[BAD] EK9 - Abstracts

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.

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.

Chronic unpredictable stress exacerbates allergic airway inflammation in mice
G. Dragunas*1,2,3, M.A. de Oliveira1, W. T. de Lima1, R. Gosens2,3, C.D. Munhoz1
 
1- Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo
2- Department of Molecular Pharmacology, University of Groningen
3 - Groningen Research Institute for Asthma and COPD (GRIAC), University Medical Center Groningen (UMCG), University of Groningen
 
Introduction
It is accepted that psychological stress can lead to asthma exacerbations (1). Amid COVID-19 pandemic, exposure to chronic and, hence, deleterious forms of psychological stress has become usual. Stress can be classically defined as a real or potential threat to one’s homeostasis, generating physiological responses, such as HPA and SNS axis activation (2). Studies in the literature are widely in agreement that stress induces neuroplastic changes in psychiatric disorders and some suggest that this might also happen in conditions as asthma (3,4). However, the knowledge concerning how stress increases asthma severity and if neuronal mechanisms play a role are scarce.
 
Methods
We applied a 12 days chronic unpredictable stress (CUS) paradigm in OVA sensitized mice followed by two daily OVA challenges to induce allergic airway inflammation. 24h after the last challenge, mice had lung functional parameters analyzed, were euthanized, bronchoalveolar lavage collected and the lungs and dorsal root ganglia (DRG) harvested. The tissues were submitted to histological and molecular assays.
 
Results
Exposure to 12 day-CUS increased cellular content recovered in BAL. This was paired to increased p65 NF-kB phosphorylation, TRPV1 and P2X3 receptors expression in DRG, but not in the lungs. Exposure to CUS before acute challenge to two OVA aerosol challenges significantly increased recovered cells in BAL. Opposite outcomes were observed after a single acute restraint stress (RS), as reduced cellularity in BAL and diminished airway resistance to methacholine. OVA+CUS group displayed increased NF-kB signaling and VCAM expression in the lungs.
 
Conclusions
Exposure to chronic stress can lead to allergic airway inflammation exacerbation in mice, whereas previous acute stress led to inflammation mitigation. Future experiments will determine differential cytokine and neurotrophic factor expression in the lungs and changes in innervation in the airways in response to chronic stress.
 
References
1.          Chen E, Miller GE. Stress and inflammation in exacerbations of asthma. Brain Behav Immun. 2007;21(8):993–9.
2.          de Kloet ER, Joëls M, Holsboer F. Stress and the brain: from adaptation to disease. Nat Rev Neurosci. 2005;6(6):463–75.
3.          Dragunas G, Woest ME, Nijboer S, Bos ST, van Asselt J, de Groot AP, et al. Cholinergic neuroplasticity in asthma driven by TrkB signaling. FASEB J. 2020;34(6):7703–17.
4.          Undem BJ, Taylor-Clark T. Mechanisms underlying the neuronal-based symptoms of allergy. J Allergy Clin Immunol. 2014;133(6):1521–34.
 

Epidermal growth factor receptor in airway remodeling during allergic airway disease – divergent roles during early life and adulthood?

H. Stölting, S. A. Walker, M. C. Zarcone, F. Puttur, S. Saglani, C. M. Lloyd
National Heart and Lung Institute, Imperial College London - London (United Kingdom)

Airway remodelling is a key pathological feature of paediatric and adult asthma, but the underlying mechanisms remain poorly understood. However, their elucidation is crucial, since lung function deficits established in children with asthma persist into adulthood. Epidermal growth factor receptor (EGFR) was shown to be overexpressed in paediatric and adult asthmatics. In addition, several in vivo studies using rodent models of allergic airway disease (AAD) have described a role for EGFR signalling in driving impaired lung function and airway remodelling in adult animals. Here, we aimed to study the role of EGFR in early life AAD. 
Bronchial epithelial cells from non-asthmatic children cultured at air-liquid interface were shown to exhibit high mRNA levels for EGFR and its ligands. Exposure of these cells to the allergen house dust mite induced EGFR activation dose-dependently, as measured by Y1068 phosphorylation. qPCR analysis of flow-sorted murine lung cell populations during postnatal development similarly showed high EGFR expression in murine lung epithelial cells from neonatal and adult mice, and lung epithelial EGFR expression was confirmed by flow cytometry. Finally, a pharmacological inhibitor was used to block EGFR signalling in a neonatal model of AAD. Preliminary findings indicate that EGFR inhibition in neonatal mice resulted in worsened lung function, as measured by a 2-fold increase in airway resistance (AUC), without affecting overall inflammation, a finding we did not observe in a corresponding adult AAD model.
These results indicate that EGFR is present in lungs at all stages of life and that, in contrast to its widely described pathogenic contribution to airway remodelling of adult animals, signalling through EGFR may play a protective role during early life AAD. 

