Alternaria-induced release of IL-18 from damaged airway epithelial cells: an NF-κB dependent mechanism of Th2 differentiation?

Abstract

Background: A series of epidemiologic studies have identified the fungus Alternaria as a major risk factor for asthma. The airway epithelium plays a critical role in the pathogenesis of allergic asthma. These reports suggest that activated airway epithelial cells can produce cytokines such as IL-25, TSLP and IL-33 that induce Th2 phenotype. However the epithelium-derived products that mediate the pro-asthma effects of Alternaria are not well characterized. We hypothesized that exposure of the airway epithelium to Alternaria releasing cytokines that can induce Th2 differentiation.

Methodology/principal finding: We used ELISA to measure human and mouse cytokines. Alternaria extract (ALT-E) induced rapid release of IL-18, but not IL-4, IL-9, IL-13, IL-25, IL-33, or TSLP from cultured normal human bronchial epithelial cells; and in the BAL fluids of naïve mice after challenge with ALT-E. Both microscopic and FACS indicated that this release was associated with necrosis of epithelial cells. ALT-E induced much greater IL-18 release compared to 19 major outdoor allergens. Culture of naïve CD4 cells with rmIL-18 induced Th2 differentiation in the absence of IL-4 and STAT6, and this effect was abrogated by disrupting NF- κB p50 or with a NEMO binding peptide inhibitor.

Conclusion/significance: Rapid and specific release of IL-18 from Alternaria-exposed damaged airway epithelial cells can directly initiate Th2 differentiation of naïve CD4(+) T-cells via a unique NF-κB dependent pathway.

Figure 1. Rapid release of cytokines from epithelial cells cultured with ALT-E.

(A, B) NHBE cells were cultured with PBS, ALT-E or other allergens for 15 min, and released cytokines were quantified. T. Alder, Tag Alder; S. Ragweed, Short Ragweed (n = 5 wells per treatment group). (C) BALF cytokine levels in naïve mice 60 min after challenge with 20 µg ALT-E or PBS control (n = 4 per group).

Figure 2. Induction of epithelial cell necrosis by ALT-E.

(A) A549 cells were cultured with ALT-E for 30 min. Changes in cell morphology were monitored by live cell imaging. (B) Time course of ALT-E-induced IL-18 release from airway epithelial cells. (C) Exposure of cultured A549 epithelial cells to ALT-E induces cell necrosis. (D) ALT-E challenge of mice induces sloughing of trypan blue positive necrotic airway epithelial cells (n = 4 per group). (E) Caspase 1 inhibitor (Cas.1 inh.) reduces IL-18 release from A549 airway epithelial cells cultured with ALT-E.

Figure 3. Effect of IL-18 on induction of Th2 differentiation.

(A) Effect of IL-18 on IL-4 secreting splenocytes ELISPOTS. (B) T cell differentiation protocol used in Panels 3C–E and Fig. 4. (C, D and E) Effect of IL-18 or IL-4 on Th2 differentiation of naïve CD4+ T-cells isolated from WT mice (C), IL-4^−/− mice (D) and WT and STAT6^−/− mice (E). n = 5−7 per group.

Figure 4. Effect of disruption of NF-κB p50 on Th2 differentiation of naïve CD4 T-cells treated with IL-18.

CD4⁺ T-cells from Wild-type (WT) and NF-κB p50^−/− mice were cultured with IL-18 in the differentiation phase, restimulated with con A, and secretion of IL-4 (A), IL-5 (B) and IL-13 (C) was quantified by ELISA. n = 5−7 per group.

Figure 5. Effect of IL-18 on Th2 differentiation and GATA3 expression in naïve CD4+ T-cells.

(A,B,C). Effect of NEMO peptide on Th2 differentiation. Negatively selected naïve CD3⁺ CD4⁺ T-cells were cultured with plate bound anti-CD3 and anti-CD28 with PBS (PBS), IL-18 100 ng/ml (IL-18), IL-18 100 ng/ml and 10 µM NEMO Binding Domain Binding Peptide (IL-18+NEMO) for the initial culture period. The cells were washed and restimulated, and secretion of IL-4 (A), IL-5 (B) and IFN-γ(C) were quantified. The data was expressed as fold increase compared to cells cultured with PBS. (D,E,F). GATA3 expression in T-cells. Negatively selected naïve CD3⁺ CD4⁺ T-cells were cultured with plate bound anti-CD3 and anti-CD28 in the presence of PBS (D), IL-4 (E) or IL-18 (F) for 7 days. GATA3 expression was measured by flow cytometric analysis.