isolation-quantitative-evaluation-brush-cells-from-mouse

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...

Harvard Medical School

Cysteinyl leukotrienes (cysLTs) facilitate mucosal type 2 immunopathology by incompletely understood mechanisms. Aspirin-exacerbated respiratory disease, a severe asthma subtype, is characterized by exaggerated eosinophilic respiratory inflammation and reactions to aspirin, each involving the marked overproduction of cysLTs. Here we demonstrate that the type 2 cysLT receptor (CysLTR), which is not targeted by available drugs, is required in two different models to amplify eosinophilic airway inflammation via induced expression of IL-33 by lung epithelial cells. Endogenously generated cysLTs induced eosinophilia and expanded group 2 innate lymphoid cells (ILC2s) in aspirin-exacerbated respiratory disease-like  mice. These responses were mitigated by deletions of either  or leukotriene C synthase (). Administrations of either LTC (the parent cysLT) or the selective CysLTR agonist N-methyl LTC to allergen sensitized wild-type mice markedly boosted ILC2 expansion and IL-5/IL-13 generation in a CysLTR-dependent manner. Expansion of ILC2s and IL-5/IL-13 generation reflected CysLTR-dependent production of IL-33 by alveolar type 2 cells, which engaged in a bilateral feed-forward loop with ILC2s. Deletion of  blunted LTC-induced ILC2 expansion and eosinophilia but did not alter IL-33 induction. Pharmacological blockade of CysLTR prior to inhalation challenge of  mice with aspirin blocked IL-33-dependent mast cell activation, mediator release, and changes in lung function. Thus, CysLTR signaling, IL-33-dependent ILC2 expansion, and IL-33-driven mast cell activation are necessary for induction of type 2 immunopathology and aspirin sensitivity. CysLTR-targeted drugs may interrupt these processes.

Type 2 Cysteinyl Leukotriene Receptors Drive IL-33-Dependent Type 2 Immunopathology and Aspirin Sensitivity.

Barrier tissue dysfunction is a fundamental feature of chronic human inflammatory diseases. Specialized subsets of epithelial cells-including secretory and ciliated cells-differentiate from basal stem cells to collectively protect the upper airway. Allergic inflammation can develop from persistent activation of type 2 immunity in the upper airway, resulting in chronic rhinosinusitis, which ranges in severity from rhinitis to severe nasal polyps. Basal cell hyperplasia is a hallmark of severe disease, but it is not known how these progenitor cells contribute to clinical presentation and barrier tissue dysfunction in humans. Here we profile primary human surgical chronic rhinosinusitis samples (18,036 cells, n = 12) that span the disease spectrum using Seq-Well for massively parallel single-cell RNA sequencing, report transcriptomes for human respiratory epithelial, immune and stromal cell types and subsets from a type 2 inflammatory disease, and map key mediators. By comparison with nasal scrapings (18,704 cells, n = 9), we define signatures of core, healthy, inflamed and polyp secretory cells. We reveal marked differences between the epithelial compartments of the non-polyp and polyp cellular ecosystems, identifying and validating a global reduction in cellular diversity of polyps characterized by basal cell hyperplasia, concomitant decreases in glandular cells, and phenotypic shifts in secretory cell antimicrobial expression. We detect an aberrant basal progenitor differentiation trajectory in polyps, and propose cell-intrinsic, epigenetic and extrinsic factors that lock polyp basal cells into this uncommitted state. Finally, we functionally demonstrate that ex vivo cultured basal cells retain intrinsic memory of IL-4/IL-13 exposure, and test the potential for clinical blockade of the IL-4 receptor α-subunit to modify basal and secretory cell states in vivo. Overall, we find that reduced epithelial diversity stemming from functional shifts in basal cells is a key characteristic of type 2 immune-mediated barrier tissue dysfunction. Our results demonstrate that epithelial stem cells may contribute to the persistence of human disease by serving as repositories for allergic memories.

Allergic inflammatory memory in human respiratory epithelial progenitor cells.

Respiratory epithelial cells (EpCs) orchestrate airway mucosal inflammation in response to diverse environmental stimuli, but how distinct EpC programs are regulated remains poorly understood. Here, we report that inhalation of aeroallergens leads to expansion of airway brush cells (BrCs), specialized chemosensory EpCs and the dominant epithelial source of interleukin-25 (IL-25). BrC expansion was attenuated in mice lacking either LTC synthase, the biosynthetic enzyme required for cysteinyl leukotriene (CysLT) generation, or the EpC receptor for leukotriene E (LTE), CysLTR. LTE inhalation was sufficient to elicit CysLTR-dependent BrC expansion in the murine airway through an IL-25-dependent but STAT6-independent signaling pathway. Last, blockade of IL-25 attenuated both aeroallergen and LTE-elicited CysLTR-dependent type 2 lung inflammation. These results demonstrate that CysLTR senses the endogenously generated lipid ligand LTE and regulates airway BrC number and function.

Eri Yoshimoto

Division of Rheumatology,
Allergy and Immunology,
Brigham and Women's Hospital,
Division of Rheumatology, Allergy and Immunology, Brigham and Women's Hospital

Harvard Medical School

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Eri Yoshimoto

Division of Rheumatology,Allergy and Immunology,Brigham and Women's Hospital,Division of Rheumatology, Allergy and Immunology, Brigham and Women's Hospital

Harvard Medical School

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Division of Rheumatology,
Allergy and Immunology,
Brigham and Women's Hospital,
Division of Rheumatology, Allergy and Immunology, Brigham and Women's Hospital