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Forkhead Box Protein 3 (FoxP3) Demethylation Is Associated with Tolerance Induction in Peanut-Induced Intestinal Allergy
AB176 Abstracts
579
Impaired Efferocytosis and Production of Mitochondrial Reactive Oxygen Species (mitoROS) By Monocytes in Human Chronic Granulomatous Disease (CGD) Is Reversed By Treatment with the Ppargamma Agonist Pioglitazone (Pio)
SUNDAY
Donna Bratton, MD1, Ruby Fernandez-Boyanapalli, PhD1, Emilia Liana Falcone2, Christa Zerbe2, Beatrix Marciano2, Steven M. Holland, MD3; 1 National Jewish Health, Denver, CO, 2NIAID, 3Laboratory of Clinical Infectious Diseases, NIAID/NIH, Bethesda, MD. RATIONALE: Efferocytosis, the clearance of dying cells, by murine CGD monocytes (Mo) and macrophages (M4) is impaired and associated with exaggerated inflammation. Pio treatment of CGD mice alters Mo/MF metabolism (increases mitoROS) and normalizes efferocytic capability, and enhances resolution of inflammation. We hypothesized that human CGD Mo (vs. normal Mo) would also have impaired efferocytosis and production of mitoROS reversible by Pio treatment in vitro and in vivo. METHODS: Human CGD and normal Mo were tested for efferocytic capability and stimulated mitoROS production with and without Pio treatment. RESULTS: CGD Mo of several genotypes were tested for efferocytic capability either fresh or following brief culture. In comparison to normal Mo, CGD Mo were significantly impaired in their efferocytosis of apoptotic Jurkat cells or carboxylated beads (apoptotic cell mimics). Overnight culture of CGD Mo with Pio reversed the impairment. MitoROS was assessed in stimulated Mo and found to be significantly diminished in CGD Mo, but was restored following culture with Pio. Mo were then isolated from two CGD patients before and during Pio treatment for inflammatory bowel disease (IBD). CGD/IBD Mo showed impaired efferocytosis compared to normal Mo at baseline. Pio treatment of the CGD/IBD patients resulted in enhanced efferocytic capability over time of Pio treatment. MitoROS production was similarly restored in Mo from CGD/IBD patients during treatment with Pio. CONCLUSIONS: Pio alters Mo/MF mitoROS/metabolism and efferocytic capability in human CGD as in murine CGD. PPARg agonism may be useful in reducing exaggerated inflammation in CGD and may restore some oxidant production in CGD phagocytes.
580
Forkhead Box Protein 3 (FoxP3) Demethylation Is Associated with Tolerance Induction in PeanutInduced Intestinal Allergy
Meiqin Wang, MD, PhD1, Ivana Yang, PhD2, Elizabeth J. Davidson, BA2, Anthony Joetham1, Jordan K. Abbott, MD1, Brian P. O’Connor, PhD1, Erwin W. Gelfand, MD, FAAAAI1; 1National Jewish Health, Denver, CO, 2Department of Medicine, University of Colorado School of Medicine, Denver, CO. RATIONALE: The role of T regulatory cells (Tregs) in the control of peanut sensitivity and tolerance induction is not well-defined. METHODS: High (0.5 mg/gram body weight) or low doses (0.05 mg/ gram body weight) of peanut extract (PE) were administered to pups every day for 2 weeks prior to peanut sensitization and challenge. Anti-CD25 was administered prior to peanut sensitization and challenge. Symptoms, intestinal inflammation, and Treg numbers in mesenteric lymph nodes (MLN) were monitored. FoxP3 methylation in genomic DNA from MLN CD4+T cells was detected by bisulfite pyrosequencing. RESULTS: Feeding high but not low doses of peanut prior to sensitization induced tolerance to peanut protein, prevented diarrhea and intestinal inflammation and increased the percentage of CD4+CD25+FoxP3+cells in MLN. Mice treated with anti-CD25 reversed established tolerance with reappearance of all manifestations of sensitivity. FoxP3 methylation within the intronic 1 region was significantly increased in sham-fed and PE sensitized and challenged mice compared to non-sensitized but challenged controls. In parallel to induction of tolerance, levels of FoxP3 methylation in 7 of the 9 CpG sites in the intronic 1 region were significantly reduced, to
J ALLERGY CLIN IMMUNOL FEBRUARY 2016
baseline levels, in mice fed high doses of peanut prior to PE sensitization and challenge. CONCLUSIONS: Tregs play an important role in the regulation of peanut sensitivity and maintenance of immune homeostasis. FoxP3 demethylation was associated with tolerance induction to peanut protein. This work was supported by AI-77609. MW was supported by Eugene F. and Easton M. Crawford Charitable Lead Unitrust.
