A single early-in-life antibiotic course increases susceptibility to DSS-induced colitis

Abstract

Background: There is increasing evidence that the intestinal microbiota plays a crucial role in the maturation of the immune system and the prevention of diseases during childhood. Early-life short-course antibiotic use may affect the progression of subsequent disease conditions by changing both host microbiota and immunologic development. Epidemiologic studies provide evidence that early-life antibiotic exposures predispose to inflammatory bowel disease (IBD).

Methods: By using a murine model of dextran sodium sulfate (DSS)-induced colitis, we evaluated the effect on disease outcomes of early-life pulsed antibiotic treatment (PAT) using tylosin, a macrolide and amoxicillin, a beta-lactam. We evaluated microbiota effects at the 16S rRNA gene level, and intestinal T cells by flow cytometry. Antibiotic-perturbed or control microbiota were transferred to pups that then were challenged with DSS.

Results: A single PAT course early-in-life exacerbated later DSS-induced colitis by both perturbing the microbial community and altering mucosal immune cell composition. By conventionalizing germ-free mice with either antibiotic-perturbed or control microbiota obtained 40 days after the challenge ended, we showed the transferrable and direct effect of the still-perturbed microbiota on colitis severity in the DSS model.

Conclusions: The findings in this experimental model provide evidence that early-life microbiota perturbation may increase risk of colitis later in life.

Keywords: Childhood antibiotic use; DSS-induced colitis; Gastrointestinal microbiota; Macrolide; Pulsed antibiotic treatment.

Fig. 1

Effect of early-life antibiotic exposure on the severity of DSS-induced colitis. a Schematic of early DSS experiment, using a single 5-day antibiotic course (PAT). C57BL/6 mouse study groups were control/H2O (n = 15), PAT/H2O (n = 16), control/DSS (n = 16), and PAT/DSS (n = 16). Nursing dams received either tylosin or non-acidified drinking water when their pups were between 5 and 10 days old (P5-P10), and pups were exposed to tylosin or not through their mother’s milk. Experimental colitis was induced at P25 by adding 2% DSS to the pup’s drinking water or not for 7 days, and mice were sacrificed at P34. b Normalized percent weight decrease between the groups, measured from P25, the first day of the DSS challenge. c Presence of blood during and after the DSS challenge was scored as 0 (no blood), 1 (hemoccult positive), 2 (hemoccult positive and visual pellet bleeding), or 4 (gross bleeding, blood around the anus). d Stool consistency during and after the DSS challenge was scored as [0 (normal), 2 (loose stool), or 4 (diarrhea)] e Fecal lipocalin-2 levels (ng/mL) at P34; control/H2O (n = 6), PAT/H2O (n = 12), control/DSS (n = 16), and PAT/DSS (n = 16). f Histology scores (total of inflammation, epithelium damage, atrophy, and dysplasia scores); control/H2O (n = 4), PAT/H2O (n = 4), control/DSS (n = 4), and PAT/DSS (n = 3) g Colonic lamina propria Th17 and Treg cells shown as absolute cell numbers and as percent of total CD4 cells; control/H2O (n = 3), PAT/H2O (n = 3), control/DSS (n = 3), and PAT/DSS (n = 3). Two-way ANOVA, Kruskal-Wallis non-parametric test and Dunn’s multiple comparison testing were used for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 2

Effect of PAT and DSS-challenge on colonic gene expression. Colonic samples were obtained at sacrifice from the mice in Fig. 1, and RNA extracted. RT-qPCR was performed using primers for four genes affected by the inflammatory process (TNFα; IL-22; Muc2; Muc4). Group sizes were control/H2O (n = 4), PAT/H2O (n = 6), control/DSS (n = 5), and PAT/DSS (n = 6). Groups were also collapsed into DSS− (n = 10) and DSS+ (n = 11) and comparisons shown by dashed lines; Mann-Whitney test for the collapsed analysis. Kruskal-Wallis non-parametric test and Dunn’s multiple comparison testing were used for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Effect of PAT and DSS-challenge on the intestinal microbial community. a Phylogenetic diversity (PD) scores over time. Group sizes were control/H2O (n = 7), PAT/H2O (n = 8), control/DSS (n = 7), and PAT/DSS (n = 8). b Comparison of microbial community structure between PAT and control groups at P21 (weaning), and between all groups at P25 (before start of DSS), P28, and P32 (end of the DSS challenge) based on unweighted UnifFrac distances as visualized by PCoA. Intra- and inter-group mean pairwise UniFrac distances are shown as bars. Intergroup community distances remain significantly greater than from P21 until day P32 for both the control and PAT-exposed groups. c Heat map showing significantly different taxa between the control/DSS (blue) and PAT/DSS (red) groups, using the linear discriminant analysis Effect Size (LEfSe) tool. Samples were from feces (P21, P25, P28, P31), or from the cecum (ce), colon (co), or ileum (il) at P34 sacrifice. Two-way ANOVA and Kruskal-Wallis tests were used for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Lasting effects of early-life antibiotic exposure on DSS-induced colitis severity and on intestinal microbial communities. a Schematic of the late time point DSS experiment. Sample sizes were the control/H2O (n = 9), PAT/H2O (n = 14), control/DSS (n = 18), and PAT/DSS (n = 26) mice for a–d. For lipocalin analyses (e) sample sizes were the control/H2O (n = 5), PAT/H2O (n = 4), control/DSS (n = 15), and PAT/DSS (n = 23). For histology scoring (f) sample sizes were the control/H2O (n = 4), PAT/H2O (n = 4), control/DSS (n = 4), and PAT/DSS (n = 4). For microbiome analyses (g, h) sample sizes were the control/H2O (n = 6), PAT/H2O (n = 8), control/DSS (n = 8), and PAT/DSS (n = 8). Tylosin exposure or not and study design was exactly as in Fig. 1, except experimental colitis by the DSS challenge was induced at P40, 30 days after PAT ended instead of P25. b–f used the same measurements and criteria as in Fig. 1, except at the time points reflecting the different study design. Fecal lipocalin-2 levels were measured at P50. g Unweighted UniFrac distances between the PAT and control groups at P40 (before start of DSS), and mean pairwise UniFrac distances within and between groups. h LEfSe cladograms indicating significantly differential taxa in control and PAT mice at P40 (30 days after PAT and immediately before the DSS challenge) and P47 (following the DSS challenge). Two-way ANOVA and Kruskal-Wallis tests were used for multiple comparisons. *p < 0.05, **p < 0.01, ****p < 0.0001

Fig. 5

Effect of transferring PAT-altered microbiota to germ-free mice on DSS-induced colitis and intestinal microbial community. a Schematic of the transfer experiment. Six-week old germ-free (GF) mice were gavaged with either PAT-perturbed (n = 5) or control (n = 4) cecal contents from P40 donor mice (30 days after the end of the PAT or control exposure). The now-conventionalized recipient mice (3 PAT and 2 control) were challenged with DSS 5 days after the cecal transfer. b–e Used the same measurements and criteria as in Fig. 1, except at the time points reflecting the different study designs. f Histology of the colon in control and PAT recipients. Magnification × 10, H&E staining. Individual scores for inflammation, epithelial injury, atrophy, and dysplasia. g Representatives of differences in mean colon length between control and PAT recipients. h Intestinal permeability measured by fluorescein isothiocyanate (FITC)-dextran in blood. Two-way ANOVA and unpaired t test were used for comparisons. *p < 0.05, **p < 0.01, ****p < 0.0001