Thymic stromal lymphopoietin (TSLP)-mediated dermal inflammation aggravates experimental asthma

Abstract

Individuals with one atopic disease are far more likely to develop a second. Approximately half of all atopic dermatitis (AD) patients subsequently develop asthma, particularly those with severe AD. This association, suggesting a role for AD as an entry point for subsequent allergic disease, is a phenomenon known as the "atopic march." Although the underlying cause of the atopic march remains unknown, recent evidence suggests a role for the cytokine thymic stromal lymphopoietin (TSLP). We have established a mouse model to determine whether TSLP plays a role in this phenomenon, and in this study show that mice exposed to the antigen ovalbumin (OVA) in the skin in the presence of TSLP develop severe airway inflammation when later challenged with the same antigen in the lung. Interestingly, neither TSLP production in the lung nor circulating TSLP is required to aggravate the asthma that was induced upon subsequent antigen challenge. However, CD4 T cells are required in the challenge phase of the response, as was challenge with the sensitizing antigen, demonstrating that the response was antigen specific. This study, which provides a clean mouse model to study human atopic march, indicates that skin-derived TSLP may represent an important factor that triggers progression from AD to asthma.

Figure 1

Intradermal administration of TSLP aggravates airway inflammation and mucus secretion in experimental asthma. (A) Experimental protocol. Mice received injections of TSLP and OVA (5 μg) four times for 12 days and rested for 9 days. To induce experimental asthma, mice were OVA challenged (50 μg in 30 μL saline) on 4 consecutive days (D21–D24) by intranasal instillation. Mice were killed on D25 for analysis. Total serum IgE (B) and OVA-specific IgE (C) on D25 (n = 5, two independent experiments). (D) Cell counts in the BAL fluid. The significance between two groups was determined by two-tailed Student’s t test. (n = 5, two independent experiments). (E) Representative lung tissue cross sections stained with PAS to visualize mucus-producing goblet cells and airway inflammation. Bar, 100 μm.

Figure 2

Airway inflammation does not require systemic TSLP. (A) No locally induced TSLP. Quantitative RT-PCR analyses of cytokines and chemokines in lungs on D25. n ≥ 4 mice per group. The significance of gene expression between two groups was determined by two-tailed Student’s t test. (B) and (C) No circulating TSLP. Serum TSLP levels not enhanced on D14 (B) and D25 (C). K5-TSLP mice were treated on 1mg/ml of doxycycline for 3 weeks and used as positive control. n ≥ 4 mice per group. N.D., < 7.8 pg/ml.

Figure 3

TSLP is dispensable once skin inflammation develops. (A) Total number and differential cell counts of cells isolated from BAL taken from TSLP–deficient (TSLP-KO) or WT BALB/c mice treated with MSA or TSLP plus OVA followed by OVA challenge. n ≥ 5 mice per group from two independent experiments. (B) Acute blockade of TSLP after skin sensitization does not inhibit allergic asthma. Cell counts in the BAL fluid. n ≥ 4 mice per group from two independent experiments. The significance between two groups was determined by two-tailed Student’s t test.

Figure 4

CD4 T cells are required for TSLP-mediated airway inflammation. (A) Cell counts in BAL fluid from WT BALB/c mice treated i.p. with rIgG or anti-CD4 Ab (GK1.5), to acutely deplete CD4 T cells after skin sensitization. n ≥ 4 mice per group from two independent experiments. (B) DO11.10 CD4+ T cells were transferred to WT BALB/c mice and then sensitized with TSLP+OVA (solid squares) or MSA+OVA (open squares). On D14, CD4+ T cells were isolated and negatively selected prior to transfer of 2 ×10⁷ cells/mouse. Recipient mice were intranasally challenged with OVA starting 24 hours after transfer, and airway symptom were monitored as above. Cell counts in BAL fluid. n = 5 mice per group. The significance between two groups was determined by two-tailed Student’s t test. (C) Skin-draining LN cells from TSLP+OVA treated mice transfer disease to naïve mice. Skin-draining LN cells from mice treated with TSLP+OVA (solid square) and MSA+OVA (open square) were isolated, and cells were cultured with OVA (100 μg/mL) for 72 hours prior to washing and intravenous transfer to naïve mice (2 × 10⁷ cells/mouse). Cell counts in BAL fluid. n = 5 mice per group. The significance between two groups was determined by two-tailed Student’s t test.

Figure 5

Airway inflammation is antigen-specific. To test the antigen specificity of atopic model, low endotoxin BSA used to sensitize and challenge the mice. (A) Total cell counts in BAL fluid. (B) Differential cell counts in BAL. (C) Serum OVA and BSA-specific IgE on D25. n ≥ 4 mice per group from two independent experiments.

Figure 6

Antigen feeding before, but not after, skin sensitization suppresses asthma-like inflammatory response. Mice were administered a 1% OVA solution in drinking water for 5 consecutive days (days –7 to –2, Tol -7; or days 14 to 19, Tol 14). (A) Cell counts in BAL fluid. (B) Total and (C) OVA-specific serum IgE on D25. n = 4 mice per group from two independent experiments.

Figure 7

Mice overexpressing TSLP and antigen in the skin develop aggravated asthma-like airway inflammation. (A) OVA-specific T cells are primed in K5-TGO mice. K5-TGO mice received 1 mg/ml of doxycycline for 3 weeks of treatment. Profile of DO11.10 CD4 T cells transferred into K5-TGO (left) and control (right) mice were determined in inguinal lymph node (upper) and spleen (lower). (B) Total cell counts and differential cell counts in the BAL (n = 4). (C) H&E staining of skin (upper) and PAS staining of lung (lower) from K5-TSLP/TGO mice (left) and K5-TGO mice (right). Bar, 100 μm.