581
Non Type-2 Severe Asthma Has Increased Bronchoalveolar Mast Cell Mediator Release and Health Care Utilization
Merritt L. Fajt, MD1, John Trudeau, BA2, Fernando Holguin, MD, MPH2, Lawrence B. Schwartz, MD, PhD, FAAAAI3, Sally E. Wenzel, MD, FAAAAI2; 1University of Pittsburgh Medical Center, Division of Pulmonary, Allergy and Critical Care Medicine, Pittsburgh, PA, 2The University of Pittsburgh Asthma Institute at UPMC and the University of Pittsburgh School of Medicine, Department of Pulmonary, Allergy and Critical Care Medicine, Pittsburgh, PA, 3Virginia Commonwealth University, Richmond, VA. RATIONALE: Severe asthma (SA) involves multiple cells. We reported increased epithelial and luminal mast cells (MCs) in severe vs. milder asthma. In response to anti-human IgE, bronchoalveolar lavage (BAL) MCs degranulate and generate eicosanoids. (Flint, 1985; Pearce, 1987) However, functional responses of SA MCs in relation to ‘‘Type-2’’ inflammation have not been explored. We hypothesized that Type-2high-SA BAL MCs would have increased response to stimulation vs. nonType-2-SA and greater healthcare utilization. METHODS: BAL cells from 57 SA (39 on systemic corticosteroids (sCS)) from the Severe Asthma Research Program were analyzed for tryptase mRNA by qPCR. In a subset (n528), BAL cells in media were stimulated with anti-human IgE (Dako) for 20 minutes. Supernatant PGD2 and tryptase were measured by enzyme immunoassay. Type-2 inflamma_300/milliliter) and tion was grouped by peripheral blood eosinophilia (> analyzed in relation to health care utilization. Data were analyzed parametrically. RESULTS: BAL cell tryptase mRNA was increased in Type-2-high vs non-Type-2-SA (p50.008). Following anti-IgE stimulation, Type-2-high released less tryptase/mRNA and generated less PGD2/mRNA than nonType-2-SA [Mean fold change/tryptase mRNA (tryptase:0.07 vs 0.95, p50.003); (PGD2:0.04 vs 0.54, p50.003). Compared to Type-2-high-SA, non-Type-2-SA were more likely to be on sCS and had more hospitalizations/urgent care visits in the past year (p50.003, p50.02, p50.03, respectively). They tended to have lower FEV1%predicted (p50.15). CONCLUSIONS: Although Type-2-high-SA had more baseline tryptase mRNA, BAL MC tryptase and PDG2 increases following stimulation were significantly greater in non-Type-2-SA, associated with greater health care utilization. Although sCS use may have decreased total MC numbers (nonType-2), the remaining MCs may be more active.
579
Impaired Efferocytosis and Production of Mitochondrial Reactive Oxygen Species (mitoROS) By Monocytes in Human Chronic Granulomatous Disease (CGD) Is Reversed By Treatment with the Ppargamma Agonist Pioglitazone (Pio)
SUNDAY
Donna Bratton, MD1, Ruby Fernandez-Boyanapalli, PhD1, Emilia Liana Falcone2, Christa Zerbe2, Beatrix Marciano2, Steven M. Holland, MD3; 1 National Jewish Health, Denver, CO, 2NIAID, 3Laboratory of Clinical Infectious Diseases, NIAID/NIH, Bethesda, MD. RATIONALE: Efferocytosis, the clearance of dying cells, by murine CGD monocytes (Mo) and macrophages (M4) is impaired and associated with exaggerated inflammation. Pio treatment of CGD mice alters Mo/MF metabolism (increases mitoROS) and normalizes efferocytic capability, and enhances resolution of inflammation. We hypothesized that human CGD Mo (vs. normal Mo) would also have impaired efferocytosis and production of mitoROS reversible by Pio treatment in vitro and in vivo. METHODS: Human CGD and normal Mo were tested for efferocytic capability and stimulated mitoROS production with and without Pio treatment. RESULTS: CGD Mo of several genotypes were tested for efferocytic capability either fresh or following brief culture. In comparison to normal Mo, CGD Mo were significantly impaired in their efferocytosis of apoptotic Jurkat cells or carboxylated beads (apoptotic cell mimics). Overnight culture of CGD Mo with Pio reversed the impairment. MitoROS was assessed in stimulated Mo and found to be significantly diminished in CGD Mo, but was restored following culture with Pio. Mo were then isolated from two CGD patients before and during Pio treatment for inflammatory bowel disease (IBD). CGD/IBD Mo showed impaired efferocytosis compared to normal Mo at baseline. Pio treatment of the CGD/IBD patients resulted in enhanced efferocytic capability over time of Pio treatment. MitoROS production was similarly restored in Mo from CGD/IBD patients during treatment with Pio. CONCLUSIONS: Pio alters Mo/MF mitoROS/metabolism and efferocytic capability in human CGD as in murine CGD. PPARg agonism may be useful in reducing exaggerated inflammation in CGD and may restore some oxidant production in CGD phagocytes.
