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Global Expression Profile of Aspergillus fumigatus Conidia During Interaction With Murine Macrophage
S134 Abstracts
518
SUNDAY
Lysophosphatidic Acid Potently Activates Human Mast Cells Through LPA5/GPR92 A. J. J. Lundequist; Rheumatology, Immunology & Allergy, Brigham& Women’s Hospital, Harvard Medical School, Boston, MA. RATIONALE: Lysophosphatidic acid (LPA) is a lipid growth factor that can induce mast cell proliferation and granule development in vitro. LPA induces its effects by interacting with G-protein coupled receptors (GPCRs), and to date there are five GPCRs specific for LPA. The expression and function of the most recently identified member, GPR92, has not been characterized on mast cells. Hence, we wanted to investigate if mast cells express GPR92 and determine the contribution of GPR92 to the LPA response of these cells. METHODS: The human mast cell line LAD2 was analyzed for its expression of GPR92 on both mRNA and protein level, and its response to LPA was analyzed measuring calcium flux and release of MIP-1b. To define GPR92’s specific contribution to these parameters, we used sequence-specific short hairpin RNA to knock down the expression of the receptor. RESULTS: GPR92 is the most abundantly expressed LPA receptor on LAD2 at both the protein and mRNA levels. Flow cytometry clearly showed that the receptor is found at the cell membrane. LPA potently induced calcium flux by LAD2 cells as well as release of MIP-1b. LPAdependent MIP-1b generation was strongly attenuated when the expression of GPR92 was targeted. CONCLUSION: The newly identified LPA receptor GPR92 is highly expressed by LAD2 and appears to be a key receptor for the generation and release of MIP-1b in response to LPA.
519
Synergistic Induction of IL-23 Expression in Fibroblast-like Synoviocytes by IL-17 and TNF-Alpha: A Positive Feedback Loop in Rheumatoid Arthritis M. Goldberg, O. Nadiv, N. Luknar-Gabor, P. Koch, Y. Katz; Assaf Harofeh Medical Center, Zerifin, ISRAEL. RATIONALE: In order to determine the mechanisms by which a chronic inflammatory network can be maintained in the arthritic joint, we examined whether cytokines generated in the joint could provide feedback to augment IL-23 and/or IL-17 expression in fibroblast-like synoviocytes (FLS). METHODS: FLS derived from synovial tissue and dermal fibroblasts (DF) derived from skin biopsies, were collected from rheumatoid arthritis (RA) and non-rheumatoid degenerative arthritis (non-RA) patients. These two cell types were then stimulated with 10 nanogram/ml of TNFa, IL-1b and IL-17 alone or in a combination of two out of three. Specific expression of IL-23 versus IL-12 was then quantitated by ‘‘real-time’’ PCR after normalization to the GAPDH in each sample. RESULTS: A striking tissue specific expression of IL-23 versus IL-12 was noted after IL-1b stimulation. Whereas IL-1b induced IL-23 over 30 fold in FLS as compared to DF, IL-12 p35 mRNA expression was increased only three-fold in DF, and resulted in no FLS response. Furthermore, FLS exhibited a unique synergistic response to TNFa and IL-17 resulting in the induction of IL-23 expression 300 fold. This synergistic response was composed of an initial priming step by IL-17, thus making FLS hyperresponsive to TNF-a-mediated stimulation. CONCLUSIONS: TNFa and IL-17 synergistically induce IL-23 expression in FLS. Since IL-23 induces the differentiation of naive T cells into Th17 cells with the subsequent generation of both TNFa and IL-17, this positive feedback loop helps explain the cascading unrelenting chronic inflammatory process in rheumatoid arthritis. Funding: Assaf Harofeh Medical Center
J ALLERGY CLIN IMMUNOL FEBRUARY 2008
520
