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Rhinovirus Modulation of Dendritic Cell Phenotype and Function
201
The Absence of Purinergic G Protein-Coupled Receptor 6 on Dendritic Cells Amplifies Antigen-Induced Pulmonary Inflammation Laura B. Fanning, MD, Denise Garofalo, Joshua A. Boyce, MD, FAAAAI; Harvard Medical School, Brigham and Womens Hospital, Boston, MA. RATIONALE: We previously reported that mice deficient in the type 6 purinergic (P2Y6) receptor develop more severe airway and lung tissue inflammation compared with wild-type (WT) mice after intranasal administration of dust mite extract. We hypothesized that expression of P2Y6 on dendritic cells (DCs) modulates antigen-induced pulmonary inflammation in mice. METHODS: Mice lacking P2Y6 expression on CD11c+ cells [p2ry6 (flox/flox);CD11c-cre mice] were sensitized intranasally with PBS alone or containing ovalbumin (OVA) and lipopolysaccharide (LPS) on days 0, 1, and 2, and challenged with OVA on days 14, 15, 18, and 19. Bronchoalveolar lavage (BAL) was performed and total and differential cell counts were determined. In addition, bone marrow-derived dendritic cells (BMDCs) were stimulated with LPS overnight and qPCR was performed to evaluate P2RY6 transcript. RESULTS: Mouse BMDCs expressed P2RY6 transcript that was suppressed by stimulation with LPS. After sensitization and challenge, p2ry6 (flox/flox);CD11c-cre mice had significantly increased BAL total cells and a trend toward increased BAL eosinophilia compared with WT mice. CONCLUSIONS: Our data suggest that the presence of P2Y6 on CD11c+ cells may inhibit antigen-induced lung inflammation, and that suppression of P2Y6 may be part of the mechanism for the effects of LPS in promoting allergen sensitization.
202
Rhinovirus Modulation of Dendritic Cell Phenotype and Function Aoife Cameron, Jaideep Dhariwal, MD, Sebastian L. Johnston, MD, PhD, Ross P. Walton, PhD; Imperial College London, London, United Kingdom. RATIONALE: Rhinoviruses (RV) are the major cause of asthma exacerbations. Evidence supports a synergistic relationship between viruses and allergen, with dendritic cells (DCs) mediating both viral responses and allergic inflammation. We hypothesise that during respiratory viral infection in atopic asthmatics, virus synergises with allergen exposure resulting in increased disease pathology, which is mediated through the modulation of DC populations. METHODS: Moderate atopic asthmatic patients and healthy controls were experimentally infected with RV-16. Bronchoalveolar lavage (BAL) was taken at baseline, day 3 and day 8 post infection and DC populations isolated using fluorescence activated cell sorting (FACS) with further flow cytometric analysis. RESULTS: Following RV infection in asthmatic patients, type I myeloid (m)DCs, which promote Th2 inflammation upon ex vivo allergen exposure, showed significantly increased recruitment to the airways at day 8 post infection (p50.008). This was also accompanied by an increase in plasmacytoid (p)DCs compared to healthy controls. Conversely, type II mDCs, which play a role in CD8+ T cell cross priming and the generation of subsequent anti-viral responses, were reduced in asthmatic airways at day 3 (p50.05) compared to healthy controls. CONCLUSIONS: We have observed an increase in type I mDCs and a decrease in anti-viral type II mDCs following RV infection in asthmatics, which may provide mechanistic understanding as to why asthmatics have more prolonged and severe respiratory viral infections. These findings will aid our understanding of the pathogenesis of asthma and the role of DCs in viral induced exacerbations.
