[BAD] EK9 - Abstracts



Articles

PhD Student


Credits: None available.

Itaconate regulates responses to inhaled aeroallergen

Gesa J. Albers1, Patricia P. Ogger1, Simone A. Walker1, John M. Halket2, Robert Gray2, Clare M. Lloyd1, Adam Byrne1
1National Heart and Lung Institute, Imperial College London, UK
2Mass Spectrometry Facility, King’s College London, UK

Background: Asthma is a chronic disease characterised by airway remodelling and hyperresponsiveness, mucus production and inflammation. Airway macrophages (AMs) are key sentinels of lung homeostasis and form the first line of defence against inhaled allergen. Increasing evidence suggests that changes in AM phenotype are underpinned by alterations in AM metabolism. The TCA cycle-derived metabolite itaconic acid (IA), synthesised by the enzyme aconitate decarboxylase (ACOD)-1, is a key regulator of macrophage function. Recently, we showed in human and murine models that IA limits pulmonary fibrosis, yet its role in regulating pulmonary responses to inhaled allergen is unknown. Here, we aimed to study the role of IA in murine models of allergic airway disease (AAD).
Methods: To characterise the kinetics of the Acod1/IA pathway in response to allergen, we measured lung Acod1 expression and BAL-IA levels after house dust mite (HDM) challenge. Next, we assessed Acod1 expression in murine AMs exposed to HDM ex vivo. Finally, to determine the role of IA in allergic airway responses, we treated WT or Acod1-/- mice with inhaled HDM and assessed disease pathology and inflammation.
Results: Continuous HDM exposure in mice resulted in augmented levels of BAL-IA and increased expression of lung Acod1. Ex vivo culture of AMs with HDM revealed enhanced Acod1 expression and a shift towards a more glycolytic phenotype. Finally, exposure of Acod1-/- mice to allergen led to an increased neutrophil-to-eosinophil ratio, compared to more eosinophilic WT controls, which was rescued by inhaled IA.
Conclusion: Our data indicate that the Acod1/IA pathway is highly induced during AAD and regulates the balance between neutrophil and eosinophil recruitment in response to inhaled allergen.

Author(s):

Assistant Professor


Credits: None available.

Modeling airway dysfunction in asthma using synthetic mucus biomaterials

As asthma worsens, occlusion of airways with mucus significantly contributes to airflow obstruction and reduced lung function. Recent evidence from clinical studies has shown mucus obtained from adults and children with asthma possesses altered mucin composition. However, how these changes alter the functional properties of the mucus gel is not yet fully understood. To study this, we have engineered a synthetic mucus biomaterial to closely mimic the properties of native mucus in health and disease. We demonstrate this model possesses comparable biophysical and transport properties to native mucus ex vivo collected from human subjects and in vitro isolated from human airway epithelial (HAE) tissue cultures. We found by systematically varying mucin composition that mucus gel viscoelasticity is enhanced when predominantly composed of mucin 5AC (MUC5AC), as is observed in asthma. As a result, asthma-like synthetic mucus gels are more slowly transported on the surface of HAE tissue cultures and at a similar rate to native mucus produced by HAE cultures stimulated with the type 2 cytokine IL-13, known to contribute to airway inflammation and MUC5AC hypersecretion in asthma. We also discovered the barrier function of asthma-like synthetic mucus towards influenza A virus was impaired as evidenced by the increased frequency of infection in MUC5AC-rich hydrogel coated HAE cultures. Together, this work establishes a biomaterial-based approach to understand airway dysfunction in asthma and related muco-obstructive lung diseases.

Author(s):

Graduate Student


Credits: None available.

