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Chronic LPS Exposure Reduces Accumulation of Pro-Atopic CD49d+ Neutrophils in the Airways Post-Paramyxoviral Respiratory Infection
AB148 Abstracts
480
SUNDAY
Human Bronchial Epithelial Cell-Derived Factors from Severe Asthmatics Can Stimulate Local Eosinophilopoetic Responses Steven G. Smith, PhD1, Manali Mukherjee, PhD2, Anam Irshad3, Sophie Plante4, Gail M. Gauvreau, PhD1, Parameswaran K. Nair, MD, PhD, FRCP, FRCPC2, Jamila Chakir, PhD5, Roma Sehmi, PhD, FAAAAI1; 1 McMaster University, Hamilton, ON, Canada, 2Firestone Institute for Respiratory Health, Hamilton, ON, Canada, 3McMaster University, 4Laval University, Quebec, 5Lava Univ., Saint Foy, Canada. RATIONALE: Bronchial epithelial cells activated by allergens, viruses or environmental pollutants produce cytokines including thymic stromal lymphopoietin (TSLP) and IL-33 that can initiate Th2 inflammatory processes. We hypothesize that bronchial epithelial cell-derived factors from asthmatics can stimulate the local maturation of eosinophil progenitors in the airways. METHODS: Bronchial epithelial cells supernatants (BECSN) were obtained from cultures of isolated bronchial epithelial cells from normal non-atopic controls (NC; n58), mild atopic asthmatics (AA; n59) and severe eosinophilic asthmatics (SEA; n55). Non-adherent mononuclear cells (NAMCs) and enriched CD34+ cells from the blood of mild asthmatics were co-cultured in methylcellulose to assess eosinophil/ basophil colony (Eo/B-CFU) formation after 14 days. Cultures were set up with BECSN alone or in the presence IL-5 (1 or 10 ng/ml). RESULTS: Compared to media control, BECSN from SEA and AA but _NC). In the presence not NC stimulated significant Eo/B- CFU (SEA>AA> of exogenous IL-5 (10 ng/ml), there was a significant increase in the number of Eo/B-CFUs grown in co-cultures of BECSN from SEA (p<0.05) but not AA (P50.051) or NC (560.5; 41613; 962; 763 Eo/ B-CFU respectively). Colony growth stimulated by BECSN from SEA was significantly attenuated by a neutralizing TSLPR antibody. Cultures of blood NAMNC with rhTSLP (1-100 pg/ml) alone stimulated significant Eo/Baso-CFU and this was significantly enhanced in combination with IL5 (1 ng/ml). CONCLUSIONS: Our results show that through the production of TSLP, bronchial epithelial cells can direct eosinophil differentiation and maturation from progenitor cells which in turn may perpetuate eosinophilic inflammation in patients with chronic eosinophilic asthma.
481
Vitamin D Deficiency in a Young, Atopic Pediatric Population Selene K. Bantz, MD1, Tiffany Dy, MD2, Ronit Herzog, MD, FAAAAI3; 1Yale University School of Medicine, New Haven, CT, 2Washington University School of Medicine, 3New York Presbyterian Hospital, Weill Cornell Medical Collegel, New York, NY. RATIONALE: Studies have demonstrated that low serum 25-hydroxyvitamin D levels are associated with increased severity of asthma and allergy in children, elevated IgE and eosinophil count, increased asthmarelated hospitalizations, and greater use of anti-inflammatory medications. The prevalence of vitamin D deficiency is not well-established, especially in young children. Varying amounts of vitamin D supplementation are commonly used, and there is no consensus of the exact dose of vitamin D supplementation required for normalization. METHODS: 47 atopic children with inadequate vitamin D levels were randomized to a control group receiving 400 IU/day vitamin D supplementation or treatment group receiving 1000 or 2000 IU/day, depending on levels (20-30 ng/mL or <20 ng/mL respectively). 25-hydroxyvitamin D, total IgE, immunoCAP for environmental allergens, and CBC with differential were measured. Vitamin D was supplemented for 3 months. RESULTS: At baseline, 15% of patients had sufficient vitamin D levels (>30ng/mL), 68% were insufficient (20-29ng/mL), and 17% deficient (<19ng/mL). After 3 months of supplementation, 41% achieved normal vitamin D levels, but 53% remained insufficient and 6% deficient. 38% of patients receiving 400IU normalized, 43% receiving 1000IU normalized, and 50% of those receiving 2000IU normalized. No significant trends were found in IgE, immunoCAP or eosinophil count. CONCLUSIONS: This study suggests the prevalence of vitamin D deficiency and insufficiency in young atopic patients is high, and current practices of vitamin D supplementation are insufficient in normalizing
J ALLERGY CLIN IMMUNOL FEBRUARY 2015
vitamin D levels in many children. Long-term studies are needed to establish recommended treatment dosing and to examine the effect of vitamin D normalization on allergic inflammation.
