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Differential Role Of Dendritic Cell Subsets In Shaping T-Cell Responses To Respiratory Viruses
AB284 Abstracts
980
Epigenetic Regulation Of Dendritic Cell Migration Dr. Timothy P. Moran, MD, PhD1, Dr. Hideki Nakano, PhD2, Dr. Hrisavgi Kondilis-Mangum, PhD2, Dr. Paul Wade, PhD2, Dr. Donald Cook, PhD2; 1Duke University Medical Center, Durham, NC, 2National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC. RATIONALE: The chemokine receptor CCR7 is critically important for the trafficking of lung dendritic cells (DCs) to lymph nodes (LN), where they orchestrate the development of allergic responses to inhaled allergens. Our objective was to investigate the molecular mechanisms that regulate lineage-specific CCR7 expression in DCs. METHODS: Ccr7 expression and function was evaluated in conventional DCs (cDCs) and monocyte-derived DCs (moDCs) generated from bone marrow (BM) of wild type or Ccr7(gfp) reporter mice. Chromatin immunoprecipitation assays were performed to assess epigenetic modifications of the Ccr7 locus in BM-derived DCs, primary lung DC subsets and progenitor populations. RESULTS: BM-derived cDCs expressed Ccr7 following activation with lipopolysaccharide and migrated efficiently to CCR7 ligands both in vitro and in vivo. By contrast, BM-derived moDCs did not express Ccr7 or respond to CCR7 ligands. The Ccr7 promoter in BM-derived moDCs was enriched for the transcriptionally repressive histone modification H3K27 tri-methylation, consistent with epigenetic regulation of gene expression. In the lung, similar repressive histone modifications at the Ccr7 locus were detected in CD11b(hi)Ly-6C(hi) moDCs, but not in migratory CD103+ cDCs. Furthermore, progenitors for lung cDCs and moDCs had disparate levels of H3K27 tri-methylation at the Ccr7 promoter, indicating that epigenetic regulation of Ccr7 occurs early during DC lineage commitment. CONCLUSIONS: Lineage-specific epigenetic mechanisms regulate CCR7-dependent DC migration, and thus help dictate the functional role of DC subsets. Manipulating epigenetic pathways could provide a novel strategy for the improvement of DC-based immunotherapies and the treatment of allergic diseases.
981
TUESDAY
LRBA Subcellular Localization: Evidence Of The LRBA's Role In Vesicle Trafficking From The Golgi To Cell Membrane and Endocytosis Mrs. Michelle A. Reiser, MS1, Dr. Jia-Wang Wang, PhD1, Mrs. Kunyu Li, BS1, Dr. Richard F. Lockey, MD2; 1Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida, Morsani College of Medicine, Tampa, FL, 2Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine and James A. Haley Veterans’ Affairs Hospital, Tampa, FL. RATIONALE: Mutation or deletion of lipopolysaccharide responsive beige-like anchor (LRBA) gene causes common variable immunodeficiency, autoimmunity and chronic inflammation. However, the underlying molecular mechanism by which this occurs is unknown. LRBA is similar in structure to the lysosomal trafficking regulator gene. Therefore, it is hypothesized that LRBA may function as a regulator of vesicle trafficking. Its subcellular localization may help to decipher its function. METHODS: Immunofluorescent staining of cultured H293 cells was conducted using a polyclonal antibody to LRBA and monoclonal antibodies against various organelle-specific proteins. Confocal images were obtained with an Olympus FV1000 MPE multiphoton laser scanning microscope and the accompanied software (FV10-ASW and JACop) were used for colocalization analysis. Time lapse video was obtained of live RAW264.7 cells stably transfected with LRBA-GFP using a Leica TCS SP2 laser scanning confocal microscope. RESULTS: LRBA is co-localized with three Golgi proteins (GM-130, P230, and GS-28), early endosome antigen 1 (an early endosome marker), the RIIb and RIIc subunits of protein kinase A (PKA), and the microtubule protein, tubulin. A video of a live cell stimulated with LPS shows LRBA-
J ALLERGY CLIN IMMUNOL FEBRUARY 2014
positive vesicles budding from the Golgi and moving into the cell membrane. CONCLUSIONS: LRBA is extensively associated with the endomembrane/vesicle trafficking system, which includes the Golgi complex, endosomes, lysosomes, plasma membrane and microtubules. LRBA also is associated with PKA. These results suggest that LRBA may play a role in membrane/vesicle trafficking and signal transduction required for the regulation and function of many immune molecules.
