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The TLR-independent DNA recognition pathway in murine macrophages: Ligand features and molecular signature
Abstracts / Cytokine 48 (2009) 46–90 Keiko Ueno-Shuto 1,2, Tsuyoshi Shuto 2, Kosuke Kato 2, Hiromichi Sakai 2, Tomomi Ono 2, Mary Ann Suico 2, Yuji Uchida 1, Naofumi Tokutomi 1, Hirofumi Kai 2, 1 Laboratory of Pharmacology, Division of Life Science, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan, 2 Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Global COE ‘‘Cell Fate Regulation Research and Education Unit”, Kumamoto University, Kumamoto, Japan Single Immunoglobulin IL-1R-related molecule (SIGIRR), also known as Toll-IL1R 8 (TIR8), is one of the immunoglobulin-like membrane proteins that plays a critical role for the negative regulation of IL-1 and lipopolysaccharide (LPS) signaling. Although SIGIRR gene expression has been reported to be down-regulated following LPS exposure, precise mechanism responsible for this down-regulation remains obscure. In this study, we first confirmed that LPS down-regulates SIGIRR mRNA expression in neutrophilic-differentiated HL-60 cells and monocytic RAW264.7 cells, both of which express endogenous SIGIRR mRNA. Down-regulation was induced in both a time- and a dose-dependent manner. Moreover, LPS-induced SIGIRR mRNA down-regulation was suppressed by the exogenous introduction of dominant negative form of toll-like receptor-4 (TLR4) in RAW264.7 cells, suggesting the indispensable role of TLR4 in LPS-induced SIGIRR down-regulation. Screening with specific inhibitors of TLR4 downstream signaling molecules identified p38 MAP kinase as a critical molecule for LPS-induced SIGIRR down-regulation. Consistently, treatment of the cells with p38 activator anisomycin showed dose-dependent down-regulation of SIGIRR. Further, promoter analysis revealed that LPS suppresses human SIGIRR promoter activity and there is a Sp1 binding region, adjacent to the transcriptional start site that seems critical for the basal SIGIRR gene expression. In accordance with this finding, treatment with Sp1-specific inhibitor mithramycin A showed dose-dependent down-regulation of SIGIRR mRNA and promoter activity, demonstrating the importance of Sp1 for the maintenance of endogenous SIGIRR expression. Importantly, chromatin immunoprecipitation analysis revealed that LPS treatment abolishes Sp1 binding to its critical target sequence in SIGIRR promoter, implicating that LPS-induced SIGIRR down-regulation is mainly due to disruption of Sp1 binding. Taken together, our study shows that LPS downregulates SIGIRR gene expression through TLR4 and possibly by suppressing Sp1 via p38 MAP kinase. doi:10.1016/j.cyto.2009.07.172
PP1-050 Curcumin decreases toll-like receptor 2 gene expression and function in human monocytic THP-1 and neutrophilic HL-60 cells Tomomi Ono, Tsuyoshi Shuto, Yuko Ohira, Mary Ann Suico, Tomoaki Koga, Takashi Sato, Keizo Sato, Hirofumi Kai, Poster Presentation I Curcumin decreases toll-like receptor 2 gene expression and function in human monocytic THP-1 and neutrophilic HL-60 cells Tomomi Ono 1, Tsuyoshi Shuto 1, Yuko Ohira 1, Mary Ann Suico 1, Tomoaki Koga 1, Takashi Sato 1, Keizo Sato 2, Hirofumi Kai 1, 1 Department of Molecular Medicine, Global COE ‘‘Cell Fate Regulation Research and Education Unit”, Japan, 2 Department of Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan
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PP1-051 The TLR-independent DNA recognition pathway in murine macrophages: Ligand features and molecular signature Evren Karayel, Tilmann Bürckstümmer, Martin Bilban, Gerhard Dürnberger, Stefan Weitzer, Javier Martinez, Giulio Superti-Furga, Poster Presentation I The TLR-independent DNA recognition pathway in murine macrophages: Ligand features and molecular signature Evren Karayel 1, Tilmann Bürckstümmer 1, Martin Bilban 2, Gerhard Dürnberger 1, Stefan Weitzer 3, Javier Martinez 3, Giulio Superti-Furga 1, 1 Research Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM), Lazarettgasse, Vienna, Austria, 2 Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria, 