PGD2 Suppresses Human NK Cell Functions Via Signaling Through D Prostanoid (DP) Receptor Y. Chen1, P. Bice1, K. S. Campbell2; 1THOMAS JEFFERSON UNIVERSITY, Philadelphia, PA, 2Fox Chase Cancer Center, Philadelphia, PA. RATIONALE: Prostaglandin D2 (PGD2) plays a critical role in the development of allergic diseases by promoting type-2 and suppressing type1 immune responses. Since activities of Natural Killer (NK) cells are often impaired in these diseases, we examined the effect of PGD2 on the functions of human NK cells and explored the involved signaling pathway. METHODS: Primary human NK cells were incubated with PGD2 (up to 10-7M) and/or specific agonist or antagonist of PGD2 receptors for varying periods of time. The cytotoxicity, cytokine production, chemotaxis, and signaling of NK cells were then measured and compared with those of vehicle (DMSO) treated cells. RESULTS: 1) Human NK cells express the DP receptor for PGD2, as detected by mRNA analysis and western blot; 2) PGD2 inhibits cytotoxicity, chemotaxis and type-1 cytokine production of human NK cells via signaling through DP; 3) PGD2 signaling via DP receptor elevates intracellular cAMP levels and the inhibitory effects on NK cells are cAMP-dependent; 4) PGD2 binding to DP suppresses Ca21 mobilization triggered by the cross-linking of the activating receptor, CD16. CONCLUSIONS: PGD2 functions through DP receptor to suppress type1 and cytolytic functions of human NK cells, thus promoting a type-2 immune response. Through this mechanism, PGD2 may contribute to the impairment of NK cell functions in allergic diseases. Funding: NIH Grant AI055842
521
Global Expression Profile of Aspergillus fumigatus Conidia During Interaction With Murine Macrophage J. Shankar1,2, S. K. Upadhyay1,3, S. Gupta1, S. F. Basir3, P. U. Sarma4, T. Madan1,5; 1Institute of Genomics and Integrative Biology, Delhi, INDIA, 2 Stanford University, Stanford, CA, 3Jamia Millia Islamia, New Delhi, INDIA, 4Indian Agricultural Research Institute, New Delhi, INDIA, 5 National Institute for Research in Reproductive Health, Mumbai, INDIA. RATIONALE: Genome sequencing of various fungi including Aspergillus fumigatus facilitate the transcriptomic profile of tissue of interest, therefore, we deduced the transcriptomic profile of A. fumigatus against host innate immune cells such as macrophage that may shed light on its complex biological behavior. METHODS: A. fumigatus specific oligo-arrays (Version I) from The Institute for Genomic Research, MD, USA, were used to obtain the transcriptomic profiles of A. fumigatus conidia incubated alone and with murine macrophage (J774A.1) in tissue culture flask containing RPMI media for 2.5 hours at 37 8C. Dye swap hybridization from two independent biological replicates was performed. Direct intensity-based normalization by median was used to normalize the data. Differentially expressed genes, either up or down 2 folds, were functionally classified based on yeast catalogue developed by the Munich Information Centre for Protein Sequences (MIPS). RESULTS: Up-regulation of signal transduction pathway (No of genes, N 5 12), transcriptional regulation (29), carbohydrate metabolism (29), secondary metabolism (36), DNA/RNA metabolism (70), transportation (35), and hypothetical proteins (200) were observed. Proteins metabolism (52), carbon-6 metabolism (57), amino acid metabolism (18), urea metabolism (7), allergens (15), chromosomal packaging (4) and hypothetical proteins (69) were down-regulated. Real-time RT-PCR of selected genes such as Polyketide synthase type I (348 folds up) and Monooxygenase gene (3 folds up) Vs Actin as a housekeeping gene showed good correlation with microarray data. CONCLUSIONS: Present data provides platform to study the molecular network during host-pathogen interactions that include signaling pathway, transcriptional regulation of gene, secondary metabolites, and also on several hypothetical proteins with unknown function. Funding: Institute of Genomics and Integrative Biology (SMM0003)
