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Convergence of Clinical and Cellular Phenotypes Among Patients with STAT3 and ERBB2IP Mutations
AB152 Abstracts
496
SUNDAY
Lipopolysaccharide-Responsive Beige-like Anchor Is Required for Both Activation and Deactivation of NFkB Jia-Wang Wang, PhD1, Michelle A. Reiser, MS1, Kunyu Li, BS1, Bangmei Wang1, Richard F. Lockey, MD1,2; 1Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida, Morsani College of Medicine, Tampa, FL, 2James Haley Veterans’ Hospital, Tampa, FL. RATIONALE: The molecular mechanisms why mutations of the lipopolysaccharide-responsive beige-like anchor (LRBA) paradoxically cause both autoimmunity and common variable immunodeficiency (CVID) are unknown. LRBA regulates vesicle trafficking and signal transduction required for the regulation and function of many immune molecules. It is hypothesized that LRBA deficiency attenuates both activation and deactivation of nuclear factor kappa beta (NFkB) resulting in immunodeficiency and autoimmunity. METHODS: LRBA was knocked down or repressed in Raji lymphoma cells by the short hairpin RNA (shRNA) or dominant negative mutant (DNM) techniques. The transcription activity and phosphorylated levels of NFkB was analyzed using the luciferase reporter assay, Western blot and flow cytometry. RESULTS: LRBA repression attenuates both NFkB activation and deactivation, inhibits NFkB nuclear translocation and increases tumor necrosis factor alpha (TNFa) and cell survival. The attenuated NFkB activation may result from inhibited NFkB nuclear translocation, while the attenuated NFkB deactivation may result in the increased TNFa and cell survival. CONCLUSIONS: These results suggest that the attenuation of NFkB activation may explain LRBA deficient immunodeficiency, while the prolonged NFkB activity and the increased proinflammatory cytokine TNFa and cell survival may explain LRBA deficient autoimmunity. This activation and deactivation (AnDA) model reveals a novel regulation mode for NFkB. It also provides a molecular mechanism for the paradoxical association of immunodeficiency and autoimmunity, which is a fundamental question in the immune system.
497
Convergence of Clinical and Cellular Phenotypes Among Patients with STAT3 and ERBB2IP Mutations Jonathan J. Lyons, MD1, Xiaomin Yu, PhD1, Kendal A. Karpe, BS1, Shirin M. Treadwell, MS1, Chi A. Ma, PhD1, Michael P. O’Connell, PhD1, Guangping Sun, MD1, Jason D. Hughes, PhD2, Huseyin Mehmet, PhD2, Joshua McElwee, PhD2, Steven M. Holland, MD3, Alexandra F. Freeman, MD4, Joshua D. Milner, MD1; 1Laboratory of Allergic Diseases, NIAID/NIH, Bethesda, MD, 2Merck Research Laboratories, Merck & Co. Inc., Boston, MA, 3Laboratory of Clinical Infectious Diseases, NIAID/NIH, Bethesda, MD, 4NIH/NIAID, Laboratory of Clinical Infectious Diseases, Bethesda, MD. RATIONALE: A number of congenital syndromes with connective tissue abnormalities are also associated with allergic disease, some of which are caused by altered TGF-beta signaling. Patients with loss of STAT3 function share multiple clinical features with these syndromes. We therefore sought to identify common cellular changes among patients with heterozygous loss-of-function ERBB2IP mutations, a negative regulator of TGF-beta signaling, and patients with dominant negative STAT3 mutations. METHODS: Patient-derived na€ıve CD4+ T-cells were differentiated under non-skewing conditions. RNA silencing of pathway genes in normal T-cells was performed. Advanced flow cytometric techniques, immunoblotting, and qRT-PCR were employed to characterize cellular responses. RESULTS: Common clinical features included atopy, elevated IgE, eosinophilia, and connective tissue abnormalities characterized by vascular abnormalities, retained primary dentition, joint hypermobility and high arched palate. FoxP3+ CD4+ T-cells were enriched among memory CD45RO+ T-cells among both cohorts of patients ex vivo. AntiCD3-mediated priming of na€ıve CD4+ T-cells from both cohorts in the presence of IL-2 alone led to the development of a spontaneous FoxP3+ CD25+ population in significant excess of controls. Knockdown of
J ALLERGY CLIN IMMUNOL FEBRUARY 2015
ERBB2IP or STAT3 in normal na€ıve CD4+ T-cells recapitulated this common cellular phenotype. CONCLUSIONS: These data suggest impaired STAT3 signaling and enhanced TGF-beta signaling converge to cause similar cellular phenotypes. Exploration of this association may help cast light on the link between connective tissue abnormalities and atopy in a variety of genetic settings.
