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Soybean Isoflavones Regulate Dendritic Cell-mediated Effector Functions
AB92 Abstracts
343
SUNDAY
Early B Lineage Cells Display Class Switch Preference To Ige D. R. Wesemann1, C. Boboila2, M. Gallagher2, F. W. Alt2; 1 Brigham and Women’s Hospital, Boston, MA, 2Children’s Hospital Boston, Boston, MA. RATIONALE: The mechanism behind the elevated IgE in some types of primary immune deficiency diseases is not clear. This study seeks to uncover the role of B cell properties that may affect class switch preference to IgE. METHODS: Primary splenic and bone marrow mouse cells were derived and cultured from S723C Rag1 mutant and wild type mice. Early B lineage cells were prepared from culturing mouse embryonic liver cells on IL7secreting fibroblasts. Class switching assays were done using FACS and ELISA analysis of single hybridoma clones. RNA was quantified using a probe-based qPCR and semi-quantitative PCR approach. RESULTS: Splenic B lineage cells from mice with the Omenn syndromelike S723C hypomorphic Rag1 mutation are skewed toward an early developmental phenotype and display class switch preference to IgE over IgG1 when stimulated with anti-CD40 and IL4. Early B lineage cells isolated from wild type mice display a similar isotype switch preference to IgE compared to more mature B cells. Anti-CD40/IL4 treatment leads to increased levels of germline e switch region transcripts in early B lineage cells compared to adult splenic B cells, despite an attenuated Stat6 phosphorylation in the former. The early B lineage cells also show evidence of increased frequency of direct m to e switch region recombination at the DNA level. CONCLUSIONS: B lineage cell maturity influences isotype switch preference, with earlier B lineage cells favoring IgE and B lineage cell immaturity found in some types of primary immune deficiencies may be a contributory mechanism leading to increased IgE levels in these disorders.
344
Prostaglandin I2 Receptor (IP) Signaling Inhibits TLR4 Cell Surface Expression and Reduces LPS-induced Lung Inflammation S. Toki1, W. Zhou1, K. Goleniewska1, G. A. FitzGerald2, R. S. Peebles, Jr, 1; 1 Vanderbilt University, Nashville, TN, 2University of Pennsylvania, Philadelphia, PA. RATIONALE: Our laboratory has previously shown that prostaglandin I2 (PGI2) analogs down-regulated inflammatory cytokine production and maturation of dendritic cells (DCs) stimulated with LPS. However, the mechanisms of this down-regulation are not yet fully understood. We hypothesized that PGI2 negatively regulates Toll-like receptor (TLR) 4 cell surface expression on immune cells. In this study, we investigated TLR4 expression on bone marrow derived dendritic cells (BMDCs) treated with PGI2 analogs, and the role of IP signaling in LPS-induced lung inflammation. METHODS: BMDCs were obtained from wild-type (WT) C57BL/6 and IP KO mice. The cells were treated with PGI2 analogs (iloprost and cicaprost) for 24 hours, and expression of TLR4 was analyzed by flow cytometry. WT and IP KO mice were injected intranasally with LPS, and 6 hours later, cytokine production in the lung was measured by ELISA. RESULTS: Both iloprost and cicaprost decreased the number of TLR4 positive cells from WT mice in a dose dependent manner, while TLR4 expression on BMDCs from IP KO mice was not affected by PGI2 analogs. Following intranasal administration of LPS, IP KO mice had significantly greater concentrations of IL-6, KC, and TNF alpha in lung homogenates compared to WT mice (p<0.05). CONCLUSIONS: PGI2 analogs decreased BMDC TLR4 expression in vitro and PGI2 -IP signaling inhibited inflammatory cytokine production in vivo. These results suggest that PGI2 -IP signaling may promote antiinflammatory effect by modifying of TLR4 expression.
J ALLERGY CLIN IMMUNOL FEBRUARY 2011
345
IL-10 Differentially Regulates Components of the TSLP Receptor Complex on Blood Dendritic Cells R. Agrawal, P. W. Wright, J. A. Woodfolk; University of Virginia, Charlottesville, VA. RATIONALE: The cytokine, thymic stromal lymphopoietin (TSLP), mediates its Th2-promoting effects primarily through dendritic cells by a pathway reported to involve OX40 ligand (OX40L). Defining regulatory pathways that modulate expression of the TSLP receptor heterodimer complex (TSLP receptor (TSLPR) and IL-7Ralpha chains) and downstream molecules could yield new targets for inhibiting Th2-driven diseases. Our objective was to examine mechanisms regulating TSLP receptor complex on blood DCs. METHODS: Purified blood DCs isolated from patients with and without atopic dermatitis (AD) were cultured in the presence or absence of different immune cells, allergen, or cytokines (24 hours), and phenotyped by multicolor flow cytometry. RESULTS: Initial PBMC cultures revealed upregulation of TSLPR chain on myeloid DCs in response to diverse allergens (Fel d 1, Der p 1, Der p 2) which was most marked in AD. Surprisingly, TSLPR was spontaneously upregulated on purified DCs in the absence of allergen irrespective of disease status. Co-culture with monocytes, but not T cells, partially inhibited this process. Moreover, IL-10 similarly suppressed spontaneous upregulation of TSLPR, and downregulated OX40L, but had no effect on IL-7Ralpha. Allergen partially counteracted this effect. Conversely, IL-7Ralpha was downregulated by IL-7, but this cytokine did not modulate TSLPR. Neither component of the TSLP receptor complex was influenced by other cytokines that are relevant to T cell function (IL-2, IL-4 or IL-6), though IL-4 and IL-6 did suppress OX40L. CONCLUSION: Interleukin-10 is a key regulator of TSLPR chain on blood DCs. Cross-talk between DCs and inhibitory monocytes could provide a novel pathway for regulating TSLP-driven Th2 responses.