518
SUNDAY
Lysophosphatidic Acid Potently Activates Human Mast Cells Through LPA5/GPR92 A. J. J. Lundequist; Rheumatology, Immunology & Allergy, Brigham& Women’s Hospital, Harvard Medical School, Boston, MA. RATIONALE: Lysophosphatidic acid (LPA) is a lipid growth factor that can induce mast cell proliferation and granule development in vitro. LPA induces its effects by interacting with G-protein coupled receptors (GPCRs), and to date there are five GPCRs specific for LPA. The expression and function of the most recently identified member, GPR92, has not been characterized on mast cells. Hence, we wanted to investigate if mast cells express GPR92 and determine the contribution of GPR92 to the LPA response of these cells. METHODS: The human mast cell line LAD2 was analyzed for its expression of GPR92 on both mRNA and protein level, and its response to LPA was analyzed measuring calcium flux and release of MIP-1b. To define GPR92’s specific contribution to these parameters, we used sequence-specific short hairpin RNA to knock down the expression of the receptor. RESULTS: GPR92 is the most abundantly expressed LPA receptor on LAD2 at both the protein and mRNA levels. Flow cytometry clearly showed that the receptor is found at the cell membrane. LPA potently induced calcium flux by LAD2 cells as well as release of MIP-1b. LPAdependent MIP-1b generation was strongly attenuated when the expression of GPR92 was targeted. CONCLUSION: The newly identified LPA receptor GPR92 is highly expressed by LAD2 and appears to be a key receptor for the generation and release of MIP-1b in response to LPA.
519
Synergistic Induction of IL-23 Expression in Fibroblast-like Synoviocytes by IL-17 and TNF-Alpha: A Positive Feedback Loop in Rheumatoid Arthritis M. Goldberg, O. Nadiv, N. Luknar-Gabor, P. Koch, Y. Katz; Assaf Harofeh Medical Center, Zerifin, ISRAEL. RATIONALE: In order to determine the mechanisms by which a chronic inflammatory network can be maintained in the arthritic joint, we examined whether cytokines generated in the joint could provide feedback to augment IL-23 and/or IL-17 expression in fibroblast-like synoviocytes (FLS). METHODS: FLS derived from synovial tissue and dermal fibroblasts (DF) derived from skin biopsies, were collected from rheumatoid arthritis (RA) and non-rheumatoid degenerative arthritis (non-RA) patients. These two cell types were then stimulated with 10 nanogram/ml of TNFa, IL-1b and IL-17 alone or in a combination of two out of three. Specific expression of IL-23 versus IL-12 was then quantitated by ‘‘real-time’’ PCR after normalization to the GAPDH in each sample. RESULTS: A striking tissue specific expression of IL-23 versus IL-12 was noted after IL-1b stimulation. Whereas IL-1b induced IL-23 over 30 fold in FLS as compared to DF, IL-12 p35 mRNA expression was increased only three-fold in DF, and resulted in no FLS response. Furthermore, FLS exhibited a unique synergistic response to TNFa and IL-17 resulting in the induction of IL-23 expression 300 fold. This synergistic response was composed of an initial priming step by IL-17, thus making FLS hyperresponsive to TNF-a-mediated stimulation. CONCLUSIONS: TNFa and IL-17 synergistically induce IL-23 expression in FLS. Since IL-23 induces the differentiation of naive T cells into Th17 cells with the subsequent generation of both TNFa and IL-17, this positive feedback loop helps explain the cascading unrelenting chronic inflammatory process in rheumatoid arthritis. Funding: Assaf Harofeh Medical Center
J ALLERGY CLIN IMMUNOL FEBRUARY 2008
520