3 Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr Gasse, Vienna, Austria Recognition of foreign DNA by cytosolic innate immune receptors triggers the production of IFN-b. However, it is unclear whether different types of DNA ligands are recognized by similar receptors and whether the resulting response is distinct from the endosomal TLR response. To address these questions, we compared the two most commonly used types of DNA ligands (IFN-stimulatory DNA (ISD) and poly(dAdT)) and assessed the minimal structural requirements for stimulatory capacity in RAW264.7 cells. Gene expression signatures and competition experiments suggest that ISD and poly(dAdT) are qualitatively indistinguishable and differ from the CpG-containing oligonucleotides triggering the TLR9 pathway. Structure–activity relationship analyses revealed that a minimal length of two helical turns is sufficient for ISD-mediated IFN-b induction, while phosphorylation at the 5 0-end is dispensable. Altogether, our data suggest that, in murine macrophages, only one major cytosolic DNA recognition pathway is operational. doi:10.1016/j.cyto.2009.07.174
PP1-052 TRIL is a novel modulator of TLR4 with enriched expression in the brain Thaddeus Carlson, Susan Carpenter, Ying Gao, Karen Percival, Scott H. Schelling, Amha G. Hewet, Wen Kuang, Padma Reddy, Debra G. Goodwin, Cheryl Nickerson-Nutter, Mayra Senices, Deborah Young, Leila Bradley, Lih-Ling Lin, James D. Clark, Ai, Poster Presentation I TRIL is a novel modulator of TLR4 with enriched expression in the brain Thaddeus Carlson 1, Susan Carpenter 5, Ying Gao 2, Karen Percival 3, Scott H. Schelling 3, Amha G. Hewet 1, Wen Kuang 4, Padma Reddy 4, Debra G. Goodwin 1, Cheryl Nickerson-Nutter 1, Mayra Senices 1, Deborah Young 1, Leila Bradley 1, Lih-Ling Lin 1, James D. Clark 1, Aisling Dunne 6, Luke A.J. O’Neill 5, Mary Collins 1, Katherine J. Seidl 1, 1 Wyeth Inflammation, Cambridge, MA, USA, 2 Neuroscience, Princeton, NJ, USA, 3 Exploratory Drug Safety, Andover, MA, USA, 4 Biological Technologies, Cambridge, MA, USA, 5 School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland, 6 Opsona Therapeutics, Institute for Molecular Medicine, St. James’s Hospital, Dublin 8, Ireland
Toll-like receptor-2 (TLR2) is a pattern recognition receptor that senses many types of bacterial components such as peptidoglycan (PGN) and activates signaling pathways that lead to induction of inflammatory cytokines. Although TLR2-dependent immune response is required for host defense if its expression and function are properly controlled, hyperresponsiveness to pathogens caused by increased expression of TLR2 trigger exaggeration of inflammatory diseases such as cystic fibrosis (CF) and inflammatory bowel disease. Therefore, identification of the agents that control TLR2 expression and their signaling is urgently needed for the treatment of TLR2-dependent hyperinflammatory diseases. Here, we showed that curcumin, a well known anti-inflammatory agent derived from the curry spice turmeric, inhibits TLR2 expression in various TLR2-expressing cells such as human monocytic THP-1 cells and neutrophilic-differentiated HL-60 cells (dHL60) and primary peripheral blood polymorphonuclear neutrophils (PMN). Strong suppression of TLR2 gene expression was specifically observed at concentrations of curcumin in the range 40–100 lM. Furthermore, we showed that bacterial-derived PGN and gram-negative bacterium lipopolysaccharide (LPS), specific ligand for TLR2 and TLR4 respectively, strongly induce IL-8 mRNA in those cells and only PGN-induced TLR2-dependent IL-8 induction is markedly inhibited by curcumin treatment (40 lM). Finally, we determined the importance of curcumin-dependent radical production for the suppressive effect of curcumin on TLR2 expression since this suppression was attenuated by free radical scavenger N-acetyl cystein (NAC) treatment. Thus, our data demonstrate that curcumin inhibits TLR2 gene expression in various cells and suggest the possible application of curcumin treatment on regulating TLR2-dependent hyperinflammatory responses.
