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The RNA-Binding Protein Hur Is Required to Control Cytokine Expression in CD4+ T Cells
Abstracts AB399
J ALLERGY CLIN IMMUNOL VOLUME 133, NUMBER 2
L4
The RNA-Binding Protein Hur Is Required to Control Cytokine Expression in CD4+ T Cells Dr. Ulus Atasoy, MD, FAAAAI1, Dr. Patsharaporn Techasintana2, Dr. Joseph Magee2, Dr. Matt Gubin3, Mr. Joe Rowles2; 1Surgery, University of MO-Columbia, Columbia, MO, 2University of Missouri, 3 Washington University. RATIONALE: During T cell activation, up to 50% of changes occur at the posttranscriptional level, which is mediated by RNA-binding proteins (RBPs). HuR is a stabilizing RBP which regulates mRNA target expression via AU-rich elements present in mRNA 3’UTRs. It is permissive for Th2 and Th17 differentiation. METHODS: Previously, we showed HuR regulates GATA-3, IL-4, IL-13 and other Th2 restricted transcripts, as well as IL-17. We hypothesized that HuR is required for Th2 differentiation and Th2 associated disease such as asthma. To avoid defects in T cell development, we utilized a HuR KO mouse model in which HuR is ablated in mature CD4+ T cells, using distal Lck-Cre-ROSA-HuRfl/fl. RESULTS: Activated or Th2 polarized HuR KO CD+4 T cells showed striking increases in IL-2 secretion and an inability to shut off IL-2 expression. These also mice had profound reductions in Th2 and Th17 cytokine expression. We used the ova challenge model of airway inflammation to further investigate HuR KO under antigenic conditions. HuR KO mice had profound reductions in BAL IL-4 and IL-13, and in pulmonary cellular infiltration with suppression of neutrophils, lymphocytes and eosinophils. The levels of cellular infiltration in HuR KO mice resembled un-immunized controls. CONCLUSIONS: HuR appears to be required for Th2 and Th17 but not Th1 differentiation and cytokine production. Furthermore, HuR functions as an off switch for IL-2 expression during T cell activation. These results may have implications for understanding mechanisms of tolerance under antigen challenge as well as in allergen driven airway lung inflammation. Whole-Exome Sequencing Reveals IKBKB As a Cause of Combined Immunodeficiency Dr. Talal Mousallem, MD1,2, Dr. Jialong Yang, PhD1, Dr. Thomas J. Urban, PharmD, PhD2, Mrs. Hongxia Wang, MS1,3, Mrs. Roberta E. Parrott, BS1, Dr. Joseph L. Roberts, MD, PhD1, Dr. Rebecca H. Buckley, MD, FAAAAI1, Dr. Xiaoping Zhong, MD, PhD1, Dr. David B. Goldstein, PhD2; 1Department of Pediatrics, Duke University Medical Center, Durham, NC, 2Center for Human Genome Variation, Duke University Medical Center, Durham, NC, 3Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, China. RATIONALE: There is considerable interest in the use of next generation sequencing to identify molecular defects in patients with primary immunodeficiency. METHODS: To identify the cause of Combined Immunodeficiency in 2 patients from 2 different consanguineous Qatari families who had a similar clinical and immunologic phenotype, we used whole-exome sequencing. The patients presented at an early age with fungal, viral and bacterial infections. The patients had hypogammaglobulinemia, normal numbers of B and T cells (most of which were CD45RA positive) and low numbers of NK cells. T cell proliferative responses to mitogens were normal, but were absent to antigens and anti-CD3. We looked for homozygous variants that were present in the patients but absent in controls and public databases. Additional studies included IKKb, IKBa, and phospho-IKBa immunoblotting, retroviral mediated reconstitution of IKKb, and cell growth in an immortalized patient B cell line. RESULTS: We found the same homozygous nonsense mutation in IKBKB (R286X) in both patients. IKKb acts as part of the IKK complex in NF-kB activation and phosphorylates the NF-kB inhibitor, IKBa. The mutation co-segregated with disease in both families. Western blotting showed absence of detectable IKKb, and retroviral mediated reconstitution with wild type IKKb corrected cell growth and restored IKBa phosphorylation and IKBa degradation in an EBV transformed patient B cell line. CONCLUSION: These data indicate that mutated IKBKB is the likely cause of immunodeficiency in these two patients. To our knowledge, this is the first report of combined immunodeficiency caused by a mutation in IKBKB.
