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Infection and Immunity, June 2005, p. 3228-3241, Vol. 73, No. 6
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.6.3228-3241.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Comparison of Salmonella enterica Serovar Typhimurium Colitis in Germfree Mice and Mice Pretreated with Streptomycin

Bärbel Stecher,1 Andrew J. Macpherson,2,{dagger} Siegfried Hapfelmeier,1 Marcus Kremer,3 Thomas Stallmach,4 and Wolf-Dietrich Hardt1*

Institute of Microbiology, D-BIOL, ETH Zürich, Wolfgang-Paulistrasse 10, CH-8093 Zürich, Switzerland,1 Institute of Experimental Immunology, Universitätsspital Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland,2 Institute of Pathology, Technische Universität München, Ismaninger Strasse 22, D-81675 München, Germany,3 Institute of Clinical Pathology, Universitätsspital Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland4

Received 31 August 2004/ Returned for modification 21 October 2004/ Accepted 2 February 2005


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ABSTRACT
 
Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of bacterial enterocolitis. Mice are generally protected from Salmonella serovar Typhimurium colonization and enterocolitis by their resident intestinal microflora. This phenomenon is called "colonization resistance" (CR). Two murine Salmonella serovar Typhimurium infection models are based on the neutralization of CR: (i) in specific-pathogen-free mice pretreated with streptomycin (StrSPF mice) antibiotics disrupt the intestinal microflora; and (ii) germfree (GF) mice are raised without any intestinal microflora, but their intestines show distinct physiologic and immunologic characteristics. It has been unclear whether the same pathogenetic mechanisms trigger Salmonella serovar Typhimurium colitis in GF and StrSPF mice. In this study, we compared the two colitis models. In both of the models Salmonella serovar Typhimurium efficiently colonized the large intestine and triggered cecum and colon inflammation starting 8 h postinfection. The type III secretion system encoded in Salmonella pathogenicity island 1 was essential in both disease models. Thus, Salmonella serovar Typhimurium colitis is triggered by similar pathogenetic mechanisms in StrSPF and GF mice. This is remarkable considering the distinct physiological properties of the GF mouse gut. One obvious difference was more pronounced damage and reduced regenerative response of the cecal epithelium in GF mice. Overall, StrSPF mice and GF mice provide similar but not identical models for Salmonella serovar Typhimurium colitis.


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INTRODUCTION
 
Salmonella enterica serovar Typhimurium infects a variety of mammalian hosts and can essentially cause two different types of disease. In humans and cattle Salmonella serovar Typhimurium infection leads to enteric disease associated with diarrhea. Bovine infection models have helped to identify Salmonella serovar Typhimurium virulence factors triggering enterocolitis (55). In susceptible mice, Salmonella serovar Typhimurium causes a typhoid-like disease characterized by rapid multiplication of bacteria in the liver and spleen but little intestinal pathology (12, 39). The intestinal tract of conventional specific-pathogen-free (SPF) mice is only poorly colonized by Salmonella serovar Typhimurium (~104 CFU/g of contents) (2, 9) and other pathogenic bacteria upon oral infection (27, 42). This phenomenon, termed "colonization resistance" (CR) (49) or "microbial interference" (31), has been described as a complex mechanism in which host and the resident microflora cooperate to prevent the growth of other potentially pathogenic bacteria. The following different mechanisms for CR have been discussed: (i) the production of inhibitory substances, like bacteriocins, by indigenous microbes (36, 47), (ii) competition for nutrients and adhesion to mucin receptor sites (3, 13, 17, 25), (iii) a potential immunomodulatory effect of the commensal microflora on host defense mechanisms (e.g., defensin secretion stimulated by bacterial products like lipopolysaccharide, lipoteichoic acid, and muramyl-dipeptide) (1, 23, 35, 37), (iv) the inhibition of toxin production by indigenous microorganisms (7, 10, 11), and (v) the generation of a physiologically restrictive environment (pH, toxic metabolites) (4, 5, 29, 30).

