Early Life Antibiotics Influence In Vivo and In Vitro Mouse Intestinal Epithelium Maturation and Functioning

Abstract

Background & aims: The use of antibiotics (ABs) is a common practice during the first months of life. ABs can perturb the intestinal microbiota, indirectly influencing the intestinal epithelial cells (IECs), but can also directly affect IECs independent of the microbiota. Previous studies have focused mostly on the impact of AB treatment during adulthood. However, the difference between the adult and neonatal intestine warrants careful investigation of AB effects in early life.

Methods: Neonatal mice were treated with a combination of amoxicillin, vancomycin, and metronidazole from postnatal day 10 to 20. Intestinal permeability and whole-intestine gene and protein expression were analyzed. IECs were sorted by a fluorescence-activated cell sorter and their genome-wide gene expression was analyzed. Mouse fetal intestinal organoids were treated with the same AB combination and their gene and protein expression and metabolic capacity were determined.

Results: We found that in vivo treatment of neonatal mice led to decreased intestinal permeability and a reduced number of specialized vacuolated cells, characteristic of the neonatal period and necessary for absorption of milk macromolecules. In addition, the expression of genes typically present in the neonatal intestinal epithelium was lower, whereas the adult gene expression signature was higher. Moreover, we found altered epithelial defense and transepithelial-sensing capacity. In vitro treatment of intestinal fetal organoids with AB showed that part of the consequences observed in vivo is a result of the direct action of the ABs on IECs. Lastly, ABs reduced the metabolic capacity of intestinal fetal organoids.

Conclusions: Our results show that early life AB treatment induces direct and indirect effects on IECs, influencing their maturation and functioning.

Keywords: Antibiotic Treatment; Fetal Organoids; Neonatal Intestine.

Graphical abstract

Figure 1

In vivo growth and macroscopic assessment of small intestine, liver, and spleen. (A) Experimental design of in vivo antibiotic treatment of pups between P10 and P20. Antibiotic mix: amoxicillin, metronidazole, and vancomycin. All analyses were performed at P20. (B) Weight of pups was measured every 2 days during antibiotic mix treatment between P10 and P20. (C) Small intestine weight, relative to body weight. (D) Small intestine length, relative to body weight. (E) Liver weight, relative to body weight. (F) Spleen weight, relative to body weight. Statistical analysis was performed by a (B) 2-way analysis of variance test with the Sidak multiple comparisons test or a (C–F) 2-tailed unpaired t test. Error bars indicate means ± SD. Levels of significance are indicated: ∗P < .05, ∗∗P < .01. n = 9–12 pups per group.

Figure 2

Early life antibiotics affect intestinal barrier function in vivo, particularly in the distal small intestine. (A) H&E staining of proximal and distal small intestine. Black triangles indicate vacuolated enterocytes and white triangles indicate nonvacuolated enterocytes. Scale bars: 100 μm. (B) Quantification of the number of vacuoles per villi in the distal small intestine. (C) Permeability assay assessed by FITC-dextran concentration in serum 4 hours after oral gavage. (D) Villi length in proximal and distal small intestine. (E) Crypt depth in proximal and distal small intestine. Immunohistochemistry of (F) proliferation markers Ki67 and (G) phosphorylated histone H3 in proximal and distal small intestine. Scale bars: 100 μm. (H) Serum concentration of IgG and IgA in control and antibiotic mix–treated pups at P20 compared with adult mice. Statistical analysis was performed by the Mann–Whitney test because data were not normally distributed when assessed by the (B) D’Agostino and Pearson normality test, the (C–E and G) 2-tailed unpaired t test or (H) 1-way analysis of variance with the Tukey multiple comparisons test. Error bars indicate (B) medians with interquartile range or (C–E, G, and H) means ± SEM. Levels of significance are indicated: ∗P < .05, ∗∗P < .01, ∗∗∗P < .0001, ∗∗∗∗P < .0001. (B–E and G) n = 9–12 pups per group and (H) n = 6–9 P20 samples per group and n = 1–3 adult samples.

