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Research paper thumbnail of Activation and Regulation of the NLRP3 Inflammasome

Research paper thumbnail of IFI16 is an innate immune sensor for intracellular DNA

Nature Immunology, 2010

The detection of intracellular microbial DNA is critical to an appropriate innate immune response... more The detection of intracellular microbial DNA is critical to an appropriate innate immune response, however current knowledge on how such DNA is sensed is limited.

Research paper thumbnail of Recruitment and Endo-Lysosomal Activation of TLR9 in Dendritic Cells Infected with Trypanosoma cruzi

The Journal of Immunology, 2008

TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypa... more TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-B-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes. Abbreviations used in this paper: ODN, oligodeoxynucleotide; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; DOTAP, 1,2-dioleoyloxy-3-(trimethylammonium)propane; RFP, red fluorescent protein; WT, wild type; YFP, yellow fluorescent protein.

Research paper thumbnail of Identification of a Novel Human MD-2 Splice Variant That Negatively Regulates Lipopolysaccharide-Induced TLR4 Signaling

The Journal of Immunology, 2010

Myeloid differentiation factor 2 (MD-2) is a secreted gp that assembles with TLR4 to form a funct... more Myeloid differentiation factor 2 (MD-2) is a secreted gp that assembles with TLR4 to form a functional signaling receptor for bacterial LPS. In this study, we have identified a novel alternatively spliced isoform of human MD-2, termed MD-2 short (MD-2s), which lacks the region encoded by exon 2 of the MD-2 gene. Similar to MD-2, MD-2s is glycosylated and secreted. MD-2s also interacted with LPS and TLR4, but failed to mediate LPS-induced NF-kB activation and IL-8 production. We show that MD-2s is upregulated upon IFN-g, IL-6, and TLR4 stimulation and negatively regulates LPS-mediated TLR4 signaling. Furthermore, MD-2s competitively inhibited binding of MD-2 to TLR4. Our study pinpoints a mechanism that may be used to regulate TLR4 activation at the onset of signaling and identifies MD-2s as a potential therapeutic candidate to treat human diseases characterized by an overly exuberant or chronic immune response to LPS.

Research paper thumbnail of Natural Loss-of-function Mutation of Myeloid Differentiation Protein 88 Disrupts Its Ability to Form Myddosomes

Journal of Biological Chemistry, 2011

Myeloid differentiation protein 88 (MyD88) is a key signaling adapter in Toll-like receptor (TLR)... more Myeloid differentiation protein 88 (MyD88) is a key signaling adapter in Toll-like receptor (TLR) signaling. MyD88 is also one of the most polymorphic adapter proteins. We screened the reported nonsynonymous coding mutations in MyD88 to identify variants with altered function. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knock-out mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. Unlike wild-type MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. We observed that IRAK4 co-localizes with MyD88 in these aggregates, and thus these foci appear to be "Myddosomes." The MyD88 S34Y loss-of-function mutant demonstrates how proper cellular localization of MyD88 to the Myddosome is a feature required for MyD88 function.

Research paper thumbnail of TLR9 and the Recognition of Self and Non-Self Nucleic Acids

Annals of the New York Academy of Sciences, 2006

Toll-like receptors (TLRs) are involved in the innate recognition of foreign material and their a... more Toll-like receptors (TLRs) are involved in the innate recognition of foreign material and their activation leads to both innate and adaptive immune responses directed against invading pathogens. TLR9 is intracellularly expressed in the endo-lysosomal compartments of specialized immune cells. TLR9 is activated in response to DNA, in particular DNA containing unmethylated CpG motifs that are more prevalent in microbial than mammalian DNA. By detecting foreign DNA signatures TLR9 can sense the presence of certain viruses or bacteria inside the cell and mount an immune response. However, under certain conditions, TLR9 can also recognize self-DNA and this may promote immune pathologies with uncontrolled chronic inflammation. The autoimmune disease systemic lupus erythematosis (SLE) is characterized by the presence of immune stimulatory complexes containing autoantibodies against endogenous DNA and DNA- and RNA-associated proteins. Recent evidence indicates that the autoimmune response to these complexes involves TLR9 and the related single-stranded RNA-responsive TLRs 7 and 8, and therefore some breakdown in the normal ability of these TLRs to distinguish self and foreign DNA. Evidence suggests that immune cells use several mechanisms to discriminate between stimulatory and nonstimulatory DNA; however, it appears that TLR9 itself binds rather indiscriminately to a broad range of DNAs. We therefore propose that there is an additional recognition step by which TLR9 senses differences in the structures of bound DNA.