Recently, we found that SPF mice pretreated with a single dose (20 mg/animal per os) of streptomycin (StrSPF mice) provide an animal model for Salmonella serovar Typhimurium colitis (2). The cecum and colon are inflamed, and only a little inflammation is observed in the ileum. Salmonella serovar Typhimurium colitis in StrSPF mice is associated with epithelial damage, polymorphonuclear granulocyte (PMN) infiltration, edema, and crypt abscesses, which are most evident at day 3 postinfection (p.i.) (2, 19). Similar inflammatory changes have been observed in several documented human cases (6) and in the bovine small and large intestines (48, 51). In contrast to the bovine or typical noncomplicated human infections, Salmonella serovar Typhimurium colitis in mice is associated with a parallel systemic infection, and susceptible natural resistance-associated macrophage protein 1-deficient mice (C57BL/6, BALB/c) die of this systemic disease on day 5 or 6 p.i. However, the systemic spread does not seem to cause or alter intestinal inflammation at 1 to 3 days postinfection (G. Paesold and W.-D. Hardt, unpublished results). Overall, StrSPF mice provide an interesting murine model for basic research on molecular aspects of acute Salmonella serovar Typhimurium colitis. Nevertheless, it should be kept in mind that direct transfer of results from any animal model to a human disease should be done with great caution.

In accordance with data from bovine models for Salmonella enterocolitis (50, 56), the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (TTSS) is a key virulence factor in StrSPF mice (2, 18). SPI-1 function enables bacteria to penetrate the intestinal epithelial barrier and reach the underlying tissues (e.g., lamina propria) (14, 19, 50). This process is associated with cytokine release and inflammation.

Germfree (GF) mice lack any intestinal microflora and are highly susceptible to colonization with various bacteria (52, 53), including Salmonella spp. (9, 31).

Due to the lack of the intestinal microflora, the intestine of GF mice has several distinct features, including altered mucus secretion (8), an underdeveloped gut-associated immune system, reduced immunoglobulin A (IgA) production (43), altered numbers of M cells (41), and reduced expression of antimicrobial peptides (21, 22). Reassociation of GF mice with a normal SPF flora can lead to acute self-limiting colitis (33), and colonization of GF mice with Salmonella serovar Typhimurium was found to cause diarrheal disease (26, 46). Due to the distinct physiological features of GF mice, it has not been clear whether Salmonella serovar Typhimurium colitis in GF mice differs from the disease in StrSPF mice.

Here, we compared StrSPF and GF mice as models for Salmonella serovar Typhimurium colitis. We studied colonization and the histopathologic features of cecal inflammation in StrSPF and GF mice. Wild-type and Salmonella serovar Typhimurium strains with a disrupted SPI-1 TTSS were included in the analyses to investigate which pathogenetic mechanisms trigger disease.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. For mouse infections, Salmonella serovar Typhimurium wild-type strain SL1344 (20) and its isogenic derivative SB161 (SL1344 {Delta}invG [24]) were grown for 12 h at 37°C in Luria-Bertani broth containing 0.3 M NaCl, diluted 1:20 in fresh medium, and subcultured for 4 h with mild aeration. Bacteria were washed in ice-cold phosphate-buffered saline (PBS) and resuspended in cold PBS (5 x 107 CFU/50 µl).

Animal experiments. Animal experiments were performed in individually ventilated cages at the BZL (Universität Zürich) as described previously (2), using SPF female C57BL/6 mice (6 to 9 weeks old) from Harlan (Horst, Netherlands) or GF C57BL/6 mice raised in a germfree isolator at BZL Zürich.

In the StrSPF model, water and food were withdrawn 4 h prior to treatment with 20 mg of streptomycin intragastrically. After this, animals were supplied with water and food ad libitum. Twenty hours after streptomycin treatment, water and food were withdrawn again for 4 h before the mice were infected with 5 x 107 CFU of serovar Typhimurium (50 µl of a suspension in PBS intragastrically by gavage). GF mice were raised in a germfree isolator and fed sterile chow. They were removed from the isolator and transferred into individually ventilated cages without water and food 2 to 4 h prior to infection. Under these conditions mice remained germfree for at least 10 to 20 h, as determined by plating of fecal pellets on appropriate culture media (data not shown). GF mice were infected with 5 x 107 CFU of serovar Typhimurium (50 µl of a suspension in PBS intragastrically). To both GF and streptomycin-pretreated mice, water was offered ad libitum immediately after infection, and food was supplied 2 h p.i. At different times after infection, the mice were sacrificed by cervical dislocation, and tissue samples from the cecum, spleen, and liver were removed for analysis. Animal experiments were approved by and were performed as required by Swiss national and institutional regulations.

