Epithelial Dysregulation in Chronic Rhinosinusitis with Nasal Polyposis (CRSwNP) and Aspirin-Exacerbated Respiratory Disease (AERD)

The airway epithelium plays a central role in the initiation and perpetuation of type 2 (T2) inflammation in eosinophilic chronic rhinosinusitis with nasal polyps (eCRSwNP) and aspirin-exacerbated respiratory disease (AERD). T2 cytokine-driven airway epithelial remodeling leads to overexpression of proinflammatory products, loss of protective mucosal factors, and dysregulated anti-viral programs that sustain tissue inflammation. While an emerging literature demonstrates heterogeneous endotypes in both CRSwNP and AERD, the discussion here will review recent findings on airway epithelial programs in the dominant T2 endotype, which itself is heterogeneous, but can be successfully treated with complete sinus surgery and biologics targeting T2 inflammation.

AIRWAY EPITHELIAL REMODELING IN CHRONIC INFLAMMATION

Barrier epithelial cells (EpCs) display remarkable plasticity which is essential for maintenance of homeostatic function in response to tissue stress and restoration of barrier integrity in wound repair. This plasticity also endows them with the capacity to adapt tissue programs and remodel the tissue microenvironment for productive ends. Thus, in response to helminth-elicited IL-13 generation, extensive goblet cell metaplasia in the intestine contributes to mucus production that is required for successful pathogen elimination. While epithelial adaptation to temporally- and spatially-restricted environmental cues underlies productive tissue patterning, which features of remodeling are beneficial in the context of diverse barrier stresses and how these programs are regulated in the face of chronic inflammation are poorly understood.

In the respiratory tract, significant alterations in the abundance of airway EpC subsets are observed in diverse respiratory diseases associated with chronic inflammation. These changes extend beyond goblet cell metaplasia in asthma and include an expansion of ciliated cells in cystic fibrosis, a novel mucociliary state detected in asthma, an increase in IL-25-secreting tuft cells in allergic fungal rhinosinusitis, an increase in neuroendocrine cells in asthma and eosinophilic granuloma/pulmonary Langerhans cell histiocytosis, loss of glandular epithelium in CRSwNP, and alterations in basal cell numbers and phenotype in chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) (Table 1). In nasal polyposis, chemosensory/tuft cells (1) and basal cells (2) are more abundant and basal cells fail to differentiate normally, endowing the epithelium with the potential for enhanced proinflammatory function, as detailed below.

Table 1.

Altered abundance of airway epithelial subsets in diverse respiratory diseases associated with chronic inflammation

Cell Type	Findings	Reference
Basal cell	Expansion of basal cell number in chronic rhinosinusitis with nasal polyposis	Ordovas-Montanes et al. Nature 2018;560:649–654
	Detection of a novel basaloid cell in patients with IPF and COPD	Adams et al. Sci. Adv 2020;6: eaba1983
	Reviews the origins of basal cell hyperplasia in COPD	Crystal. Am J Respir Crit Care Med 2014;190(12):1355–1362
Chemosensory Epithelial Cells (tuft, brush, solitary chemosensory cells)	Expansion of solitary chemosensory cells in the inflamed sinus tissue from patients with allergic fungal rhinosinusitis	Patel et al. Int Forum Allergy Rhinol 2019;9(7):730–737
	Expansion of IL-25 producing solitary chemosensory cells in CRSwNP	Patel et al. Int Forum of Allergy Rhinol 2018;8(8):900–906 Kohanski et al. J Allergy Clin Immunol 2018; 142(2):460–469
	Expansion of lung tuft cells in the recovery phase of murine influenza	Rane et al, Am J Physiol Lung Cell Mol Physiol. 2019;316(6):L1141-L9.
	Expansion of tracheal tuft cells in murine models of allergen-elicited inflammation (house dust mite and Alternaria)	Bankova et al. Sci Immunol. 2018;3(28).
Ciliated Cells	Expansion of ciliated cells in cystic fibrosis	Carraro et al. Nat Med 2021;27: 806–814
	Development of a novel muco-ciliated cell state in asthma	Vieira-Braga et al. Nat Med 2018;25:1153–1163
Pulmonary Neuroendocrine Cells	Expansion of pulmonary neuroendocrine cells in lung biopsies of patients with eosinophilic granuloma /pulmonary langerhans cell histiocytosis (PLCH)	Aguayo et al. J Clin Invest 1990;86(3):838–44.
	Expansion of pulmonary neuroendocrine cells in asthma	Sui et al. Science 2018;360: eaan8546
Glandular EpCs	Loss of glandular epithelium in CRSwNP	Seshadri et al. Allergy 2012;67(7):920–8.