498
Thymic Stromal Lymphopoietin Secretion As a Function of Genotype Claudia C. K. Hui, PhD1, Ashley Yu, BSc1, Delia Heroux, BSC1, Loubna Akhabir2, Andrew Sandford, PhD3, Helen Neighbour4, Judah A. Denburg, MD, FRCPC, FAAAAI5; 1Division of Clinical Immunology & Allergy, McMaster University, Hamilton, ON, Canada, 2University of British Columbia, James Hogg Research Centre, Providence Heart + Lung Institute, BC, Canada, 3University of British Columbia, James Hogg Research Centre, Providence Heart + Lung Institute, Vancouver, BC, Canada, 4Firestone Institute for Respiratory Health, McMaster University, Hamilton, ON, Canada, 5Division of Clinical Immunology and Allergy, Department of Medicine, McMaster University, Hamilton, ON, Canada. RATIONALE: Recent candidate gene and genome-wide association studies have identified a single nucleotide polymorphism (SNP) rs1837253 of thymic stromal lymphopoietin (TSLP) to be inversely associated with asthma and related-traits. Rs1837253 is suggested to have functional consequences on TSLP expression due to the absence of linkage disequilibrium to other SNPs. The objective of this study was to evaluate the expression and secretion of TSLP as a function of rs1837253 genotype using nasal epithelial cells (NEC) cultured from non-atopic and atopic individuals. METHODS: Genomic DNA was isolated from mouthwash samples and genotyped for rs1837253 using TaqManÒ genotyping assay. NEC from the nasal turbinate were collected, expanded and cultured, and the induction of TSLP by polyI:C was examined. RESULTS: The data were in Hardy Weinberg equilibrium. Atopic sensitization itself did not affect basal or polyI:C-mediated secretion of TSLP from NEC. Stratifying by genotype, stimulation with polyI:C resulted in decreased TSLP secretion in NEC obtained from heterozygous (CT; 1.8-fold) and homozygous minor allele (TT; 2.5-fold) individuals compared to NEC from homozygous major allele individuals (CC: p<0.05). CONCLUSIONS: We show for the first time that SNP rs1837253 in TSLP may be involved in the regulation of TSLP secretion, which may help explain its protective associations with asthma, atopic asthma and airway hyper-responsiveness. Identifying functional consequences of SNPs in genes with previously reported clinical associations will eventually pave the way for novel therapies targeting the source of inflammation rather than life-long therapies aimed at dampening inflammation and easing symptoms.
496
SUNDAY
Lipopolysaccharide-Responsive Beige-like Anchor Is Required for Both Activation and Deactivation of NFkB Jia-Wang Wang, PhD1, Michelle A. Reiser, MS1, Kunyu Li, BS1, Bangmei Wang1, Richard F. Lockey, MD1,2; 1Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida, Morsani College of Medicine, Tampa, FL, 2James Haley Veterans’ Hospital, Tampa, FL. RATIONALE: The molecular mechanisms why mutations of the lipopolysaccharide-responsive beige-like anchor (LRBA) paradoxically cause both autoimmunity and common variable immunodeficiency (CVID) are unknown. LRBA regulates vesicle trafficking and signal transduction required for the regulation and function of many immune molecules. It is hypothesized that LRBA deficiency attenuates both activation and deactivation of nuclear factor kappa beta (NFkB) resulting in immunodeficiency and autoimmunity. METHODS: LRBA was knocked down or repressed in Raji lymphoma cells by the short hairpin RNA (shRNA) or dominant negative mutant (DNM) techniques. The transcription activity and phosphorylated levels of NFkB was analyzed using the luciferase reporter assay, Western blot and flow cytometry. RESULTS: LRBA repression attenuates both NFkB activation and deactivation, inhibits NFkB nuclear translocation and increases tumor necrosis factor alpha (TNFa) and cell survival. The attenuated NFkB activation may result from inhibited NFkB nuclear translocation, while the attenuated NFkB deactivation may result in the increased TNFa and cell survival. CONCLUSIONS: These results suggest that the attenuation of NFkB activation may explain LRBA deficient immunodeficiency, while the prolonged NFkB activity and the increased proinflammatory cytokine TNFa and cell survival may explain LRBA deficient autoimmunity. This activation and deactivation (AnDA) model reveals a novel regulation mode for NFkB. It also provides a molecular mechanism for the paradoxical association of immunodeficiency and autoimmunity, which is a fundamental question in the immune system.