Analysis of serovar Typhimurium loads in the intestine, mLN, spleen, and liver. To analyze colonization, the spleen, liver, and mesenteric lymph nodes (mLN) were removed aseptically and homogenized in 4°C PBS containing 0.5% Tergitol and 0.5% bovine serum albumin as described previously (2). The bacterial loads were determined by plating on MacConkey agar plates containing streptomycin (50 µg/ml). The minimal detectable levels were 10 CFU/organ in the mLN, 20 CFU/organ in the spleen, and 100 CFU/organ in the liver. Cecal contents were collected at different times after infection, and the bacterial loads were determined by plating. The minimal detectable level was 10 CFU per sample.

Histological procedures. Tissue samples were embedded in OCT (Sakura, Torrance, CA), snap-frozen in liquid nitrogen, and stored at –80°C. Cryosections (5 µm) were mounted on glass slides, air dried for 2 h at room temperature, and stained with hematoxylin and eosin (H&E). As indicated below, cecal tissues were fixed in 4% formalin and embedded in paraffin prior to sectioning and staining.

Cecal pathology was independently evaluated by two pathologists in a blinded manner using 5-µm-thick H&E-stained sections and the following histopathological scoring scheme, as previously described (2).

(i) Submucosal edema. Submucosal edema (expressed as a percentage) was deduced from the extension of the submucosa and was scored by morphometric analysis according to the following formula: submucosal edema = (b – a)/c, where a is the area enclosed by the mucosa (mucosa and intestinal lumen), b is the area enclosed by the borderline between the submucosa and the tunica muscularis (submucosa, mucosa, and intestinal lumen), and c is the area enclosed by the outer edge of the tunica muscularis (tunica muscularis, submucosa, mucosa, and lumen; area of the whole cecal cross section). The scores for submucosal edema were as follows: 0, no pathological changes; 1, detectable edema (submucosal edema, <10%); 2, moderate edema (submucosal edema, 10 to 40%); 3, profound edema (submucosal edema, ≥40%).

(ii) PMN infiltration into the lamina propria. PMN in the lamina propria were enumerated in 10 high-power fields (magnification, x400; field diameter, 420 µm), and the average number of PMN per high-power field was calculated. The scores were determined as follows: 0, less than 5 PMN per high-power field; 1, 5 to 20 PMN per high-power field; 2, 21 to 60 PMN per high-power field; 3, 61 to 100 PMN per high-power field; 4, more than 100 PMN per high-power field.

(iii) Goblet cells. The average number of goblet cells per high-power field (magnification, x400) was calculated from 10 different regions of the cecal epithelium. The scores were determined as follows: 0, more than 28 goblet cells per high-power field (in the cecum of the normal SPF mice we observed an average of 6.4 crypts per high-power field, and the average crypt consisted of 35 to 42 epithelial cells, 25 to 35% of which were differentiated into goblet cells); 1, 11 to 28 goblet cells per high-power field;. 2, 1 to 10 goblet cells per high-power field; 3, less than 1 goblet cell per high-power field.

(iv) Epithelial integrity. Epithelial integrity was scored as follows: 0, no pathological changes detectable in 10 high-power fields (magnification, x400); 1, epithelial desquamation; 2, erosion of the epithelial surface (gaps of 1 to 10 epithelial cells per lesion); 3, epithelial ulceration (gaps of >10 epithelial cells per lesion) (at this stage, there was generally granulation tissue below the epithelium).

