Rhinitis, sinusitis, and ocular allergy
Cystatin SN is a potent upstream initiator of epithelial-derived type 2 inflammation in chronic rhinosinusitis

https://doi.org/10.1016/j.jaci.2022.04.034Get rights and content

Background

Cystatin SN (CST1) and cystatin SA (CST2) are cysteine protease inhibitors that protect against allergen, viral, and bacterial proteases. Cystatins are overexpressed in the setting of allergic rhinitis and chronic rhinosinusitis with nasal polyps (CRSwNP); however, their role in promoting type 2 inflammation remains poorly characterized.

Objective

The purpose of this study was to use integrated poly-omics and a murine exposure model to explore the link between cystatin overexpression in CRSwNP and type 2 inflammation.

Methods

In this institutional review board– and institutional animal care and use committee–approved study, we compared tissue, exosome, and mucus CST1 and CST2 between CRSwNP and controls (n = 10 per group) by using matched whole exome sequencing, transcriptomic, proteomic, posttranslational modification, histologic, functional, and bioinformatic analyses. C57/BL6 mice were dosed with 3.9 μg/mL of CST1 or PBS intranasally for 5 to 18 days in the presence or absence of epithelial ABCB1a knockdown. Inflammatory cytokines were quantified by using Quansys multiplex assays or ELISAs.

Results

Of the 1305 proteins quantified, CST1 and CST2 were among the most overexpressed protease inhibitors in tissue, exosome, and mucus samples; they were localized to the epithelial layer. Multiple posttranslational modifications were identified in the polyp tissue. Exosomal CST1 and CST2 were strongly and significantly correlated with eosinophils and Lund-Mackay scores. Murine type 2 cytokine secretion and TH2 cell infiltration increased in a time-dependent manner following CST1 exposure and was abrogated by epithelial knockdown of ABCB1a, a regulator of epithelial cytokine secretion.

Conclusion

CST1 is a potent upstream initiator of epithelial-derived type 2 inflammation in CRSwNP. Therapeutic strategies targeting CST activity and its associated posttranslational modifications deserve further interrogation.

Section snippets

Patient tissue and mucus sampling

Tissue and mucus sampling were approved by the Massachusetts Eye and Ear Infirmary institutional review board. Matched tissue and mucus exosomal samples were taken from patients who were undergoing sinonasal surgery and had not been exposed to antibiotics or any topical/systemic steroids for at least 4 weeks. In accordance with the inclusion criteria, the study population consisted of patients diagnosed with CRSwNP (as specified by the international consensus statement on allergy and rhinology

Proteomic and transcriptomic expression analyses

Among the entire 1,305 protein SOMAscan array of biologically relevant immune and inflammatory proteins, CST1 and CST2 were the seventh and second most overexpressed mucosal proteins between the samples from patients with CRSwNP (mean ± SD = 52,214 ± 23,857 relative fluorescence units [RFU] and 67,820 ± 52,932 RFU, respectively) and the controls (mean ± SD = 7,735 ± 14,377 RFU and 4,427 ± 10,345 RFU, respectively) (fold change [FC] = 6.75 [P = 6.70 × 10–5] and 15.31 [P = 1.72 × 10–4],

Discussion

Integrated poly-omics refers to a biologic analysis in which multiple omics data sets may be combined through bioinformatics approaches to identify biomarkers and unravel the complex geno-pheno-enviro–type relationships even with low sample numbers.40,41 Our analysis confirmed that both CST1 and CST2 are profoundly overexpressed in CRSwNP at both the protein and mRNA levels, are localized to the epithelial surface, and may be sampled from tissue, whole mucus, and mucus-derived exosomes.

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    Supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke (grant 1R01NS108968-01 [to B.S.B.]).

    Disclosure of potential conflict of interest: B. S. Bleier has received grants from the MEEI Curing Kids Fund, Cook Medical, Medtronics; has consultant arrangements with Olympus, Medtronics, 3D Matrix, Third Wave Therapeutics, Bear-ENT, and Karl Storz; has provided expert testimony on ear, nose, and throat–related cases; has a patent for P-gp and cystatin inhibition for chronic rhinosinusitis and receives royalties from this patent; and has stock and/or stock options in Interscope, Inquis Medical, and Diceros Therapeutics. The rest of the authors declare that they have no relevant conflicts of interest.

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