Cannabis compounds have both anti-inflammatory and pro-inflammatory activities in lung epithelial and macrophages while substantially increasing phagocytosis in vitro

Seegehalli M Anil1+, Nurit Shalev1+, Ajjampura C Vinayaka1+, Stalin Nadarajan1+, Dvory Namdar1, Eduard Belausov1, Irit Shoval2, Karthik Ananth Mani3, Guy Mechrez3, Hinanit Koltai1*
1 Institute of Plant Science, Agriculture Research Organization, Volcani Center, Rishon LeZion 7528809, Israel
2 The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel.
3 Institute for Postharvest and Food Science, Agriculture Research Organization, Volcani Center, Rishon LeZion 7528809, Israel
 
+ These authors contributed equally to this work
Cannabis sativa is used worldwide for medical purposes and is known to have anti-inflammatory activity, yet the potential for use of C. sativa compounds against Coronavirus disease 2019 (COVID-19)-like inflammation is unexplored. The purpose of this study was to examine the anti-inflammatory activity of cannabis on markers of immune responses associated with COVID-19 inflammation. An extract fraction from high cannabidiol (CBD) cannabis strain (FCBD) substantially reduced dose dependently interleukin 6 (IL-6) and interleukin-8 (IL-8) levels in an alveolar epithelial (A549) cell line. FCBD contained CBD, cannabigerol (CBG) and tetrahydrocannabivarin (THCV), and multiple terpenes. Treatments with FCBD and phytocannabinoid standards that compose FCBD (FCBD:std) reduced in a dose dependent way IL-6, IL-8, C-C Motif Chemokine Ligands (CCLs) 2 and 7 in the A549 cell line. It also reduced expression of angiotensin I converting enzyme 2 (ACE2), a receptor for SARS-CoV-2. Treatment with FCBD induced macrophage (differentiated KG1 cell line) polarization and phagocytosis in vitro, and increased expression of scavenger receptor CD36 and that of type II receptor for the Fc region of IgG (FcγRII). FCBD treatment also substantially increased IL-6 and IL-8 expression in macrophages. FCBD:std, while maintaining the anti-inflammatory activity in alveolar epithelial cells, led to reduced pro-inflammatory IL secretion in macrophages in comparison to FCBD and reduced level of phagocytosis. The phytocannabinoid mixture may show superior activity for reduction of lung inflammation over that of the cannabis fraction. Yet, as for now, users and healthcare personnel should avoid the use of cannabis for COVID-19 prevention or treatment.

MicroRNA regulation of airway mucus production

Siddiqui S*1,2, Johansson K*1,2,3, Joo A1,2, Bonser LR4,5, Koh KD4,5, Le Tonqueze O4,5, Bolourchi S1,2, Bautista RA1,2, Zlock L6, Roth TL3,7,8,9, Marson A3,9,10,11,12,13, Bhakta NR1, Ansel KM2,3, Finkbeiner WE6, Erle DJ4,5, Woodruff PG1,2,5
1 Department of Medicine, Division of Pulmonary and Critical Care Medicine, 2Sandler Asthma Basic Research Center, 3Department of Microbiology and Immunology, 4Lung Biology Center,  5Cardiovascular Research Institute, 6Department of Pathology, 7Biomedical Sciences Graduate Program,  8Diabetes Center, 9Innovative Genomics Institute, University of California, Berkeley, 10J. David Gladstone Institutes, 11Department of Medicine, Division of Infectious Diseases, 12Parker Institute for Cancer Immunotherapy and 13Chan Zuckerberg Biohub, San Francisco, CA, USA
* Equal contribution
 
Rational: Interleukin (IL)-13-induced goblet cell metaplasia contributes to airway remodeling and pathological mucus hypersecretion in asthma. MicroRNAs (miRNAs) are a distinct class of noncoding RNAs, about 20-22 nucleotides long, that mediate sequence-specific repression of target mRNAs. Cellular responses are modulated by miRNAs but their role in mucus regulation is largely unexplored. We hypothesized that airway epithelial miRNAs play a role in IL-13-induced mucus regulation.
Methods: We performed CRISPR/Cas9-editing of primary human bronchial epithelial cells (HBECs) to target a specific miRNA candidate, miR-141, by delivery of MIR141-targeting guide RNAs (gRNA) via electroporation. HBECs that received MIR141 gRNAs or non-targeting gRNA control were differentiated at air-liquid-interface (ALI) and epithelial mucus was induced by IL-13 stimulation.
Results: miR-141, a member of the miR-141/200 family, was identified as one of the most highly expressed miRNAs in human airway epithelium by miRNA sequencing. Analysis of bronchial brushings from asthmatic subjects revealed that miR-141 is reduced at baseline in asthma but is induced shortly after airway allergen challenge. Knock down of miR-141 resulted in decreased goblet cells frequency, intracellular MUC5AC and total secreted mucus. These effects correlated with a reduction in a goblet cell gene expression signature and enrichment of a basal cell gene expression signature defined by single cell RNA sequencing. Furthermore, intranasal administration of a sequence-specific miR-141 inhibitor in mice decreased Aspergillus-induced secreted mucus and mucus-expressing cells in the lung, and reduced airway hyper-responsiveness without affecting cellular inflammation.
Conclusions: We have identified a miRNA that regulates pathological airway mucus production in human cells and in mice and is amenable to therapeutic manipulation through an inhaled route.  

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.

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.

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.

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.