Decreased expression of colonic Slc26a3 and carbonic anhydrase iv as a cause of fatal infectious diarrhea in mice

Abstract

Citrobacter rodentium causes epithelial hyperplasia and colitis and is used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections. Little or no mortality develops in most inbred strains of mice, but C3H and FVB/N mice exhibit fatal outcomes of infection. Here we test the hypothesis that decreased intestinal transport activity during C. rodentium infection results in fatality in C3H/HeOu and FVB/N mice. Susceptible strains were compared to resistant C57BL/6 mice and to inbred strains SWR and SJL of Swiss origin, which have not been previously characterized for outcomes of C. rodentium infection. Mortality in susceptible strains C3H/HeOu and FVB/N was associated with significant fluid loss in feces, a remarkable downregulation of Slc26a3 and carbonic anhydrase IV (CAIV) message and protein expression, retention of chloride in stool, and hypochloremia, suggesting defects in intestinal chloride absorption. SWR, SJL, and C57BL/6 mice were resistant and survived the infection. Fluid therapy fully prevented mortality in C3H/HeOu and FVB/N mice without affecting clinical disease. Common pathogenic mechanisms, such as decreased levels of expression of Slc26a3 and CAIV, affect intestinal ion transport in C. rodentium-infected FVB and C3H mice, resulting in profound electrolyte loss, dehydration, and mortality. Intestinal chloride absorption pathways are likely a potential target for the treatment of infectious diarrhea.

FIG. 1.

Experimental inoculation of animals with C. rodentium causes severe disease in C3H and FVB mice but not in SWR, SJL, or C57BL/6 mice. (A) Mortality of infected C3H mice and FVB mice, but not infected SWR, SJL, and C57BL/6 mice, was observed after 7 dpi (P < 0.0001). (B) Significant body weight (BW) loss was observed for infected C3H mice and FVB mice by 9 dpi (P < 0.005). Data were normalized to body weights of uninfected mice of the corresponding strains and are presented as mean differences ± SEM. (C) Fecal bacterial counts were highest in C3H mice and lowest in FVB mice at 3 dpi. Levels of fecal bacterial shedding were comparable between the strains on days 9 and 12, except for SJL mice, which had a delayed clearance of infection (P < 0.0001). Data were log transformed and are presented as mean differences ± SEM. *, P < 0.05; ***, P < 0.001 (compared with three other inbred strains). #, P < 0.05 compared with one or two other inbred strains.

FIG. 2.

Colonic loads of C. rodentium and extraintestinal dissemination of infection. (A to C) Bacterial counts in colon increased more slowly in FVB mice, consistent with a delayed increase in fecal bacterial shedding. By 9 dpi, colonic counts were lower for C57BL/6 mice than for C3H and SWR but not FVB mice. C3H mice demonstrated significantly higher levels of bacterial translocation to the liver at 4 and 9 dpi and to spleen at 4 dpi, whereas C57BL/6 mice had the lowest spleen counts at 9 dpi. Data were log transformed and are presented as mean differences ± SEM. The dashed line indicates the limit of detection. *, P < 0.05; **, P < 0.01 (compared with three other inbred strains). #, P < 0.05 compared with one or two other inbred strains. (D) Levels of serum endotoxin were low and not different between groups (P > 0.05). Data are presented as mean differences ± SEM. EU, endotoxin units.

FIG. 3.

Colonic lesions in C. rodentium-infected mice. (A) Increased colon weight in C. rodentium-infected mice. Data were normalized to body weight and are presented as mean differences ± SEM. Groups indicated by different letters (a, b, and c) are significantly different (P < 0.05). (B) Cumulative disease indices of colonic lesions. Median lines and ranges are presented. Groups indicated by different letters (a, b, and c) are significantly different (P < 0.0001). (C to F) Microscopic lesions in C. rodentium-infected mice. (C) Normal colon from an uninoculated C3H mouse. (D and E) Transmural colitis, ulcer, and blood congestion in C3H (D) and FVB (E) mice at 9 dpi. (F and G) Hyperplasia with mild to moderate colitis in SWR (F) and C57BL/6 (G) mice at 9 dpi. (H) Although SJL mice had colonic lesions comparable to those of SWR and C57BL/6 mice (data not shown), they also developed mild to moderate liver lesions characterized by focal to multifocal hepatocellular coagulative necrosis at 14 dpi. Shown is hematoxylin and eosin staining. Original magnifications are ×100.

