Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses

Abstract

The lower intestine of adult mammals is densely colonized with nonpathogenic (commensal) microbes. Gut bacteria induce protective immune responses, which ensure host-microbial mutualism. The continuous presence of commensal intestinal bacteria has made it difficult to study mucosal immune dynamics. Here, we report a reversible germ-free colonization system in mice that is independent of diet or antibiotic manipulation. A slow (more than 14 days) onset of a long-lived (half-life over 16 weeks), highly specific anticommensal immunoglobulin A (IgA) response in germ-free mice was observed. Ongoing commensal exposure in colonized mice rapidly abrogated this response. Sequential doses lacked a classical prime-boost effect seen in systemic vaccination, but specific IgA induction occurred as a stepwise response to current bacterial exposure, such that the antibody repertoire matched the existing commensal content.

Fig. 1

Reversible E. coli HA107 colonization induces specific mucosal IgA. (A) Germ-free Swiss-Webster mice were analyzed for fecal shedding of live E. coli by bacterial plating and enrichment culture in m-DAP- and D-Ala-supplemented media of fecal material at indicated times after gavage of 10¹⁰ CFU of HA107 (n=6) or wild type parent strain JM83 (E. coli K-12; n=6). Data points represent individual mice from one experiment. Bars indicate medians. (B) Germ-free SwissWebster mice treated as in (A) with HA107 (n=9) or E. coli K-12 (n=3) and analyzed after 48h. Data from one of two independent experiments are shown. (C) Germ-free Swiss-Webster mice were gavaged 6 times over 14 days with doses of 10¹⁰ CFU of HA107, and after a further 14 days their germ-free status was confirmed by bacterial culture from feces and intestinal contents and culture-independent bacterial staining (2). n.d., not detected by enrichment culture from cecal or fecal material (<10¹ CFU). Data from n=4 mice from one of 7 independent experiments are shown. (D-F) Germ-free Swiss-Webster mice were gavaged 6 times over 2 weeks (10¹⁰ CFU per dose, panel E, “HA107”, n=4) or colonized with a sentinel colonized mouse containing an E. coli-free altered Schaedler flora (panel F, “ASF”, n=3) and compared to age-matched germ-free controls (panel D, “GF”, n=3). Sections of duodenum were stained with a FITC-mouse-IgA antibody (green) and DAPI (blue) as a nuclear counterstain. Insets indicate numbers of IgA plasma cells per intestinal villus (mean ± S.D.). (G-J) Live bacterial flow cytometric analysis of IgA-bacterial binding using IgA-containing intestinal washes from the mice depicted in D-F (see fig. S3 for technical details). Blue squares, HA107 gavaged mice (HA107); red triangles, ASF sentinel colonized mice (ASF); black circles, germ-free control mice (GF). Images and curves in panels D-J represent individual mice from one of 7 independent experiments.

Fig. 2

Germ-free mice require a large dose of live bacteria for IgA production. (A) Germ-free Swiss-Webster mice were gavaged 6 times over 14 days with the indicated amounts of HA107 or PBS vehicle control only, or kept as germ-free controls (GF control), and analyzed at day 14. Tissue sections were stained for mouse-IgA to determine the numbers of duodenal IgA plasma cells. (B) Live bacterial flow cytometric analysis of intestinal washes from the mice described in (A) Data points and curves in A and B represent individual mice from one of two experiments. (C) Doses of 10⁹ or 10¹⁰ CFU of either live or heat-killed (15 min autoclaving) HA107 were gavaged into germ-free C57BL/6 and Swiss-Webster mice (6 times over 2 weeks) and analyzed by live bacterial flow cytometry on day 14. Black circles, germ-free control kept in the same isolator. Curves represent individual mice from one experiment. (D-F) Doses of 10¹⁰ CFU of live or peracidic acid-killed HA107 were gavaged into germ-free Swiss-Webster mice (6 times over 2 weeks) and analyzed by flow cytometric analysis of duodenal leukocytes (D), IgA-specific ELISA (E) and live bacterial flow cytometry (F) on day 14. Germ-free controls are depicted as black circles. Data points and curves represent individual mice from one experiment. Horizontal bars indicate means; *, statistically significant (p<0.05; 1-way ANOVA); ns, non-significant.

Fig. 3

Additive induction of intestinal IgA in proportion to bacterial exposure. Germ-free C57BL/6 mice were (A, B) given 1, 2 or 6 doses of 10¹⁰ CFU HA107 over the course of 1 week and analyzed at day 14. (C, D) In parallel, 3 groups of mice were given 6 doses in intervals of 1, 2-3 or 7 days apart, and analyzed on day 42. Intestinal washes were analyzed by IgA specific ELISA and live bacterial flow cytometry. -LogEC50 values of IgA-E. coli binding were calculated as described in Materials and Methods (; A, B), and numbers of duodenal IgA plasma cells were determined from 7 μm frozen sections stained for mouse IgA (C, D). Bars indicate means; *, statistically significant p<0.05 (1-way ANOVA); ns, non-significant. Data points represent individual mice from one of two experiments.

Fig. 4

Commensal-induced specific IgA is long-lived in germ-free mice, but rapidly lost in the face of ongoing IgA induction in microbiota-colonized mice. (A) Germ-free Swiss-Webster mice were gavaged 6 times over 2 weeks with 10¹⁰ CFU HA107 and kept germ-free for up to 112 days after discontinuation of HA107 treatment. Intestinal washes taken at the indicated time points were analyzed by IgA specific ELISA and live bacterial flow cytometry, and -LogEC50 values of IgA-E. coli binding were calculated. (B) Tissue sections from the experimental mice described in (A) were stained for mouse IgA to determine the numbers of IgA plasma cells in HA107-treated and germ-free control mice. (C) Colonized mice containing an E. coli-free ASF microbiota were treated as described in (A) and kept under barrier conditions for up to 119 days after discontinuation of HA107 treatment, and analyzed as above at the indicated time points. Bars indicate means; *, statistically significant (p<0.05; 1-way ANOVA). Data points in A and B, respectively in C represent individual mice from one of two independent experiments.