TLR4 is activated by bacterial LPS and endogenous ‘‘danger” signals, and is a critical component of the innate immune response. Toll-like receptor 4 Interactor with Leucine rich repeats (TRIL) is a novel protein containing 13 leucine rich repeats (LRRs), a type III fibronectin domain, and a putative transmembrane domain. It has structural homology to the TLR family and CNS-enriched LRR-containing proteins such as LINGO and NOGO. TRIL binds both TLR4 and LPS, and siRNA knockdown attenuates LPS signaling and cytokine production in cell lines, suggesting that TRIL may modulate LPS signaling through TLR4 (Carpenter et al., J Immunol, in press). We studied mice with genetic deletion of the TRIL gene and showed that TRIL KO mice have normal absolute numbers of T, B, NK, and myeloid cells in spleen, thymus and bone marrow, suggesting that critical cellular components of the immune system are unaltered. Furthermore, quantitative (q)RT-PCR detected no compensatory changes in TLR4 or CD14 in a variety of tissues and cells. After injection with LPS and D-galactosamine, TRIL KO mice had decreased serum IL-12p40 at ninety minutes, and increased GM-CSF, IL-1b and IFNc after three hours compared with WT littermates, supporting the notion that TRIL is a modulator of LPS signaling. Expression of TRIL mRNA was moderate in spleen, lymph node, thymus, and bone marrow, and was upregulated by LPS stimulation in thioglycollate-elicited peritoneal macrophages. However, TRIL is most highly expressed in the central nervous system in several cell types including astrocytes, neurons, and oligodendrocytes. These data suggest that although TRIL plays a role in LPS responses outside the CNS, it may be more important for inflammation within the CNS. Interestingly, TRIL KO mice showed decreased clinical severity in a model of chronic EAE. Future experiments focus on the potential role of TRIL in the astrocyte LPS response and in CNS inflammation.
doi:10.1016/j.cyto.2009.07.173
doi:10.1016/j.cyto.2009.07.175
PP1-050 Curcumin decreases toll-like receptor 2 gene expression and function in human monocytic THP-1 and neutrophilic HL-60 cells Tomomi Ono, Tsuyoshi Shuto, Yuko Ohira, Mary Ann Suico, Tomoaki Koga, Takashi Sato, Keizo Sato, Hirofumi Kai, Poster Presentation I Curcumin decreases toll-like receptor 2 gene expression and function in human monocytic THP-1 and neutrophilic HL-60 cells Tomomi Ono 1, Tsuyoshi Shuto 1, Yuko Ohira 1, Mary Ann Suico 1, Tomoaki Koga 1, Takashi Sato 1, Keizo Sato 2, Hirofumi Kai 1, 1 Department of Molecular Medicine, Global COE ‘‘Cell Fate Regulation Research and Education Unit”, Japan, 2 Department of Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan
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PP1-051 The TLR-independent DNA recognition pathway in murine macrophages: Ligand features and molecular signature Evren Karayel, Tilmann Bürckstümmer, Martin Bilban, Gerhard Dürnberger, Stefan Weitzer, Javier Martinez, Giulio Superti-Furga, Poster Presentation I The TLR-independent DNA recognition pathway in murine macrophages: Ligand features and molecular signature Evren Karayel 1, Tilmann Bürckstümmer 1, Martin Bilban 2, Gerhard Dürnberger 1, Stefan Weitzer 3, Javier Martinez 3, Giulio Superti-Furga 1, 1 Research Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM), Lazarettgasse, Vienna, Austria, 2 Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria, 3 Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr Gasse, Vienna, Austria Recognition of foreign DNA by cytosolic innate immune receptors triggers the production of IFN-b. However, it is unclear whether different types of DNA ligands are recognized by similar receptors and whether the resulting response is distinct from the endosomal TLR response. To address these questions, we compared the two most commonly used types of DNA ligands (IFN-stimulatory DNA (ISD) and poly(dAdT)) and assessed the minimal structural requirements for stimulatory capacity in RAW264.7 cells. Gene expression signatures and competition experiments suggest that ISD and poly(dAdT) are qualitatively indistinguishable and differ from the CpG-containing oligonucleotides triggering the TLR9 pathway. Structure–activity relationship analyses revealed that a minimal length of two helical turns is sufficient for ISD-mediated IFN-b induction, while phosphorylation at the 5 0-end is dispensable. Altogether, our data suggest that, in murine macrophages, only one major cytosolic DNA recognition pathway is operational. doi:10.1016/j.cyto.2009.07.174
