Anti il 1β

AT2 cells were isolated from naïve mice as previously described (28 (link), 53 , 75 ). Briefly, mouse lungs were perfused with chilled PBS and intratracheally instilled with 1mL of dispase II (15U/mL, Roche), tying off the trachea and cutting away the lobes from the mainstem bronchi. Lungs were incubated in 4mL of 15U/mL dispase II for 45min while shaking at room temperature, followed by mechanical dissociation with an 18G needle. Following passage through a 100μm filter, lungs underwent 10min of DNase I digestion (50μg/mL) and filtered through 70μm filter prior to RBC lysis. Single-cell suspensions were subject to CD45 depletion using microbeads (Miltenyi), incubated with anti-FcgRIII/II (Fc block) and stained with CD45, EpCAM, MHC-II, and viability die (see antibody details in Supplementary Table 2). Fluorescence assisted cell sorting was performed on the BD Influx cell sorter to isolate AT2 cells as described previously (75 ) and collected in 500μL DMEM + 40% FBS + 2% P/S. Sorted AT2 cells (200,000 per well) were plated in a 96-well plate in DMEM/F12 + 10% FBS and cultured at 37°C, 5% CO₂ for 3 days prior to harvest. For in vitro neutralization anti-IL-1β (B122; BioXCell) was used at 10μg/mL and for stimulation, IL-1β (Peprotech) was used at 20ng/mL.

CFA-induced inflammation was induced by i.p. injection of 200–400 μl of a 1:1 emulsion of DPBS and CFA (Sigma-Aldrich). Mice were analyzed at the stated time points after CFA injection. PolyI:C treatments were performed intravenously at 1.4 mg/Kg/d. LTβR signaling was blocked with 200 μg of LTβR-Ig consisting of the LTβR ectodomain fused with the Fc domain of a mouse IgG1 antibody specific for hen egg lysozyme (Hel-Ig). Control experiments were performed by treatment with 200 μg HEL-Ig. Inflammatory cytokines TNF-α, IFN-γ, and IL-1β were blocked with anti-TNF (#BE0058; Bio-X-Cell), anti-INF-γ (#BE0055; Bio-X-Cell), or anti-IL-1β (#BE0246; Bio-X-Cell) antibodies at 100 μg/mouse injected intravenously via the tail vein or retro-orbital sinus immediately before CFA injection. For LTβR-Ig treatment at steady state, mice were injected with 100 μg LTβR-Ig i.v. once a week for 3 wk and analyzed 3 wk after the first injection. In vivo BrdU incorporation was performed by BrdU injection i.v. (1 mg/mouse). Flow cytometry detection of incorporated BrdU was performed with FITC or APC BrdU Flow Kit (BD Biosciences) by following the manufacturer’s protocol.