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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.

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Background Prostaglandin D2 (PGD2) is the dominant cyclooxygenase product of mast cells and is an effector of aspirin-induced respiratory reactions in aspirin-exacerbated respiratory disease (AERD). Objective We evaluated the role of the innate cytokine thymic stromal lymphopoietin (TSLP) acting on mast cells to generate PGD2 and facilitate tissue eosinophilia and nasal polyposis in AERD. Methods Urinary eicosanoids were measured in aspirin-tolerant controls and patients with AERD. Nasal polyp specimens from subjects with AERD and chronic rhinosinusitis were analyzed via qPCR, western blot, and immunohistochemistry. Human cord blood-derived and peripheral blood-derived mast cells were stimulated with TSLP in vitro to assess PGD2 generation. Results Urinary levels of a stable PGD2 metabolite (uPGD-M) were 2-fold higher in subjects with AERD relative to controls, and increased further during aspirin-induced reactions. Peak uPGD-M levels during aspirin reactions correlated with reductions in blood eosinophil counts and lung function, and with increases in nasal congestion. Mast cells sorted from nasal polyps expressed PGD2 synthase (hPGDS) mRNA at higher levels than did eosinophils from the same tissue. Whole nasal polyp TSLP mRNA expression correlated strongly with mRNA encoding hPGDS (r = .75), the mast cell-specific marker carboxypeptidase A3 (r = .74), and uPGD-M (r=0.74). The cleaved, active form of TSLP was increased in AERD nasal polyps relative to aspirin-tolerant controls. Recombinant TSLP induced PGD2 generation by cultured human mast cells. Conclusions Our study demonstrates that mast cell-derived PGD2 is a major effector of type 2 immune responses driven by TSLP, and suggests that dysregulation of this innate system contributes significantly to the pathophysiology of AERD.

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Background: The cause of severe nasal polyposis in aspirinexacerbated respiratory disease (AERD) is unknown. Elevated antibody levels have been associated with disease severity in nasal polyps, but upstream drivers of local antibody production in nasal polyps are undetermined. Objective: We sought to identify upstream drivers and phenotypic properties of local antibody-expressing cells in nasal polyps from subjects with AERD. Methods: Sinus tissue was obtained from subjects with AERD, chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), CRS without nasal polyps, and controls without CRS. Tissue antibody levels were quantified via ELISA and immunohistochemistry and were correlated with disease severity. Antibody-expressing cells were profiled with single-cell RNA sequencing, flow cytometry, and immunofluorescence, with IL-5Ra function determined through IL-5 stimulation and subsequent RNA sequencing and quantitative PCR. Results: Tissue IgE and IgG4 levels were elevated in AERD compared with in controls (P < .01 for IgE and P < .001 for IgG4 vs CRSwNP). Subjects with AERD whose nasal polyps recurred rapidly had higher IgE levels than did subjects with AERD, with slower regrowth (P 5 .005). Single-cell RNA sequencing revealed increased IL5RA, IGHG4, and IGHE in antibodyexpressing cells from patients with AERD compared with antibody-expressing cells from patients with CRSwNP. There were more IL-5Ra 1 plasma cells in the polyp tissue from those with AERD than in polyp tissue from those with CRSwNP (P 5 .026). IL-5 stimulation of plasma cells in vitro induced changes in a distinct set of transcripts. Conclusions: Our study identifies an increase in antibodyexpressing cells in AERD defined by transcript enrichment of IL5RA and IGHG4 or IGHE, with confirmed surface expression of IL-5Ra and functional IL-5 signaling. Tissue IgE and IgG4 levels are elevated in AERD, and higher IgE levels are associated with faster nasal polyp regrowth. Our findings suggest a role for IL-5Ra 1 antibody-expressing cells in facilitating local antibody production and severe nasal polyps in AERD.

Mast cells (MCs) play a pathobiologic role in type 2 (T2) allergic inflammatory diseases of the airway, including asthma and chronic rhinosinusitis with nasal polyposis (CRSwNP). Distinct MC subsets infiltrate the airway mucosa in T2 disease, including subepithelial MCs expressing the proteases tryptase and chymase (MCTC) and epithelial MCs expressing tryptase without chymase (MCT). However, mechanisms underlying MC expansion and the transcriptional programs underlying their heterogeneity are poorly understood. Here, we use flow cytometry and single-cell RNA-sequencing (scRNA-seq) to conduct a comprehensive analysis of human MC hyperplasia in CRSwNP, a T2 cytokine–mediated inflammatory disease. We link discrete cell surface phenotypes to the distinct transcriptomes of CRSwNP MCT and MCTC, which represent polarized ends of a transcriptional gradient of nasal polyp MCs. We find a subepithelial population of CD38highCD117high MCs that is markedly expanded during T2 inflammation. These CD38highCD117high MCs exhibit an intermediate phenotype relative to the expanded MCT and MCTC subsets. CD38highCD117high MCs are distinct from circulating MC progenitors and are enriched for proliferation, which is markedly increased in CRSwNP patients with aspirin-exacerbated respiratory disease, a severe disease subset characterized by increased MC burden and elevated MC activation. We observe that MCs expressing a polyp MCT–like effector program are also found within the lung during fibrotic diseases and asthma, and further identify marked differences between MCTC in nasal polyps and skin. These results indicate that MCs display distinct inflammation-associated effector programs and suggest that in situ MC proliferation is a major component of MC hyperplasia in human T2 inflammation.