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. 2004 Jun;72(6):3315-24.
doi: 10.1128/IAI.72.6.3315-3324.2004.

Clearance of Citrobacter rodentium requires B cells but not secretory immunoglobulin A (IgA) or IgM antibodies

Christian Maaser  1 , Michael P Housley, Mitsutoshi Iimura, Jennifer R Smith, Bruce A Vallance, B Brett Finlay, John R Schreiber, Nissi M Varki, Martin F Kagnoff, Lars Eckmann
Affiliations

Clearance of Citrobacter rodentium requires B cells but not secretory immunoglobulin A (IgA) or IgM antibodies

Christian Maaser et al. Infect Immun. 2004 Jun.

Abstract

Citrobacter rodentium, a murine model pathogen for human enteropathogenic Escherichia coli, predominantly colonizes the lumen and mucosal surface of the colon and cecum and causes crypt hyperplasia and mucosal inflammation. Mice infected with C. rodentium develop a secretory immunoglobulin A (IgA) response, but the role of B cells or secretory antibodies in host defense is unknown. To address this question, we conducted oral C. rodentium infections in mice lacking B cells, IgA, secreted IgM, polymeric Ig receptor (pIgR), or J chain. Normal mice showed peak bacterial numbers in colon and feces at 1 week and bacterial eradication after 3 to 4 weeks. B-cell-deficient mice were equally susceptible initially but could not control infection subsequently. Tissue responses showed marked differences, as infection of normal mice was accompanied by transient crypt hyperplasia and mucosal inflammation in the colon and cecum at 2 but not 6 weeks, whereas B-cell-deficient mice had few mucosal changes at 2 weeks but severe epithelial hyperplasia with ulcerations and mucosal inflammation at 6 weeks. The functions of B cells were not mediated by secretory antibodies, since mice lacking IgA or secreted IgM or proteins required for their transport into the lumen, pIgR or J chain, cleared C. rodentium normally. Nonetheless, systemic administration of immune sera reduced bacterial numbers significantly in normal and pIgR-deficient mice, and depletion of IgG abrogated this effect. These results indicate that host defense against C. rodentium depends on B cells and IgG antibodies but does not require production or transepithelial transport of IgA or secreted IgM.

