Airway epithelial SPDEF integrates goblet cell differentiation and pulmonary Th2 inflammation

Abstract

Epithelial cells that line the conducting airways provide the initial barrier and innate immune responses to the abundant particles, microbes, and allergens that are inhaled throughout life. The transcription factors SPDEF and FOXA3 are both selectively expressed in epithelial cells lining the conducting airways, where they regulate goblet cell differentiation and mucus production. Moreover, these transcription factors are upregulated in chronic lung disorders, including asthma. Here, we show that expression of SPDEF or FOXA3 in airway epithelial cells in neonatal mice caused goblet cell differentiation, spontaneous eosinophilic inflammation, and airway hyperresponsiveness to methacholine. SPDEF expression promoted DC recruitment and activation in association with induction of Il33, Csf2, thymic stromal lymphopoietin (Tslp), and Ccl20 transcripts. Increased Il4, Il13, Ccl17, and Il25 expression was accompanied by recruitment of Th2 lymphocytes, group 2 innate lymphoid cells, and eosinophils to the lung. SPDEF was required for goblet cell differentiation and pulmonary Th2 inflammation in response to house dust mite (HDM) extract, as both were decreased in neonatal and adult Spdef(-/-) mice compared with control animals. Together, our results indicate that SPDEF causes goblet cell differentiation and Th2 inflammation during postnatal development and is required for goblet cell metaplasia and normal Th2 inflammatory responses to HDM aeroallergen.

Figure 4. SPDEF is required for AHR, Th2, and TH17 inflammatory responses to HDM in postnatal mice.

HDM (50 μg) was administered i.n. daily for 3 days to PND15 control and Spdef^–/– mice, and the mice were sacrificed 2 days later. (A) Decreased mucous metaplasia and inflammatory responses to HDM were observed in Spdef^–/– mice. (A and C) BALF cells (n = 4) were stained with Diff-Quik, demonstrating eosinophilic and neutrophilic infiltrates. Goblet cell differentiation (A) and increased Acta2 mRNA caused by HDM challenge (Table 4) were modestly decreased in Spdef^–/– mice. (B) Spdef^–/– mice were less responsive to HDM (green) than were controls (red line). Data represent the mean ± SEM, using 2-way Anova with Bonferroni’s post tests. n = 6/group. (D and E) Flow analysis of lung cells from Spdef^+/+ (white bars) and Spdef^–/– mice (black bars) after HDM challenge. SiglecF⁺CCR3⁺ eosinophils were decreased, and ST2⁺, IL-17RB⁺, ICOS⁺ ILCs and CD3⁺ cells were unaltered. CD3⁺ IL-4⁺ and CD3⁺IL-17⁺ T cells were decreased in Spdef^–/– mice, and CD3⁺IFN-γ⁺ T cells were unaltered. Gating strategies are depicted in Supplemental Figure 1. Data represent the mean ± SEM. *P < 0.05 compared with controls using an unpaired, 2-tailed Student’s t test. n = 4/group.

Figure 3. SPDEF is required for Th2 inflammatory responses, AHR, and recruitment of ST2⁺ ILCs and IL-4⁺, IL-13⁺, and IL-17⁺ T cells in response to HDM extract in adult mice.

HDM extract (100 μg) was administered i.n. daily for 3 days to adult control and Spdef^–/– mice, which were sacrificed 2 days later. (A) Decreased mucous metaplasia and inflammatory responses to HDM were seen in Spdef^–/– mice. (A and C) BALF cells (n = 4) were stained with Diff-Quik, which showed eosinophilic and neutrophilic infiltrates, and differential cell counts were quantified. Goblet cell differentiation (A) and the increase in Acta2 mRNA (Table 3) caused by HDM challenge were modestly decreased in Spdef^–/– mice. (B) AHR was decreased in Spdef^–/– mice. Data represent the mean ± SEM. Significance was analyzed using 2-way ANOVA with Bonferroni’s post tests. n = 6/group. (D and E) Flow analysis of lung cells from Spdef^+/+ (white bars) and Spdef^–/– mice (black bars) after HDM challenge. CD45⁺ and CD3⁺ cell numbers were not altered, and ST2⁺ ILC numbers were decreased. (E) Numbers of IL-4⁺, IL-13⁺, and IL-17⁺ T cells were decreased in Spdef^–/– mice. Gating strategies are depicted in Supplemental Figure 1. Data represent the mean ± SEM. *P < 0.05 compared with controls using an unpaired, 2-tailed Student’s t test. n = 4/group.

Figure 2. Conditional expression of FOXA3 in airway epithelial cells causes pulmonary inflammation and goblet cell metaplasia.

(A–C) Control littermates (white bars) and Scgb1ab-rtTA OTet7-Foxa3 dams (black bars) were placed on doxycycline from E16.5 to P15. Lung tissue was obtained from the pups at P15. Scgb1ab-rtTA OTet7-Foxa3 pups (black bars) developed spontaneous eosinophilic inflammation, goblet cell metaplasia, and AHR. Diff-Quik staining of BALF (n = 4) demonstrated eosinophilic infiltration. (E) Flow cytometric analysis of lung cells obtained at P15 demonstrated increased numbers of SiglecF⁺CCR3⁺, (D–F) ST2⁺, IL-17RB⁺, and ICOS⁺ ILCs and CD3⁺ IL-4–producing T cells. IFN-γ–producing CD3⁺ T cells were unaltered. (G) pDCs (CD11b^–CD11c⁺B220⁺), CD103⁺ mDCs (CD11b⁺CD11c⁺B220^–) were increased and mDCs were unchanged in lung from Scgb1a1-rtTA OTet7-Foxa3 pups. Activation markers (I-A/E and CD86) on pDCs and mDCs are shown in G. Data represent the mean ± SEM. *P < 0.05 compared with controls using an unpaired, 2-tailed Student’s t test. n = 4/group.

Figure 1. SPDEF causes goblet cell differentiation, pulmonary inflammation, and AHR.

Dams of Scgb1ab-rtTA TRE-Spdef (black bars) and control littermate single-transgenic mice (white bars) were placed on doxycycline from E16.5 to P15. (A) At P15, lung histology of the pups was assessed by H&E (n = 8) and BALF (n = 4) staining with Diff-Quik. Immunostaining for SPDEF, FOXA3, and MUC5B demonstrated extensive goblet cell differentiation (n = 8). (A and C) Cells isolated from BALF were stained with Diff-Quik and showed eosinophilic infiltrates. Data represent the mean ± SEM. *P < 0.05 compared with controls using an unpaired, 2-tailed Student’s t test. (B) AHR is represented as Penh in response to methacholine. Data represent the mean ± SEM of 6 mice per group. *P < 0.05 by 2-way ANOVA. (E) Flow cytometric analysis of lung cells obtained at P15 demonstrated increased numbers of SiglecF⁺CCR3⁺ in Scgb1a1-rtTA Spdef mice (black bar) compared with cell numbers detected in littermate control mice (white bar). (D and E) ST2⁺, IL-17RB⁺, and ICOS⁺ ILCs and total CD3⁺ and CD3⁺ IL-4–producing T cells were increased, and IFN-γ–producing CD3⁺ T cells were unaltered. Data represent the mean ± SEM. *P < 0.05 compared with controls using an unpaired, 2-tailed Student’s t test. n = 4/group. Examples of FACS analyses are provided in Supplemental Figure 1.