203
Impact of Sublingual and Oral Immunotherapy for Peanut Allergy on Blood Dendritic Cells Mark Gorelik, MD1, Satya Narisety, MD2, Kristin Chichester, MS2, Anthony Guerrerio, MD, PhD3, Anja P. Bieneman, BS4, Corinne Keet, MD PhD3, Robert G. Hamilton, PhD, D.ABMLI, FAAAAI3, Robert A. Wood, MD5, John T. Schroeder, PhD4, Pamela Frischmeyer Guerrerio, MD, PhD6; 1Johns Hopkins Medical Institute, Baltimore, MD, 2Johns Hopkins University School of Medicine, 3Johns Hopkins University School of Medicine, Baltimore, MD, 4Johns Hopkins University, Baltimore, MD, 5Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA, 6National Institute of Allergy and Infectious Diseases, Bethesda, MD. RATIONALE: Dendritic cells (DCs) are key antigen presenting cells that direct tolerogenic and effector T cell functions. We evaluated how peanut sublingual (SLIT) and oral (OIT) immunotherapy altered DC responses. METHODS: Blood was obtained from subjects at baseline and at multiple timepoints during a placebo-controlled trial comparing peanut OIT to SLIT. Plasmacytoid (pDC) and myeloid (mDC) were purified and cultured with autologous CD4+T cells. Allergen-induced cytokine secretion was measured in co-cultures by multiplexing technology, and expression of MHC II and costimulatory molecules on DCs by flow cytometry. RESULTS: Peanut-induced secretion of TH2 cytokines decreased in pDCT and mDC-T co-cultures after 12 months of maintenance dosing with both OIT and SLIT (p<0.05). Levels of CD40, HLA-DR, and CD86 also decreased on DCs, while expression of CD80 increased (p<0.05). Effects were most striking in mDC-T cell co-cultures from subjects receiving OIT. These markers of immunologic suppression often reversed within weeks following withdrawal from IT, in some cases during ongoing maintenance therapy. Peanut-induced release of other effector cytokines, including IFN-g and IL-10, changed similarly to TH2 cytokines. Changes in DC-T cell responses did not correlate with clinical outcomes. Stimulation of co-cultures with dust mite led to cytokine changes that mimicked those seen with peanut. CONCLUSIONS: OIT and SLIT for peanut allergy induced marked suppression of dendritic cell activation and Th2 cytokine responses during the initial phases of IT in an antigen non-specific manner. While there was substantial inter-individual variation, in many subjects suppression appeared to be transient, even while on maintenance dosing.
SATURDAY
Abstracts AB63
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2
The Absence of Purinergic G Protein-Coupled Receptor 6 on Dendritic Cells Amplifies Antigen-Induced Pulmonary Inflammation Laura B. Fanning, MD, Denise Garofalo, Joshua A. Boyce, MD, FAAAAI; Harvard Medical School, Brigham and Womens Hospital, Boston, MA. RATIONALE: We previously reported that mice deficient in the type 6 purinergic (P2Y6) receptor develop more severe airway and lung tissue inflammation compared with wild-type (WT) mice after intranasal administration of dust mite extract. We hypothesized that expression of P2Y6 on dendritic cells (DCs) modulates antigen-induced pulmonary inflammation in mice. METHODS: Mice lacking P2Y6 expression on CD11c+ cells [p2ry6 (flox/flox);CD11c-cre mice] were sensitized intranasally with PBS alone or containing ovalbumin (OVA) and lipopolysaccharide (LPS) on days 0, 1, and 2, and challenged with OVA on days 14, 15, 18, and 19. Bronchoalveolar lavage (BAL) was performed and total and differential cell counts were determined. In addition, bone marrow-derived dendritic cells (BMDCs) were stimulated with LPS overnight and qPCR was performed to evaluate P2RY6 transcript. RESULTS: Mouse BMDCs expressed P2RY6 transcript that was suppressed by stimulation with LPS. After sensitization and challenge, p2ry6 (flox/flox);CD11c-cre mice had significantly increased BAL total cells and a trend toward increased BAL eosinophilia compared with WT mice. CONCLUSIONS: Our data suggest that the presence of P2Y6 on CD11c+ cells may inhibit antigen-induced lung inflammation, and that suppression of P2Y6 may be part of the mechanism for the effects of LPS in promoting allergen sensitization.