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Figures

FIG. 1.
FIG. 1.
C. rodentium infection of B-cell-deficient mice. B-cell-deficient mice (○) and C57BL/6 controls (•) were infected orally with 5 × 108 C. rodentium cells, and bacterial numbers (CFU) in the stool were determined weekly. Data are means ± standard errors (n = 7 to 11 mice per time point). *, P < 0.01 (by rank sum test) relative to C57BL/6 controls at the same time point. The dashed line represents the detection limit of the CFU assay.
FIG. 2.
FIG. 2.
Histological analysis of colon and cecum in C. rodentium-infected mice. C57BL/6 control mice (A to E and K to O) and B-cell-deficient mice (B cell KO) (F to J and P to T) were left uninfected (week 0; A, F, K, and P) or were infected orally with C. rodentium and sacrificed after 2 weeks (B, D, E, G, L, N, O, and Q) or 6 weeks (C, H, I, J, M, R, S, and T). Paraffin sections of colon (A to J) and cecum (K to T) were prepared and stained with hematoxylin and eosin. The colons and ceca of C57BL/6 mice show crypt hyperplasia, increased mitotic activity in the epithelium, and a loss of goblet cells at 2 weeks after infection (B and L). The submucosa is edematous and infiltrated with mostly mononuclear cells, while mucosal infiltration with inflammatory cells is modest (B, E, L, and O). The epithelial surface is intact with a normal-appearing brush border (D and N). In B-cell-deficient mice, extensive crypt hyperplasia in the colon and cecum is seen at 6 weeks, which is accompanied by submucosal inflammation and edema (H, J, R, and T). In parallel, the epithelial surface exhibits erosions and apoptotic/necrotic epithelial cells (I and S; arrows) and is covered by a pseudomembrane of coagulated proteinaceous material and dead epithelial cells (H, I, and S; asterisks). The magnifications are identical for the following groups of panels (with the applicable size bar given in parentheses): A to C and F to H (size bar shown in panel A); D, E, I, and J (size bar in panel D); K to M and P to R (size bar in panel K); N, O, S, and T (size bar in panel N).
FIG. 3.
FIG. 3.
Morphometric analysis of crypt depths after C. rodentium infection. Paraffin sections from uninfected (week 0) and C. rodentium-infected (weeks 2 and 6 postinfection [p.i.]) control (C57BL/6) and B-cell-deficient mice (B cell KO) were stained with hematoxylin and eosin. Crypt depths in colon and cecum were determined microscopically. Data are means ± standard errors (n ≥ 3 mice per time point). *, P < 0.05 (by rank sum test) relative to uninfected mice of the same strain.
FIG. 4.
FIG. 4.
Histological analysis of spleen and liver after oral C. rodentium infection. C57BL/6 control mice (A to C and G to I) and B-cell-deficient mice (B cell KO) (D to F and J to L) were left uninfected (week 0; A, D, G, and J) or were infected orally with C. rodentium and sacrificed after 2 weeks (B, E, H, and K) or 6 weeks (C, F, I, and L). Paraffin sections of spleen (A to F) and liver (G to L) were prepared and stained with hematoxylin and eosin. Each spleen section shows areas of the white pulp (WP) and red pulp (RP). In panel B, the normally narrow marginal zone (MZ) between white and red pulp is widened, with numerous enlarged, irregular-shaped mononuclear cells (arrows). Representative examples of these cells (enclosed by the rectangle) are shown at higher magnification in the inset. In panel F, numerous neutrophils are present in clusters and microabscesses within the marginal zone and red pulp (arrows). Examples of these cells (enclosed by the rectangle) are shown at higher magnification in the inset (mouse neutrophils have ring-shaped, nonsegmented nuclei). For the liver, each section is centered around a portal triad containing a portal venule (PV). In panels H and L, mononuclear cell infiltrates are seen in a mostly periportal location (arrows). The magnifications are identical for panels A to F and G to L.
FIG. 5.
FIG. 5.
C. rodentium infection of mice deficient in secretory antibody production or transport into the intestinal lumen. Mice deficient for IgA (A; ○), secreted IgM (B; ○), pIgR (C; ○), or J chain (D; ○) and the appropriate controls—B6129 mice (A; •) for IgA-deficient, secreted IgM-deficient, and pIgR-deficient mice and BALB/c (D; •) for J chain-deficient mice—were infected orally with C. rodentium, and bacterial numbers in the feces were determined weekly. Data are means ± standard errors (n ≥ 6 mice per time point). *, P < 0.05 (by rank sum test) relative to control mice at the same time point. The gray lines in panels B and C represent the means of the data from B6129 mice in panel A and are only shown for comparison. The dashed lines represent the detection limit of the CFU assay.
FIG. 6.
FIG. 6.
Infection of IgG3-deficient mice with C. rodentium. Mice lacking IgG3 (○) and BALB/c controls (•) were infected orally with C. rodentium, and fecal bacterial numbers were determined weekly. Data are means ± standard error (n = 5 to 6 mice per time point). The dashed line represents the detection limit of the CFU assay.
FIG. 7.
FIG. 7.
Passive immune protection against C. rodentium. Mice deficient for pIgR were infected orally with C. rodentium (○) or left uninfected (•), and sera were collected after 6 weeks and tested for anti-C. rodentium antibodies by isotype-specific ELISA (A). Naive C57BL/6 mice (B and D) or pIgR-deficient mice (C) were infected orally with C. rodentium and injected intraperitoneally on days 4 to 7 and 11 to 14 with identical volumes of immune (Imm Ig), preimmune (Pre Ig), or IgG-depleted immune sera (Imm Ig-IgG). Bacterial numbers (CFU) in the feces were determined on days 7 (B) and 14 to 18 (B to D). Results are the mean ± standard deviation of quadruplicate samples (A) or mean ± standard error of six or more mice (B to D). OD450, optical density at 450 nm. *, P < 0.05 (by t test) relative to preimmune Ig-injected mice at the same time point.
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