FIG. 4.

Effects of C. rodentium infection on expression of genes involved in colonic ion and water transport. The expression of genes was normalized to that for uninoculated FVB mice. Data are presented as median (center line), upper and lower quartile (box), and range. Groups indicated by different letters (a, b, and c) are significantly different (P < 0.05 after log transformation).

FIG. 5.

Expression of CAIV and DRA in situ. (A) Expression of the CAIV and DRA proteins in colonic segments was detected using semiquantitative analysis as described in Materials and Methods. Uninoculated mice of all strains had normal levels of expression and were excluded from the analysis. CAIV was normally expressed throughout the whole large intestine (regions “a” through “d”), whereas DRA was usually not expressed in the most proximal colon (region “d”). Susceptible C3H and FVB mice were characterized by a more significant loss of CAIV and DRA expression throughout the intestine than resistant SWR and C57BL/6 mice during C. rodentium infection (P < 0.0001; n = 3 mice per group). Medians and ranges are shown. *, P < 0.05; ***, P < 0.001; ns, not significant. (B) Representative images for grading of CAIV (a through d) and DRA (e through h) protein expression. Grades “0” (a and e), “1” (b and f), “2” (c and g), and “3” (d and h) are presented. Arrows indicate apical DRA expression. Original magnifications are ×200.

FIG. 6.

C. rodentium infection induces chloride diarrhea in susceptible mice. (A and B) Infection resulted in chloride retention in feces (A) and hypochloremia (B) in C3H and FVB mice at 9 and 12 dpi, respectively (P < 0.001). Resistant SWR and C57BL/6 mice did not demonstrate abnormal fecal or serum chloride concentrations. Data are presented as mean differences ± SEM. (C) C3H and FVB mice demonstrated significant water loss in stool, reaching more than a 20% loss of initial fecal water content as disease progressed. The diarrheal response of resistant strains was comparable and less dramatic (P < 0.005). Data were normalized to water content in uninfected mice of the corresponding strains and are presented as mean differences ± SEM. The dashed line indicates the 20% water loss threshold. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with three other inbred strains). #, P < 0.05 compared with one or two other inbred strains.

FIG. 7.

Effects of fluid therapy on disease outcome for susceptible mice. (A) Mortality of C. rodentium-infected C3H and FVB mice was fully prevented by fluid administration (P < 0.0001). (B) Body weight loss in infected C3H and FVB mice was not affected by fluid administration (P > 0.05), although it was delayed in infected FVB mice (P < 0.05 at 5 to 10 dpi). Data are presented as mean differences ± SEM. (C) Levels of fecal bacterial shedding were comparable between fluid-treated and untreated groups for both strains (P > 0.05). C. rodentium counts were lower in FVB mice at 3 dpi and higher at 18 dpi than in C3H mice (*, P < 0.05). Data were log transformed and are presented as mean differences ± SEM. The dashed line indicates the limit of detection. (D) No differences in stool water losses were observed between infected fluid-treated and untreated animals (P > 0.05), although the diarrheal response was more rapid in C3H mice than in FVB mice (***, P < 0.001). In addition, uninfected C3H mice had lower stool water contents in the beginning of the experiment than did uninoculated FVB mice (*, P < 0.05). Data are presented as mean differences ± SEM. (E) Fluid therapy did not affect bacterial recovery from tissues of C3H mice (P > 0.05). Data were log transformed, and each dot corresponds to an individual animal. Lines indicate mean values. (F) Mucosal permeability was measured by the detection of FITC-dextran in serum after administration by intragastric intubation. No differences between groups of C3H mice were found (P > 0.05). Each dot corresponds to an individual animal, and lines indicate mean values. C, uninoculated controls; I, infected untreated mice; IF, infected mice treated with fluid therapy intervention.