ADAR-mediated editing of miR-200b-3p in airway cells is associated with moderate-to-severe asthma

Magnaye KM(1), Naughton KA(1), Huffman J(1), Hogarth DK(2), Naureckas ET(2), White SR(2), Ober C(1)
1. Department of Human Genetics, University of Chicago, Chicago, IL
2. Department of Medicine, University of Chicago, Chicago, IL

Asthma is a chronic lung disease characterized by persistent airway inflammation and bronchial hyperresponsiveness. Altered microRNA-mediated gene silencing in bronchial epithelial cells has been reported in asthma, yet microRNA adenosine to inosine (A-to-I) editing in asthma remains unexplored. We performed the first genome-wide analysis of ADAR-mediated microRNA editing using microRNA-seq in primary bronchial epithelial cells from 142 adult asthma cases and non-asthma controls. Of 19 A-to-I edited sites detected in these microRNAs, 16 were in seed regions. Four of the 16 edited sites were observed in >10 individuals and were tested for differential editing (% A-to-I) between groups. One site at position 5 of miR-200b-3p was edited less frequently in asthma cases compared to controls (P = 0.013). A-to-I editing of this site was then compared between asthma severity groups (mild, moderate and severe) based on lung function and medication use. The moderate (P = 0.037) and severe (P = 0.00031), but not mild (P = 0.77), asthma cases had significantly less A-to-I editing of the 5th position of miR-200b-3p compared to controls. Bioinformatic prediction revealed 232 in silico target genes of the edited miR-200b-3p, which were enriched for both IL-4 and interferon gamma signaling pathways and included the SOCS1 (suppressor of cytokine signaling 1) gene. SOCS1 was more highly expressed in moderate (P = 0.017) and severe (P = 0.0054) asthma cases compared to controls. Moreover, both miR-200b-3p editing and SOCS1 were associated with BAL eosinophil levels and an epithelial cell signature of Type 2 asthma. Overall, reduced ADAR-mediated editing of the 5th position of miR-200b-3p in lower airway cells from moderate-to-severe asthmatics may lead to overexpression of a centrally important negative regulator of cytokine signaling, SOCS1. We proposed ADAR-mediated editing as an epigenetic mechanism contributing to features of moderate-to-severe asthma in adulthood. Supported by U19 AI095230. KMM is supported by F31 HL143891.

Author(s):

Instructor in Medicine


Credits: None available.

Defining the functions of tissue-resident and circulating memory Th2 cells in allergic asthma

Rod A. Rahimi1,2,3, Keshav Nepal1,2,4, Murat Cetinbas5,6, Ruslan I. Sadreyev5,7, and Andrew D. Luster1,2,4

1Airway Immunity Research Program, 2Center for Immunology and Inflammatory Diseases, 3Division of Pulmonary and Critical Care Medicine, 4Division of Rheumatology, Allergy, and Immunology, 5Department of Molecular Biology, 6Department of Genetics, 7Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA

Tissue-resident memory T (Trm) cells are a unique population of memory T cells that are durably parked in non-lymphoid tissues and play an important role in host defense against recurrent infection and malignancy. CD4+ T helper type 2 (Th2) cells play a central role in allergic diseases, including asthma, but the biology of Th2 Trm cells in recurrent allergic inflammation is not well defined. Specifically, the mechanisms whereby Th2 Trm cells and circulating memory Th2 cells promote allergic asthma pathogenesis remain unclear. Using a house dust mite (HDM) model of allergic asthma and parabiosis, we demonstrate that Th2 Trm cells and circulating memory Th2 cells perform distinct functions in vivo. Upon HDM rechallenge, circulating memory Th2 cells trafficked into the lung parenchyma and ignited perivascular inflammation to promote inflammatory cell recruitment, including CD4+ T cells, eosinophils, and dendritic cells. In contrast, Th2 Trm cells proliferated near airways and were critical in promoting mucus metaplasia, airway hyperresponsiveness, and airway eosinophil activation. Transcriptional analysis revealed that Th2 Trm cells and circulating memory Th2 cells share a core Th2 gene signature, but also exhibit distinct transcriptional profiles. Th2 Trm cells express a tissue-adaptation signature, including genes involved in extracellular matrix biology and lipid metabolism. Our findings demonstrate that Th2 Trm cells and circulating memory Th2 cells are functionally and transcriptionally distinct subsets with unique roles in vivo, with the establishment of Th2 Trm cells being critical for the full manifestation of allergic airway disease. Defining the unique mechanisms regulating the development and maintenance of Th2 Trm cells within the lungs has the potential to yield novel therapeutic approaches for allergic asthma.

