Reduced Expression of Antimicrobial Protein Secretory Leukoprotease Inhibitor and Clusterin in Chronic Rhinosinusitis with Nasal Polyps

Abstract

Introduction: Antimicrobial peptides and proteins (AMPs) constitute the first line of defense against pathogenic microorganisms in the airway. The association between AMPs and chronic rhinosinusitis with nasal polyps (CRSwNP) requires further investigations. This study is aimed at investigating the expression and regulation of major dysregulated AMPs in the nasal mucosa of CRSwNP.

Methods: The expression of AMPs was analyzed in nasal tissue from patients with eosinophilic (E) CRSwNP and nonECRSwNP and healthy subjects using RNA sequencing. The 10 most abundant AMPs expressed differentially in CRSwNP patients were verified by real-time PCR, and of these, the expression and regulation of secretory leukoprotease inhibitor (SLPI) and clusterin (CLU) were investigated further.

Results: The 10 most abundant AMPs expressed differentially in CRSwNP compared to healthy control, regardless of subtypes, included BPIFA1, BPIFB1, BPIFB2, CLU, LTF, LYZ, and SLPI, which were downregulated, and S100A8, S100A9, and HIST1H2BC, which were upregulated. ELISA and immunofluorescence confirmed the decreased expression of SLPI and CLU levels in CRSwNP. SLPI is expressed in both nasal epithelial cells and glandular cells, whereas CLU is mainly expressed in glandular cells. AB/PAS staining further demonstrated that both SLPI and CLU were mainly produced by mucous cells in submucosal glands. Furthermore, the numbers of submucosal glands were significantly decreased in nasal polyp tissue of CRSwNP compared to nasal tissue of controls. SLPI was downregulated by TGF-β1 and IL-4 in cultured nasal tissues in vitro, while CLU expression was inhibited by TGF-β1. Glucocorticoid treatment for 2 weeks significantly increased the expression of all downregulated AMPs, but not LYZ. Additionally, budesonide significantly increased the expression of SLPI and CLU in cultured nasal tissues.

Conclusion: The expression of major antimicrobial proteins is significantly decreased in nasal tissue of CRSwNP. The expression of SLPI and CLU is correlated with the numbers of submucosal glands and regulated by inflammatory cytokines and glucocorticoids.

Figure 1

Expression of top abundant AMPs in nasal tissues. (a) Expression of the 10 most abundant AMPs in nasal tissues from ECRSwNP, nonECRSwNP, and healthy controls identified by RNA sequencing. The color coding of heat maps represents the gene expression level normalized to the control group, which is calculated based on Transcripts Per Million (TPM). A significant difference in the expression of BPIFA1 was found between the ECRSwNP and nonECRSwNP groups. (b–h) The expression of AMPs (BPIFA1, SLPI, BPIFB1, LYZ, LTF, BPIFB2, and CLU) in nasal tissue of ECRSwNP, nonECRSwNP, and healthy controls verified by real-time PCR. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. AMPs: antimicrobial peptides and proteins; CRSwNP: chronic rhinosinusitis with nasal polyps; ECRSwNP: eosinophilic CRSwNP; BPIFA: BPI fold-containing family A member; BPIFB: BPI fold-containing family B member; CLU: clusterin; LTF: lactoferrin; LYZ: lysozyme; SLPI: secretory leukoprotease inhibitor.

Figure 2

Expression of SLPI and CLU at the protein level in nasal tissues of CRSwNP. (a) Immunofluorescence staining of SLPI and CLU in nasal tissues from ECRSwNP, nonECRSwNP, and healthy controls. Epithelium area and submucosal glands were displayed at high magnification. Bars = 25 μm. (b, c) The expression of SLPI (b) and CLU (c) in nasal secretions of ECRSwNP, nonECRSwNP, and healthy controls detected by ELISA. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. CRSwNP: chronic rhinosinusitis with nasal polyps; ECRSwNP: eosinophilic CRSwNP; nonECRSwNP: noneosinophilic CRSwNP; CLU: clusterin; SLPI: secretory leukoprotease inhibitor.

Figure 3

Evaluation of sequential staining for AB/PAS, SLPI, and CLU in nasal tissues. AB/PAS staining and immunohistochemical staining for SLPI and CLU were performed on serial sections of nasal tissues from ECRSwNP, nonECRSwNP, and healthy controls. Bars = 50 μm. ECRSwNP: eosinophilic CRSwNP; nonECRSwNP: noneosinophilic CRSwNP; CLU: clusterin; SLPI: secretory leukoprotease inhibitor.

Figure 4

The expression of AMPs in nasal tissues of CRSwNP in response to glucocorticoid treatment. Patients with CRSwNP were treated with 2-week oral glucocorticoid. The expression of AMPs (BPIFA1, SLPI, BPIFB1, LYZ, LTF, BPIFB2, and CLU) was detected in nasal polyp tissues before and after glucocorticoid treatment by real-time PCR. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. AMPs: antimicrobial peptides and proteins; CRSwNP: chronic rhinosinusitis with nasal polyps; BPIFA: BPI fold-containing family A member; BPIFB: BPI fold-containing family B member; CLU: clusterin; LTF: lactoferrin; LYZ: lysozyme; SLPI: secretory leukoprotease inhibitor.

Figure 5

The expression of SLPI and CLU is correlated with the numbers of submucosal glands. (a) The number of submucosal glands per high power field (×200 magnifications) in the nasal mucosa of ECRSwNP, nonECRSwNP, and healthy controls. (b, c) Correlations between the gland counts and the expression of SLPI and CLU. (d) AB/PAS staining shows goblet cell hyperplasia in the epithelial mucosa of ECRSwNP, nonECRSwNP, and healthy controls. Bars = 50 μm. ^∗∗∗P < 0.001. ECRSwNP: eosinophilic CRSwNP; nonECRSwNP: noneosinophilic CRSwNP.

Figure 6

Regulation of SLPI and CLU by cytokines and glucocorticoid. (a–d) The expression level of SLPI (a, c) and CLU (b, d), detected by real-time PCR, in cultured human nasal tissues and A549 cells after 48 hours of stimulation by cytokines or unstimulated control. (e) The upregulated mRNA level of SLPI and CLU in cultured nasal tissues after 48 hours of stimulation by budesonide. (f) Potential glucocorticoid response element in the upstream region of SLPI and CLU. ^∗P < 0.05; CLU: clusterin; SLPI: secretory leukoprotease inhibitor; BUD: budesonide; U.S.: unstimulated control; GRE: glucocorticoid response element; TSS: transcription start.