982
Differential Role Of Dendritic Cell Subsets In Shaping T-Cell Responses To Respiratory Viruses Dr. Meera Rani Gupta, MD, Dr. Deepthi Kolli, PhD, Dr. Antonella Casola, MD, Dr. Roberto P. Garofalo, MD; University of Texas Medical Branch, Galveston, TX. RATIONALE: Respiratory syncytial virus (RSV) and Human Metapneumovirus (hMPV) are significant respiratory pathogens. The cellular response after infection likely determines the short- and longterm sequela of disease. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping adaptive immune responses in the respiratory tract; however, few studies have examined how interactions of RSV or hMPV with individual mDC subsets affects skewing of anti-viral responses. METHODS: BDCA-1+ and BDCA-3+ mDCs were isolated from peripheral blood of healthy adults using FACS sorting, infected with RSV or hMPV (MOI55) for 24 hours, and co-cultured with allogenic CD4+ T-cells. After 7 days, the percentage of CD4+T-cells expressing IFN-g (Th1), IL-4 (Th2), IL-17A (Th17), CD25 and FOXP3 (Tregs) was examined using flow cytometry. Statistically significant differences in subset distribution between uninfected and infected co-cultures were determined using student’s paired t-test. RESULTS: RSV-infected BDCA-1+ mDCs induced expansion of Th1 cells (p50.03); hMPV-infected BDCA-1+ mDCs tended towards expansion of Th1 (p50.08) and Th17 populations (p50.09). RSV-infected BDCA-3+ mDCs induced expansion of Th2 cells (p50.01) and Tregs (p50.04), whereas hMPV-infected BDCA-3+ mDCs induced expansion of Th17 cells (p50.003). CONCLUSIONS: These results demonstrate a subset-specific role for BDCA-1+ and BDCA-3+mDCs in regulating T-cell responses during RSV infection, as well as a virus-specific effect of RSVon skewing anti-viral immune responses. Identifying the characteristics of adaptive immune responses during infection helps understand the implications for long-term disease. Defining the role mDC subsets play in shaping adaptive immune responses is critical for developing vaccine and treatment strategies against these respiratory pathogens.
980
Epigenetic Regulation Of Dendritic Cell Migration Dr. Timothy P. Moran, MD, PhD1, Dr. Hideki Nakano, PhD2, Dr. Hrisavgi Kondilis-Mangum, PhD2, Dr. Paul Wade, PhD2, Dr. Donald Cook, PhD2; 1Duke University Medical Center, Durham, NC, 2National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC. RATIONALE: The chemokine receptor CCR7 is critically important for the trafficking of lung dendritic cells (DCs) to lymph nodes (LN), where they orchestrate the development of allergic responses to inhaled allergens. Our objective was to investigate the molecular mechanisms that regulate lineage-specific CCR7 expression in DCs. METHODS: Ccr7 expression and function was evaluated in conventional DCs (cDCs) and monocyte-derived DCs (moDCs) generated from bone marrow (BM) of wild type or Ccr7(gfp) reporter mice. Chromatin immunoprecipitation assays were performed to assess epigenetic modifications of the Ccr7 locus in BM-derived DCs, primary lung DC subsets and progenitor populations. RESULTS: BM-derived cDCs expressed Ccr7 following activation with lipopolysaccharide and migrated efficiently to CCR7 ligands both in vitro and in vivo. By contrast, BM-derived moDCs did not express Ccr7 or respond to CCR7 ligands. The Ccr7 promoter in BM-derived moDCs was enriched for the transcriptionally repressive histone modification H3K27 tri-methylation, consistent with epigenetic regulation of gene expression. In the lung, similar repressive histone modifications at the Ccr7 locus were detected in CD11b(hi)Ly-6C(hi) moDCs, but not in migratory CD103+ cDCs. Furthermore, progenitors for lung cDCs and moDCs had disparate levels of H3K27 tri-methylation at the Ccr7 promoter, indicating that epigenetic regulation of Ccr7 occurs early during DC lineage commitment. CONCLUSIONS: Lineage-specific epigenetic mechanisms regulate CCR7-dependent DC migration, and thus help dictate the functional role of DC subsets. Manipulating epigenetic pathways could provide a novel strategy for the improvement of DC-based immunotherapies and the treatment of allergic diseases.
