Application of AREG in chronic sinusitis with rhinopolyp eosinophilic inflammation

Abstract

The invention relates to the field of biological medicine, in particular to application of AREG in chronic sinusitis with nasal polyp eosinophilic inflammation. According to the invention, the nasal epithelial cell, especially the basal cell, is found for the first time, is a cell source of the AREG and is also one of important target cells acted by the AREG. AREG promotes proliferation of epithelial basal cells and inhibits differentiation of the basal cells into cilia cells, resulting in structural abnormality and function impairment of human CRSwNP tissue epithelial barriers. The AREG chemotactices and activates human peripheral blood eosinophilic granulocytes, and the receptor EGFR antagonist Erlotinib of the AREG can antagonize the effect of the AREG on the eosinophilic granulocytes. The AREG secreted by neutralizing epithelial cells or other inflammatory cells can effectively relieve the eosinophilic inflammatory reaction of the nasal mucosa of the CRSwNP mouse model and recover the damaged epithelial barrier function of the nasal mucosa of the mouse. It is proved that when the neutralizing antibody of AREG or EGFR or shRNA is used as the antagonist, the eosinophilic inflammatory response of CRSwNP can be relieved by inhibiting the effect of AREG.

Description

Technical Field

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

[0002] Chronic rhinosinusitis with nasal polyps (CRSwNP) is a clinical phenotype of chronic sinusitis, with the main symptoms including nasal congestion, runny nose, head / facial pain and hyposmia. According to the number of eosinophils in the tissue, it is divided into eosinophilic CRSwNP (ECRSwNP) and non-eosinophilic CRSwNP (Non-ECRSwNP). In my country, about half of CRSwNP patients are ECRSwNP. ECRSwNP patients often have more severe symptoms, higher rates of concomitant asthma and postoperative recurrence, which imposes a heavy burden on the socioeconomic situation.

[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 and participate in various biological processes of epithelial cells, such as proliferation, differentiation, and tissue repair. Recent studies have found that AREG can be secreted by a variety of immune cells, such as eosinophils, mast cells, and type 2 innate lymphocytes, and is closely related to airway remodeling in eosinophilic inflammatory responses. AREG released by memory Th2 (T helper 2 cell) cells can recruit eosinophils and mediate the fibrotic response of eosinophilic airway inflammation.

[0004] Existing studies have shown that AREG is expressed at increased levels in ECRSwNP tissues, suggesting that AREG may be involved in the eosinophilic inflammatory response of CRSwNP. The infiltration of eosinophils near the epithelium of CRSwNP patients is closely related to the severity of the disease, and no studies have suggested that epithelial cells can secrete AREG to recruit eosinophils to aggravate the inflammatory response; subepithelial inflammatory cells can also secrete AREG, and there is no evidence that using neutralizing antibodies or shRNA against AREG or EGFR as antagonists can reduce the eosinophilic inflammatory response of 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 of AREG or inhibiting the action of AREG in relieving 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 of AREG or inhibiting the 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 anti-AREG neutralizing antibody, EGFR receptor antagonist, anti-EGFR neutralizing antibody, and AREG-shRNA.

[0011] In certain embodiments, the preparation that inhibits the expression and action of AREG has the following effects in treating chronic sinusitis with nasal polyps: reducing the size of polyps, inhibiting the infiltration of eosinophils and other inflammatory cells, and restoring 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, resulting in structural abnormalities and functional damage to the epithelial barrier of CRSwNP patients. AREG chemotactic and activates 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 :Results of the air-liquid culture model of CRSwNP epithelial cells stimulated by AREG;

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

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

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

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

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

[0022] Figure 4B :The effect of intranasal administration of anti-AREG neutralizing antibody on eosinophilic inflammatory response in the mouse CRSwNP model;

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

[0024] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, 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, see 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, see 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 the control group, used an air-liquid culture model to simulate the structure of human epithelial cells, added AREG for 24 hours on the 28th day after the model was established, and then detected the expression of epithelial tight junction protein (ZO-1) 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, see Figure 2A .

[0029] The inventors collected epithelial cells from CRSwNP patients and the control group, fused them with matrix gel, and cultured 3D organoids in vitro. AREG was added for stimulation at the middle (14th day) and the end (28th day) of the culture, 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 stimulated CRSwNP organoids, basal cells with proliferation ability (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 by magnetic bead sorting, and established a transwell model, that is, 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 experiments. The absolute number of eosinophils chemotactic to the lower chamber of the transwell was counted by counting magnetic beads by flow cytometry. The results showed that AREG can chemotactic eosinophils, and its receptor EGFR blocker Erlotinib can effectively inhibit the chemotactic effect of AREG on eosinophils, see 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] These results indicate 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 nasal mucosa in mouse CRSwNP model

[0035] The inventors used anti-AREG neutralizing antibodies in the mouse CRSwNP model to study the possible therapeutic effect of AREG in CRSwNP. H&E staining of nasal tissue sections showed that after repeated exposure to ovalbumin and Aspergillus oryzae protease, multiple polypoid lesions appeared in the nasal cavity of mice, indicating that the CRSwNP mouse model was successfully established. The number of polypoid lesions in the nasal cavity of the mouse CRSwNP model was significantly reduced by intranasal administration of anti-AREG neutralizing antibodies, see 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 in the CRSwNP model of mice, see Figure 4B However, in the CRSwNP model of mice treated with anti-IgG isotype control, the above eosinophilic inflammatory response indicators did not change significantly, suggesting that neutralizing AREG in nasal mucosal tissue can effectively reduce the eosinophilic inflammatory response of the mouse nasal mucosa.

[0037] Compared with the anti-IgG isotype control treatment, the intranasal administration of anti-AREG neutralizing antibody significantly increased the expression of epithelial tight junction protein Occludin in the mouse CRSwNP model, suggesting that antagonizing the role of AREG improves the barrier function of CRSwNP epithelial cells, see 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 nasal mucosa in mice.

[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 the scope of protection of the present invention.

Claims

1. A use of AREG as a target in eosinophilic inflammation of chronic sinusitis with nasal polyps.

2. A use of AREG as a target in the preparation of a drug for the treatment of chronic sinusitis with nasal polyps and eosinophilic inflammation.

3. Use of a preparation that inhibits AREG expression or AREG action in relieving eosinophilic inflammatory response in chronic sinusitis with nasal polyps, characterized in that: The use is for in vitro non-therapeutic purposes.

4. The use according to claim 3, characterized in that The models include an epithelial cell air-liquid culture model, an organoid culture model, and a chronic sinusitis with nasal polyps mouse model.

5. Use of a preparation that inhibits the expression of AREG or the action of AREG in the preparation of a drug for treating chronic sinusitis with nasal polyps.

6. The use according to claim 5, characterized in that: The preparation for inhibiting AREG expression or inhibiting AREG action includes one or more of anti-AREG neutralizing antibody, EGFR receptor antagonist, anti-EGFR neutralizing antibody, and AREG-shRNA.

7. The use according to claim 5, characterized in that: The preparation for inhibiting the expression and action of AREG has the following effects in treating chronic sinusitis with nasal polyps: reducing the volume of polyps, inhibiting the infiltration of eosinophils and other inflammatory cells, and restoring the epithelial barrier function.

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