PGD2 Suppresses Human NK Cell Functions Via Signaling Through D Prostanoid (DP) Receptor Y. Chen1, P. Bice1, K. S. Campbell2; 1THOMAS JEFFERSON UNIVERSITY, Philadelphia, PA, 2Fox Chase Cancer Center, Philadelphia, PA. RATIONALE: Prostaglandin D2 (PGD2) plays a critical role in the development of allergic diseases by promoting type-2 and suppressing type1 immune responses. Since activities of Natural Killer (NK) cells are often impaired in these diseases, we examined the effect of PGD2 on the functions of human NK cells and explored the involved signaling pathway. METHODS: Primary human NK cells were incubated with PGD2 (up to 10-7M) and/or specific agonist or antagonist of PGD2 receptors for varying periods of time. The cytotoxicity, cytokine production, chemotaxis, and signaling of NK cells were then measured and compared with those of vehicle (DMSO) treated cells. RESULTS: 1) Human NK cells express the DP receptor for PGD2, as detected by mRNA analysis and western blot; 2) PGD2 inhibits cytotoxicity, chemotaxis and type-1 cytokine production of human NK cells via signaling through DP; 3) PGD2 signaling via DP receptor elevates intracellular cAMP levels and the inhibitory effects on NK cells are cAMP-dependent; 4) PGD2 binding to DP suppresses Ca21 mobilization triggered by the cross-linking of the activating receptor, CD16. CONCLUSIONS: PGD2 functions through DP receptor to suppress type1 and cytolytic functions of human NK cells, thus promoting a type-2 immune response. Through this mechanism, PGD2 may contribute to the impairment of NK cell functions in allergic diseases. Funding: NIH Grant AI055842
521
Global Expression Profile of Aspergillus fumigatus Conidia During Interaction With Murine Macrophage J. Shankar1,2, S. K. Upadhyay1,3, S. Gupta1, S. F. Basir3, P. U. Sarma4, T. Madan1,5; 1Institute of Genomics and Integrative Biology, Delhi, INDIA, 2 Stanford University, Stanford, CA, 3Jamia Millia Islamia, New Delhi, INDIA, 4Indian Agricultural Research Institute, New Delhi, INDIA, 5 National Institute for Research in Reproductive Health, Mumbai, INDIA. RATIONALE: Genome sequencing of various fungi including Aspergillus fumigatus facilitate the transcriptomic profile of tissue of interest, therefore, we deduced the transcriptomic profile of A. fumigatus against host innate immune cells such as macrophage that may shed light on its complex biological behavior. METHODS: A. fumigatus specific oligo-arrays (Version I) from The Institute for Genomic Research, MD, USA, were used to obtain the transcriptomic profiles of A. fumigatus conidia incubated alone and with murine macrophage (J774A.1) in tissue culture flask containing RPMI media for 2.5 hours at 37 8C. Dye swap hybridization from two independent biological replicates was performed. Direct intensity-based normalization by median was used to normalize the data. Differentially expressed genes, either up or down 2 folds, were functionally classified based on yeast catalogue developed by the Munich Information Centre for Protein Sequences (MIPS). RESULTS: Up-regulation of signal transduction pathway (No of genes, N 5 12), transcriptional regulation (29), carbohydrate metabolism (29), secondary metabolism (36), DNA/RNA metabolism (70), transportation (35), and hypothetical proteins (200) were observed. Proteins metabolism (52), carbon-6 metabolism (57), amino acid metabolism (18), urea metabolism (7), allergens (15), chromosomal packaging (4) and hypothetical proteins (69) were down-regulated. Real-time RT-PCR of selected genes such as Polyketide synthase type I (348 folds up) and Monooxygenase gene (3 folds up) Vs Actin as a housekeeping gene showed good correlation with microarray data. CONCLUSIONS: Present data provides platform to study the molecular network during host-pathogen interactions that include signaling pathway, transcriptional regulation of gene, secondary metabolites, and also on several hypothetical proteins with unknown function. Funding: Institute of Genomics and Integrative Biology (SMM0003)