Figure 3

Genome-wide gene expression analysis of sorted intestinal epithelial cells. (A) Experimental design of genome-wide gene expression analysis of fluorescence-activated cell sorter (FACS)-sorted P20 intestinal epithelial cells. (B) Percentage of EpCAM stained cells in the total number of FACS-sorted intestinal epithelial cells per condition. (C) Principal component analysis (PCA) of sorted epithelial cells from P20 proximal and distal small intestine treated with the antibiotic mix or PBS (control). (D) Volcano plots of microarray analysis showing genes differentially expressed between control and antibiotic-treated FACS-sorted P20 intestinal epithelial cells. Green dots identify down-regulated genes and red dots identify up-regulated genes. Statistical analysis by empirical Bayes analysis of variance, P < .05 cut-off. n = 4 samples per group.

Figure 4

Differential gene expression analysis and GSEA of sorted intestinal epithelial cells after antibiotic treatment. Curated heatmaps of selected genes from top down-regulated and top up-regulated genes, based on biological interest and grouped according to function (A) maturation and (B) intracellular digestion in proximal and distal epithelial cells. The colored bar represents the expression level from low (green) to high (red). GSEA plots comparing control and antibiotic-treated distal SI epithelial cells against gene sets of (C) GO vacuole organization and (D) HALLMARK fatty acid metabolism. Enrichment score (ES), normalized enrichment score (NES), and P values are indicated in the image. Curated heatmaps of selected genes from top down-regulated and top up-regulated genes, based on biological interest and grouped according to function (E) (innate) defense in distal epithelial cells and (F) (trans)epithelial sensing in proximal and distal epithelial cells. The colored bar represents the expression level from low (green) to high (red). GSEA plots comparing (G) distal SI control and antibiotic-treated epithelial cells against a published gene set of distal SI enterocytes and (H) comparing proximal SI control and antibiotic-treated epithelial cells against a proximal SI enteroendocrine cell signature. ES, NES, and P values are indicated in the image. Statistical analysis by empirical Bayes analysis of variance, P < .05 cut-off. n = 4 samples per group.

Figure 5

Early life antibiotics induce in vivo precocious maturation of the intestinal epithelium. (A) Whole-tissue real-time qPCR analysis of adult maturation markers Sis and Arg2 and of fetal maturation markers FcRn and Ass1 in proximal and distal small intestine. Relative expression to reference genes Cyp and Ppib. Immunohistochemistry of (B) adult marker Sis and (C) fetal marker Ass1 in proximal and distal small intestine. Black triangles indicate positive cells and white triangles indicate negative cells. Scale bars: 100 μm. Statistical analysis was performed by 1-tailed unpaired t test (Sis, FcRn, and Ass1) or Mann–Whitney test because data were not distributed normally when assessed by the D’Agostino and Pearson normality test (Arg2). Error bars indicate means ± SD (Sis, FcRn, and Ass1) or medians with interquartile range (Arg2). Levels of significance are indicated: ∗P < .05, ∗∗P < .01. n = 10–12 pups per group.

Figure 6

Treatment with early life antibiotics in vivo decreases expression of intracellular digestion markers and increases expression of innate defense and enteroendocrine cell markers. Whole-tissue real-time qPCR analysis of (A) intracellular digestion markers CtsL, CtsZ, CtsA, Dab2, and Mcoln3 and of (B) innate defense markers Lyz1, Reg3β, and Reg3ɣ in distal small intestine. (C) Immunohistochemistry of LYZ1 and quantification of amount of lysozyme-1–positive cells per crypt in distal small intestine. Black arrowheads indicate positively stained cells. Scale bars: 100 μm. (D) Alcian blue and periodic acid-Schiff (PAS) staining of proximal and distal small intestine. Scale bars: 100 μm. (E) Whole-tissue real-time qPCR analysis of (trans)epithelial sensing markers Gip, Nts, Gcg, Pyy, Sst, Sct, and Cck in proximal small intestine. Relative expression to reference genes Cyp and Ppib. Statistical analysis was performed by 1-tailed unpaired t test (CtsZ, Dab2, Lyz, Reg3ɣ, Gip, Nts, Gcg, Pyy, Sst, Sct, and Cck) or the Mann–Whitney test because data were not distributed normally when assessed by the D’Agostino and Pearson normality test (CtsL, CtsA, Mcoln3, and Reg3β). Error bars indicate means ± SD (CtsZ, Dab2, Lyz, Reg3ɣ, Gip, Nts, Gcg, Pyy, Sst, Sct, and Cck) or medians with interquartile range (CtsL, CtsA, Mcoln3, and Reg3β). Levels of significance are indicated: ∗P < .05, ∗∗P < .01. n = 8–12 pups per group.

