The use of AREG in eosinophilic inflammation of chronic sinusitis with nasal polyps

Abstract

The present invention relates to the field of biomedicine, and specifically, the present invention relates to the use of AREG in eosinophilic inflammation of chronic sinusitis with nasal polyps. The present invention discovered for the first time that nasal epithelial cells, especially basal cells, are both the cell source of AREG and one of the important target cells of AREG. AREG promotes the proliferation of epithelial basal cells and inhibits the differentiation of basal cells into ciliated cells, resulting in structural abnormalities and functional damage to the epithelial barrier of human CRSwNP tissue. AREG chemotaxis and activation of human peripheral blood eosinophils, and its receptor EGFR antagonist Erlotinib can antagonize the effect of AREG on eosinophils. Neutralizing AREG secreted by epithelial cells or other inflammatory cells can effectively reduce the eosinophilic inflammatory response of the nasal mucosa in the CRSwNP mouse model and restore the damaged epithelial barrier function of the mouse nasal mucosa. It is proved that the use of neutralizing antibodies or shRNA against AREG or EGFR as antagonists can reduce the eosinophilic inflammatory response of CRSwNP by inhibiting the action of AREG.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of AREG in treating eosinophilic inflammation of chronic sinusitis with nasal polyps. Background Art

[0002] Chronic rhinosinusitis with nasal polyps (CRSwNP) is a clinical phenotype of chronic rhinosinusitis, with primary symptoms including nasal congestion, runny nose, head / facial pain, and hyposmia. Based on the number of eosinophils in tissues, CRSwNP is categorized as either eosinophilic CRSwNP (ECRSwNP) or non-eosinophilic CRSwNP (non-ECRSwNP). In my country, approximately half of CRSwNP patients have ECRSwNP. ECRSwNP patients often have more severe symptoms, asthma, and postoperative recurrence rates, placing a significant socioeconomic burden.

[0003] Amphiregulin (AREG) is a member of the epidermal growth factor (EGF) family. It binds to the epidermal growth factor receptor (EGFR) to activate downstream signaling pathways, participating in various biological processes in epithelial cells, such as proliferation, differentiation, and tissue repair. Recent studies have found that AREG can be secreted by various immune cells, including eosinophils, mast cells, and type 2 innate lymphocytes, and is closely associated with airway remodeling in eosinophilic inflammatory responses. AREG released by memory Th2 (T helper 2) cells can recruit eosinophils and mediate the fibrotic response of eosinophilic airway inflammation.

[0004] Existing studies have shown that AREG expression is elevated in ECRSwNP tissue, suggesting that AREG may be involved in the eosinophilic inflammatory response in CRSwNP. Eosinophil infiltration near the epithelium of CRSwNP patients is closely associated with disease severity, but no studies have yet suggested that epithelial cells can secrete AREG to recruit eosinophils and exacerbate the inflammatory response. Subepithelial inflammatory cells can also secrete AREG, but there is currently no evidence that neutralizing antibodies or shRNA targeting AREG or EGFR as antagonists can reduce the eosinophilic inflammatory response in CRSwNP by inhibiting the action of AREG. Summary of the Invention

[0005] The present invention first provides a use of AREG as a target in eosinophilic inflammation of chronic sinusitis with nasal polyps.

[0006] The present invention also provides a use of AREG as a target in the preparation of a drug for treating chronic sinusitis with nasal polyps and eosinophilic inflammation.

[0007] The present invention also provides the use of a preparation for inhibiting the expression or function of AREG in alleviating the eosinophilic inflammatory response of chronic sinusitis with nasal polyps, wherein the use is for non-therapeutic purposes in vitro.

[0008] In certain embodiments, the models include an epithelial cell air-liquid culture model, an organoid culture model, and a chronic sinusitis with nasal polyps mouse model.

[0009] The present invention also provides the use of a preparation for inhibiting the expression or action of AREG in preparing a medicine for treating chronic sinusitis with nasal polyps.

[0010] In certain embodiments, the agent for inhibiting AREG expression comprises one or more of an anti-AREG neutralizing antibody, an EGFR receptor antagonist, an anti-EGFR neutralizing antibody, and AREG-shRNA.

