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. 2022 Nov;26(5):347.
doi: 10.3892/mmr.2022.12863. Epub 2022 Sep 30.

Downregulation of deubiquitinating enzyme USP25 promotes the development of allergic rhinitis by enhancing TSLP signaling in the nasal epithelium

Wenchuan Chang #  1 , Hao Lv #  1 , Lu Tan  1 , Ziang Gao  1 , Peiqiang Liu  1 , Danxue Qin  1 , Wei Zhang  1 , Yu Xu  1
Affiliations

Downregulation of deubiquitinating enzyme USP25 promotes the development of allergic rhinitis by enhancing TSLP signaling in the nasal epithelium

Wenchuan Chang et al. Mol Med Rep. 2022 Nov.

Abstract

Ubiquitin‑specific peptidase 25 (USP25) is a key deubiquitylase belonging to the USP superfamily that is primarily involved in inflammation and the immune response. Thymic stromal lymphopoietin (TSLP) is an epithelial‑derived cytokine that is regarded as the master switch that initiates and maintains the type 2 immune response in allergic rhinitis (AR). However, the molecular mechanisms by which USP25 regulates TSLP signaling in the nasal epithelium in AR remain unclear. The present study assessed the protein expression levels of USP25 in the nasal epithelium of patients with AR. Moreover, USP25 knockout (KO) and wild‑type (WT) mice were treated with ovalbumin (OVA) to establish a model of AR. The results of western blotting and immunohistochemistry in the present study demonstrated that the protein expression levels of USP25 were significantly decreased in the nasal mucosa of patients with AR and AR mice, whereas the protein expression levels of TSLP were significantly increased. Allergic inflammation was more severe in USP25 KO mice compared with WT mice exposed to OVA, as demonstrated by increased nose scratching and sneezing, increased eosinophil infiltration, goblet cell hyperplasia and enhanced T helper type 2 (Th2) cytokine production. The results of in vitro experiments demonstrated that silencing or overexpression of USP25 decreased or increased TNF receptor‑associated factor 3 (TRAF3) protein expression levels, respectively, in human nasal epithelial cells, whereas TSLP protein expression levels were negatively associated with the expression of USP25 and TRAF3. In summary, USP25 downregulation enhanced TSLP signaling in the nasal mucosal epithelium via decreased TRAF3 expression, thereby exacerbating inflammation in AR. Therefore, USP25 may act as a novel therapeutic target in AR.

Keywords: allergic rhinitis; deubiquitylase; epithelial‑derived cytokine; thymic stromal lymphopoietin; ubiquitin‑specific peptidase 25.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
USP25 protein expression levels are decreased in the nasal mucosa of patients with AR. (A) Representative immunohistochemical images of USP25 (magnification, ×400). (B) Protein expression of USP25, TRAF3 and TSLP in nasal mucosal tissue was assessed using western blotting. (C) Semi-quantitative analysis of western blot band intensity. Data are presented as the mean ± standard deviation (n=6). Each experiment was repeated three times. Scale bar, 50 µm. Statistical analysis was performed using independent Student's t-test. *P<0.05. USP25, ubiquitin-specific peptidase 25; AR, allergic rhinitis; TRAF3, TNF receptor-associated factor 3; TSLP, thymic stromal lymphopoietin; Ctrl, control.
Figure 2.
Figure 2.
USP25 knockdown increases protein expression levels of TSLP in the nasal mucosa of OVA-induced mice. (A) Schematic experimental protocol for OVA-induced AR in USP25 KO and WT mice. (B) Representative immunohistochemical images of USP25 and TSLP staining. (C) Protein expression levels of (D) USP25, (E) TRAF3 and (F) TSLP in nasal mucosal tissue were assessed using western blotting. Semi-quantitative analysis of band intensity. Data are presented as the mean ± standard deviation (n=6). Each experiment was repeated three times. Scale bar, 50 µm. Statistical analysis was performed using two-way ANOVA followed by Tukey's post hoc test. *P<0.05. USP25, ubiquitin-specific peptidase 25; TSLP, thymic stromal lymphopoietin; OVA, ovalbumin; AR, allergic rhinitis; TRAF3, TNF receptor-associated factor 3; i.p., intraperitoneal; i.n., intranasal; KO, knockout; WT, wild-type.
Figure 3.
Figure 3.
USP25 knockdown exacerbates OVA-induced inflammatory responses in nasal mucosa and nasal symptoms. (A) H&E and PAS staining were used to assess eosinophil infiltration and goblet cell metaplasia, respectively. Red arrows indicate eosinophils. Blue arrows indicate PAS-positive goblet cells. Quantitative analysis of (B) eosinophils and (C) goblet cells. Frequency of (D) sneezing and (E) nose scratching was counted for 10 min following final treatment with OVA. Data are presented as the mean ± standard deviation (n=6). Each experiment was repeated three times. Scale bar, 50 µm. Statistical analysis was performed using two-way ANOVA followed by Tukey's post hoc test. *P<0.05, **P<0.01 and ***P<0.001. USP25, ubiquitin-specific peptidase 25; OVA, ovalbumin; H&E, hematoxylin and eosin; PAS, periodic acid-Schiff; KO, knockout; WT, wild-type.
Figure 4.
Figure 4.
USP25 knockdown promotes expression of pro-inflammatory cytokines in NLF and serum of OVA-induced mice. IFN-γ, IL-4, IL-5, IL-10 and IL-13 protein expression levels were assessed in (A) serum and (B) NLF using ELISA. Data are presented as the mean ± standard deviation (n=6). Each experiment was repeated three times. Statistical analysis was performed using two-way ANOVA followed by Tukey's post hoc test. *P<0.05, **P<0.01 and ***P<0.001. USP25, ubiquitin-specific peptidase 25; NLF, nasal lavage fluid; OVA, ovalbumin; KO, knockout; WT, wild-type.
Figure 5.
Figure 5.
USP25 knockdown increases TSLP protein expression levels via inhibition of TRAF3 in vitro. (A) Protein expression levels of (B) USP25, (C) TRAF3 and (D) TSLP were assessed using western blotting in HDM-stimulated nasal epithelial cells following transfection with si-USP25 and pcDNA-TRAF3 alone or in combination. Semi-quantitative analysis of band intensity. (E) TSLP protein expression levels in cell culture supernatant were assessed using ELISA. Data are presented as the mean ± standard deviation (n=3). Each experiment was repeated three times. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. *P<0.05. USP25, ubiquitin-specific peptidase 25; TSLP, thymic stromal lymphopoietin; TRAF3, TNF receptor-associated factor 3; HDM, house dust mite; si, small interfering.
Figure 6.
Figure 6.
USP25 overexpression decreases TSLP protein expression levels by increasing TRAF3 protein expression levels in vitro. (A) Protein expression levels of (B) USP25, (C) TRAF3 and (D) TSLP were assessed using western blotting in HDM-stimulated nasal epithelial cells following transfection with pcDNA-USP25 and si-TRAF3 alone or in combination. Semi-quantitative analysis of band intensity. (E) TSLP levels in cell culture supernatant were assessed using ELISA. Data are presented as the mean ± standard deviation (n=3). Each experiment was repeated three times. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. *P<0.05. USP25, ubiquitin-specific peptidase 25; TSLP, thymic stromal lymphopoietin; TRAF3, TNF receptor-associated factor 3; HDM, house dust mite; si, small interfering.
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Grants and funding

The present study was supported by the National Natural Science Foundation of China (grant no. 82071017), the National Natural Science Foundation of Hubei Province (grant no. 2021CFB125) and the Fundamental Research Funds for the Central Universities (grant no. 2042021kf0093).