Author(s):

PhD Student


Credits: None available.

Butyrate selectively inhibits metabolic reprogramming of inflammatory eosinophils in allergic asthma

Rossana Azzoni1, Tara Sutherland1, Tracy Hussell1, Joanne Konkel1, Yashaswini Kannan2, Edith Hessel3 and John Grainger1

1Faculty of Medicine, Biology and Health, University of Manchester, Lydia Becker Institute, Manchester,UK
2Adaptive Immunity Research Unit, Glaxosmithkline, Stevenage, UK
3 Mestag Therapeutics, London, UK

Background: Allergic asthma is characterised by dominant Th2 responses to innocuous environmental antigens, such as house dust mite (HDM), resulting in significant airway eosinophilia. Eosinophil activation exerts a central role in asthma pathogenesis through the release of cytotoxic granules and cytokines. As well as these effector functions, eosinophils have been shown to promote homeostatic responses at steady-state and disease, which may explain why eosinophil-targeted therapies have had mixed results. Two different populations of eosinophils have recently been identified in the lungs of murine models of allergic inflammation, and have been described as Siglec-FintCD11bint and Siglec-FhiCD11bhi eosinophils. 

Aims: It is still not clear how Siglec-FintCD11bint and Siglec-FhiCD11bhi eosinophils originate, and further develop during allergic disease, how they are maintained, what their phenotype is and if specific factors mediate these processes. Therefore, our aim was to better define these mechanisms to understand whether we could bias towards targeting one population or the other from a therapeutic standpoint. 

Methods: We have used a murine model of acute allergic airway inflammation induced by intranasal exposure to HDM. Sensitisation (day 0) with HDM was followed by 5 consecutive challenges (day 7-11) and euthanasia 24 hours after the last challenge.     

Results: We detected Siglec-FintCD11bint and Siglec-FhiCD11bhi eosinophils in the lungs and blood of HDM-treated mice. We found that Siglec-FhiCD11bhi but not Siglec-FintCD11bint eosinophils contributed to the inflammatory response. This was mediated via enhanced CD98 expression and metabolic activity, which represented universal features of Th2 inflammatory responses. We also established that, following HDM administration, the short-chain fatty butyrate could specifically decrease levels of Siglec-FhiCD11bhi eosinophils as well as their metabolic activity in the lung and that this mechanism may be mediated by GPR109a. Thus, selective targeting of Siglec-FhiCD11bhi eosinophils and their metabolism may be beneficial in ameliorating allergic lung inflammation. 

Author(s):

Lecturer


Credits: None available.

B cell IgM isotype regulates airway smooth muscle contraction in allergic asthma
Authors and Affiliations: 
Sabelo Hadebe*1, Jermaine Khumalo1,2, Katelyn Jones1, Anca Savulescu4, Sandisiwe Mangali1,2, Amkele Ngomti1,2,Martyna Scibiorek1,2, Frank Kirstein1, Frank Brombacher*1,2,3  
1Division of Immunology, and South African Medical Research Council (SAMRC) Immunology of Infectious Diseases, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa  
2International Centre for Genetic Engineering and Biotechnology (ICGEB) and Institute of Infectious Diseases and Molecular Medicine (IDM), Division of Immunology, Health Science Faculty, University of Cape Town, Cape Town, South Africa  
3Wellcome Centre for Infectious Diseases Research in Africa (CIDRI-Africa), Institute of Infectious Diseases and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town, 7925, South Africa 
4Division of Chemical, Systems & Synthetic Biology, Faculty of Health Sciences, Institute of Infectious Disease & Molecular Medicine, University of Cape Town, Cape Town, South Africa
Abstract:
Allergic asthma is a disease driven by T helper 2 (TH2)-type cells secreting interleukin (IL-) 4, 5 and 13. It is characterized by eosinophils, airway hyperresponsiveness (AHR) and IgE secreting B cells. B cells play a role in allergic asthma in an allergen load dependent manner. IgM isotype secreted by naïve B cells is important for class switching. It is currently unclear how IgM isotype contributes in the development of allergic asthma. We investigated the role of IgM isotype in a house dust mite (HDM)-induced TH2 allergic asthma model. We sensitised wild type (wt), IgM-deficient (IgM-/-) and B cell-deficient (uMT-/-) mice with high (>10 ug) and low (<3 ug) dose of HDM extracts. We found IgM to be essential in IgE production and in AHR, but not in TH2 airway inflammation or eosinophilia. Transfer of wild type serum, but not B cells into IgM-/-mice could restore class switching but not AHR. RNA seq suggested that IgM regulated AHR through modulating airway smooth muscle (ASM) genes, particularly those associated with acetylcholine and contraction. Using single cell force cytometry (FLECS) and CRISPR-Cas9 technology we validate the importance of some of these genes in human ASM contraction. These unprecedented findings suggest that IgM has a unique function in allergic asthma and regulates AHR by interacting with structural cells.

