Fig. 5: Butyrate suppresses neonatal ILC2 responses via upregulation of IFN1 signaling.

a,b Metabolomics analysis was performed to evaluate the metabolites in breastmilk and serum from Abx-treated dams and control dams. a Volcano plot showing metabolites with differential abundance between Abx-treated dams and the controls (n = 3). The two-sided Wilcoxon rank-sum test was employed to identify differences. The Benjamini-Hochberg method was applied for multiple testing correction. b Targeted metabolomics analysis of SCFAs in serum (n = 5)/breast milk (n = 3) of Abx-treated dams and controls. c ILC2s from neonatal lungs were cultured in vitro with 100 ng/mL IL-33, 20 ng/mL IL-2 and 20 ng/mL IL-7 for 3 days, in the presence of butyrate (But, 2 mM) or medium control (Med). The mRNA expression of Gata3, Il5, and Il13 was determined by qRT-PCR (n = 4). d GF newborn mice were intraperitoneally injected with butyrate (100 mg/kg) daily for 5 consecutive days, and pups were sacrificed at postnatal day 7 (n = 5). The absolute numbers of ILC2s, the frequency of Ki-67⁺ ILC2s and IL-5⁺IL-13⁺ ILC2s, as well as eosinophils in lungs were evaluated by flow cytometry. e–g Lung ILC2s from neonatal mice were cultured with 100 ng/mL IL-33, 20 ng/mL IL-2, and 20 ng/mL IL-7 for 3 days in the presence or absence of butyrate (2 mM). e Phosphorylation of STAT1 and STAT2 was determined by flow cytometry (n = 5). f mRNA expression of Ifnar1, Irf7, Mx1, Isg15 and Ifnb1 was determined by qRT-PCR (n = 4). g Lung ILC2s from neonates were cultured with IL-2, IL-7, and IL-33 for 3 days in the presence or absence of butyrate (2 mM), fludarabine (Flu, 5 μM), or hydrocortisone (Hydro, 0.4 mg/mL) for 12 h. Amounts of IL-5 and IL-13 in supernatants were determined by ELISA (n = 4). h Expression of GPR41 on neonatal ILC2s was evaluated by flow cytometry (left) and immunofluorescence. Scale bar, 10 μm (right). i–j Gpr41^−/− pregnant mice and WT control were subjected to antibiotic treatment; pups were intraperitoneally injected with butyrate (100 mg/kg) daily for 5 consecutive days and were sacrificed at PND7 (n = 4). Absolute numbers of ILC2s (i-left) and the frequency of IL-5⁺IL-13⁺ ILC2s (i-middle) and absolute numbers of eosinophils (i-right) were evaluated by flow cytometry. Amounts of IFNβ in lung homogenates were measured by ELISA (j). In Fig. 5e and 5j, the data are presented as the mean ± SEM values, by unpaired two-tailed Student’s t test. For box plots, the data are shown as “Min to Max, show all points”. For box plots, the midline represents the median; box represents the interquartile range (IQR) between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles (b, c, d, f, g, i). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired two-tailed Student’s t test (d, g, i) or Mann–Whitney U test (b, c, f). Data are presentative of 2-3 independent experiments (c–j). Statistical source data are provided in Source Data.