497
Convergence of Clinical and Cellular Phenotypes Among Patients with STAT3 and ERBB2IP Mutations Jonathan J. Lyons, MD1, Xiaomin Yu, PhD1, Kendal A. Karpe, BS1, Shirin M. Treadwell, MS1, Chi A. Ma, PhD1, Michael P. O’Connell, PhD1, Guangping Sun, MD1, Jason D. Hughes, PhD2, Huseyin Mehmet, PhD2, Joshua McElwee, PhD2, Steven M. Holland, MD3, Alexandra F. Freeman, MD4, Joshua D. Milner, MD1; 1Laboratory of Allergic Diseases, NIAID/NIH, Bethesda, MD, 2Merck Research Laboratories, Merck & Co. Inc., Boston, MA, 3Laboratory of Clinical Infectious Diseases, NIAID/NIH, Bethesda, MD, 4NIH/NIAID, Laboratory of Clinical Infectious Diseases, Bethesda, MD. RATIONALE: A number of congenital syndromes with connective tissue abnormalities are also associated with allergic disease, some of which are caused by altered TGF-beta signaling. Patients with loss of STAT3 function share multiple clinical features with these syndromes. We therefore sought to identify common cellular changes among patients with heterozygous loss-of-function ERBB2IP mutations, a negative regulator of TGF-beta signaling, and patients with dominant negative STAT3 mutations. METHODS: Patient-derived na€ıve CD4+ T-cells were differentiated under non-skewing conditions. RNA silencing of pathway genes in normal T-cells was performed. Advanced flow cytometric techniques, immunoblotting, and qRT-PCR were employed to characterize cellular responses. RESULTS: Common clinical features included atopy, elevated IgE, eosinophilia, and connective tissue abnormalities characterized by vascular abnormalities, retained primary dentition, joint hypermobility and high arched palate. FoxP3+ CD4+ T-cells were enriched among memory CD45RO+ T-cells among both cohorts of patients ex vivo. AntiCD3-mediated priming of na€ıve CD4+ T-cells from both cohorts in the presence of IL-2 alone led to the development of a spontaneous FoxP3+ CD25+ population in significant excess of controls. Knockdown of
J ALLERGY CLIN IMMUNOL FEBRUARY 2015
ERBB2IP or STAT3 in normal na€ıve CD4+ T-cells recapitulated this common cellular phenotype. CONCLUSIONS: These data suggest impaired STAT3 signaling and enhanced TGF-beta signaling converge to cause similar cellular phenotypes. Exploration of this association may help cast light on the link between connective tissue abnormalities and atopy in a variety of genetic settings.
498
Thymic Stromal Lymphopoietin Secretion As a Function of Genotype Claudia C. K. Hui, PhD1, Ashley Yu, BSc1, Delia Heroux, BSC1, Loubna Akhabir2, Andrew Sandford, PhD3, Helen Neighbour4, Judah A. Denburg, MD, FRCPC, FAAAAI5; 1Division of Clinical Immunology & Allergy, McMaster University, Hamilton, ON, Canada, 2University of British Columbia, James Hogg Research Centre, Providence Heart + Lung Institute, BC, Canada, 3University of British Columbia, James Hogg Research Centre, Providence Heart + Lung Institute, Vancouver, BC, Canada, 4Firestone Institute for Respiratory Health, McMaster University, Hamilton, ON, Canada, 5Division of Clinical Immunology and Allergy, Department of Medicine, McMaster University, Hamilton, ON, Canada. RATIONALE: Recent candidate gene and genome-wide association studies have identified a single nucleotide polymorphism (SNP) rs1837253 of thymic stromal lymphopoietin (TSLP) to be inversely associated with asthma and related-traits. Rs1837253 is suggested to have functional consequences on TSLP expression due to the absence of linkage disequilibrium to other SNPs. The objective of this study was to evaluate the expression and secretion of TSLP as a function of rs1837253 genotype using nasal epithelial cells (NEC) cultured from non-atopic and atopic individuals. METHODS: Genomic DNA was isolated from mouthwash samples and genotyped for rs1837253 using TaqManÒ genotyping assay. NEC from the nasal turbinate were collected, expanded and cultured, and the induction of TSLP by polyI:C was examined. RESULTS: The data were in Hardy Weinberg equilibrium. Atopic sensitization itself did not affect basal or polyI:C-mediated secretion of TSLP from NEC. Stratifying by genotype, stimulation with polyI:C resulted in decreased TSLP secretion in NEC obtained from heterozygous (CT; 1.8-fold) and homozygous minor allele (TT; 2.5-fold) individuals compared to NEC from homozygous major allele individuals (CC: p<0.05). CONCLUSIONS: We show for the first time that SNP rs1837253 in TSLP may be involved in the regulation of TSLP secretion, which may help explain its protective associations with asthma, atopic asthma and airway hyper-responsiveness. Identifying functional consequences of SNPs in genes with previously reported clinical associations will eventually pave the way for novel therapies targeting the source of inflammation rather than life-long therapies aimed at dampening inflammation and easing symptoms.