Research paper thumbnail of Erratum: Ligand-induced conformational changes allosterically activate Toll-like receptor 9 (Nature Immunology (2007) vol. 8 (772-779))

Research paper thumbnail of 9

Research paper thumbnail of Corrigendum: Ligand-induced conformational changes allosterically activate Toll-like receptor 9

Nature Immunology, 2007

In the version of this article initially published, the distance values reported in Tables 1 and ... more In the version of this article initially published, the distance values reported in Tables 1 and 2 are incorrect. The correct values are provided in the revised tables. Accordingly, line 6 on p 774 should read "7.3 nm"; line 11 on p 774 should read "a 12% decrease"; line 12 on p 777 ...

Research paper thumbnail of Ligand-induced conformational changes allosterically activate Toll-like receptor 9

Nature Immunology, 2007

Microbial and synthetic DNA rich in CpG dinucleotides stimulates Toll-like receptor 9 (TLR9), whe... more Microbial and synthetic DNA rich in CpG dinucleotides stimulates Toll-like receptor 9 (TLR9), whereas DNA lacking CpG either is inert or can inhibit TLR9 activation. The molecular mechanisms by which TLR9 becomes activated or is inhibited are not well understood. Here we show that TLR9 bound to stimulatory and inhibitory DNA; however, only stimulatory DNA led to substantial conformational changes in the TLR9 ectodomain. In the steady state, 'inactive' TLR9 homodimers formed in an inactivated conformation. Binding of DNA containing CpG, but not of DNA lacking CpG, to TLR9 dimers resulted in allosteric changes in the TLR9 cytoplasmic signaling domains. In endosomes, conformational changes induced by DNA containing CpG resulted in close apposition of the cytoplasmic signaling domains, a change that is probably required for the recruitment of signaling adaptor molecules. Our results indicate that the formation of TLR9 dimers is not sufficient for its activation but instead that TLR9 activation is regulated by conformational changes induced by DNA containing CpG.

Research paper thumbnail of Erratum: Local increase in thymic stomal lymphopoietin induces systemic alterations in B cell development

Nature Immunology, 2007

These links to content published by NPG are automatically generated. ... The Seeker, the non-prof... more These links to content published by NPG are automatically generated. ... The Seeker, the non-profit Citrus Research and Development Foundation, desires proposals for compo… ... The ultimate goal of this Challenge is to facilitate a sustainable increase in the treatment ...

Research paper thumbnail of Corrigendum: Toll-like receptor 9—dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE

Research paper thumbnail of Toll-like receptor 9–dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE

Nature Immunology, 2007

Absorbance (450 nm) Absorbance (450 nm) Absorbance (450 nm)

Research paper thumbnail of NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals

Research paper thumbnail of Recruitment and Endo-Lysosomal Activation of TLR9 in Dendritic Cells Infected with Trypanosoma cruzi

The Journal of Immunology, 2008

TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypa... more TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-B-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes. Abbreviations used in this paper: ODN, oligodeoxynucleotide; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; DOTAP, 1,2-dioleoyloxy-3-(trimethylammonium)propane; RFP, red fluorescent protein; WT, wild type; YFP, yellow fluorescent protein.

Research paper thumbnail of RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

Journal of Experimental Medicine, 2013

Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammal... more Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE-DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo.