L5
L6
Sputum Cytokines and Adult Asthma Endotypes Dr. Ioana O. Agache1, Dr. Costel Agache2, Mrs. Beate R€uckert3, Prof. Liliana Rogozea1, Dr. Cristina Ciobanu2, Prof. Cezmi A. Akdis, MD, FAAAAI4; 1Transylvania University Brasov, Romania, 2Theramed Medical Center, Romania, 3Swiss Institute of Allergy and Asthma Research, Switzerland, 4Swiss Institute of Allergy and Asthma Research, Davos, Switzerland. RATIONALE: Asthma clusters can be separated according to their endotype by combining visible properties and sputum cytokines. METHODS: The Local Ethics Committee approved the study. Induced sputum sampled outside an exacerbation was processed by dialysis (D-Tube Dialyzers Midi, Novagen) followed by ultrafiltration (Amicon Ultra 3K, Millipore, 3000 NMWL). Sputum cytokines were measured with the 27 Plex Human Cytokine Group I Bioplex (Biorad). Hierarchical cluster analysis with a wide array of variables was used. Difference between clusters was assesed by One-way Anova. RESULTS: 60 adult asthmatics, mean age 44614 years old, 52% females were included. Three clusters were separated, significantly different for all sputum cytokines (except RANTES and FGF-b), asthma severity (GINA), onset of asthma, ACT score, lung function (LF), AHR, exacerbation frequency, near-fatal asthma (NFA), corticosteroid-resistance, blood eosinophils. Cluster 1 (n527, 67% males) had the lowest frequency of asthma exacerbations, the best LF, predominant macrophages and lymphocytes in induced sputum and the highest sputum IL-4, IL-1RA, VEGF and IL-7. Cluster 2 (n514, 64.3% females) had the longest asthma duration, the highest AHR and frequency of asthma exacerbations, corticosteroid-resistance and NFA, the worst LF and ACT score, the highest sputum and blood eosinophilia and the highest sputum cytokines. Cluster 3 (n519, 63.2% females), had the shortest asthma duration, the highest FeNO and total serum IgE, the highest incidence of LF decline and atopy, sputum neutrophilia and the lowest values for all sputum cytokines. CONCLUSION: Deep phenotyping incorporating phenotypic traits, longitudinal data and biomarkers of inflammation stratifies asthma into subclasses according to their biological basis.
L7
Omalizumab Therapy Is Associated with Reduced Circulating Basophil Populations in Asthmatic Children Dr. David A. Hill, MD, PhD1, Dr. Mark Siracusa, PhD2, Kathryn Ruymann1, Dr. Elia Tait Wojno, PhD2, Dr. David Artis, PhD2, Dr. Jonathan M. Spergel, MD, PhD, FAAAAI1; 1The Children’s Hospital of Philadelphia, Philadelphia, PA, 2University of Pennsylvania, Philadelphia, PA. RATIONALE: Basophils have been implicated in promoting the early development of TH2 cell responses in some murine models of TH2 cytokine-associated inflammation. However, the specific role of basophils in allergic asthma remains an active area of research. Recent studies in animal models and human subjects suggest that IgE may regulate the homeostasis of human basophil populations. Here, we examine basophil populations in children with severe asthma before and during therapy with the IgE directed monoclonal antibody omalizumab. METHODS: Subjects were selected based on age between 5 and 18 years, body weight and IgE level compatible with the omalizumab dosing table, and severe asthma. Asthma symptoms assessments were administered during therapy. Peripheral blood samples were obtained before and during therapy and basophil frequencies and numbers were monitored using flow cytometric techniques. RESULTS: Omalizumab therapy was associated with a significant reduction in circulating basophil frequencies and numbers (p<0.05, Wilcoxon Signed Rank Test), a finding that correlated with improved clinical outcomes. CONCLUSIONS: The observation that circulating basophils are reduced following omalizumab therapy supports a mechanistic link between IgE levels and circulating basophil populations and may provide new insights into one mechanism by which this drug improves asthma symptoms.