[0011] In certain embodiments, the preparation that inhibits the expression and action of AREG in the treatment of chronic sinusitis with nasal polyps can reduce the size of polyps, inhibit the infiltration of eosinophils and other inflammatory cells, and restore epithelial barrier function.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects:

[0013] The present invention discovered for the first time that nasal epithelial cells, especially basal cells, are both the cell source of AREG and one of the important target cells of AREG. AREG stimulates the CRSwNP epithelium, promotes the proliferation of epithelial basal cells and inhibits the differentiation of basal cells into ciliated cells, leading to structural abnormalities and functional damage of the epithelial barrier in CRSwNP patients. AREG chemotaxis and activation of human peripheral blood eosinophils, and its receptor EGFR antagonist Erlotinib can antagonize the effect of AREG on eosinophils. Neutralizing AREG secreted by epithelial cells or other inflammatory cells can effectively reduce the eosinophilic inflammatory response of the nasal mucosa in the CRSwNP mouse model and restore the damaged epithelial barrier function of the mouse nasal mucosa. It is proved that the use of neutralizing antibodies or shRNA of AREG or EGFR as antagonists can reduce the eosinophilic inflammatory response of CRSwNP by inhibiting the action of AREG. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A:The expression of amphiregulin AREG and its receptor EGFR in epithelial cells of CRSwNP tissue;

[0015] Figure 1B : To analyze the expression of amphiregulin AREG and its receptor EGFR in different epithelial cell types of CRSwNP tissue;

[0016] Figure 2A :The results of the air-liquid culture model of CRSwNP epithelial cells stimulated by AREG;

[0017] Figure 2B :AREG stimulates cell proliferation in normal mucosa and CRSwNP organoid models;

[0018] Figure 2C :The results of AREG stimulating cell differentiation in normal mucosa and CRSwNP organoid models;

[0019] Figure 3A :Effect of AREG on eosinophil chemotaxis;

[0020] Figure 3B :The effect of AREG on eosinophil activation was mainly evaluated by the expression of cell surface molecule CD11b;

[0021] Figure 4A : Effects of intranasal administration of anti-AREG neutralizing antibodies on the number of nasal polypoid lesions in the mouse CRSwNP model;

[0022] Figure 4B Effects of intranasal administration of anti-AREG neutralizing antibodies on eosinophilic inflammatory responses in a mouse CRSwNP model;

[0023] Figure 5 : Effects of intranasal administration of anti-AREG neutralizing antibodies on epithelial tight junction proteins in a mouse CRSwNP model. DETAILED DESCRIPTION

[0024] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0025] Example 1: Quantitative Detection of AREG and Its Receptor EGFR

[0026] The inventors detected the protein expression levels of AREG and its receptor EGFR in the epithelium of control mucosa, ECRSwNP tissue, and Non-ECRSwNP tissue by immunoblotting and found that the expression of AREG and EGFR in the epithelium of CRSwNP, especially ECRSwNP tissue, was significantly higher than that in normal mucosa. Figure 1A, suggesting that ECRSwNP epithelial cells secrete more AREG and are also one of the important target cells for AREG. Flow cytometry was used to further compare the expression of AREG and its receptor EGFR in different epithelial cell subtypes of CRSwNP, and it was found that AREG and EGFR were mainly expressed in epithelial basal cells, followed by secretory cells and ciliated cells. Figure 1B The results showed that compared with normal mucosa, the expression of amphiregulin AREG and its receptor EGFR was increased in the epithelium of human CRSwNP tissue, suggesting that nasal epithelial cells, especially basal cells, are both the cell source of AREG and one of the important target cells of AREG.

[0027] Example 2: AREG destroys epithelial tight junctions, promotes basal cell proliferation and inhibits basal cell differentiation

[0028] The inventors collected epithelial cells from CRSwNP patients and a control group, used an air-liquid culture model to simulate the structure of human epithelial cells, and added AREG for 24 hours on the 28th day after the model was established. Afterwards, the expression of epithelial tight junction protein (ZO-1) was detected by immunofluorescence. The results showed that AREG could significantly reduce the expression area of ​​ZO-1, indicating that AREG has a destructive effect on epithelial tight junctions. Figure 2A .