The combined pathological score for each tissue sample was determined by adding the averaged scores described above, and the scores indicated the following: 0, intestine intact without any signs of inflammation; 1 to 2, minimal signs of inflammation which were not signs of disease (this was frequently found in the cecum of SPF mice); 3 to 4, slight inflammation; 5 to 8 moderate inflammation; 9 to 13, profound inflammation.

Immunofluorescence experiments. Cryosections (7 µm) were mounted on glass slides and air dried for 2 h at room temperature prior to immunostaining. Sections were fixed in 4% paraformaldehyde for 30 min, washed in PBS, permeabilized with Triton X-100 (0.1% in PBS, 10 min, room temperature), washed, and blocked in 10% (wt/vol) normal goat serum in PBS for 1 h. The sections were stained for 1 h with polyclonal rabbit anti-class I and II cytokeratin (1:100; Biomedical Technologies, Stoughton, Mass.), polyclonal rabbit anti-Ki-67 (1:100; Abcam, Cambridge, United Kingdom), or monoclonal rat anti-CD18 (1:100) and hamster anti-ICAM-1 (1:100; Becton Dickinson) in PBS containing 10% (wt/vol) goat serum. Fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (1:100), Cy5-conjugated anti-rat IgG (1:100), and Cy3-conjugated anti-hamster IgG (1:100) polyclonal antibodies (Dianova, Germany) diluted in PBS containing 10% (wt/vol) goat serum served as secondary antibodies. DNA was stained with DAPI (4',6'-diamidino-2-phenylindole) (0.5 µg/ml; Sigma). F-actin was visualized by staining with tetramethylrhodamine (TRITC)-conjugated phalloidin (Molecular Probes). Stained slides were mounted with Vectashield (Vector Laboratories).

Images were recorded at a magnification of x40 using a Perkin-Elmer Ultraview confocal imaging system and a Zeiss Axiovert 200 microscope. Red, green, and Cy5 fluorescence was recorded confocally, while DAPI fluorescence was imaged by epifluorescence microscopy. Images were combined using the Adobe Photoshop version 7.0.1 software, ensuring that all panels from each figure were processed in the same way.

Statistical analysis. Statistical analysis of the individual pathological scores for submucosal edema, PMN infiltration, loss of goblet cells, and epithelial integrity and of the combined pathological score was performed using the exact Mann-Whitney U test and the SPSS version 11.0 software, as described previously (2). P values of <0.05 were considered statistically significant. Bacterial colonization was analyzed in a similar manner. To allow statistical analysis of the bacterial loads, the values for animals yielding "no CFU" were set to the minimal detectable value (for mLN, 10 CFU; for spleens, 20 CFU; for livers, 100 CFU; for intestinal contents, between 67 and 400 CFU [see above]). After this, the median values were calculated using Microsoft Excel XP, and statistical analysis was performed using the exact Mann-Whitney U test and the SPSS version 11.0 software. P values of <0.05 were considered statistically significant.


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RESULTS
 
Time course of serovar Typhimurium colitis in GF and StrSPF mice. StrSPF and GF mice are animal models for Salmonella serovar Typhimurium-induced colitis (2, 31). However, these two disease models have never been compared side by side. In a pilot experiment we compared the kinetics of colonization by Salmonella serovar Typhimurium and inflammation in StrSPF and GF mice. SPF mice harboring a normal undisturbed intestinal microflora served as a control. We infected seven SPF mice, six StrSPF mice (mice pretreated with streptomycin 24 h prior to infection), and five GF mice that were kept under germfree conditions until 2 h prior to infection with the Salmonella serovar Typhimurium wild-type strain (5 x 107 CFU intragastrically). At 4 h, 8 h, and 20 h p.i. the numbers of animals indicated below were sacrificed, and we analyzed colonization of the cecum, as well as pathological changes in the large intestine (as described in Materials and Methods). At most times we used two SPF, two GF, and two StrSPF mice. One mouse was used to examine the results at 8 h for GF mice, and three mice were used to test SPF mice at 20 h p.i. (Fig. 1).



