The enteric microbiota regulates jejunal Paneth cell number and function without impacting intestinal stem cells

Abstract

Paneth cells (PCs) are epithelial cells found in the small intestine, next to intestinal stem cells (ISCs) at the base of the crypts. PCs secrete antimicrobial peptides (AMPs) that regulate the commensal gut microbiota. In contrast, little is known regarding how the enteric microbiota reciprocally influences PC function. In this study, we sought to characterize the impact of the enteric microbiota on PC biology in the mouse small intestine. This was done by first enumerating jejunal PCs in germ-free (GF) versus conventionally raised (CR) mice. We next evaluated the possible functional consequences of altered PC biology in these experimental groups by assessing epithelial proliferation, ISC numbers, and the production of AMPs. We found that PC numbers were significantly increased in CR versus GF mice; however, there were no differences in ISC numbers or cycling activity between groups. Of the AMPs assessed, only Reg3γ transcript expression was significantly increased in CR mice. Intriguingly, this increase was abrogated in cultured CR versus GF enteroids, and could not be re-induced with various bacterial ligands. Our findings demonstrate the enteric microbiota regulates PC function by increasing PC numbers and inducing Reg3γ expression, though the latter effect may not involve direct interactions between bacteria and the intestinal epithelium. In contrast, the enteric microbiota does not appear to regulate jejunal ISC census and proliferation. These are critical findings for investigators using GF mice and the enteroid system to study PC and ISC biology.

Keywords: Paneth cell; Reg3γ; enteroid; germ-free; intestinal stem cell; microbiota.

Figure 1.

Paneth cell (PC) number is decreased in germ-free (GF) mice. A: Representative H&E images of PCs located at the base of the crypts from the jejunum of conventionally raised (CR) and GF mice (60x magnification, scale bar = 20 μm). At least 2 jejunal sections were prepared per mouse, from 7–8 mice/group. B: Quantitative analysis of PC number per crypt from CR and GF mice. At least 10 crypts/mouse were evaluated (***P < 0.001). C: Representative flow cytometry plots gating Lysozyme (Lyz)⁺ epithelial cells from CR and GF mice. D: Quantitative analysis of PCs as a percentage of the total epithelium in CR and GF mice (n = 5 pools of mice/group, with 4 mice/pool, *P < 0.05). All quantitative data are represented as scatter plots with a line representing the mean.

Figure 2.

Conventionally raised (CR) and germ-free (GF) mice have similar numbers of total proliferating cells within the intestinal epithelium. A: EdU immunofluorescence (IF) staining from the jejunum of CR and GF mice (10x magnification, single section from 1 mouse/group). B: Representative flow cytometry plots showing total number of proliferating cells (EdU⁺) in CR and GF mice. C: Quantitative analysis of proliferating cells as a percentage of the total epithelium in CR and GF mice (n = 5 pools of mice/group, with 4 mice/pool, P = 0.5). N.S. = not significant. Quantitative data are represented as scatter plots with a line representing the mean.

Figure 3.

Conventionally raised (CR) and germ-free (GF) mice have similar numbers of intestinal stem cells (ISCs). A: Representative flow cytometry plots showing total ISC numbers (CD24^loLyz^–) in CR and GF mice. B: Quantitative analysis of ISCs as a percentage of the total epithelium in CR and GF mice (n = 5 pools of mice/group, with 4 mice/pool, P = 0.5). C: Representative flow cytometry plots evaluating the cycling status of ISCs (CD24^loLyz^–EdU⁺) in CR and GF mice. Lyz⁺ cells have been excluded from these gates. D: Quantitative analysis of cycling ISCs as a percentage of total ISCs (n = 5 pools of mice/group, with 4 mice/pool, P = 0.8). N.S. = not significant. Quantitative data are represented as scatter plots with a line representing the mean.

Figure 4.

Conventionally raised (CR) and germ-free (GF) jejunal enteroids show no differences in crypt survival or proliferation. A: Representative images of day 6 jejunal enteroids from CR and GF mice. Arrowheads (white) indicate PCs. B: Efficiency of enterosphere (day 1) and enteroid (day 6) formation from cultured CR and GF crypts (n = 6-7 mice/group, P > 0.2 for both measures). Represented as percentage of plated crypts surviving. C: Lateral bud counts (n = 6-7 mice/group) and area (n = 3 mice/group) of CR and GF enteroids on day 6 of culture (P > 0.4 for both measures). For all enteroid studies, 200–400 crypts were plated per mouse. N.S. = not significant. Quantitative data are represented as scatter plots with a line representing the mean.

Figure 5.

The enteric microbiota selectively regulates jejunal Paneth cell (PC) Reg3γ expression. Transcript levels of the primary antimicrobial peptide classes in C57BL/6J mice were measured in conventionally raised (CR) and germ-free (GF) jejunal crypts, including: lysozyme (Lyz), regenerating islet-derived protein 3 gamma (Reg3γ), angiogenin 4 (Ang4), global α-defensins (PanCrp), and cryptdin-related sequence 1c (Crs1c). Data are shown as target gene expression normalized to β-actin (scatter plot with mean, ***P < 0.001). N.S. = not significant.

Figure 6.

Elevated Reg3γ expression of conventionally raised (CR) crypts is rapidly lost in culture. Scatter plot (with mean) shows Reg3γ transcript expression of freshly isolated CR and germ-free (GF) crypts, and corresponding enteroids harvested after 6 days of culture. Data are depicted as target gene expression normalized to β-actin. Two-way ANOVA showed highly significant main effects of microbial status (P < 0.0001) and culture (P < 0.0001) as well as the interaction of the two variables (P < 0.0001). Pairwise Mann-Whitney comparisons showed the Reg3γ mRNA in CR enteroids to be significantly less than those in CR crypts (***P < 0.002) and to be not significantly (N.S.) different from those in GF crypts (P > 0.94).