Author(s):

Sr. Research Associate


Credits: None available.

Pediatric asthma, viral trends and public health outcomes during the COVID-19 pandemic
Samir Sayeda*, Kiara Taquechela*, Avantika R. Diwadkarb*, Jesse W. Dudleyc, Robert W. Grundmeierc,d, Chén C. Kenyond,e, David A. Hilla,d,f,‡, Blanca E. Himesb,‡ and Sarah E. Henricksona,d,f,‡
 
a Division of Allergy and Immunology, Children’s Hospital of Philadelphia, Philadelphia, PA
b Department of Biostatistics, Epidemiology and Informatics, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, PA
c Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA
d Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
e Center for Pediatric Clinical Effectiveness and PolicyLab, Children’s Hospital of
Philadelphia, Philadelphia, PA
f Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
* These authors contributed equally to this work.
‡ These authors contributed equally to this work.

The coronavirus disease 2019 (COVID-19) pandemic caused dramatic changes in daily routines in the United States that might have influenced viral transmission patterns during this time. In the Philadelphia area, March 17th, 2020 marked the date on which public health measures were enacted to reduce transmission of COVID-19. Asthma-related encounters and respiratory viral testing data were extracted from Children’s Hospital of Philadelphia (CHOP) electronic health records for the last five years between mid-January to mid-May (2015 to 2020) in order to assess the impact of those measures. Changes in viral testing patterns, patient encounter details and air pollution before and after March 17th were assessed and compared with data from 2015 to 2019 as a historical reference. We found decreased asthma-related systemic steroid prescriptions and decreased frequency of positive rhinovirus test results but air pollution levels did not substantially change compared with historical trends. We are now collecting Fall 2020 data to further our understanding on the effects COVID-19 has had on the presentation of pediatric asthma.

Author(s):

Assistant Professor


Credits: None available.