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  		FIG. 1. Preliminary time course study of Salmonella serovar Typhimurium infection in nonpretreated SPF, StrSPF, and GF mice. Seven nonpretreated SPF mice, six StrSPF mice, and five GF mice were infected with 5 x 107 CFU of wild-type SL1344 intragastrically. At 4 h, 8 h, and 20 h p.i. one, two, or three animals were sacrificed, as indicated in panel A. (A) Bacterial loads in the cecal contents. (B) Histopathological analyses: H&E-stained sections of cryo-embedded cecal tissue were scored with respect to edema in the submucosa, PMN infiltration (PMN inf.), reduction in the number of goblet cells, and desquamation, erosion, and ulceration of the epithelial layer (see Materials and Methods). Scores are plotted as stacked vertical bars. The dotted line indicates the limit of detection (cecal contents).

In the SPF mice, the cecal colonization levels were low (105 to 106 CFU per g cecal contents) at 4 h and 8 h p.i. and decreased at 20 h p.i. (101, 103, and 106 CFU per g cecal contents) (Fig. 1A). For the StrSPF and GF mice only one of the two mice in each group showed cecal colonization at 4 h p.i. (105 to 106 CFU/g) (Fig. 1A). At 8 h and 20 h p.i., serovar Typhimurium had colonized the ceca of both StrSPF and GF mice at high densities (108 to 109 CFU/g). This is in line with earlier studies with StrSPF and GF mice (2, 9, 44, 46) and indicated that Salmonella serovar Typhimurium colonized StrSPF and GF mice with similar kinetics.

No inflammation was detected in ceca of infected SPF mice at any of the times investigated (the pathological scores ranged from 1 to 2) (Fig. 1B and 2A and D). In contrast, StrSPF and GF mice developed pronounced intestinal inflammation characterized by mucosal edema, PMN influx, and destruction of the cecal epithelium within 20 h (the pathological score for the cecum ranged from 6.5 to 9.5) (Fig. 1B and 2B, C, E, F, G, and H). Crypt elongation was observed in the StrSPF mice but not in the GF mice. This was investigated in more detail, as described below.



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  		FIG. 2. Pathological changes in ceca of nonpretreated SPF, StrSPF, and GF mice infected with Salmonella serovar Typhimurium. Thin sections (5 µm) of cryo-embedded cecal tissues of mice from the experiment described in the legend to Fig. 1 were stained with H&E as described in Materials and Methods. Nonpretreated SPF mice (A and D), StrSPF mice (B, E, and G), and GF mice (C, F, and H) were infected with wild-type strain SL1344 for 20 h. Panels D, E, and F are enlargements of the sections indicated by the boxes in panels A, B, and C, respectively. Panels G and H are enlargements of the sections indicated by the boxes in panels E and F, respectively. The arrows in panels G and H indicate PMN. e, submucosal edema; L, intestinal lumen; er, erosion. (A, B, and C) Scale bars = 200 µm. (D, E, and F) Scale bars = 100 µm. (G and H) Scale bars = 20 µm.

Thus, Salmonella serovar Typhimurium colonized the ceca of StrSPF and GF mice with similar efficiencies. Severe cecal and colonic inflammation developed in both of these groups of mice within 20 h but not in SPF mice. This is in line with the notion that the mere lack of bacterial microflora is sufficient to allow Salmonella serovar Typhimurium to colonize the murine large intestine and cause colitis.

SPI-1 is required for colitis in GF and StrSPF mice. Salmonella serovar Typhimurium colitis was observed in GF and StrSPF mice. However, it was not clear whether the disease was attributable to similar mechanisms in the two cases. Was inflammation due to the reduced epithelial barrier function observed in GF animals? Might this have allowed penetration of bacterial products or living bacteria into the intestinal tissue? Or was it attributable to Salmonella serovar Typhimurium-specific virulence factors? The latter phenomenon has been observed in StrSPF mice, in which inflammation is strongly dependent on the SPI-1 TTSS (2, 18, 19). We hypothesized that Salmonella serovar Typhimurium strains with a disrupted SPI-1 TTSS should be attenuated in both mouse models if Salmonella serovar Typhimurium colitis is attributable to the same pathogenetic mechanism.

