Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice

Abstract

Mice with targeted deletion of the gene for interleukin-10 (IL-10) spontaneously develop enterocolitis when maintained in conventional conditions but develop only colitis when kept in specific-pathogen-free (SPF) environments. This study tested the hypothesis that enteric bacteria are necessary for the development of spontaneous colitis and immune system activation in IL-10-deficient mice. IL-10-deficient mice were maintained in either SPF conditions or germfree conditions or were populated with bacteria known to cause colitis in other rodent models. IL-10-deficient mice kept in SPF conditions developed colitis in all segments of the colon (cecum and proximal and distal colon). These mice exhibited immune system activation as evidenced by increased expression of CD44 on CD4(+) T cells; increased mesenteric lymph node cell numbers; and increased production of immunoglobulin A (IgA), IgG1, and IL-12 p40 from colon fragment cultures. Mice populated with bacterial strains, including Bacteroides vulgatus, known to induce colitis in other rodent models had minimal colitis. Germfree IL-10-deficient mice had no evidence of colitis or immune system activation. We conclude therefore that resident enteric bacteria are necessary for the development of spontaneous colitis and immune system activation in IL-10-deficient mice.

FIG. 1

Resident enteric bacteria are required for colon inflammation in IL-10-deficient mice. Representative photomicrographs from the middle colon of (a) an IL-10-deficient SPF mouse, (b) an IL-10-deficient germfree mouse, and (c) a wild-type SPF mouse. Note the marked mucosal hyperplasia, lamina propria cellular infiltrates, and crypt abscess (arrow) shown in panel a that are absent in panels b and c. Magnification, ×58.

FIG. 1

Resident enteric bacteria are required for colon inflammation in IL-10-deficient mice. Representative photomicrographs from the middle colon of (a) an IL-10-deficient SPF mouse, (b) an IL-10-deficient germfree mouse, and (c) a wild-type SPF mouse. Note the marked mucosal hyperplasia, lamina propria cellular infiltrates, and crypt abscess (arrow) shown in panel a that are absent in panels b and c. Magnification, ×58.

FIG. 2

Mean colon inflammatory scores of germfree or SPF IL-10-deficient mice, SPF wild-type mice, and germfree heterozygote mice. Sections of colon representing at least three areas per mouse were scored in a blinded manner on a scale from 0 (no inflammation) to 4 (severe inflammation), and the scores were averaged to calculate mean scores. Median values, 25th and 75th percentiles (boxes), 10th and 90th percentiles (error bars), outlying values (circles), and means (dashed lines) are represented by box plots. A statistically different (P < 0.05) mean (from all other means) is indicated by the asterisk. gf, germfree; ko, IL-10 deficient; het, heterozygous; wt, wild type.

FIG. 3

Cecal inflammation is more severe in germfree IL-10-deficient mice colonized as adults with a Helicobacter-negative SPF flora than in IL-10-deficient mice colonized at weaning. Representative histologies from (a) a wild-type SPF mouse, (b) an IL-10-deficient mouse (15 weeks old) maintained in SPF conditions since 3 weeks of age, (c) an IL-10-deficient mouse raised germfree and then moved to SPF conditions as an adult and sacrificed 5 to 7 weeks later are shown. Note the marked mucosal hyperplasia, transmural cellular infiltrates with aggressive submucosal inflammation, and crypt abscess (arrow) shown in panel c compared with the mild mucosal hyperplasia and infiltration limited to the lamina propria shown in panel b. Magnification, ×58.

FIG. 3

Cecal inflammation is more severe in germfree IL-10-deficient mice colonized as adults with a Helicobacter-negative SPF flora than in IL-10-deficient mice colonized at weaning. Representative histologies from (a) a wild-type SPF mouse, (b) an IL-10-deficient mouse (15 weeks old) maintained in SPF conditions since 3 weeks of age, (c) an IL-10-deficient mouse raised germfree and then moved to SPF conditions as an adult and sacrificed 5 to 7 weeks later are shown. Note the marked mucosal hyperplasia, transmural cellular infiltrates with aggressive submucosal inflammation, and crypt abscess (arrow) shown in panel c compared with the mild mucosal hyperplasia and infiltration limited to the lamina propria shown in panel b. Magnification, ×58.

FIG. 4

Mean immunoglobulin concentrations in supernatants of cultured colon fragments from mice with different bacterial stimulation. Segments of colon were minced and cultured, and supernatants were collected 18 h later for immunoglobulin quantitation by ELISA. All values are expressed on an equivalent weight basis. Median values, 25th and 75th percentiles (boxes), 10th and 90th percentiles (error bars), outlying values (circles), and means (dashed lines inside boxes) are represented by box plots. Statistically different medians (P < 0.05) (from all other medians in the respective panel) are indicated by asterisks (a) IgG2a concentrations; (b) IgG1 concentrations; (c) IgA concentrations. Abbreviations are the same as those in the legend for Fig. 2.