Der f 31, a novel allergen from Dermatophagoides farinae, activates epithelial cells and enhances lung-resident group 2 innate lymphoid cells

Abstract

Airway epithelial cell-derived thymic stromal lymphopoietin (TSLP) and IL-33 can enhance lung-resident group 2 innate lymphoid cells (ILC2s), and they play an important role in the development of allergic diseases. This study tests the hypothesis that Der f 31 (Dermatophagoides farinae-31), an allergen, modulates airway epithelial cell functions and increases the frequency of lung ILC2s. Our previous research identified cofilin (Der f 31) as a novel allergen. In this study, we found that recombinant Der f 31 (r-Der f 31) upregulated the expression of co-stimulatory molecules in DCs and promoted Th2-skewed polarization. The levels of TSLP and IL-33 in epithelial cells were upregulated by r-Der f 31 via the activation of Toll-like receptor 2. Furthermore, in in vivo studies, r-Der f 31 induced eosinophil-like airway allergy and increased the number of lung-resident ILC2s. In summary, Der f 31 can modulate the functions of airway epithelial cells and increase levels of lung-resident ILC2s.

Figure 1

Co-stimulatory molecules are up-regulated by Der f 31. DC2.4 cells were seeded into 6-well plates for overnight culture and then stimulated with Der f 31 (20 μg/ml) or LPS (1 μg/ml) for 24 hours. The expression levels of CD40 (A), CD80 (B) and CD83 (C) were tested by flow cytometry. These data (means ± SEMs) were generated from three independent experiments and processed with GraphPad software. The significant differences between two groups were tested by two-tailed T-test. *p < 0.05. **p < 0.01.

Figure 2

Der f 31 induces Th2-skewed polarization. BMDCs were collected from BALB/c mice, and cultured in medium with CSF2 and IL-4 for 8 days. BMDCs and spleen cells were co-cultured at 1:10 for 3 days, and proliferation and differentiation were evaluated via flow cytometry. (A) The differentiation of CD4+ T cells. (B) The proliferation of CD4+ T cells. These data (means ± SEMs) were generated from three independent experiments and processed with GraphPad software. The significant differences between two groups were tested by two-tailed T-test. **p < 0.01. ***p < 0.001. ns, no significant difference.

Figure 3

Der f 31 up-regulates the expression levels of TSLP and IL-33 in epithelial cells via TLR2. A549 cells were seeded into 6-well plates overnight and treated with different concentrations of Der f 31. At 4 hours, total RNA was extracted to analyze the expression levels of TSLP and IL-33 by RT-PCR. (A) The results of RT-PCR. The lung tissues from normal BALB/c mice were digested, and the cells were incubated with anti-TLR2 antibody, IgG1, κ isotype control (IC) or TLR4 inhibitor for 2 hours or 6 hours and then treated with Der f 31. At 96 hours, the supernatants were collected to perform ELISA. (B) The expression levels of TSLP. (C) The expression levels of IL-33. These data (means ± SEMs) were generated from three independent experiments and processed with GraphPad software. The significant differences between two groups were tested by two-tailed T-test. **p < 0.01. ***p < 0.001. ns, no significant difference.

Figure 4

Airway allergy is induced by Der f 31. Four- to 7-week-old female BALB/c mice were immunized with PBS, Der f 31 or Der f 1 and then challenged with Der f 31 or Der f 1 for one week. (A) The scheme of establishment of mouse model. (B) Total IgE, allergen-specific IgE, IgG1 and IgG2a were detected by ELISA by optical density (OD). (C) The cytokines in BALF. (D) The cytokines in the lung homogenates. E, Histological sections of lung tissues. These data (means ± SEMs) were generated from one of three experiments (n = 6) and processed with GraphPad software. The significant differences between two groups were tested by two-tailed T-test. **p < 0.01. ***p < 0.001. ns, no significant difference.

Figure 5

Der f 31 induces Th2-type response in spleen. The spleen cells were cultured with Der f 31 and Der f 1 for 3 days, then analyzed by FACS. (A) The different subtypes of T helper cells. (B) The proliferation of CD4+ T cells in the spleen. (C) The cytokines in the spleen cell supernatant. These data (means ± SEMs) were generated from one of three experiments (n = 6), processed with GraphPad software. The significant differences between two groups were tested by two-tailed T-test. **p < 0.01. ***p < 0.001. ns, no significant difference.

Figure 6

Der f 31 triggers an eosinophilia. (A) representative flow profile of Siglec-F+ eosinophils in BALF. (B) statistics chart of FACS. (C) the total cell counts in the BALF. (D) Differential cell counts in the BALF. These data (means ± SEMs) were generated from one of three experiments (n = 4–6) and processed with GraphPad software. The significant differences between two groups were tested by two-tailed T-test. **p < 0.01. ***p < 0.001. ns, no significant difference.

Figure 7

The cytokines from BALF and lung homogenates. (A) The cytokines in BALF. The lung tissues were homogenized by sonication on ice and then centrifuged to obtain supernatants to test the expression levels of cytokines. (B) The cytokines from lung homogenates. The results are means ± SEMs (n = 4–6), processed with GraphPad software. The significant differences between two groups were tested by two-tailed T-test. *p < 0.05. **p < 0.01. ***p < 0.001. ns, no significant difference.

Figure 8

Lung-resident ILC2s are enhanced by Der f 31. (A) Lung ILC2s identified by FACS. (B) Number of lung-resident ILC2s. (C) The mRNA levels of TSLP, IL-33 and IL -13 in the lung. These data (Means ± SEMs) were generated from one of three experiments (n = 4–6) and processed with GraphPad software. The significant differences between two groups were tested by two-tailed T-test. *p < 0.05. **p < 0.01.