Research paper thumbnail of Activation of TLR9 in Dendritic Cells Recruitment and Endo-Lysosomal

Research paper thumbnail of Activation and Regulation of the NLRP3 Inflammasome

Research paper thumbnail of IFI16 is an innate immune sensor for intracellular DNA

Nature Immunology, 2010

The detection of intracellular microbial DNA is critical to an appropriate innate immune response... more The detection of intracellular microbial DNA is critical to an appropriate innate immune response, however current knowledge on how such DNA is sensed is limited.

Research paper thumbnail of Recruitment and Endo-Lysosomal Activation of TLR9 in Dendritic Cells Infected with Trypanosoma cruzi

The Journal of Immunology, 2008

TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypa... more TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-B-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes. Abbreviations used in this paper: ODN, oligodeoxynucleotide; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; DOTAP, 1,2-dioleoyloxy-3-(trimethylammonium)propane; RFP, red fluorescent protein; WT, wild type; YFP, yellow fluorescent protein.

Research paper thumbnail of Identification of a Novel Human MD-2 Splice Variant That Negatively Regulates Lipopolysaccharide-Induced TLR4 Signaling

The Journal of Immunology, 2010

Myeloid differentiation factor 2 (MD-2) is a secreted gp that assembles with TLR4 to form a funct... more Myeloid differentiation factor 2 (MD-2) is a secreted gp that assembles with TLR4 to form a functional signaling receptor for bacterial LPS. In this study, we have identified a novel alternatively spliced isoform of human MD-2, termed MD-2 short (MD-2s), which lacks the region encoded by exon 2 of the MD-2 gene. Similar to MD-2, MD-2s is glycosylated and secreted. MD-2s also interacted with LPS and TLR4, but failed to mediate LPS-induced NF-kB activation and IL-8 production. We show that MD-2s is upregulated upon IFN-g, IL-6, and TLR4 stimulation and negatively regulates LPS-mediated TLR4 signaling. Furthermore, MD-2s competitively inhibited binding of MD-2 to TLR4. Our study pinpoints a mechanism that may be used to regulate TLR4 activation at the onset of signaling and identifies MD-2s as a potential therapeutic candidate to treat human diseases characterized by an overly exuberant or chronic immune response to LPS.

Research paper thumbnail of Natural Loss-of-function Mutation of Myeloid Differentiation Protein 88 Disrupts Its Ability to Form Myddosomes

Journal of Biological Chemistry, 2011

Myeloid differentiation protein 88 (MyD88) is a key signaling adapter in Toll-like receptor (TLR)... more Myeloid differentiation protein 88 (MyD88) is a key signaling adapter in Toll-like receptor (TLR) signaling. MyD88 is also one of the most polymorphic adapter proteins. We screened the reported nonsynonymous coding mutations in MyD88 to identify variants with altered function. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knock-out mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. Unlike wild-type MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. We observed that IRAK4 co-localizes with MyD88 in these aggregates, and thus these foci appear to be "Myddosomes." The MyD88 S34Y loss-of-function mutant demonstrates how proper cellular localization of MyD88 to the Myddosome is a feature required for MyD88 function.

Research paper thumbnail of TLR9 and the Recognition of Self and Non-Self Nucleic Acids

Annals of the New York Academy of Sciences, 2006

Toll-like receptors (TLRs) are involved in the innate recognition of foreign material and their a... more Toll-like receptors (TLRs) are involved in the innate recognition of foreign material and their activation leads to both innate and adaptive immune responses directed against invading pathogens. TLR9 is intracellularly expressed in the endo-lysosomal compartments of specialized immune cells. TLR9 is activated in response to DNA, in particular DNA containing unmethylated CpG motifs that are more prevalent in microbial than mammalian DNA. By detecting foreign DNA signatures TLR9 can sense the presence of certain viruses or bacteria inside the cell and mount an immune response. However, under certain conditions, TLR9 can also recognize self-DNA and this may promote immune pathologies with uncontrolled chronic inflammation. The autoimmune disease systemic lupus erythematosis (SLE) is characterized by the presence of immune stimulatory complexes containing autoantibodies against endogenous DNA and DNA- and RNA-associated proteins. Recent evidence indicates that the autoimmune response to these complexes involves TLR9 and the related single-stranded RNA-responsive TLRs 7 and 8, and therefore some breakdown in the normal ability of these TLRs to distinguish self and foreign DNA. Evidence suggests that immune cells use several mechanisms to discriminate between stimulatory and nonstimulatory DNA; however, it appears that TLR9 itself binds rather indiscriminately to a broad range of DNAs. We therefore propose that there is an additional recognition step by which TLR9 senses differences in the structures of bound DNA.

