM2 inflammasome and IFI16 receptor.

Immunity 2012 Apr 5;36(4):561-71. Epub 2012 Apr 5.

Structural Immunobiology Unit, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0430, USA.

Recognition of DNA by the innate immune system is central to antiviral and antibacterial defenses, as well as an important contributor to autoimmune diseases involving self DNA. AIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16) have been identified as DNA receptors that induce inflammasome formation and interferon production, respectively. Here we present the crystal structures of their HIN domains in complex with double-stranded (ds) DNA. Non-sequence-specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone. An intramolecular complex of the AIM2 Pyrin and HIN domains in an autoinhibited state is liberated by DNA binding, which may facilitate the assembly of inflammasomes along the DNA staircase. These findings provide mechanistic insights into dsDNA as the activation trigger and oligomerization platform for the assembly of large innate signaling complexes such as the inflammasomes.
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http://dx.doi.org/10.1016/j.immuni.2012.02.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334467PMC
April 2012

Innate immune recognition of an AT-rich stem-loop DNA motif in the Plasmodium falciparum genome.

Immunity 2011 Aug 4;35(2):194-207. Epub 2011 Aug 4.

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.

Although Toll-like receptor 9 (TLR9) has been implicated in cytokine and type I interferon (IFN) production during malaria in humans and mice, the high AT content of the Plasmodium falciparum genome prompted us to examine the possibility that malarial DNA triggered TLR9-independent pathways. Over 6000 ATTTTTAC ("AT-rich") motifs are present in the genome of P. falciparum, which we show here potently induce type I IFNs. Parasite DNA, parasitized erythrocytes and oligonucleotides containing the AT-rich motif induce type I IFNs via a pathway that did not involve the previously described sensors TLR9, DAI, RNA polymerase-III or IFI16/p204. Rather, AT-rich DNA sensing involved an unknown receptor that coupled to the STING, TBK1 and IRF3-IRF7 signaling pathway. Mice lacking IRF3, IRF7, the kinase TBK1 or the type I IFN receptor were resistant to otherwise lethal cerebral malaria. Collectively, these observations implicate AT-rich DNA sensing via STING, TBK1 and IRF3-IRF7 in P. falciparum malaria.
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http://dx.doi.org/10.1016/j.immuni.2011.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3162998PMC
August 2011

Mice lacking Tbk1 activity exhibit immune cell infiltrates in multiple tissues and increased susceptibility to LPS-induced lethality.

J Leukoc Biol 2010 Dec 22;88(6):1171-80. Epub 2010 Jul 22.

Department of Inflammation, Wyeth Research, Cambridge, MA 02140, USA.

TBK1 is critical for immunity against microbial pathogens that activate TLR4- and TLR3-dependent signaling pathways. To address the role of TBK1 in inflammation, mice were generated that harbor two copies of a mutant Tbk1 allele. This Tbk1(Δ) allele encodes a truncated Tbk1(Δ) protein that is catalytically inactive and expressed at very low levels. Upon LPS stimulation, macrophages from Tbk1(Δ/Δ) mice produce normal levels of proinflammatory cytokines (e.g., TNF-α), but IFN-β and RANTES expression and IRF3 DNA-binding activity are ablated. Three-month-old Tbk1(Δ/Δ) mice exhibit mononuclear and granulomatous cell infiltrates in multiple organs and inflammatory cell infiltrates in their skin, and they harbor a 2-fold greater amount of circulating monocytes than their Tbk1(+/+) and Tbk1(+/Δ) littermates. Skin from 2-week-old Tbk1(Δ/Δ) mice is characterized by reactive changes, including hyperkeratosis, hyperplasia, necrosis, inflammatory cell infiltrates, and edema. In response to LPS challenge, 3-month-old Tbk1(Δ/Δ) mice die more quickly and in greater numbers than their Tbk1(+/+) and Tbk1(+/Δ) counterparts. This lethality is accompanied by an overproduction of several proinflammatory cytokines in the serum of Tbk1(Δ/Δ) mice, including TNF-α, GM-CSF, IL-6, and KC. This overproduction of serum cytokines in Tbk1(Δ/Δ) mice following LPS challenge and their increased susceptibility to LPS-induced lethality may result from the reactions of their larger circulating monocyte compartment and their greater numbers of extravasated immune cells.
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http://dx.doi.org/10.1189/jlb.0210071DOI Listing
December 2010

The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses.