Figure 7

Appearance and budding quantification of proximal and distal fetal intestinal organoids treated with early life antibiotics over time. (A) Experimental design of in vitro treatment of mouse fetal intestinal organoids with the antibiotic mix. Organoids were analyzed on days 3, 13, 20, and 27 of culture. (B) Microscopic images of control and antibiotic mix–treated organoids on days 3, 13, 20, and 27 of culture. Scale bars: 500 μm. (C) Quantification of the number of buds per organoid of proximal and distal cultures in control and antibiotic mix conditions. Statistical analysis was performed by a 1-tailed paired t test. Error bars indicate means ± SD. Levels of significance are indicated: ∗P < .05, ∗∗P < .01). n = 18–57 organoids per condition and day of 4 independent cultures.

Figure 8

Antibiotic treatment accelerates fetal organoid maturation in vitro. (A) Relative expression of adult maturation markers Sis and Arg2 detected by real-time qPCR in proximal and distal fetal organoids. Relative expression to reference genes Rpl32 and TbP. (B) Immunohistochemistry and quantification of proximal and distal fetal organoids of SIS. Scale bars: 50 μm. Relative expression by real-time qPCR of (C) fetal maturation markers FcRn and Ass1 in proximal and distal fetal organoids and of (D) intracellular digestion markers CtsL, CtsZ, CtsA, Dab2, and Mcoln3 in distal fetal organoids. Relative expression to reference genes Rpl32 and TbP. Statistical analysis was performed by 2-way analysis of variance with the (A, C, and D) Sidak multiple comparisons test or the (B) 2-tailed unpaired t test. Error bars indicate means ± SD. Levels of significance are indicated: ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. (A, C, and D) n = 3 individual wells from a representative organoid culture of 4–6 independent cultures and (B) n = 8–12 organoids of 2 independent cultures.

Figure 9

Antibiotic treatment of in vitro fetal organoids induces differentiation of Paneth cells and enteroendocrine cells. (A) Real-time qPCR analysis and (B) immunohistochemistry of innate defense marker Lyz1/LYZ1 in distal fetal organoids. Relative expression to reference genes Rpl32 and TbP. Black arrowheads indicate Paneth cells positively stained for LYZ1 and white arrows indicate Paneth cells not stained for LYZ1. Scale bars: 50 μm. Real-time qPCR analysis of (C) innate defense markers Reg3β and Reg3ɣ in distal fetal organoids and (D) (trans)epithelial sensing markers Gip, Nts, Gcg, Pyy, Sst, Sct, and Cck in proximal fetal organoids. Relative expression to reference genes Rpl32 and TbP. (E) Immunofluorescence of whole proximal fetal organoids for GIP and quantification of the amount of GIP-positive cells per organoid area. Statistical analysis was performed by (A, C, and D) 2-way analysis of variance with the Sidak multiple comparisons test or (E) 2-tailed unpaired t test. Error bars indicate means ± SD. Levels of significance are indicated: ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. (A, C, and D) n = 3 individual wells from a representative organoid culture of 4–6 independent cultures and (G) n = 40 organoids of 2 independent cultures.

Figure 10

Respiration capacity of distal fetal organoids decreases upon in vitro early life antibiotics. (A) Experimental design of seahorse experiments to measure mitochondrial respiration and glycolysis in mouse fetal intestinal organoids after 5 days of antibiotic mix treatment. (B) Graphic representation of key parameters measured by OCR. Real-time respiration levels in the supernatant of (C) proximal and (D) distal fetal organoids measured as OCR, and basal respiration, ATP production, and maximal respiration rates calculated using OCR levels determined by seahorse assay. Statistical analysis was performed by the 1-tailed unpaired t test. Error bars indicate means ± SEM (line graphs) or means ± SD (column graphs). Levels of significance are indicated: ∗P < .05. n = 4–5 individual wells, representative of 2–4 independent experiments.

Figure 11

Glycolytic capacity of fetal organoids decreases upon in vitro early life antibiotics. (A) Graphic representation of key parameters measured by ECAR. Real-time glycolysis levels in the supernatant of (B) proximal and (C) distal fetal organoids measured as ECAR, and glycolysis and glycolytic capacity calculated using ECAR levels determined by seahorse assay. Statistical analysis was performed by the 1-tailed unpaired t test. Error bars indicate means ± SEM (line graphs) or means ± SD (column graphs). Levels of significance are indicated: ∗P < .05, ∗∗P < .01. n = 4–5 individual wells, representative of 2–4 independent experiments.