J ALLERGY CLIN IMMUNOL VOLUME 133, NUMBER 2
L4
The RNA-Binding Protein Hur Is Required to Control Cytokine Expression in CD4+ T Cells Dr. Ulus Atasoy, MD, FAAAAI1, Dr. Patsharaporn Techasintana2, Dr. Joseph Magee2, Dr. Matt Gubin3, Mr. Joe Rowles2; 1Surgery, University of MO-Columbia, Columbia, MO, 2University of Missouri, 3 Washington University. RATIONALE: During T cell activation, up to 50% of changes occur at the posttranscriptional level, which is mediated by RNA-binding proteins (RBPs). HuR is a stabilizing RBP which regulates mRNA target expression via AU-rich elements present in mRNA 3’UTRs. It is permissive for Th2 and Th17 differentiation. METHODS: Previously, we showed HuR regulates GATA-3, IL-4, IL-13 and other Th2 restricted transcripts, as well as IL-17. We hypothesized that HuR is required for Th2 differentiation and Th2 associated disease such as asthma. To avoid defects in T cell development, we utilized a HuR KO mouse model in which HuR is ablated in mature CD4+ T cells, using distal Lck-Cre-ROSA-HuRfl/fl. RESULTS: Activated or Th2 polarized HuR KO CD+4 T cells showed striking increases in IL-2 secretion and an inability to shut off IL-2 expression. These also mice had profound reductions in Th2 and Th17 cytokine expression. We used the ova challenge model of airway inflammation to further investigate HuR KO under antigenic conditions. HuR KO mice had profound reductions in BAL IL-4 and IL-13, and in pulmonary cellular infiltration with suppression of neutrophils, lymphocytes and eosinophils. The levels of cellular infiltration in HuR KO mice resembled un-immunized controls. CONCLUSIONS: HuR appears to be required for Th2 and Th17 but not Th1 differentiation and cytokine production. Furthermore, HuR functions as an off switch for IL-2 expression during T cell activation. These results may have implications for understanding mechanisms of tolerance under antigen challenge as well as in allergen driven airway lung inflammation. Whole-Exome Sequencing Reveals IKBKB As a Cause of Combined Immunodeficiency Dr. Talal Mousallem, MD1,2, Dr. Jialong Yang, PhD1, Dr. Thomas J. Urban, PharmD, PhD2, Mrs. Hongxia Wang, MS1,3, Mrs. Roberta E. Parrott, BS1, Dr. Joseph L. Roberts, MD, PhD1, Dr. Rebecca H. Buckley, MD, FAAAAI1, Dr. Xiaoping Zhong, MD, PhD1, Dr. David B. Goldstein, PhD2; 1Department of Pediatrics, Duke University Medical Center, Durham, NC, 2Center for Human Genome Variation, Duke University Medical Center, Durham, NC, 3Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, China. RATIONALE: There is considerable interest in the use of next generation sequencing to identify molecular defects in patients with primary immunodeficiency. METHODS: To identify the cause of Combined Immunodeficiency in 2 patients from 2 different consanguineous Qatari families who had a similar clinical and immunologic phenotype, we used whole-exome sequencing. The patients presented at an early age with fungal, viral and bacterial infections. The patients had hypogammaglobulinemia, normal numbers of B and T cells (most of which were CD45RA positive) and low numbers of NK cells. T cell proliferative responses to mitogens were normal, but were absent to antigens and anti-CD3. We looked for homozygous variants that were present in the patients but absent in controls and public databases. Additional studies included IKKb, IKBa, and phospho-IKBa immunoblotting, retroviral mediated reconstitution of IKKb, and cell growth in an immortalized patient B cell line. RESULTS: We found the same homozygous nonsense mutation in IKBKB (R286X) in both patients. IKKb acts as part of the IKK complex in NF-kB activation and phosphorylates the NF-kB inhibitor, IKBa. The mutation co-segregated with disease in both families. Western blotting showed absence of detectable IKKb, and retroviral mediated reconstitution with wild type IKKb corrected cell growth and restored IKBa phosphorylation and IKBa degradation in an EBV transformed patient B cell line. CONCLUSION: These data indicate that mutated IKBKB is the likely cause of immunodeficiency in these two patients. To our knowledge, this is the first report of combined immunodeficiency caused by a mutation in IKBKB.