REMODELING IN NASAL POLYPOSIS AUGMENTS THE INFLAMMATORY CAPACITY OF THE EPITHELIUM

Basal cells produce the innate T2 cytokine thymic stromal lymphopoietin (TSLP) which is dysregulated in both CRSwNP and AERD. TSLP levels are higher in polyp tissue from AERD as compared to CRS (3). Single cell RNA-sequencing (scRNA-seq) demonstrates basal cells are the dominant site of TSLP expression and bulk sequencing of sorted basal cells demonstrates a > 8-fold upregulation of TSLP in basal cells from CRSwNP and AERD as compared to CRSsNP (2). As TSLP induces mast cell generation of prostaglandin D₂ (PGD₂) and is correlated with both mast cell number and PGD₂ metabolites detected in the urine of patients with AERD, these results suggest that increases in basal cell number and cytokine expression promote mast cell activation in polyp tissue (3).

IL-33 is also generated in basal cells, and increased in basal cells from patients with CRSwNP and AERD, as compared to CRSsNP (2). While IL-33 levels are reported to be elevated in some, but not all studies of CRSwNP, a large genome-wide association study from Iceland and the United Kingdom reported a gain-of-function variant in the regulatory region of IL33 that is associated with nasal polyposis (4), suggesting a pathobiologic role in disease. Through this expression, basal cells have the potential to activate polyp mast cells, pathogenic effector Th2 cells, and type 2 innate lymphoid cells (ILC2s), all of which express the IL-33 receptor. Notably, basal cell expression of IL33 and TSLP in CRSwNP and AERD is highly correlated with transcription factors that regulate basal cell differentiation, suggesting a close link between basal cell state and proinflammatory potential that deserves further attention.

Chemosensory/tuft EpCs are also expanded in CRSwNP, where they are the dominant site of IL-25 expression (1). These cells also have the capacity to generate cysteinyl leukotrienes (CysLTs) (5) which promote vascular permeability and inflammation and are detected at higher levels in the sinus tissue and urine of patients with AERD and CRSwNP, as compared to controls. Notably, clinically relevant fungal antigens including Aspergillus and Alternaria (6) drive tuft cell hyperplasia in an innate and IgE-independent fashion, and murine models demonstrate tuft cell expansion in the lung and trachea in response to influenza and allergens respectively (Table 1), suggesting that heterogeneous drivers of chemosensory cell expansion may promote airway remodeling and a common pro-inflammatory T2 epithelial program.

An additional proinflammatory axis that emerges in nasal polyposis is ALOX15 and its 15-lipoxygenase protein which are expressed in EpCs, and more abundantly expressed in EpCs from AERD > CRSwNP > controls (2, 7, 8). This enzyme regulates expression of CCL26 (7), providing a mechanism by which EpCs can recruit eosinophils and promote goblet cell metaplasia. Furthermore, subjects with a loss of function variant in ALOX15 are protected from development of CRSwNP (4), highlighting the importance of this lipid biosynthetic pathway in these diseases. Notably, EpC 15-lipoxygenase generates 15-hydroxyeicosatetraenoic acid (15-HETE) which is metabolized by mast cells to 15-oxo-eicosatetraenoic acid, a metabolite detected at higher levels in AERD and CRSwNP, as compared to controls (8). This suggests a third epithelial-mast cell axis in nasal polyposis and highlights that detection of transcellular interactions will be essential to understanding the novel inflammatory pathways that develop in remodeled epithelium. The application of new technologies such as spatial transcriptomics should greatly advance our detection of these novel stromal – immune interactions that emerge in disease.

EPC RESPONSIVENESS TO IL-4 AND IL-13

IL-4 and IL-13 stimulation of the airway epithelium can expand basal and chemosensory cell numbers and upregulate TSLP, IL33, and ALOX15, suggesting a prominent role for IL-4 and IL-13 in ‘driving’ functional changes in the epithelium that are germane to disease. Moreover, expression analysis demonstrates a shared IL-4/IL-13 gene signature that is enriched in airway EpCs from CRSwNP and AERD, as compared to CRSsNP, and a reduction in EpC proinflammatory genes after successful treatment with Dupilumab (2). However, key data suggests that some epithelial changes are not simply the result of increased IL-4 or IL-13, but rather EpC-intrinsic defects of a genetic or epigenetic fashion that promote exaggerated responses to these T2 cytokines. For example, IL-13 elicits expansion of chemosensory EpCs and IL-25 generation in epithelial air-liquid interface cultures derived from nasal polyposis but not from control tissues (1). Similarly, basal cells from patients with polyposis, but not control, exhibit increased expression of IL-4- and IL-13-elicited Wnt pathway genes and altered proliferation despite weeks of ex vivo passage (2), again signaling a defect in airway EpCs that persists outside the inflamed environment of the sinonasal mucosa. Finally, variants in epithelial genes such as ALOX15 and IL33 are associated with risk of nasal polyposis (4), also suggesting that intrinsic defects in the epithelium likely influence responsiveness to the T2 cytokine environment. Ultimately, careful studies of epithelial epigenetics in response to common immune and environmental stresses will be needed to tease out many of these associations.

