Mice lacking T and B lymphocytes develop transient colitis and crypt hyperplasia yet suffer impaired bacterial clearance during Citrobacter rodentium infection

Abstract

The bacterial pathogen Citrobacter rodentium belongs to a family of gastrointestinal pathogens that includes enteropathogenic and enterohemorrhagic Escherichia coli and is the causative agent of transmissible colonic hyperplasia in mice. The molecular mechanisms used by these pathogens to colonize host epithelial surfaces and form attaching and effacing (A/E) lesions have undergone intense study. In contrast, little is known about the host's immune response to these infections and its importance in tissue pathology and bacterial clearance. To address these issues, wild-type mice and mice lacking T and B lymphocytes (RAG1 knockout [KO]) were infected with C. rodentium. By day 10 postinfection (p.i.), both wild-type and RAG1 KO mice developed colitis and crypt hyperplasia, and these responses became more exaggerated in wild-type mice over the next 2 weeks, as they cleared the infection. By day 24 p.i., bacterial clearance was complete, and the colitis had subsided; however, crypt heights remained increased. In contrast, inflammatory and crypt hyperplastic responses in the RAG1 KO mice were transient, subsiding after 2 weeks. By day 24 p.i., RAG1 KO mice showed no signs of bacterial clearance and infection was often fatal. Surprisingly, despite remaining heavily infected, tissues from RAG1 KO mice surviving the acute colitis showed few signs of disease. These results thus emphasize the important contribution of the host immune response during infection by A/E bacterial pathogens. While T and/or B lymphocytes are essential for host defense against C. rodentium, they also mediate much of the tissue pathology and disease symptoms that occur during infection.

FIG. 1.

(A) Both wild-type (solid circles) and RAG1 KO mice (solid squares) lose body weight during the first 24 days of a C. rodentium infection, compared to uninfected wild-type (open circles) and RAG1 KO (open squares) mice. Weight data from one representative experiment out of three are shown. Error bars represent standard errors. Each data point represents average weight data pooled from 10 mice and is expressed as the percentage of the initial body weight. Infected wild-type mice weighed significantly less than uninfected mice from day 2 until day 24 p.i. Infected RAG1 KO mice weighed less than uninfected mice on day 2 and days 14 to 21 p.i. ∗, P < 0.05. (B) Survival of wild-type mice (solid circles) is greater than that of RAG1 KO mice (open circles) over the first 24 days of a C. rodentium infection. Survival data from one representative experiment out of three are shown. Each data point represents the percentage of surviving mice from an initial population of 10 mice.

FIG. 2.

Histology showing the colonic crypt hyperplasia and inflammation that occur during the course of C. rodentium infection in both wild-type and RAG1 KO mice. Control colonic tissue taken from a wild-type mouse (A), showing the normal appearance of the colon and basal crypt heights. Uninfected RAG1 KO tissues were similar in size and appearance. By day 10, lengthening of the crypts and significant infiltration of inflammatory cells were observed in both wild-type (B) and RAG1 KO (C) mice. The original magnification for panels A to C was ×40. Under higher magnification, (originally ×200), the dramatic increase in crypt heights and crypt cellularity can be more clearly seen when comparing an uninfected wild-type colon (D) to tissues taken from day 10 p.i. wild-type (E) and RAG1 KO (F) mice. Note the massive inflammatory infiltrate in the RAG1 KO tissue (arrows). The infiltrate was comprised predominantly of neutrophils, as seen by enlarging the submucosal region (G). A common observation during infection of wild-type mice was the presence of enlarged or hypertrophic colonic patches as shown in panel H (original magnification, ×40).

FIG. 3.

Both wild-type (solid bars) and RAG1 KO (open bars) mice undergo significant increases in colonic crypt heights over the first 24 days of a C. rodentium infection. However, the mean crypt heights (in micrometers) in the colons of wild-type mice were significantly greater than those in RAG1 KO mice on days 18, 21, and 24 p.i., at the later stages of infection. The data represent the mean of three independent experiments in which each group contained five mice. ∗, P < 0.05. Error bars represent standard errors.

FIG. 4.

Colonic tissues taken from wild-type (wt) and RAG1 KO mice express IFN-γ mRNA during C. rodentium infection, but only wild-type mice showed increased expression during infection. RT-PCR for IFN-γ shows expression significantly increases in the tissues of wild-type mice, from undetectable levels in uninfected mice to a progressive elevation in expression seen on days 6, 10, and 18 p.i. In contrast, as the infection progressed, IFN-γ mRNA expression progressively decreased in the colons of RAG1 KO mice at each of days 6, 10, and 18 p.i. GAPDH was used as the housekeeping gene control.

FIG. 5.

Following oral infection, both wild-type and RAG1 KO mice were heavily colonized by C. rodentium. The numbers of bacteria recovered from the colons of individual infected wild-type mice (solid circles) and RAG1 KO mice (open circles) are presented on days 6, 10, 14, 18, 21, and 24 p.i. Each group contained four to five mice. The asterisk denotes the recovery of significantly more bacteria (mean value) recovered from RAG1 KO mice than from wild-type mice at days 18, 21, and 24 p.i. ∗, P < 0.05.

FIG. 6.

Immunofluorescence staining showing the presence of C. rodentium on the lumenal surface of the colons of wild-type mice (day 10 p.i.). Staining for C. rodentium LPS is seen in red, and host cell F-actin is seen in green, while host cell nuclei are blue. In panel A, the colonic lumen is at the top, and the mucosa is at the bottom (original magnification, ×630). Note the jagged appearance of the intestinal epithelium with projections jutting into the lumen, a common feature of the colonic hyperplasia seen during C. rodentium infection. Adherent bacteria (red) are seen coating the surface of most epithelial cells. In addition, several epithelial cells, some with bacteria still adhering (see arrows) are seen being sloughed into the colonic lumen. Note that the F-actin staining is reduced on the sloughed cells. An enlarged view of the infected epithelium is seen in panel B. Note the numerous bacteria covering the epithelial surface. While many appear adherent (arrows), others appear to form a thick coating on the epithelial surface. As well, F-actin rearrangements seen as areas of intense green (arrowheads) are found across the surface of the infected cells. Where the bacteria and the rearrangements are on the same plane, the rearrangements are found underneath many of the adhering bacteria. As such, we believe these to be A/E lesions.

FIG. 7.

Histology showing the presence of C. rodentium on the lumenal surface of the colons of both wild-type and RAG1 KO mice during infection. Note the ragged appearance of the epithelial surface and the epithelial cells sloughing into the lumen on day 10 p.i. in a wild-type mouse (A). Many of the cells being sloughed are still covered with adherent bacteria (see arrows). While similar observations were made in RAG1 KO tissues (B), the crypts in RAG1 KO tissues were almost always filled with bacteria (arrows). By day 24 p.i., wild-type mice had cleared the infection, with no C. rodentium found on the superficial surface of the colon (C), while at the same time point, RAG1 KO mice remained heavily infected with large numbers of C. rodentium cells coating the mucosal surface and in the colonic lumen (arrows) (D) Magnification for panels A to D, ×400.