482
Cat Dander Extract Require TLR4/MD2 to Induce Both ROS Generation and Neutrophil Recruitment Koa Hosoki, MD, PhD, Istvan Boldogh, PhD, Sanjiv Sur, MD; University of Texas Medical Branch, Galveston, TX. RATIONALE: Airway neutrophilia is a hallmark of severe and suddenonset asthma. Cat dander is a major indoor antigen that induces an early wave of neutrophil recruitment in asthmatic subjects (PMID16815143). However, the innate immune mechanisms that contribute to this early neutrophil recruitment induced by cat dander are unknown. METHODS: WT mice, Tlr4 knockout (KO) mice and Cd14KO mice were intranasally challenged with cat dander extract. BALF levels of neutrophils were quantified 16 h later. Three HEK 293 cell lines (cells that do not express TLR4, CD14 or MD2 (TLR4Null), cells that overexpress TLR4, but not CD14 and MD2 (TLR4Hi), and cells that overexpress TLR4, CD14 and MD2 (TCMHi) were stimulated with cat dander extract, and intracellular ROS generation and IL-8 secretion were quantified. RESULTS: Intranasal cat dander extract challenge in WT mice increased recruitment of neutrophils. These effects were attenuated in Tlr4KO mice, but not in Cd14KO. Stimulation with cat dander extract induced CXCL secretion in TCMHi cells but not TLR4Null cells or TLR4Hi cells. TLR4Hi cells transfected with a plasmid to overexpress MD2 secreted IL-8 by stimulation with cat dander extract. Suppression of Md2 in lungs by intravenous siRNA administration prior to allergen challenge attenuated cat dander extract-induced neutrophil recruitment. Cat dander extract also increased intracellular ROS in TCMHi cells but not TLR4Null cells or TLR4Hi cells. Cat dander required TLR4 to increase GSSG levels in airways. CONCLUSIONS: Cat dander extracts utilizes TLR4/MD2 to induce IL-8 secretion, neutrophil recruitment and induce oxidative stress in the airways.
483
Chronic LPS Exposure Reduces Accumulation of Pro-Atopic CD49d+ Neutrophils in the Airways Post-Paramyxoviral Respiratory Infection Matthew T. Perkovich, Jennifer L. Santoro, BS, Erika Buell, Dorothy S. Cheung, MD, FAAAAI, Mitchell H. Grayson, MD, FAAAAI; Medical College of Wisconsin, Milwaukee, WI. RATIONALE: Viral upper respiratory infections increase the risk of developing atopic disease. Using Sendai virus (SeV, a paramyxovirus), we previously showed that post-viral atopic disease is depends upon accumulation of CD49d+ neutrophils in the airway. A single exposure to high dose endotoxin (LPS, 3 mg) prevents this accumulation, suggesting an interaction of the hygiene and viral hypotheses. The hygiene hypothesis posits that chronic LPS exposure decreases risk of atopy. We thus sought to determine the effect of chronic LPS exposure on SeV-mediated accumulation of CD49+neutrophils in the airways. METHODS: C57BL6 mice were given 0.1 mg LPS, 0.3 mg LPS, 3.0 mg LPS or PBS intranasally daily. On day 8, all mice were infected with 2x105 pfu SeV. On day 11, bronchoalveolar lavage (BAL) fluid was analyzed for CD49d+neutrophils by flow cytometry. RESULTS: The frequency of CD49d+ neutrophils in the airways of PBS treated and SeV infected mice was 31.0%64.2% (n54). There was a trend for chronic LPS exposure to reduce the frequency of these cells (20.7%66.9%, p50.119; 18.6%63.9%, p50.069; and 23.8%61.3%, _2). p50.077, for 0.1 mg, 0.3 mg, and 3.0 mg LPS, respectively, n> CONCLUSIONS: Chronic low dose exposure to LPS appears able to reduce SeV-mediated accumulation of CD49d+ neutrophils in the airways. These data suggest the hygiene hypothesis may mitigate virus-induced risk of atopic disease. Future studies will examine whether chronic exposure to LPS reduces development of atopic disease and whether longer duration LPS exposure has a similar effect.