PP1-052 TRIL is a novel modulator of TLR4 with enriched expression in the brain Thaddeus Carlson, Susan Carpenter, Ying Gao, Karen Percival, Scott H. Schelling, Amha G. Hewet, Wen Kuang, Padma Reddy, Debra G. Goodwin, Cheryl Nickerson-Nutter, Mayra Senices, Deborah Young, Leila Bradley, Lih-Ling Lin, James D. Clark, Ai, Poster Presentation I TRIL is a novel modulator of TLR4 with enriched expression in the brain Thaddeus Carlson 1, Susan Carpenter 5, Ying Gao 2, Karen Percival 3, Scott H. Schelling 3, Amha G. Hewet 1, Wen Kuang 4, Padma Reddy 4, Debra G. Goodwin 1, Cheryl Nickerson-Nutter 1, Mayra Senices 1, Deborah Young 1, Leila Bradley 1, Lih-Ling Lin 1, James D. Clark 1, Aisling Dunne 6, Luke A.J. O’Neill 5, Mary Collins 1, Katherine J. Seidl 1, 1 Wyeth Inflammation, Cambridge, MA, USA, 2 Neuroscience, Princeton, NJ, USA, 3 Exploratory Drug Safety, Andover, MA, USA, 4 Biological Technologies, Cambridge, MA, USA, 5 School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland, 6 Opsona Therapeutics, Institute for Molecular Medicine, St. James’s Hospital, Dublin 8, Ireland
Toll-like receptor-2 (TLR2) is a pattern recognition receptor that senses many types of bacterial components such as peptidoglycan (PGN) and activates signaling pathways that lead to induction of inflammatory cytokines. Although TLR2-dependent immune response is required for host defense if its expression and function are properly controlled, hyperresponsiveness to pathogens caused by increased expression of TLR2 trigger exaggeration of inflammatory diseases such as cystic fibrosis (CF) and inflammatory bowel disease. Therefore, identification of the agents that control TLR2 expression and their signaling is urgently needed for the treatment of TLR2-dependent hyperinflammatory diseases. Here, we showed that curcumin, a well known anti-inflammatory agent derived from the curry spice turmeric, inhibits TLR2 expression in various TLR2-expressing cells such as human monocytic THP-1 cells and neutrophilic-differentiated HL-60 cells (dHL60) and primary peripheral blood polymorphonuclear neutrophils (PMN). Strong suppression of TLR2 gene expression was specifically observed at concentrations of curcumin in the range 40–100 lM. Furthermore, we showed that bacterial-derived PGN and gram-negative bacterium lipopolysaccharide (LPS), specific ligand for TLR2 and TLR4 respectively, strongly induce IL-8 mRNA in those cells and only PGN-induced TLR2-dependent IL-8 induction is markedly inhibited by curcumin treatment (40 lM). Finally, we determined the importance of curcumin-dependent radical production for the suppressive effect of curcumin on TLR2 expression since this suppression was attenuated by free radical scavenger N-acetyl cystein (NAC) treatment. Thus, our data demonstrate that curcumin inhibits TLR2 gene expression in various cells and suggest the possible application of curcumin treatment on regulating TLR2-dependent hyperinflammatory responses.
TLR4 is activated by bacterial LPS and endogenous ‘‘danger” signals, and is a critical component of the innate immune response. Toll-like receptor 4 Interactor with Leucine rich repeats (TRIL) is a novel protein containing 13 leucine rich repeats (LRRs), a type III fibronectin domain, and a putative transmembrane domain. It has structural homology to the TLR family and CNS-enriched LRR-containing proteins such as LINGO and NOGO. TRIL binds both TLR4 and LPS, and siRNA knockdown attenuates LPS signaling and cytokine production in cell lines, suggesting that TRIL may modulate LPS signaling through TLR4 (Carpenter et al., J Immunol, in press). We studied mice with genetic deletion of the TRIL gene and showed that TRIL KO mice have normal absolute numbers of T, B, NK, and myeloid cells in spleen, thymus and bone marrow, suggesting that critical cellular components of the immune system are unaltered. Furthermore, quantitative (q)RT-PCR detected no compensatory changes in TLR4 or CD14 in a variety of tissues and cells. After injection with LPS and D-galactosamine, TRIL KO mice had decreased serum IL-12p40 at ninety minutes, and increased GM-CSF, IL-1b and IFNc after three hours compared with WT littermates, supporting the notion that TRIL is a modulator of LPS signaling. Expression of TRIL mRNA was moderate in spleen, lymph node, thymus, and bone marrow, and was upregulated by LPS stimulation in thioglycollate-elicited peritoneal macrophages. However, TRIL is most highly expressed in the central nervous system in several cell types including astrocytes, neurons, and oligodendrocytes. These data suggest that although TRIL plays a role in LPS responses outside the CNS, it may be more important for inflammation within the CNS. Interestingly, TRIL KO mice showed decreased clinical severity in a model of chronic EAE. Future experiments focus on the potential role of TRIL in the astrocyte LPS response and in CNS inflammation.
doi:10.1016/j.cyto.2009.07.173
doi:10.1016/j.cyto.2009.07.175