ADDITIONAL EFFECTS OF T2 CYTOKINES ON THE AIRWAY EPITHELIUM

IL-13 has additional well-described roles on airway EpCs that are highly relevant to nasal polyposis disease pathogenesis. This includes downregulation of prostaglandin E₂ (PGE₂) generation and other immunoregulatory proteins, reduction in innate immune proteins, reduced expression of tight junction proteins required for barrier defense, and impairments in antiviral immunity (Table 2). While these defects may not drive proinflammatory pathways directly, they may play a role in microbial colonization such as with Staphylococcus aureus or susceptibility to viral infections, each of which is posited to play a role in disease pathogenesis. Notably, single cell RNA-sequencing analysis of nasal EpC air-liquid interface cultures with chronic (>11 days) IL-13 treatment demonstrates an increase in interferon signaling-associated gene expression (Table 2). These results are consistent with the observation that those with allergic rhinitis or asthma had an increased interferon response following exposure to a TLR7/8 agonist (9) and that individuals with AERD have elevated levels of IFN-γ (10), suggesting a complex feedback between interferon regulated pathways and T2 cytokine signaling that requires further understanding.

Table 2.

Effects of IL-13 On Epithelial Innate, Barrier, and Antiviral Immunity

Effect	Findings	Reference
PGE2 Generation	IL-13 reduces expression of PGE2 biosynthetic enzymes (COX-2 and PGE synthase 1), and upregulates PGE2 metabolizing enzymes (15-PG dehydrogenase), consistent with a reduction in PGE2 in the supernatants of IL-13-treated air liquid interface cultures derived from bronchial brushings.	Trudeau et al. J Allergy Clin Immunol 2006;117(6):1446–54
Innate immunity	Using scRNA-seq and bulk sequencing of ALI cultures, this group demonstrated that IL-13 reduces transcripts encoding innate immune proteins such as S100A8, S100A9, SCGB1A1, BPIFA1, LTF, each of which was previously reported to be reduced in nasal polyposis.	Jackson et al. Cell Rep 2020;32(1): 107872
Antiviral responses	The same paper reported that while acute IL-13 stimulation of air-liquid interface cultures downregulates interferon signaling, ‘chronic’ 11-day stimulation increases it, highlighting the complex feedback networks existing between these pathways even in epithelial cells from control subjects.	Jackson et al. Cell Rep 2020;32(1): 107872
Antiviral responses	IL-13 reduces EpC expression of STING leading to impaired antiviral responses and enhanced IL-13 signaling.	Wang et al. J Allergy Clin Immunol, 2020;147(5):1692–1703
Barrier function	IL-13 reduces tight junction proteins in lung cell lines grown in air-liquid interface cultures	Ahdieh et al. Am J Physiol Cell Physiol 2001;281(6):C2029–38 Saatian et al, Tissue Barriers 2013;1(2):e2433

CONCLUSIONS

Airway EpC remodeling in CRSwNP and AERD extends far beyond goblet cell metaplasia, producing a network of dysregulated inflammatory pathways, alterations in EpC subsets, and an EpC-mast cell crosstalk that sustains T2 inflammation. Genetic, functional, and clinical studies demonstrate a major role for IL-4 and IL-13 in driving epithelial remodeling and suggest that patients with these diseases have increased EpC responsiveness to this signaling axis. While targeted therapy with Dupilumab has remarkable therapeutic efficacy, discontinuation leads to relapse within weeks, suggesting that durable local signals persist that recall effector cells to the sinus mucosa to reestablish inflammation and reestablish the remodeled airway. Thus, defining the transcriptional, epigenetic, and functional EpC changes that persist in treated patients will be essential to move from chronic therapy to cure.

Figure 1:

T2 Cytokine-Dependent Airway Remodeling in Nasal Polyposis. The airway epithelial barrier includes ciliated cells (tan), secretory club and goblet cells (pink), tuft/chemosensory EpCs (blue) and basal EpC progenitors (plum). Under the influence of IL-4 and IL-13, basal cells and tuft cells are expanded, increasing the epithelial potential to generate IL-33, TSLP, IL-25, and CysLTs. Additionally, ALOX15 is induced, which can promote tissue eosinophilia. Diverse immunoregulatory molecules are downregulated by IL-4 and IL-13, and detected at reduced levels in nasal polyp EpCs from CRSwNP and AERD. These include PGE₂ and STING.