Six or seven GF mice (kept in a germfree isolator until 2 h prior to infection) or six or seven StrSPF mice were used for each experimental group. For intragastric infection we used either wild-type Salmonella serovar Typhimurium strain SL1344 (5 x 107 CFU; seven mice), an isogenic Salmonella serovar Typhimurium mutant (SB161 [= SL1344 {Delta}invG]; 5 x 107 CFU; nonfunctional SPI-1 TTSS [24]; seven mice), or PBS (mock infection; six mice). At 24 h p.i., mice were sacrificed, and we analyzed cecal colonization and pathology, as well as bacterial loads in the mLN, spleen, and liver.

Both Salmonella serovar Typhimurium strains had efficiently colonized the cecal lumina and mesenteric lymph nodes but not the spleens of GF and StrSPF mice at 24 h p.i. (Fig. 3A, B, and C). Liver colonization was observed in several animals infected with SB161 (Fig. 3D). This is in line with previous data for StrSPF mice (2). Wild-type Salmonella serovar Typhimurium caused severe colitis in GF and StrSPF animals, while SB161 did not (Fig. 3E and Table 1) (P << 0.05). Thus, Salmonella serovar Typhimurium colitis in GF and StrSPF mice required a functional SPI-1 TTSS.



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  		FIG.3. Infection of GF and StrSPF mice with Salmonella serovar Typhimurium wild-type strain and strain SB161 for 24 h. Groups of five StrSPF mice or GF mice were infected intragastrically for 24 h with 5 x 107 CFU of serovar Typhimurium strain SL1344 (wild type) or SB161 or were mock infected with PBS (A to D) (solid circles). In an independent experiment, groups of two StrSPF mice or two GF mice were infectedintragastrically for 24 h with 5 x 107 CFU of serovar Typhimurium strain SL1344 (wild type) or SB161. One StrSPF mouse and one GF mouse were mock infected with PBS. Tissue colonization was determined, and cecal tissues were fixed in 4% formalin and embedded in paraffin. The latter mice are indicated by open circles in panels A to D and by plus signs in panel E. (A to D) Bacterial loads in the cecal contents (A), in the mLN (B), in the spleen (C), and in the liver (D). (E) Histopathological analyses. H&E-stained sections of cryo-embedded (not labeled) or paraffin-embedded (plus signs) cecal tissues were scored with respect to edema in the submucosa, PMN infiltration (PMN inf.), reduction in the number of goblet cells, and desquamation, erosion, and ulceration of the epithelial layer (see Materials and Methods). The dotted lines indicate the limit of detection. The solid lines indicate the median. n.s., not statistically significant (P ≥ 0.05); S. Tm, Salmonella serovar Typhimurium; stat. anal., statistical analysis; wt, wild type.


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  		TABLE 1. Comparison of disease parameters for mice shown in Fig. 3 receiving PBS or SB161 and for mice receiving SB161 or wild-type strain SL1344a

Detailed histopathological analysis revealed that wild-type Salmonella serovar Typhimurium caused slightly more pronounced colitis in GF mice than in StrSPF mice (pathological scores, 9 to 12 and 5 to 8.5, respectively) (Fig. 3E and Table 2). While PMN infiltration and submucosal edema did not differ (P > 0.05), the loss of goblet cells (P = 0.001) and disruption of the intestinal epithelium (P = 0.002) were more pronounced in GF mice than in StrSPF mice (Table 2 and Fig. 3E and 4C, F, L, and O). In GF mice infected with wild-type Salmonella serovar Typhimurium cecal crypts were extremely shallow and the epithelium showed erosion and ulceration, while StrSPF mice displayed merely epithelial desquamation (Fig. 4, compare panels L and O with panels C and F).