Nat Immunol 2010 May 28;11(5):395-402. Epub 2010 Mar 28.

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA .

Inflammasomes regulate the activity of caspase-1 and the maturation of interleukin 1beta (IL-1beta) and IL-18. AIM2 has been shown to bind DNA and engage the caspase-1-activating adaptor protein ASC to form a caspase-1-activating inflammasome. Using Aim2-deficient mice, we identify a central role for AIM2 in regulating caspase-1-dependent maturation of IL-1beta and IL-18, as well as pyroptosis, in response to synthetic double-stranded DNA. AIM2 was essential for inflammasome activation in response to Francisella tularensis, vaccinia virus and mouse cytomegalovirus and had a partial role in the sensing of Listeria monocytogenes. Moreover, production of IL-18 and natural killer cell-dependent production of interferon-gamma, events critical in the early control of virus replication, were dependent on AIM2 during mouse cytomegalovirus infection in vivo. Collectively, our observations demonstrate the importance of AIM2 in the sensing of both bacterial and viral pathogens and in triggering innate immunity.
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http://dx.doi.org/10.1038/ni.1864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2887480PMC
May 2010

Cell type-specific recognition of human metapneumoviruses (HMPVs) by retinoic acid-inducible gene I (RIG-I) and TLR7 and viral interference of RIG-I ligand recognition by HMPV-B1 phosphoprotein.

J Immunol 2010 Feb 30;184(3):1168-79. Epub 2009 Dec 30.

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Human metapneumoviruses (HMPVs) are recently identified Paramyxoviridae that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful defense against virus infection relies on early detection by germ line-encoded pattern recognition receptors and activation of cytokine and type I IFN genes. Recently, the RNA helicase retinoic acid-inducible gene I (RIG-I) has been shown to sense HMPV. In this study, we investigated the abilities of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I IFNs. Despite the abilities of both HMPV-A1 and HMPV-B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5' triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells displayed a unique ability to sense both HMPV-A1 and HMPV-B1 and in this case sensing was via TLR7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell type-specific manners.
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http://dx.doi.org/10.4049/jimmunol.0902750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2834787PMC
February 2010

Phagosomal retention of Francisella tularensis results in TIRAP/Mal-independent TLR2 signaling.

J Leukoc Biol 2010 Feb 4;87(2):275-81. Epub 2009 Nov 4.

Department of Microbiology and Immunology, University of Maryland, Baltimore, Baltimore, MD 21201, USA.

TLR2 plays a central role in the activation of innate immunity in response to Ft, the causative agent of tularemia. We reported previously that Ft LVS elicited strong, dose-dependent NF-kappaB reporter activity in TLR2-expressing human embryo kidney 293 T cells and that Ft LVS-induced murine macrophage proinflammatory cytokine gene and protein expression is TLR2-dependent. We demonstrated further that Ft can signal through TLR2 from within the phagosome and that phagosomal retention of Ft leads to greatly increased expression of a subset of proinflammatory genes. The two adaptor proteins associated with TLR2-mediated signaling are MyD88 and TIRAP. Although MyD88 is absolutely required for the Ft-induced macrophage cytokine response, the requirement for TIRAP can be overcome through retention of Ft within the phagosome. TIRAP-independent signaling was observed whether Ft was retained in the phagosome as a result of bacterial mutation (LVSDeltaiglC) or BFA-mediated inhibition of phagosome acidification. The requirement for TIRAP in TLR2 signaling could also be overcome by increasing the concentrations of synthetic bacterial TLR2 agonists. Taken together, these data suggest that prolonging or enhancing the interaction between TLR2 and its agonist overcomes the "bridging" function ascribed previously to TIRAP.
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http://dx.doi.org/10.1189/jlb.0909619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812562PMC
February 2010

NOD2, RIP2 and IRF5 play a critical role in the type I interferon response to Mycobacterium tuberculosis.