346
Soybean Isoflavones Regulate Dendritic Cell-mediated Effector Functions M. Masilamani, J. Wei, H. A. Sampson; Mount Sinai School of Medicine, New York, NY. RATIONALE: Soybeans are the most common source of isoflavones in the human diet. Isoflavones have been shown to possess anti-inflammatory and anti-oxidant properties. We hypothesized that active isoflavones in the gut milieu modulate immune responses to dietary antigens by regulating dendritic cell (DC) function. We have addressed this hypothesis in an in vitro DC activation model. METHODS: Human monocyte derived DCs (MDDC) were activated with lipopolysaccharide (LPS) or cholera toxin (CT) in the presence of isoflavones: genistein or daidzein. The surface expression levels of DC activation markers were analyzed by flow cytometry. LPS or CT activated DCs 1/- isoflavones were washed and cultured with freshly isolated allogenic na€ıve CD41 T cells for 5 days or with autologous NK cells for 2h. IFNg and IL-13 were determined in the DC-T cell supernatants. NK cell degranulation and DC cytotoxicity were measured by flow cytometry. RESULTS: Isoflavones significantly suppressed (>50%) the activationinduced expression of DC maturation markers and MHC class I but not MHC class II molecules in vitro. Isoflavone treatment inhibited the ability of LPS-DCs to induce IFN-g and CT-DCs to induce IL-13 by 32% and 77 % respectively. NK cell degranulation and the percentage of dead DCs was increased by 60% and 90% respectively in isoflavone-treated LPS-DC-NK co-culture experiments. No significant change in NK induced cell death was observed in CT-DCs treated with isoflavones. CONCLUSIONS: These results demonstrate that soybean isoflavones suppress DC-maturation and its subsequent DC-mediated effector cell functions. We propose that these mechanisms may play a role in preventing sensitization to food allergens.
343
SUNDAY
Early B Lineage Cells Display Class Switch Preference To Ige D. R. Wesemann1, C. Boboila2, M. Gallagher2, F. W. Alt2; 1 Brigham and Women’s Hospital, Boston, MA, 2Children’s Hospital Boston, Boston, MA. RATIONALE: The mechanism behind the elevated IgE in some types of primary immune deficiency diseases is not clear. This study seeks to uncover the role of B cell properties that may affect class switch preference to IgE. METHODS: Primary splenic and bone marrow mouse cells were derived and cultured from S723C Rag1 mutant and wild type mice. Early B lineage cells were prepared from culturing mouse embryonic liver cells on IL7secreting fibroblasts. Class switching assays were done using FACS and ELISA analysis of single hybridoma clones. RNA was quantified using a probe-based qPCR and semi-quantitative PCR approach. RESULTS: Splenic B lineage cells from mice with the Omenn syndromelike S723C hypomorphic Rag1 mutation are skewed toward an early developmental phenotype and display class switch preference to IgE over IgG1 when stimulated with anti-CD40 and IL4. Early B lineage cells isolated from wild type mice display a similar isotype switch preference to IgE compared to more mature B cells. Anti-CD40/IL4 treatment leads to increased levels of germline e switch region transcripts in early B lineage cells compared to adult splenic B cells, despite an attenuated Stat6 phosphorylation in the former. The early B lineage cells also show evidence of increased frequency of direct m to e switch region recombination at the DNA level. CONCLUSIONS: B lineage cell maturity influences isotype switch preference, with earlier B lineage cells favoring IgE and B lineage cell immaturity found in some types of primary immune deficiencies may be a contributory mechanism leading to increased IgE levels in these disorders.