L5
L6
Sputum Cytokines and Adult Asthma Endotypes Dr. Ioana O. Agache1, Dr. Costel Agache2, Mrs. Beate R€uckert3, Prof. Liliana Rogozea1, Dr. Cristina Ciobanu2, Prof. Cezmi A. Akdis, MD, FAAAAI4; 1Transylvania University Brasov, Romania, 2Theramed Medical Center, Romania, 3Swiss Institute of Allergy and Asthma Research, Switzerland, 4Swiss Institute of Allergy and Asthma Research, Davos, Switzerland. RATIONALE: Asthma clusters can be separated according to their endotype by combining visible properties and sputum cytokines. METHODS: The Local Ethics Committee approved the study. Induced sputum sampled outside an exacerbation was processed by dialysis (D-Tube Dialyzers Midi, Novagen) followed by ultrafiltration (Amicon Ultra 3K, Millipore, 3000 NMWL). Sputum cytokines were measured with the 27 Plex Human Cytokine Group I Bioplex (Biorad). Hierarchical cluster analysis with a wide array of variables was used. Difference between clusters was assesed by One-way Anova. RESULTS: 60 adult asthmatics, mean age 44614 years old, 52% females were included. Three clusters were separated, significantly different for all sputum cytokines (except RANTES and FGF-b), asthma severity (GINA), onset of asthma, ACT score, lung function (LF), AHR, exacerbation frequency, near-fatal asthma (NFA), corticosteroid-resistance, blood eosinophils. Cluster 1 (n527, 67% males) had the lowest frequency of asthma exacerbations, the best LF, predominant macrophages and lymphocytes in induced sputum and the highest sputum IL-4, IL-1RA, VEGF and IL-7. Cluster 2 (n514, 64.3% females) had the longest asthma duration, the highest AHR and frequency of asthma exacerbations, corticosteroid-resistance and NFA, the worst LF and ACT score, the highest sputum and blood eosinophilia and the highest sputum cytokines. Cluster 3 (n519, 63.2% females), had the shortest asthma duration, the highest FeNO and total serum IgE, the highest incidence of LF decline and atopy, sputum neutrophilia and the lowest values for all sputum cytokines. CONCLUSION: Deep phenotyping incorporating phenotypic traits, longitudinal data and biomarkers of inflammation stratifies asthma into subclasses according to their biological basis.
L7
Omalizumab Therapy Is Associated with Reduced Circulating Basophil Populations in Asthmatic Children Dr. David A. Hill, MD, PhD1, Dr. Mark Siracusa, PhD2, Kathryn Ruymann1, Dr. Elia Tait Wojno, PhD2, Dr. David Artis, PhD2, Dr. Jonathan M. Spergel, MD, PhD, FAAAAI1; 1The Children’s Hospital of Philadelphia, Philadelphia, PA, 2University of Pennsylvania, Philadelphia, PA. RATIONALE: Basophils have been implicated in promoting the early development of TH2 cell responses in some murine models of TH2 cytokine-associated inflammation. However, the specific role of basophils in allergic asthma remains an active area of research. Recent studies in animal models and human subjects suggest that IgE may regulate the homeostasis of human basophil populations. Here, we examine basophil populations in children with severe asthma before and during therapy with the IgE directed monoclonal antibody omalizumab. METHODS: Subjects were selected based on age between 5 and 18 years, body weight and IgE level compatible with the omalizumab dosing table, and severe asthma. Asthma symptoms assessments were administered during therapy. Peripheral blood samples were obtained before and during therapy and basophil frequencies and numbers were monitored using flow cytometric techniques. RESULTS: Omalizumab therapy was associated with a significant reduction in circulating basophil frequencies and numbers (p<0.05, Wilcoxon Signed Rank Test), a finding that correlated with improved clinical outcomes. CONCLUSIONS: The observation that circulating basophils are reduced following omalizumab therapy supports a mechanistic link between IgE levels and circulating basophil populations and may provide new insights into one mechanism by which this drug improves asthma symptoms.