480
SUNDAY
Human Bronchial Epithelial Cell-Derived Factors from Severe Asthmatics Can Stimulate Local Eosinophilopoetic Responses Steven G. Smith, PhD1, Manali Mukherjee, PhD2, Anam Irshad3, Sophie Plante4, Gail M. Gauvreau, PhD1, Parameswaran K. Nair, MD, PhD, FRCP, FRCPC2, Jamila Chakir, PhD5, Roma Sehmi, PhD, FAAAAI1; 1 McMaster University, Hamilton, ON, Canada, 2Firestone Institute for Respiratory Health, Hamilton, ON, Canada, 3McMaster University, 4Laval University, Quebec, 5Lava Univ., Saint Foy, Canada. RATIONALE: Bronchial epithelial cells activated by allergens, viruses or environmental pollutants produce cytokines including thymic stromal lymphopoietin (TSLP) and IL-33 that can initiate Th2 inflammatory processes. We hypothesize that bronchial epithelial cell-derived factors from asthmatics can stimulate the local maturation of eosinophil progenitors in the airways. METHODS: Bronchial epithelial cells supernatants (BECSN) were obtained from cultures of isolated bronchial epithelial cells from normal non-atopic controls (NC; n58), mild atopic asthmatics (AA; n59) and severe eosinophilic asthmatics (SEA; n55). Non-adherent mononuclear cells (NAMCs) and enriched CD34+ cells from the blood of mild asthmatics were co-cultured in methylcellulose to assess eosinophil/ basophil colony (Eo/B-CFU) formation after 14 days. Cultures were set up with BECSN alone or in the presence IL-5 (1 or 10 ng/ml). RESULTS: Compared to media control, BECSN from SEA and AA but _NC). In the presence not NC stimulated significant Eo/B- CFU (SEA>AA> of exogenous IL-5 (10 ng/ml), there was a significant increase in the number of Eo/B-CFUs grown in co-cultures of BECSN from SEA (p<0.05) but not AA (P50.051) or NC (560.5; 41613; 962; 763 Eo/ B-CFU respectively). Colony growth stimulated by BECSN from SEA was significantly attenuated by a neutralizing TSLPR antibody. Cultures of blood NAMNC with rhTSLP (1-100 pg/ml) alone stimulated significant Eo/Baso-CFU and this was significantly enhanced in combination with IL5 (1 ng/ml). CONCLUSIONS: Our results show that through the production of TSLP, bronchial epithelial cells can direct eosinophil differentiation and maturation from progenitor cells which in turn may perpetuate eosinophilic inflammation in patients with chronic eosinophilic asthma.
481
Vitamin D Deficiency in a Young, Atopic Pediatric Population Selene K. Bantz, MD1, Tiffany Dy, MD2, Ronit Herzog, MD, FAAAAI3; 1Yale University School of Medicine, New Haven, CT, 2Washington University School of Medicine, 3New York Presbyterian Hospital, Weill Cornell Medical Collegel, New York, NY. RATIONALE: Studies have demonstrated that low serum 25-hydroxyvitamin D levels are associated with increased severity of asthma and allergy in children, elevated IgE and eosinophil count, increased asthmarelated hospitalizations, and greater use of anti-inflammatory medications. The prevalence of vitamin D deficiency is not well-established, especially in young children. Varying amounts of vitamin D supplementation are commonly used, and there is no consensus of the exact dose of vitamin D supplementation required for normalization. METHODS: 47 atopic children with inadequate vitamin D levels were randomized to a control group receiving 400 IU/day vitamin D supplementation or treatment group receiving 1000 or 2000 IU/day, depending on levels (20-30 ng/mL or <20 ng/mL respectively). 25-hydroxyvitamin D, total IgE, immunoCAP for environmental allergens, and CBC with differential were measured. Vitamin D was supplemented for 3 months. RESULTS: At baseline, 15% of patients had sufficient vitamin D levels (>30ng/mL), 68% were insufficient (20-29ng/mL), and 17% deficient (<19ng/mL). After 3 months of supplementation, 41% achieved normal vitamin D levels, but 53% remained insufficient and 6% deficient. 38% of patients receiving 400IU normalized, 43% receiving 1000IU normalized, and 50% of those receiving 2000IU normalized. No significant trends were found in IgE, immunoCAP or eosinophil count. CONCLUSIONS: This study suggests the prevalence of vitamin D deficiency and insufficiency in young atopic patients is high, and current practices of vitamin D supplementation are insufficient in normalizing
J ALLERGY CLIN IMMUNOL FEBRUARY 2015
vitamin D levels in many children. Long-term studies are needed to establish recommended treatment dosing and to examine the effect of vitamin D normalization on allergic inflammation.