Obesity dysregulates immunometabolic status in pediatric asthma and impacts vaccine responses
Sarah E. Henrickson1,2, Peyton Conrey2, Sasikanth Manne1,3, , Samir Sayed2, Kaitlin C. O’Boyle3 Bertram Bengsch1,†  , Ting Qian4, Ramin S. Herati1,5††­­†, Laura A. Vella1,6, Allison R. Greenplate1,3, Sam J.  McCright1,7, Cécile Alanio1,3, 12, Frank Mentch11, Kenneth E. Schmader8, Christopher F. Pastore9, Li-Yin Hun9, Scott E. Hensley1,10, De’Broski Herbert9, Aaron J. Masino4, Jorge Henao-Mejia1,7, Hakon Hakonarson11, Joshua D. Rabinowitz12, Susan E. Coffin6 and E. John Wherry1,3,12 
1Institute for Immunology, University of Pennsylvania, Philadelphia, PA.
2Division of Allergy-Immunology, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA.
3Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA.
4Department of Biomedical and Health Informatics, The Children’s Hospital of Philadelphia, Philadelphia, PA.
5Department of Medicine, University of Pennsylvania Perelman School of Medicine 
6Division of Infectious Disease, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA. 
7Department of Pathology, The Children’s Hospital of Philadelphia, Philadelphia, PA.
8Division of Geriatrics, Department of Medicine, Duke University Medical Center and Geriatric Research, Education, and Clinical Center, Durham VA Medical Center, Durham, NC.
9School of Veterinary Medicine, Department of Pathobiology, University of Pennsylvania, Philadelphia. PA
10Department of Microbiology, University of Pennsylvania, Philadelphia, PA.
11Center for Applied Genomics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA.
12Parker Institute for Cancer Immunotherapy at University of Pennsylvania
13Department of Chemistry, Princeton University, Princeton, NJ.
†1Department of Medicine II, Gastroenterology, Hepatology, Endocrinology, and Infectious Diseases, University Medical Center Freiburg, Faculty of Medicine, Freiburg, Germany, and Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany
††Department of Medicine, New York University, Grossman School of Medicine, New York City, NY.
 
Asthma and obesity are two of the most common chronic childhood diseases worldwide, with dramatically increasing prevalence over the last few decades. These diseases impact morbidity and mortality and strain health care systems financially. Asthma risk increases as body mass index (BMI) increases, suggesting a pathophysiological link. Both asthma and obesity are independently linked to altered immune status, however, it remains unclear how these diseases converge to affect pediatric immune function. To address this question, we investigated the immunometabolic profile in obese asthmatic (OA), non-obese asthmatic (A), obese non-asthmatic (O), and healthy control (HC) children using mass cytometry, serum metabolomics, cytokine analysis and clinical history. This multi-modal approach revealed two major forms of immune dysfunction in pediatric allergic OA: altered baseline T cell activation state (exhaustion-like) and increased type 2 immunity. OA had increased Th2 differentiation and decreased Th17 differentiation and these changes were associated with altered blood metabolites, including increased glutamate and decreased acetate. A mouse model of OA confirmed increased exhausted-like CD8 T cells compared to A and HC mice. Finally, immunometabolic dysregulation and altered T cell activation status in O and OA patients was linked to prolonged retention of humoral vaccine responses. These insights into the mechanistic links between metabolic alterations and immune dysfunction in OA may improve understanding of the severe asthma exacerbations secondary to viral upper respiratory tract infections seen in OA and provide opportunities for novel therapeutic approaches.

Author(s):

Head-Clinical Research


Credits: None available.

Toll-like receptors, Wnt receptors and Calcium-activated Chloride/Potassium Channels as promising immunomodulators of allergic airway inflammation, airway hyper-responsiveness and asthma: translational research impact
Saumya Pandey (M.Sc., Ph.D.)
Department of Clinical Research, Indira IVF Hospital, Udaipur, India

