The Role of CD40 in Allergic Rhinitis and Airway Remodelling

Abstract

Background: Allergic rhinitis (AR) affects millions of people and is lack of effective treatment. CD40 is an important costimulatory molecule in immunity. However, few studies have focused on the role of CD40 in AR.

Methods: In this study, we built mouse model of chronic AR. The mice were divided into the AR, control, intravenous CD40 siRNA, and nasal CD40 siRNA groups (n = 6 each). We detected OVA-sIgE, IL-4, IL-5, IL-13, IL-10, IFN-γ, and TGF-β levels in serum and supernatant by ELISA, CD40⁺ splenic DCs, and Foxp3⁺ Tregs by flow cytometry and CD40 mRNA by RT²-PCR. We also used PAS and MT stains to assess tissue remodelling.

Results: (1) The OVA-sIgE, IL-4, IL-5, and IL-13 levels in the serum or supernatant of nasal septal membrane of AR mice were significantly higher than control. After treated with CD40 siRNA, those indicators were significantly decreased. The IFN-γ, IL-10, and TGF-β levels in AR mice were significantly lower than that in control and were increased by administration of CD40 siRNA. (2) AR mice had significantly fewer Foxp3⁺ Tregs in the spleen than control mice. After treated with CD40 siRNA, AR mice had significantly more Foxp3⁺ Tregs. (3) AR mice exhibited a significantly higher CD40 mRNA levels than control. Administration of CD40 siRNA significantly reduced the CD40 mRNA level. (4) The AR mice showed significantly greater collagen deposition than the control in MT staining. Applications of CD40 siRNA significantly reduced the collagen deposition in AR mice.

Conclusion: CD40 siRNA therapy shows promise for chronic AR as it significantly attenuated allergic symptoms and Th2-related inflammation and upregulated Foxp3⁺ Tregs. CD40 plays a role in tissue remodelling in AR, which can be inhibited by CD40 siRNA application.

Figure 1

Mouse model of chronic AR and CD40 siRNA application.

Figure 2

(a) Serum and supernatant levels of OVA-sIgE were measured by ELISA. AR mice produced significantly more OVA-sIgE than control. OVA-sIgE level in mice treated with CD40 siRNA was lower than AR mice. Mice treated nasally with CD40 siRNA produced significantly less OVA-sIgE than mice given intravenously CD40 siRNA. (b) Serum and supernatant levels of IL-5 were measured by ELISA. AR mice produced significantly more IL-5 than control. IL-5 level in mice treated with CD40 siRNA was lower than AR mice. (c) Serum and supernatant levels of IFN-γ were measured by ELISA. AR mice produced significantly less IFN-γ than control. IFN-γ level in mice treated with CD40 siRNA was higher than AR mice. Mice treated nasally with CD40 siRNA produced significantly more IFN-γ than mice given intravenously CD40 siRNA. (d) Supernatant levels of IL-4 were measured by ELISA. AR mice produced significantly more IL-4 than control. IL-4 level in mice treated with CD40 siRNA was lower than AR mice. (e) Supernatant levels of IL-13 were measured by ELISA. AR mice produced significantly more IL-13 than control. IL-13 level in mice treated with CD40 siRNA was lower than AR mice. (f) Supernatant levels of IL-10 were measured by ELISA. AR mice produced significantly less IL-10 than control. IL-10 level in mice treated with CD40 siRNA was higher than AR mice. (g) Supernatant levels of TGF-β were measured by ELISA. AR mice produced significantly less TGF-β than control. TGF-β level in mice treated with CD40 siRNA was higher than AR mice. P values <0.05 were considered as significance.

Figure 3

(a) CD40⁺ DCs and Foxp3⁺ Tregs of the spleen in control, AR, intravenous CD40 siRNA, and nasal CD40 siRNA groups were detected by flow cytometry. CD40 expression on DCs in AR mice was higher than control group. There was no difference between AR and siRNA groups. CD40 siRNA treatment resulted in inhibition of CD40 expression on DCs. There was no significant difference between intravenous and nasal siRNA group. AR mice shown significantly less Foxp3⁺ Tregs, compared with control mice. Mice nasally treated with CD40 siRNA had significantly more Foxp3⁺ Tregs, compared with AR mice. There was no significant difference between intravenous CD40 siRNA and AR group. P values <0.05 were considered as significance. (b) CD40⁺ DCs of the spleen in control, AR, intravenous CD40 siRNA, and nasal CD40 siRNA groups were detected by flow cytometry. (c) Foxp3⁺ Tregs of the spleen in control, AR, intravenous CD40 siRNA, and nasal CD40 siRNA groups were detected by flow cytometry.

Figure 4

The mRNA level of CD40 in nasal tissues was detected by RT²-PCR. AR mice expressed significantly higher CD40 mRNA compared with control group. CD40 mRNA was significantly reduced in the mice treated with CD40 siRNA, both by intravenous and nasal methods. The mice treated with nasal CD40 siRNA expressed significantly lower CD40 mRNA than mice that received intravenous CD40 siRNA. P values <0.05 were considered as significance.

Figure 5

(a) Goblet cell numbers of control, AR, intravenous CD40 siRNA, and nasal CD40 siRNA groups were detected by PAS staining. There was no significant difference in goblet cell numbers between AR, CD40 siRNA treatment, and control groups. All P values >0.05. (b) Collagen deposition of control, AR, intravenous CD40 siRNA, and nasal CD40 siRNA groups was detected by MT staining. In this study, AR mice expressed significantly more collagen deposition, compared to control group. Mice treated nasally or intravenously with CD40 siRNA expressed significantly less collagen deposition, compared with AR mice. P values <0.05 were considered as significance. (c) Goblet cell numbers of control, AR, intravenous CD40 siRNA, and nasal CD40 siRNA groups were detected by PAS staining. (d) Collagen deposition of control, AR, intravenous CD40 siRNA, and nasal CD40 siRNA groups was detected by MT staining.