PLoS Pathog 2009 Jul 3;5(7):e1000500. Epub 2009 Jul 3.

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA, USA.

While the recognition of microbial infection often occurs at the cell surface via Toll-like receptors, the cytosol of the cell is also under surveillance for microbial products that breach the cell membrane. An important outcome of cytosolic recognition is the induction of IFNalpha and IFNbeta, which are critical mediators of immunity against both bacteria and viruses. Like many intracellular pathogens, a significant fraction of the transcriptional response to Mycobacterium tuberculosis infection depends on these type I interferons, but the recognition pathways responsible remain elusive. In this work, we demonstrate that intraphagosomal M. tuberculosis stimulates the cytosolic Nod2 pathway that responds to bacterial peptidoglycan, and this event requires membrane damage that is actively inflicted by the bacterium. Unexpectedly, this recognition triggers the expression of type I interferons in a Tbk1- and Irf5-dependent manner. This response is only partially impaired by the loss of Irf3 and therefore, differs fundamentally from those stimulated by bacterial DNA, which depend entirely on this transcription factor. This difference appears to result from the unusual peptidoglycan produced by mycobacteria, which we show is a uniquely potent agonist of the Nod2/Rip2/Irf5 pathway. Thus, the Nod2 system is specialized to recognize bacteria that actively perturb host membranes and is remarkably sensitive to mycobacteria, perhaps reflecting the strong evolutionary pressure exerted by these pathogens on the mammalian immune system.
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http://dx.doi.org/10.1371/journal.ppat.1000500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2698121PMC
July 2009

Selection of molecular structure and delivery of RNA oligonucleotides to activate TLR7 versus TLR8 and to induce high amounts of IL-12p70 in primary human monocytes.

J Immunol 2009 Jun;182(11):6824-33

Institute of Clinical Chemistry and Pharmacology, University Hospital, University of Bonn, Bonn, Germany.

Detection of non-self RNA by TLRs within endosomes and by retinoic acid-inducible gene I (RIG-I)-like helicases in the cytosol is central to mammalian antiviral immunity. In this study, we used pathway-specific agonists and targeted delivery to address RNA immunorecognition in primary human immune cells. Within PBMC, plasmacytoid dendritic cells (pDC) and monocytes were found to be responsible for IFN-alpha production upon immunorecognition of RNA. The mechanisms of RNA recognition in pDC and monocytes were distinct. In pDC, recognition of ssRNA and dsRNA oligonucleotides was TLR7-dependent, whereas a 5' triphosphate moiety (RIG-I ligand activity) had no major contribution to IFN-alpha production. In monocytes, the response to RNA oligonucleotides was mediated by either TLR8 or RIG-I. TLR8 was responsible for IL-12 induction upon endosomal delivery of ssRNA oligonucleotides and RIG-I was responsible for IFN-alpha production upon delivery of 5' triphosphate RNA into the cytosol. In conclusion, the dissection of these pathways by selecting the appropriate structure and delivery of RNA reveals pDC as major producer of IFN-alpha upon TLR-mediated stimulation and monocytes as major producer of IFN-alpha upon RIG-I-mediated stimulation. Furthermore, our results uncover the potential of monocytes to function as major producers of IL-12p70, a key Th1 cytokine classically ascribed to myeloid dendritic cells that cannot be induced by CpG oligonucleotides in the human system.
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http://dx.doi.org/10.4049/jimmunol.0803001DOI Listing
June 2009

Insights into interferon regulatory factor activation from the crystal structure of dimeric IRF5.

Nat Struct Mol Biol 2008 Nov 5;15(11):1213-20. Epub 2008 Oct 5.

Department of Biochemistry and Molecular Pharmacology, 364 Plantation Street, Worcester, Massachusetts 01605, USA.