Abstract:
Introduction: Allergic airway inflammation/airway hyper-responsiveness and asthma have emerged as major public health challenges in United States of America/Asia-Pacific region. Targeting Toll-like receptor (TLR), Wnt-Frizzled receptor signaling and Calcium-activated-Chloride (ClCa)/Potassium (IKCa3.1) Channels in unraveling the cellular/molecular/genetic basis of susceptibility to inflammatory diseases in specific human patient population subset(s) is emerging as an attractive immunotherapeutic pharmacological strategy in management/prevention of asthma in the Covid-19 pandemic era. 
Objectives: My exploratory study aimed to investigate the role of TLRs, Wnt-Fzd receptors and ClCa/IKCa3.1 ion-channels in human airway smooth muscle cells, bronchial epithelial cells (ASMCs/NHBEC/BEAS-2B from ATCC), and eosinophils-derived from asthma patients of North American ethnicity.
Methods: Whole cell patch-clamp electrophysiological-recordings with stringent pH/ osmolality-checks were conducted for ClCa/IKCa3.1 ion-channel physiology and outwardly-/inwardly-rectifying currents in cultured cells (passages: P2-P5) grown on sterile cover-slips. RNA and Protein were extracted from ASMCs/NHBECs/BEAS-2B/eosinophils using Trizol and RIPA methods. Borosilicate patch-pipettes were fabricated using Sutter instrument and resistance was checked alongwith bore-diameter prior to filling bath- and pipette-solutions; micromanipulators were adjusted for gigaseal-recordings. The study was approved by Institutional Review Board. 
Results: Cell-viability assays (MTT) demonstrated >80% viability of ASMCs/NHBECs/ BEAS-2B cells and asthma patients’-isolated eosinophils. Mean age of American patients (N=7; White N=2/African-American N=4/Caucasian N=1) was 47.0 years (S.D±5.0 years). Receptor/ion channel physiology studies demonstrated the expression of TLR2/4, Wnt2/4, Fzd2 and intermediate conductance IKCa3.1/ClCa mRNA transcripts; beta-actin/GAPDH were used as internal controls. Patch-clamp electrophysiology recordings detected Chloride/Potassium channel current-spikes in cultured cells in presence of intracellular Calcium, and DIDS-Chloride channel inhibitor. My preliminary data implicates the public health impact of TLR2/4-Wnt2/4-Fzd2/ClCa-KCa regulatory networks-mediated immunomodulation in AHR and asthma management in North American cohort. Future pharmacogenetics-based epidemiology/association-studies with larger sample-size and subgroup-stratification for Covid-19 positivity status and Covid-19 relapse/recurrence rates are warranted for development of cost-effective predictive biomarkers for AHR and asthma susceptible populations of diverse ethnicities. 
Acknowledgement: Dr. Pandey acknowledges the fellowship/fund support from Department of Biomedical Sciences, School of Medicine, Creighton University, Omaha, Nebraska, USA-National Institutes of Health, Bethesda, Maryland, USA during her tenure.
Conflicts of Interest: None.

Author(s):

Associate Professor, Internal Medicine, Division Chief, Allergy Immunology, Ann Arbor VA


Credits: None available.

RNA binding protein HuR posttranscriptionally regulates CD4+ T cell inflammatory gene expression in asthma
Ulus Atasoy1, Fatemeh Fattahi1, Jason Ellis1, Kristin Bahleda1, Nerissa Reister1, Njira Lugogo1,
1 University of Michigan, Ann Arbor, MI.

Due to poor correlation between steady-state mRNA levels and protein product, transcriptomic analyses may miss critical genes controlling inflammation. Many genes are regulated posttranscriptionally at levels of mRNA stability and translation by RNA-binding proteins (RBPs) and miRNAs, however this is not well understood. Pro-inflammatory genes which play pivotal roles in airway inflammation usually have labile mRNA transcripts and are regulated posttranscriptionally. Using novel RIP-Seq methods, we have uncovered how RBP HuR (elavl1) regulates key genes involved in CD4+ Th subset differentiation since it binds to and regulates gata3 and Th2 cytokine mRNAs. HuR regulates inflammatory genes allowing for lung inflammation in asthma. We previously demonstrated that HuR overexpression in CD4+ T cells results in increases in Th2 cytokine production. Conditional ablation of HuR in T cells (distal Lck-cre HuRfl/fl), abrogates Th2 differentiation, cytokine production and lung inflammation in ova challenge model. We hypothesized that HuR may similarly regulate lung inflammation in human asthma. We discovered that HuR protein expression is greatly increased (100%) in peripheral CD4+ T cells from asthmatics (both type 2 high and low) compared with healthy individuals. Asthmatic PBLs have increased frequency and production of both Th2/Th17 signature cytokines. Using a drug (acadesine aka AICAR) which interferes with HuR function, we show that CD4+ T cells treated with acadesine have decreases in Th2/17 cytokine expression. Taken together, these data suggest that HuR plays a permissive role in both allergen and non-allergen driven airway inflammation by regulating key genes and that interfering with its function may be a novel way to treat asthma.

Author(s):