482
Cat Dander Extract Require TLR4/MD2 to Induce Both ROS Generation and Neutrophil Recruitment Koa Hosoki, MD, PhD, Istvan Boldogh, PhD, Sanjiv Sur, MD; University of Texas Medical Branch, Galveston, TX. RATIONALE: Airway neutrophilia is a hallmark of severe and suddenonset asthma. Cat dander is a major indoor antigen that induces an early wave of neutrophil recruitment in asthmatic subjects (PMID16815143). However, the innate immune mechanisms that contribute to this early neutrophil recruitment induced by cat dander are unknown. METHODS: WT mice, Tlr4 knockout (KO) mice and Cd14KO mice were intranasally challenged with cat dander extract. BALF levels of neutrophils were quantified 16 h later. Three HEK 293 cell lines (cells that do not express TLR4, CD14 or MD2 (TLR4Null), cells that overexpress TLR4, but not CD14 and MD2 (TLR4Hi), and cells that overexpress TLR4, CD14 and MD2 (TCMHi) were stimulated with cat dander extract, and intracellular ROS generation and IL-8 secretion were quantified. RESULTS: Intranasal cat dander extract challenge in WT mice increased recruitment of neutrophils. These effects were attenuated in Tlr4KO mice, but not in Cd14KO. Stimulation with cat dander extract induced CXCL secretion in TCMHi cells but not TLR4Null cells or TLR4Hi cells. TLR4Hi cells transfected with a plasmid to overexpress MD2 secreted IL-8 by stimulation with cat dander extract. Suppression of Md2 in lungs by intravenous siRNA administration prior to allergen challenge attenuated cat dander extract-induced neutrophil recruitment. Cat dander extract also increased intracellular ROS in TCMHi cells but not TLR4Null cells or TLR4Hi cells. Cat dander required TLR4 to increase GSSG levels in airways. CONCLUSIONS: Cat dander extracts utilizes TLR4/MD2 to induce IL-8 secretion, neutrophil recruitment and induce oxidative stress in the airways.
483
Chronic LPS Exposure Reduces Accumulation of Pro-Atopic CD49d+ Neutrophils in the Airways Post-Paramyxoviral Respiratory Infection Matthew T. Perkovich, Jennifer L. Santoro, BS, Erika Buell, Dorothy S. Cheung, MD, FAAAAI, Mitchell H. Grayson, MD, FAAAAI; Medical College of Wisconsin, Milwaukee, WI. RATIONALE: Viral upper respiratory infections increase the risk of developing atopic disease. Using Sendai virus (SeV, a paramyxovirus), we previously showed that post-viral atopic disease is depends upon accumulation of CD49d+ neutrophils in the airway. A single exposure to high dose endotoxin (LPS, 3 mg) prevents this accumulation, suggesting an interaction of the hygiene and viral hypotheses. The hygiene hypothesis posits that chronic LPS exposure decreases risk of atopy. We thus sought to determine the effect of chronic LPS exposure on SeV-mediated accumulation of CD49+neutrophils in the airways. METHODS: C57BL6 mice were given 0.1 mg LPS, 0.3 mg LPS, 3.0 mg LPS or PBS intranasally daily. On day 8, all mice were infected with 2x105 pfu SeV. On day 11, bronchoalveolar lavage (BAL) fluid was analyzed for CD49d+neutrophils by flow cytometry. RESULTS: The frequency of CD49d+ neutrophils in the airways of PBS treated and SeV infected mice was 31.0%64.2% (n54). There was a trend for chronic LPS exposure to reduce the frequency of these cells (20.7%66.9%, p50.119; 18.6%63.9%, p50.069; and 23.8%61.3%, _2). p50.077, for 0.1 mg, 0.3 mg, and 3.0 mg LPS, respectively, n> CONCLUSIONS: Chronic low dose exposure to LPS appears able to reduce SeV-mediated accumulation of CD49d+ neutrophils in the airways. These data suggest the hygiene hypothesis may mitigate virus-induced risk of atopic disease. Future studies will examine whether chronic exposure to LPS reduces development of atopic disease and whether longer duration LPS exposure has a similar effect.