Figure 4. NMU activates ILC2s.

(a) Gating strategy for flow cytometric analysis of bulk LPLs cytokine assays. Lineage1: CD11b, CD11c and B220 (all APC-eF780); Lineage2: CD3, CD5 (both PerCP-Cy5.5) and FcεRI PerCP-eF710.

(b,c) Concentration (Mean + SD, n= 3) of IL-13 (b) or IL-5 (c) in the culture supernatant after 4h stimulation of bulk LPLs with a control peptide or NMU as determined by ELISA (n.d. : not detectable).

(d) Bulk LPLs from Il1rl1^+/+ and Il1rl1^−/− mice were incubated in medium with or without NMU for 4h in vitro. Percentage (Mean + SD, Il1rl1^+/+ n= 5, Il1rl1^−/− n= 4) of IL-5⁺ KLRG1⁺ cells.

(e,f) LPLs from Nmur1^+/+ or Nmur1^−/− mice were analyzed by flow cytometry. Plots are gated on Lin⁻CD45⁺ lymphocytes (e). Percentage (Mean + SD, n= 3) of GATA-3⁺ KLRG1⁺ cells (f).

(g) Bulk LPLs were incubated for 30 min with DMSO or the inhibitor of Gαq proteins FR900359 in vitro. Medium, NMU or the indicated cytokine cocktail was then added and the assay was incubated for another 4h. Percentage (Mean + SD, n=3) of IL-13 YFP⁺ KLRG1⁺ cells among all KLRG1⁺ cells.

(h) Percentage (Mean + SD, n=4) of IL-5⁺ or IL-13⁺ KLRG1⁺ cells as determined by intracellular cytokine staining.

(j-l) Overnight incubation of sort-purified intestinal ILC2s or C57BL/6 mice (k,l ; n=5).

(i-k) Intestinal ILC2s from Il13^Yfp/+ mice were sort-purified and incubated in medium with or without NMU overnight in vitro. FSC (Mean + SD, n=3) (i), histogram overlay of IL-13 YFP (j) and percentage (Mean + SD ; n=3) of IL-13 YFP ⁺ ILC2s (k).

(l) ILC2s from the small intestine were sort-purified and incubated in medium without or with NMU over night in vitro. Contour plots show intracellular flow cytometry analysis for IL-5 and IL-13.

Data are representative of two (b-d, g,h) or three independent experiments (e, f, i-l) with similar results. Gating in (a) is representative for cytokine assays used in the whole study.