Research paper thumbnail of Erratum: Ligand-induced conformational changes allosterically activate Toll-like receptor 9 (Nature Immunology (2007) vol. 8 (772-779))

Research paper thumbnail of 9

Research paper thumbnail of Corrigendum: Ligand-induced conformational changes allosterically activate Toll-like receptor 9

Nature Immunology, 2007

In the version of this article initially published, the distance values reported in Tables 1 and ... more In the version of this article initially published, the distance values reported in Tables 1 and 2 are incorrect. The correct values are provided in the revised tables. Accordingly, line 6 on p 774 should read "7.3 nm"; line 11 on p 774 should read "a 12% decrease"; line 12 on p 777 ...

Research paper thumbnail of Ligand-induced conformational changes allosterically activate Toll-like receptor 9

Nature Immunology, 2007

Microbial and synthetic DNA rich in CpG dinucleotides stimulates Toll-like receptor 9 (TLR9), whe... more Microbial and synthetic DNA rich in CpG dinucleotides stimulates Toll-like receptor 9 (TLR9), whereas DNA lacking CpG either is inert or can inhibit TLR9 activation. The molecular mechanisms by which TLR9 becomes activated or is inhibited are not well understood. Here we show that TLR9 bound to stimulatory and inhibitory DNA; however, only stimulatory DNA led to substantial conformational changes in the TLR9 ectodomain. In the steady state, 'inactive' TLR9 homodimers formed in an inactivated conformation. Binding of DNA containing CpG, but not of DNA lacking CpG, to TLR9 dimers resulted in allosteric changes in the TLR9 cytoplasmic signaling domains. In endosomes, conformational changes induced by DNA containing CpG resulted in close apposition of the cytoplasmic signaling domains, a change that is probably required for the recruitment of signaling adaptor molecules. Our results indicate that the formation of TLR9 dimers is not sufficient for its activation but instead that TLR9 activation is regulated by conformational changes induced by DNA containing CpG.

Research paper thumbnail of Erratum: Local increase in thymic stomal lymphopoietin induces systemic alterations in B cell development

Nature Immunology, 2007

These links to content published by NPG are automatically generated. ... The Seeker, the non-prof... more These links to content published by NPG are automatically generated. ... The Seeker, the non-profit Citrus Research and Development Foundation, desires proposals for compo… ... The ultimate goal of this Challenge is to facilitate a sustainable increase in the treatment ...

Research paper thumbnail of Corrigendum: Toll-like receptor 9—dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE

Research paper thumbnail of Toll-like receptor 9–dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE

Nature Immunology, 2007

Absorbance (450 nm) Absorbance (450 nm) Absorbance (450 nm)

Research paper thumbnail of NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals

Research paper thumbnail of Recruitment and Endo-Lysosomal Activation of TLR9 in Dendritic Cells Infected with Trypanosoma cruzi

The Journal of Immunology, 2008

TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypa... more TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-B-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes. Abbreviations used in this paper: ODN, oligodeoxynucleotide; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; DOTAP, 1,2-dioleoyloxy-3-(trimethylammonium)propane; RFP, red fluorescent protein; WT, wild type; YFP, yellow fluorescent protein.

Research paper thumbnail of RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

Journal of Experimental Medicine, 2013

Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammal... more Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE-DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo.

Research paper thumbnail of Activation of TLR9 in Dendritic Cells Recruitment and Endo-Lysosomal