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  		TABLE 2. Comparison of disease parameters for GF and StrSPF mice: statistical analysis of disease parameters from Fig. 3a



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  		FIG. 4. Histopathological analysis, infiltration of CD18+ cells, and expression of ICAM-1 in GF and StrSPF mice at 24 h p.i. Thin sections of cecal tissues of StrSPF mice (A to I) and GF mice (J to R) from the experiment described in the legend to Fig. 2 were stained as described in Materials and Methods. (A to F and J to O) H&E-stained thin sections of paraffin-embedded tissues (see Fig. 3E). Mice were mock infected with PBS (A, D, J, and M) or infected with SB161 (B, E, K, and N) or wild-type strain SL1344 (C, F, L, and O). Panels D to F and M to O are enlargements of the areas indicated by the boxes in panels A to C and J to L, respectively. The boxes in panels F and O show PMN (arrows). (G to I and P to R) Expression of ICAM-1 and infiltration of CD18+ cells. Thin sections (7 µm) of cryo-embedded tissues were stained with DAPI, rat anti-mouse CD18, hamster anti-mouse ICAM-1, and polyclonal anti-rat IgG-Cy5 and anti-hamster IgG-Cy3 antibodies as described in Materials and Methods (DNA, blue; ICAM-1, red; CD18, green). Mice were mock infected with PBS (G and P) or were infected with SB161 (H and Q) or wild-type strain SL1344 (I and R). L, intestinal lumen; e, edema; er, erosion. The arrows indicate PMN. (A to C and J to L) Scales bars = 200 µm. (D to I and M to R) Scale bars = 50 µm.

It should be noted that the distinct mucosal architecture of GF mice may slightly affect the quantitative evaluation of inflammation (see Materials and Methods). The lamina propria and epithelium are less broad in GF mice. Thus, the pathological score for PMN infiltration (number of PMN per optical field; see Materials and Methods) might slightly underrepresent the density of PMN infiltrating the mucosa of GF animals. This should be kept in mind when pathological scores for StrSPF and GF mice are compared.

We analyzed the difference in epithelial damage between StrSPF and GF mice in more detail. Increased leukocyte infiltration might provide one possible explanation. We performed immunofluorescence analyses of CD18+ cells (e.g., PMN, macrophages, NK-cells, and CD11c+ dendritic cells) in cecal tissues. We also analyzed the expression of ICAM-1, an intercellular adhesion molecule predominantly expressed on vascular endothelium and high endothelial venules, as well as on a variety of activated lymphocytes. ICAM-1 expression is upregulated upon inflammation, mediating infiltration of CD18+ cells into target tissues. We found upregulation of ICAM-1 and concomitant infiltration of CD18+ cells in the cecal mucosa and also transmigration into the cecal lumina of StrSPF and GF mice infected with wild-type Salmonella serovar Typhimurium (Fig. 4I and R). Low expression of ICAM-1 and few CD18+ cells were detected in SB161-infected mice and mice mock infected with PBS (Fig. 4G, H, P, and Q). Thus, wild-type Salmonella serovar Typhimurium seems to trigger equivalent CD18+ infiltration and ICAM-1 upregulation in GF and StrSPF mice. Increased numbers of infiltrating CD18+ cells are not likely to account for the severe mucosal ulceration found in GF mice.

Salmonella serovar Typhimurium infection of GF and StrSPF mice for 2 days. Overall, Salmonella serovar Typhimurium colitis is similar in StrSPF and GF mice, and in both cases the SPI-1 TTSS is required for the induction of colitis on day 1 p.i. To assess whether disease progression differed between StrSPF and GF mice, we also analyzed colitis at 48 h pi. We reasoned that subtle differences between StrSPF and GF mice might lead to more obvious differences after a prolonged infection period.

Nine or 10 GF mice (kept in a germfree isolator until 2 h prior to infection) or six or seven StrSPF mice were infected with either wild-type Salmonella serovar Typhimurium SL1344 (5 x 107 CFU) or an isogenic Salmonella serovar Typhimurium mutant (SB161 [= SL1344 {Delta}invG]; 5 x 107 CFU) or were mock infected with PBS (Fig. 5). At 48 h p.i., mice were sacrificed, and we analyzed cecal colonization and pathology, as well as the bacterial loads in the mLN, spleen, and liver.