Interferon regulatory factors (IRFs) are essential in the innate immune response and other physiological processes. Activation of these proteins in the cytoplasm is triggered by phosphorylation of serine and threonine residues in a C-terminal autoinhibitory region, which stimulates dimerization, transport into the nucleus, assembly with the coactivator CBP/p300 and initiation of transcription. The crystal structure of the transactivation domain of pseudophosphorylated human IRF5 strikingly reveals a dimer in which the bulk of intersubunit interactions involve a highly extended C-terminal region. The corresponding region has previously been shown to block CBP/p300 binding to unphosphorylated IRF3. Mutation of key interface residues supports the observed dimer as the physiologically activated state of IRF5 and IRF3. Thus, phosphorylation is likely to activate IRF5 and other family members by triggering conformational rearrangements that switch the C-terminal segment from an autoinihibitory to a dimerization role.
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http://dx.doi.org/10.1038/nsmb.1496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757928PMC
November 2008

Functional characterization of murine interferon regulatory factor 5 (IRF-5) and its role in the innate antiviral response.

J Biol Chem 2008 May 10;283(21):14295-308. Epub 2008 Mar 10.

Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, MD 21231, USA.

Although the role of human IRF-5 in antiviral and inflammatory responses in vitro has been well characterized, much remains to be elucidated about murine IRF-5. Murine IRF-5, unlike the heavily spliced human gene, is primarily expressed as a full-length transcript, with only a single splice variant that was detected in very low levels in the bone marrow of C57BL/6J mice. This bone marrow variant contains a 288-nucleotide deletion from exons 4-6 and exhibits impaired transcriptional activity. The murine IRF-5 can be activated by both TBK1 and MyD88 to form homodimers and bind to and activate transcription of type I interferon and inflammatory cytokine genes. The importance of IRF-5 in the antiviral and inflammatory response in vivo is highlighted by marked reductions in serum levels of type I interferon and tumor necrosis factor alpha (TNFalpha) in Newcastle disease virus-infected Irf5(-)(/)(-) mice. IRF-5 is critical for TLR3-, TLR4-, and TLR9-dependent induction of TNFalpha in CD11c(+) dendritic cells. In contrast, TLR9, but not TLR3/4-mediated induction of type I IFN transcription, is dependent on IRF-5 in these cells. In addition, IRF-5 regulates TNFalpha but not type I interferon gene transcription in Newcastle disease virus-infected peritoneal macrophages. Altogether, these data reveal the cell type-specific importance of IRF-5 in MyD88-mediated antiviral pathways and the widespread role of IRF-5 in the regulation of inflammatory cytokines.
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http://dx.doi.org/10.1074/jbc.M800501200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2386920PMC
May 2008

A modified quantitative EMSA and its application in the study of RNA--protein interactions.

J Biochem Biophys Methods 2004 Aug;60(2):85-96

Health Science Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai Research Center of Biotechnology, 500 Caobao Road, Shanghai 200233, PR China.

Electrophoretic mobility shift assay (EMSA) is used to detect the complex of protein and nonradioisotope-labeled probe qualitatively. In this paper, we describe a modified quantitative EMSA, which uses biotin-labeled RNA in the complex formation. The RNA-protein complex is separated by agarose gel electrophoresis and capillary transferred to a positively charged nylon membrane. It is then detected through a secondary detection system using 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT) as the colorimetric precipitating substrate. After scanning and quantification by an image analysis program, ImageQuant, it was observed that the optical density of the bands was proportional to the decadic logarithm value of standard RNA quantities in the tested range. By using the standard curve of the optical densities plotted against the logarithm values of RNA quantities in the linear range, we can calculate RNA quantities according to the optical density of the band and make a quantitative analysis of EMSA. This method was applied successfully in the study of the interactions between the AU-rich element (ARE) and HuR, which is one of the human members of the (embryonic lethal abnormal vision) ELAV family. The results reveal that the biotin-labeled AUFL transcripts in the RNA-protein complex can be detected quantitatively, while maintaining the biological features of its binding ability to the HuR protein. By this method, it is possible to conduct qualitative and quantitative analyses of the EMSA in the study of RNA-protein interactions.
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http://dx.doi.org/10.1016/j.jbbm.2004.03.008DOI Listing
August 2004