202
Rhinovirus Modulation of Dendritic Cell Phenotype and Function Aoife Cameron, Jaideep Dhariwal, MD, Sebastian L. Johnston, MD, PhD, Ross P. Walton, PhD; Imperial College London, London, United Kingdom. RATIONALE: Rhinoviruses (RV) are the major cause of asthma exacerbations. Evidence supports a synergistic relationship between viruses and allergen, with dendritic cells (DCs) mediating both viral responses and allergic inflammation. We hypothesise that during respiratory viral infection in atopic asthmatics, virus synergises with allergen exposure resulting in increased disease pathology, which is mediated through the modulation of DC populations. METHODS: Moderate atopic asthmatic patients and healthy controls were experimentally infected with RV-16. Bronchoalveolar lavage (BAL) was taken at baseline, day 3 and day 8 post infection and DC populations isolated using fluorescence activated cell sorting (FACS) with further flow cytometric analysis. RESULTS: Following RV infection in asthmatic patients, type I myeloid (m)DCs, which promote Th2 inflammation upon ex vivo allergen exposure, showed significantly increased recruitment to the airways at day 8 post infection (p50.008). This was also accompanied by an increase in plasmacytoid (p)DCs compared to healthy controls. Conversely, type II mDCs, which play a role in CD8+ T cell cross priming and the generation of subsequent anti-viral responses, were reduced in asthmatic airways at day 3 (p50.05) compared to healthy controls. CONCLUSIONS: We have observed an increase in type I mDCs and a decrease in anti-viral type II mDCs following RV infection in asthmatics, which may provide mechanistic understanding as to why asthmatics have more prolonged and severe respiratory viral infections. These findings will aid our understanding of the pathogenesis of asthma and the role of DCs in viral induced exacerbations.
203
Impact of Sublingual and Oral Immunotherapy for Peanut Allergy on Blood Dendritic Cells Mark Gorelik, MD1, Satya Narisety, MD2, Kristin Chichester, MS2, Anthony Guerrerio, MD, PhD3, Anja P. Bieneman, BS4, Corinne Keet, MD PhD3, Robert G. Hamilton, PhD, D.ABMLI, FAAAAI3, Robert A. Wood, MD5, John T. Schroeder, PhD4, Pamela Frischmeyer Guerrerio, MD, PhD6; 1Johns Hopkins Medical Institute, Baltimore, MD, 2Johns Hopkins University School of Medicine, 3Johns Hopkins University School of Medicine, Baltimore, MD, 4Johns Hopkins University, Baltimore, MD, 5Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA, 6National Institute of Allergy and Infectious Diseases, Bethesda, MD. RATIONALE: Dendritic cells (DCs) are key antigen presenting cells that direct tolerogenic and effector T cell functions. We evaluated how peanut sublingual (SLIT) and oral (OIT) immunotherapy altered DC responses. METHODS: Blood was obtained from subjects at baseline and at multiple timepoints during a placebo-controlled trial comparing peanut OIT to SLIT. Plasmacytoid (pDC) and myeloid (mDC) were purified and cultured with autologous CD4+T cells. Allergen-induced cytokine secretion was measured in co-cultures by multiplexing technology, and expression of MHC II and costimulatory molecules on DCs by flow cytometry. RESULTS: Peanut-induced secretion of TH2 cytokines decreased in pDCT and mDC-T co-cultures after 12 months of maintenance dosing with both OIT and SLIT (p<0.05). Levels of CD40, HLA-DR, and CD86 also decreased on DCs, while expression of CD80 increased (p<0.05). Effects were most striking in mDC-T cell co-cultures from subjects receiving OIT. These markers of immunologic suppression often reversed within weeks following withdrawal from IT, in some cases during ongoing maintenance therapy. Peanut-induced release of other effector cytokines, including IFN-g and IL-10, changed similarly to TH2 cytokines. Changes in DC-T cell responses did not correlate with clinical outcomes. Stimulation of co-cultures with dust mite led to cytokine changes that mimicked those seen with peanut. CONCLUSIONS: OIT and SLIT for peanut allergy induced marked suppression of dendritic cell activation and Th2 cytokine responses during the initial phases of IT in an antigen non-specific manner. While there was substantial inter-individual variation, in many subjects suppression appeared to be transient, even while on maintenance dosing.
SATURDAY
Abstracts AB63
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2