Af 200 na

The design of the IL-1α secretion assay was adapted based on a previous report^{50 (link)}. T_H17 cells (1 × 10⁶) were stained with 1 mg ml⁻¹ of sulfo-NHS-LC-biotin (catalog no. ab145611, Abcam), incubated for 30 min at room temperature and then washed 3× with PBS (pH 8) supplemented with 100 mM glycine. The final washing of cells was performed with PBS supplemented with 0.5% bovine serum albumin. Cell surface biotinylation was validated with PE-labeled streptavidin (catalog no. 554061, BD Pharmingen). Purified anti-human IL-1α antibodies (AF-200-NA, R&D) were labeled with streptavidin using a Lightning-Link Streptavidin Conjugation kit (catalog no. ab102921, Abcam). For cytokine secretion, cells were stimulated with anti-CD3 and anti-CD28 for 72 h. The cells were collected and labeled with streptavidin-IL-1α and incubated for 24 h on the MACSmix tube rotator (Miltenyi Biotec). Recombinant IL-1α (Miltenyi Biotec) was added as a positive control. The cells were then stained with a PE-labeled IL-1α antibody (clone 364-3B3-14, BioLegend, 1:50).

Lung tissue sections (5 μm) were deparaffinized in xylene, rehydrated in graded alcohol, and antigen retrieval was performed in EDTA (1 mM, pH 8.0) for 10 min in a microwave. Samples were blocked in 1% bovine serum albumin (BSA) and incubated overnight at 4°C with anti‐IL‐1α (AF‐200‐NA, R&D) or an IgG1 control antibody (both 10 μg/mL, 1% BSA). Slides were washed and incubated with a fluorescein isothiocyanate–conjugated anti‐goat secondary antibody (F7367, Sigma) for 2 h at room temperature. Slides were washed and mounted in VectorShield mounting media containing DAPI (Vector Laboratories, Burlingame, CA). Images were acquired using a Leica TCS SP2 UV confocal microscope.

The cells were seeded in exactly the same way as described above for coculture experiments. After overnight attachment of the cells, neutralizing antibodies or isotype controls (100 ng/ml) were added to the cultures as recommended by the supplier. Neutralizing antibodies against IL-1α (R&D: AF-200-NA), TNF-α (R&D: AF-210-NA), CCL27 (R&D: AF-376), and CCL28 (R&D: AF-717) all had goat IgG (R&D: AB-108-C) as an isotype control. For IL-18 (R&D: D044-3), a mouse IgG1 (R&D: MAB002) was used as an isotype control. After 30 min, the culture medium was further supplemented with Hst1 (4 and 50 μM), LL-37 (2 and 4 μM), or vehicle (H₂O). 24 h after exposure, the supernatant was collected and stored at −20°C until further analysis by ELISA and cell viability was determined using the MTT assay (as described above).