Fig. 5: Breast milk activates AHR on the intestinal epithelium and protects against experimental NEC in newborn mice.

a, b qRT-PCR showing the expression of Cyp1a1 (a, n = 6, 5, 6, 5 wells of enteroids, WT saline vs WT breast milk p = 0.0096) and Tnf-α (b, n = 5, 3, 3, 3, 3 wells of enteroids, Ctrl vs WT LPS p = 0.0001, WT LPS vs WT LPS + breast milk p = 0.0325) in mouse intestinal enteroids from wild-type (WT) and Ahr^-/- mice, treated with saline or LPS in the presence or absence of breast milk. c, d LPS-induced apoptosis in enteroids measured by TUNEL assay in wild-type and Ahr^-/- mouse enteroids as quantified (c, n = 7, 5, 7, 10, 4 enteroid sections, Ctrl vs WT LPS p < 0.0001, WT LPS vs WT LPS + breast milk p < 0.0001) and revealed (d) by confocal microscopy. TUNEL, green signal; nuclei (DAPI, blue signal). e–g qRT-PCR showing the expression of Cyp1a1 (e, n = 9, 6, 9 5 mice, WT saline vs WT breast milk p = 0.0026), Il6 (f, n = 9, 6, 4, 5, 3 mice, Ctrl vs WT LPS p = 0.0039, WT LPS vs WT LPS + breast milk p = 0.0070) and Tnf-α (g, n = 5, 3, 3, 3, 3 mice, Ctrl vs WT LPS p = 0.0001, WT LPS vs WT LPS + breast milk p = 0.0325) in wild-type and Ahr^-/- mice treated with saline, LPS and/or breast milk as indicated. h–l Representative H&E- stained section (h), NEC severity score (i, n = 6, 7 mice, p = 0.0023), and the expression of Il6 (j, n = 7, 6 mice, p = 0.0056), Tnf-α (k, n = 7, 6 mice, p = 0.0221) and Tlr4 (l, n = 7, 6 mice, p = 0.0465) in wild-type and Ahr^-/- mice induced to develop NEC in the presence or absence of breast milk as indicated. Scale bars in d, 10 μm. Scale bars in h, 100 μm. All data are presented as mean values ±  SEM. *p < 0.05, **p < 0.01, ***p < 0.001, p values obtained either from two-sided t tests or one-way ANOVA followed by multiple comparisons. Each dot in graphs represents data from an individual well of enteroids, section of enteroids, or an individual mouse.