981
TUESDAY
LRBA Subcellular Localization: Evidence Of The LRBA's Role In Vesicle Trafficking From The Golgi To Cell Membrane and Endocytosis Mrs. Michelle A. Reiser, MS1, Dr. Jia-Wang Wang, PhD1, Mrs. Kunyu Li, BS1, Dr. Richard F. Lockey, MD2; 1Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida, Morsani College of Medicine, Tampa, FL, 2Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine and James A. Haley Veterans’ Affairs Hospital, Tampa, FL. RATIONALE: Mutation or deletion of lipopolysaccharide responsive beige-like anchor (LRBA) gene causes common variable immunodeficiency, autoimmunity and chronic inflammation. However, the underlying molecular mechanism by which this occurs is unknown. LRBA is similar in structure to the lysosomal trafficking regulator gene. Therefore, it is hypothesized that LRBA may function as a regulator of vesicle trafficking. Its subcellular localization may help to decipher its function. METHODS: Immunofluorescent staining of cultured H293 cells was conducted using a polyclonal antibody to LRBA and monoclonal antibodies against various organelle-specific proteins. Confocal images were obtained with an Olympus FV1000 MPE multiphoton laser scanning microscope and the accompanied software (FV10-ASW and JACop) were used for colocalization analysis. Time lapse video was obtained of live RAW264.7 cells stably transfected with LRBA-GFP using a Leica TCS SP2 laser scanning confocal microscope. RESULTS: LRBA is co-localized with three Golgi proteins (GM-130, P230, and GS-28), early endosome antigen 1 (an early endosome marker), the RIIb and RIIc subunits of protein kinase A (PKA), and the microtubule protein, tubulin. A video of a live cell stimulated with LPS shows LRBA-
J ALLERGY CLIN IMMUNOL FEBRUARY 2014
positive vesicles budding from the Golgi and moving into the cell membrane. CONCLUSIONS: LRBA is extensively associated with the endomembrane/vesicle trafficking system, which includes the Golgi complex, endosomes, lysosomes, plasma membrane and microtubules. LRBA also is associated with PKA. These results suggest that LRBA may play a role in membrane/vesicle trafficking and signal transduction required for the regulation and function of many immune molecules.
982
Differential Role Of Dendritic Cell Subsets In Shaping T-Cell Responses To Respiratory Viruses Dr. Meera Rani Gupta, MD, Dr. Deepthi Kolli, PhD, Dr. Antonella Casola, MD, Dr. Roberto P. Garofalo, MD; University of Texas Medical Branch, Galveston, TX. RATIONALE: Respiratory syncytial virus (RSV) and Human Metapneumovirus (hMPV) are significant respiratory pathogens. The cellular response after infection likely determines the short- and longterm sequela of disease. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping adaptive immune responses in the respiratory tract; however, few studies have examined how interactions of RSV or hMPV with individual mDC subsets affects skewing of anti-viral responses. METHODS: BDCA-1+ and BDCA-3+ mDCs were isolated from peripheral blood of healthy adults using FACS sorting, infected with RSV or hMPV (MOI55) for 24 hours, and co-cultured with allogenic CD4+ T-cells. After 7 days, the percentage of CD4+T-cells expressing IFN-g (Th1), IL-4 (Th2), IL-17A (Th17), CD25 and FOXP3 (Tregs) was examined using flow cytometry. Statistically significant differences in subset distribution between uninfected and infected co-cultures were determined using student’s paired t-test. RESULTS: RSV-infected BDCA-1+ mDCs induced expansion of Th1 cells (p50.03); hMPV-infected BDCA-1+ mDCs tended towards expansion of Th1 (p50.08) and Th17 populations (p50.09). RSV-infected BDCA-3+ mDCs induced expansion of Th2 cells (p50.01) and Tregs (p50.04), whereas hMPV-infected BDCA-3+ mDCs induced expansion of Th17 cells (p50.003). CONCLUSIONS: These results demonstrate a subset-specific role for BDCA-1+ and BDCA-3+mDCs in regulating T-cell responses during RSV infection, as well as a virus-specific effect of RSVon skewing anti-viral immune responses. Identifying the characteristics of adaptive immune responses during infection helps understand the implications for long-term disease. Defining the role mDC subsets play in shaping adaptive immune responses is critical for developing vaccine and treatment strategies against these respiratory pathogens.