Extended Data Fig. 5: Enteric GABAergic neurons are major source of GABA.
a, b, Fecal bacterial load calculation (a) and ELISA assay of small intestinal GABA (b) in wild-type mice fed with vehicle or antibiotics in drinking water for 14 days. n = 3 per group. c, Diagram of GABA biosynthesis. d, QPCR analysis of Gad2 gene expression in dorsal root ganglions (DRG) and enteric neurons isolated from wild-type mice. n = 3 biological independent experiments. e, Immunofluorescent microscopy of Gad2 in small intestine (up) and colon (down) from wild-type mice, and nNOS (f) and CGRP (g) in colon tissues collected from Gad2-Cre+Rosa26LSL-tdTomato mice. Scale bar, 100μm. n = 3 times of experiments were repeated independently with similar results. h, QPCR analysis of knockdown efficiency (left) and ELISA analysis of GABA production in supernatants (right) of in vitro cultured enteric neurons infected with AAVs carrying shRNAs or a control sequence. n = 3 per group. i, Scheme of Gad2 knockout mice construction and validation by DNA sequencing. j, k, Immunoblotting assay of Gad2 expression in small intestine tissues (j) and ELISA analysis of GABA production in enteric neurons (k) from Gad2+/+ and Gad2-/- mice. n = 3 per group. l, m, FACS analysis of ILC1s (l; CD3−CD19−CD45+CD127+NKp46+NK1.1+) and ILC2s (m; CD3−CD19−CD127+CD90+Sca-1+KLRG1+) in the small intestine from Gad2+/+ and Gad2-/- mice. n = 5 per group. n, Calculation of indicated ILC numbers as in l, m. n = 5 per group. o, FACS analysis of Ki-67 the proliferation (left) and IL-17A production of (right) of ILC3 subsets in the colon from Gad2+/+ and Gad2-/- mice. n = 5 per group. p, q, H&E staining assay (p) and histopathological damage evaluation (q) of colon tissues from Gad2+/+ and Gad2-/- mice intraperitoneally administrated with IL17A neutralization antibody or isotype during TNBS-induced colitis. Scale bar, 100μm. n = 5 per group. ns, not significant. Data were analyzed by an unpaired two-side Student’s t-test and shown as means ± SD. Data were representative of at least three independent experiments.