580
Forkhead Box Protein 3 (FoxP3) Demethylation Is Associated with Tolerance Induction in PeanutInduced Intestinal Allergy
Meiqin Wang, MD, PhD1, Ivana Yang, PhD2, Elizabeth J. Davidson, BA2, Anthony Joetham1, Jordan K. Abbott, MD1, Brian P. O’Connor, PhD1, Erwin W. Gelfand, MD, FAAAAI1; 1National Jewish Health, Denver, CO, 2Department of Medicine, University of Colorado School of Medicine, Denver, CO. RATIONALE: The role of T regulatory cells (Tregs) in the control of peanut sensitivity and tolerance induction is not well-defined. METHODS: High (0.5 mg/gram body weight) or low doses (0.05 mg/ gram body weight) of peanut extract (PE) were administered to pups every day for 2 weeks prior to peanut sensitization and challenge. Anti-CD25 was administered prior to peanut sensitization and challenge. Symptoms, intestinal inflammation, and Treg numbers in mesenteric lymph nodes (MLN) were monitored. FoxP3 methylation in genomic DNA from MLN CD4+T cells was detected by bisulfite pyrosequencing. RESULTS: Feeding high but not low doses of peanut prior to sensitization induced tolerance to peanut protein, prevented diarrhea and intestinal inflammation and increased the percentage of CD4+CD25+FoxP3+cells in MLN. Mice treated with anti-CD25 reversed established tolerance with reappearance of all manifestations of sensitivity. FoxP3 methylation within the intronic 1 region was significantly increased in sham-fed and PE sensitized and challenged mice compared to non-sensitized but challenged controls. In parallel to induction of tolerance, levels of FoxP3 methylation in 7 of the 9 CpG sites in the intronic 1 region were significantly reduced, to
J ALLERGY CLIN IMMUNOL FEBRUARY 2016
baseline levels, in mice fed high doses of peanut prior to PE sensitization and challenge. CONCLUSIONS: Tregs play an important role in the regulation of peanut sensitivity and maintenance of immune homeostasis. FoxP3 demethylation was associated with tolerance induction to peanut protein. This work was supported by AI-77609. MW was supported by Eugene F. and Easton M. Crawford Charitable Lead Unitrust.
581
Non Type-2 Severe Asthma Has Increased Bronchoalveolar Mast Cell Mediator Release and Health Care Utilization
Merritt L. Fajt, MD1, John Trudeau, BA2, Fernando Holguin, MD, MPH2, Lawrence B. Schwartz, MD, PhD, FAAAAI3, Sally E. Wenzel, MD, FAAAAI2; 1University of Pittsburgh Medical Center, Division of Pulmonary, Allergy and Critical Care Medicine, Pittsburgh, PA, 2The University of Pittsburgh Asthma Institute at UPMC and the University of Pittsburgh School of Medicine, Department of Pulmonary, Allergy and Critical Care Medicine, Pittsburgh, PA, 3Virginia Commonwealth University, Richmond, VA. RATIONALE: Severe asthma (SA) involves multiple cells. We reported increased epithelial and luminal mast cells (MCs) in severe vs. milder asthma. In response to anti-human IgE, bronchoalveolar lavage (BAL) MCs degranulate and generate eicosanoids. (Flint, 1985; Pearce, 1987) However, functional responses of SA MCs in relation to ‘‘Type-2’’ inflammation have not been explored. We hypothesized that Type-2high-SA BAL MCs would have increased response to stimulation vs. nonType-2-SA and greater healthcare utilization. METHODS: BAL cells from 57 SA (39 on systemic corticosteroids (sCS)) from the Severe Asthma Research Program were analyzed for tryptase mRNA by qPCR. In a subset (n528), BAL cells in media were stimulated with anti-human IgE (Dako) for 20 minutes. Supernatant PGD2 and tryptase were measured by enzyme immunoassay. Type-2 inflamma_300/milliliter) and tion was grouped by peripheral blood eosinophilia (> analyzed in relation to health care utilization. Data were analyzed parametrically. RESULTS: BAL cell tryptase mRNA was increased in Type-2-high vs non-Type-2-SA (p50.008). Following anti-IgE stimulation, Type-2-high released less tryptase/mRNA and generated less PGD2/mRNA than nonType-2-SA [Mean fold change/tryptase mRNA (tryptase:0.07 vs 0.95, p50.003); (PGD2:0.04 vs 0.54, p50.003). Compared to Type-2-high-SA, non-Type-2-SA were more likely to be on sCS and had more hospitalizations/urgent care visits in the past year (p50.003, p50.02, p50.03, respectively). They tended to have lower FEV1%predicted (p50.15). CONCLUSIONS: Although Type-2-high-SA had more baseline tryptase mRNA, BAL MC tryptase and PDG2 increases following stimulation were significantly greater in non-Type-2-SA, associated with greater health care utilization. Although sCS use may have decreased total MC numbers (nonType-2), the remaining MCs may be more active.