[0029] The inventors collected epithelial cells from CRSwNP patients and a control group, fused them with matrigel, and cultured them in 3D organoids in vitro. AREG was added at the middle (14th day) and end (28th day) of the culture for stimulation, and the organoids were collected on the 32nd day for fixation and embedding. Immunofluorescence staining was also used to analyze the effect of AREG on different epithelial cell subtypes. The results showed that after AREG stimulation of CRSwNP organoids, the proliferation of basal cells (Ki67 + P63 + ) increased in number, see Figure 2B , basal cells differentiate into ciliated cells (Foxj1 + ) is reduced, see Figure 2C These results suggest that AREG can promote the proliferation of epithelial basal cells in CRSwNP and inhibit the differentiation of basal cells into ciliated cells, and that the cell composition and function of the epithelial cell layer in human CRSwNP are abnormal.

[0030] Example 3: Effect of AREG on Eosinophils

[0031] The inventors obtained eosinophils from peripheral blood samples of CRSwNP patients through magnetic bead sorting and established a transwell model, in which eosinophils were added to the upper chamber of the transwell and AREG recombinant protein and / or EGFR blocker Erlotinib were added to the lower chamber of the transwell for chemotaxis. Flow cytometry was used to count the absolute number of eosinophils that chemotacticed to the lower chamber of the transwell using magnetic beads. The results showed that AREG could chemotacticize eosinophils, while its receptor EGFR blocker Erlotinib could effectively inhibit the chemotactic effect of AREG on eosinophils. Figure 3A .

[0032] In addition, AREG stimulated eosinophils cultured in vitro and found that the expression of CD11b, an activation marker on the surface of eosinophils, increased. After the addition of the EGFR blocker Erlotinib, the expression of CD11b decreased. Figure 3B .

[0033] This indicates that AREG can promote the chemotaxis and activation of eosinophils through its receptor EGFR, causing eosinophilic inflammatory response, while its receptor EGFR antagonist Erlotinib can antagonize the effect of AREG on eosinophils.

[0034] Example 4: Effect of neutralizing AREG on eosinophilic inflammation of the nasal mucosa in a mouse CRSwNP model

[0035] The inventors used anti-AREG neutralizing antibodies in a mouse CRSwNP model to investigate the potential therapeutic effects of AREG in CRSwNP. H&E staining of nasal tissue sections revealed that repeated exposure to ovalbumin and Aspergillus oryzae protease resulted in the development of multiple polypoid lesions in the mouse nasal cavity, demonstrating the successful establishment of a CRSwNP mouse model. Intranasal administration of anti-AREG neutralizing antibodies significantly reduced the number of polypoid lesions in the nasal cavity of the CRSwNP mouse model. Figure 4A .

[0036] By staining the eosinophils infiltrating the nasal mucosa with Sirius red, it was found that intranasal administration of anti-AREG neutralizing antibodies could significantly reduce the thickness of the nasal mucosa and the absolute number of eosinophils infiltrating the CRSwNP model in mice. Figure 4B However, in the CRSwNP model of mice treated with anti-IgG isotype control, the above eosinophilic inflammatory response indicators did not show significant changes, suggesting that neutralizing AREG in nasal mucosal tissue can effectively reduce the eosinophilic inflammatory response of the mouse nasal mucosa.

[0037] Compared with anti-IgG isotype control treatment, intranasal administration of anti-AREG neutralizing antibodies significantly increased the expression of epithelial tight junction protein Occludin in the CRSwNP mouse model, suggesting that antagonizing the effect of AREG improves the barrier function of CRSwNP epithelial cells. Figure 5 .

[0038] These results suggest that neutralizing AREG secreted by epithelial cells or other inflammatory cells can effectively reduce the eosinophilic inflammatory response of the nasal mucosa in the CRSwNP mouse model and restore the damaged epithelial barrier function of the mouse nasal mucosa.

[0039] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a preparation that inhibits the expression or action of AREG in the preparation of a drug for treating the damaged epithelial barrier function of the nasal mucosa in chronic sinusitis with nasal polyps.

2. The use according to claim 1, characterized in that The preparation for inhibiting AREG expression or inhibiting the action of AREG includes one or more of anti-AREG neutralizing antibodies, EGFR receptor antagonists, anti-EGFR neutralizing antibodies, and AREG-shRNA.

3. The use according to claim 1, characterized in that The effects of the preparation for inhibiting AREG expression or inhibiting the action of AREG in treating chronic sinusitis with nasal polyps are: reducing the volume of polyps, inhibiting the infiltration of eosinophils and other inflammatory cells, and restoring epithelial barrier function.

Images