Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Subscribe

Logo for

Science 328 (5986): 1705-1709

Copyright © 2010 by the American Association for the Advancement of Science

Reversible Microbial Colonization of Germ-Free Mice Reveals the Dynamics of IgA Immune Responses

Siegfried Hapfelmeier1,2,*, Melissa A. E. Lawson2, Emma Slack1,2, Jorum K. Kirundi1,2, Maaike Stoel2, Mathias Heikenwalder3, Julia Cahenzli2, Yuliya Velykoredko2, Maria L. Balmer1, Kathrin Endt4, Markus B. Geuking2, Roy Curtiss, 3rd5, Kathy D. McCoy2, and Andrew J. Macpherson1,2,*

1 DKF (Maurice Müller Laboratories), MEM, Universitätsklinik für Viszerale Chirurgie und Medizin (UVCM), University of Bern, 3013 Bern, Switzerland.
2 Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, ON L8N 3Z5, Canada.
3 Department of Pathology, University Hospital Zurich, 8091 Zurich, Switzerland.
4 Institute of Microbiology, Federal Institute of Technology, 8093 Zurich, Switzerland.
5 Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA.


Figure 1
View larger version (37K):
[in this window]
[in a new window]

 
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 48 hours. Data from one of two independent experiments are shown. (C) Germ-free Swiss-Webster mice were gavaged six 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 (7). n.d., not detected by enrichment culture from cecal or fecal material (<101 CFU). Data from n = 4 mice from one of seven independent experiments are shown. (D to F) Germ-free Swiss-Webster mice were gavaged six times over 2 weeks [1010 CFU of HA107 per dose, (E), n = 4] or colonized with a sentinel colonized mouse containing an E. coli-free ASF [(F), n = 3] and compared to age-matched germ-free controls [(D), GF, n = 3]. Sections of duodenum were stained with a fluorescein isothiocyanate (FITC)–mouse–IgA antibody (green) and 4',6'-diamidino-2-phenylindole (DAPI) (blue) as a nuclear counterstain. Insets indicate numbers of IgA plasma cells per intestinal villus (mean ± SD). (G to J) Live bacterial flow cytometric analysis of IgA-bacterial binding using IgA-containing intestinal washes from the mice depicted in (D) to (F) (see fig. S3 for technical details). Blue squares, HA107-gavaged mice; red triangles, ASF sentinel colonized mice; and black circles, germ-free control mice (GF). Images and curves in (D) to (J) represent individual mice from one of seven independent experiments.

 

Figure 2
View larger version (17K):
[in this window]
[in a new window]

 
Fig. 2. Germ-free mice require a large dose of live bacteria for IgA production. (A) Germ-free Swiss-Webster mice were gavaged six times over 14 days with the indicated amounts of HA107 or phosphate-buffered saline (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. FACS, fluorescence-activated cell sorting. (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 (six 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 to F) Doses of 1010 CFU of live or peracidic acid–killed HA107 were gavaged into germ-free Swiss-Webster mice (six times over 2 weeks) and analyzed by flow cytometric analysis of duodenal leukocytes (D), IgA-specific enzyme-linked immunosorbent assay (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; *P < 0.05; one-way analysis of variance (ANOVA); ns, nonsignificant.

 

Figure 3
View larger version (9K):
[in this window]
[in a new window]

 
Fig. 3. Additive induction of intestinal IgA in proportion to bacterial exposure. Germ-free C57BL/6 mice were (A and B) given one, two, or six doses of 1010 CFU HA107 over the course of 1 week and analyzed at day 14. (C and D) In parallel, three groups of mice were given six doses in intervals of 1, 2 to 3, or 7 days apart and analyzed on day 42. Intestinal washes were analyzed by IgA-specific ELISA and live bacterial flow cytometry. –LogEC50 (where EC50 indicates median effective concentration) values of IgA–E. coli binding were calculated as described in (7) [(A) and (B)], and numbers of duodenal IgA plasma cells were determined from 7-µm frozen sections stained for mouse IgA [(C) and (D)]. Bars indicate means; *P < 0.05 (one-way ANOVA); ns, nonsignificant. Data points represent individual mice from one of two experiments.

 

Figure 4
View larger version (10K):
[in this window]
[in a new window]

 
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 six times over 2 weeks with 1010 CFU of 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), 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; *P < 0.05; one-way ANOVA. Data points in (A) and (B), respectively, and in (C) represent individual mice from one of two independent experiments.

 


To Advertise     Find Products


Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882