344
Prostaglandin I2 Receptor (IP) Signaling Inhibits TLR4 Cell Surface Expression and Reduces LPS-induced Lung Inflammation S. Toki1, W. Zhou1, K. Goleniewska1, G. A. FitzGerald2, R. S. Peebles, Jr, 1; 1 Vanderbilt University, Nashville, TN, 2University of Pennsylvania, Philadelphia, PA. RATIONALE: Our laboratory has previously shown that prostaglandin I2 (PGI2) analogs down-regulated inflammatory cytokine production and maturation of dendritic cells (DCs) stimulated with LPS. However, the mechanisms of this down-regulation are not yet fully understood. We hypothesized that PGI2 negatively regulates Toll-like receptor (TLR) 4 cell surface expression on immune cells. In this study, we investigated TLR4 expression on bone marrow derived dendritic cells (BMDCs) treated with PGI2 analogs, and the role of IP signaling in LPS-induced lung inflammation. METHODS: BMDCs were obtained from wild-type (WT) C57BL/6 and IP KO mice. The cells were treated with PGI2 analogs (iloprost and cicaprost) for 24 hours, and expression of TLR4 was analyzed by flow cytometry. WT and IP KO mice were injected intranasally with LPS, and 6 hours later, cytokine production in the lung was measured by ELISA. RESULTS: Both iloprost and cicaprost decreased the number of TLR4 positive cells from WT mice in a dose dependent manner, while TLR4 expression on BMDCs from IP KO mice was not affected by PGI2 analogs. Following intranasal administration of LPS, IP KO mice had significantly greater concentrations of IL-6, KC, and TNF alpha in lung homogenates compared to WT mice (p<0.05). CONCLUSIONS: PGI2 analogs decreased BMDC TLR4 expression in vitro and PGI2 -IP signaling inhibited inflammatory cytokine production in vivo. These results suggest that PGI2 -IP signaling may promote antiinflammatory effect by modifying of TLR4 expression.
J ALLERGY CLIN IMMUNOL FEBRUARY 2011
345
IL-10 Differentially Regulates Components of the TSLP Receptor Complex on Blood Dendritic Cells R. Agrawal, P. W. Wright, J. A. Woodfolk; University of Virginia, Charlottesville, VA. RATIONALE: The cytokine, thymic stromal lymphopoietin (TSLP), mediates its Th2-promoting effects primarily through dendritic cells by a pathway reported to involve OX40 ligand (OX40L). Defining regulatory pathways that modulate expression of the TSLP receptor heterodimer complex (TSLP receptor (TSLPR) and IL-7Ralpha chains) and downstream molecules could yield new targets for inhibiting Th2-driven diseases. Our objective was to examine mechanisms regulating TSLP receptor complex on blood DCs. METHODS: Purified blood DCs isolated from patients with and without atopic dermatitis (AD) were cultured in the presence or absence of different immune cells, allergen, or cytokines (24 hours), and phenotyped by multicolor flow cytometry. RESULTS: Initial PBMC cultures revealed upregulation of TSLPR chain on myeloid DCs in response to diverse allergens (Fel d 1, Der p 1, Der p 2) which was most marked in AD. Surprisingly, TSLPR was spontaneously upregulated on purified DCs in the absence of allergen irrespective of disease status. Co-culture with monocytes, but not T cells, partially inhibited this process. Moreover, IL-10 similarly suppressed spontaneous upregulation of TSLPR, and downregulated OX40L, but had no effect on IL-7Ralpha. Allergen partially counteracted this effect. Conversely, IL-7Ralpha was downregulated by IL-7, but this cytokine did not modulate TSLPR. Neither component of the TSLP receptor complex was influenced by other cytokines that are relevant to T cell function (IL-2, IL-4 or IL-6), though IL-4 and IL-6 did suppress OX40L. CONCLUSION: Interleukin-10 is a key regulator of TSLPR chain on blood DCs. Cross-talk between DCs and inhibitory monocytes could provide a novel pathway for regulating TSLP-driven Th2 responses.
346
Soybean Isoflavones Regulate Dendritic Cell-mediated Effector Functions M. Masilamani, J. Wei, H. A. Sampson; Mount Sinai School of Medicine, New York, NY. RATIONALE: Soybeans are the most common source of isoflavones in the human diet. Isoflavones have been shown to possess anti-inflammatory and anti-oxidant properties. We hypothesized that active isoflavones in the gut milieu modulate immune responses to dietary antigens by regulating dendritic cell (DC) function. We have addressed this hypothesis in an in vitro DC activation model. METHODS: Human monocyte derived DCs (MDDC) were activated with lipopolysaccharide (LPS) or cholera toxin (CT) in the presence of isoflavones: genistein or daidzein. The surface expression levels of DC activation markers were analyzed by flow cytometry. LPS or CT activated DCs 1/- isoflavones were washed and cultured with freshly isolated allogenic na€ıve CD41 T cells for 5 days or with autologous NK cells for 2h. IFNg and IL-13 were determined in the DC-T cell supernatants. NK cell degranulation and DC cytotoxicity were measured by flow cytometry. RESULTS: Isoflavones significantly suppressed (>50%) the activationinduced expression of DC maturation markers and MHC class I but not MHC class II molecules in vitro. Isoflavone treatment inhibited the ability of LPS-DCs to induce IFN-g and CT-DCs to induce IL-13 by 32% and 77 % respectively. NK cell degranulation and the percentage of dead DCs was increased by 60% and 90% respectively in isoflavone-treated LPS-DC-NK co-culture experiments. No significant change in NK induced cell death was observed in CT-DCs treated with isoflavones. CONCLUSIONS: These results demonstrate that soybean isoflavones suppress DC-maturation and its subsequent DC-mediated effector cell functions. We propose that these mechanisms may play a role in preventing sensitization to food allergens.