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  		FIG.5. Infection of GF and StrSPF mice with wild-type Salmonella serovar Typhimurium and strain SB161 for 48 h. Groups of five StrSPF mice or seven or eight GF mice were infected intragastrically for 48 h with 5 x 107 CFU of serovar Typhimurium strain SL1344 (wild type) or SB161 or were mock infected with PBS. In an independent experiment, groups of two StrSPF mice or two GF mice were infected intragastrically for 48 h with 5 x 107 CFU of serovar Typhimurium strain SL1344 (wild type) or SB161. One StrSPF mouse or one GF mouse was mock infected with PBS. Tissue colonization was determined, and cecal tissues were fixed in 4% formalin and embedded in paraffin. These mice are represented by open circles (A to D) or by plus signs (E). We analyzed bacterial loads in the cecal contents (A), in the mLN (B), in the spleen (C), and in the liver (D) and performed histopathological analyses (E). H&E-stained sections of cryo-embedded (not labeled) or paraffin-embedded (plus signs) cecal tissues were scored with respect to edema in the submucosa, PMN infiltration (PMN inf.), reduction in the number of goblet cells, and desquamation, erosion, and ulceration of the epithelial layer (see Materials and Methods). Scores are plotted as stacked vertical bars. The dotted lines indicate the limit of detection. The solid lines indicate the median. The dagger and the asterisk indicate animals analyzed in Fig. 6M and N. n.s., not statistically significant (P ≥ 0.05); S. Tm, Salmonella serovar Typhimurium; stat. anal., statistical analysis; wt, wild type.

In GF mice, the cecum was colonized with high efficiency at days 1 and 2 postinfection by all strains analyzed (Fig. 3A and 5A). StrSPF mice were colonized with similar efficiency at day 1 p.i., but at day 2 p.i. wild-type Salmonella serovar Typhimurium was present at significantly lower numbers than SB161 (Fig. 5A). This was also observed in one other study (19) but not in our first study of the streptomycin-pretreated mouse model (2). The reason for this variability is currently unclear. We speculate that reduction of the Salmonella serovar Typhimurium density by the onset of an unidentified element of the inflammatory response might play a role. In any case, at day 3 p.i. the level of wild-type Salmonella serovar Typhimurium increased and reached 109 to 1010 CFU/g at day 4 p.i., which was approximately 10-fold higher than the concentration in mice infected with SB161 (19).

At macroscopic inspection, the ceca and colons of mice infected for 2 days with wild-type Salmonella serovar Typhimurium appeared to be small, white, and edematous and were filled with a purulent exudate. The ceca and colons of all SB161- and mock-infected mice did not show overt signs of disease. Histopathological examination of the cecal tissue of wild-type Salmonella serovar Typhimurium-infected GF and StrSPF mice revealed that the overall degrees of the inflammation differed slightly but significantly (P = 0.044) in GF and StrSPF mice at 48 h p.i. (Fig. 5E and 6C, F, I, and L). Individual scoring parameters showed pronounced differences (Fig. 5E and Table 3). As found at 24 h p.i., the epithelial damage and loss of goblet cells were more pronounced in GF mice (P < 0.05) (Table 3 and Fig. 6F and L), while PMN influx and submucosal edema did not differ significantly between GF and StrSPF animals. Overall, the level of inflammation at 48 h p.i. was low in mice inoculated with PBS or SB161 (Table 4 and Fig. 5E and 6A, B, D, E, G, H, J, and K). Similar scores are routinely observed in noninfected SPF animals and are not considered signs of colitis.



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  		FIG. 6. Cecal pathology at 48 h p.i. Thin sections of paraffin-embedded cecal tissues of StrSPF mice and GF mice from the experiment described in the legend to Fig. 5E (plus signs) were stained as described in Materials and Methods. Mice were mock infected with PBS (A, D, G, and J) or were infected with SB161 (B, E, H, and K) or wild-type strain SL1344 (C, F, I, and L). Panels D to F and J to L are enlargements of the areas indicated by boxes in panels A to C and G to I, respectively. The boxes in panels F and L show PMN (arrows). (M and N) Cryo-embedded tissue sections of the apical and medial part of the cecum of two wild-type SL1344-infected GF mice (indicated by a dagger and an asterisk in Fig. 5E) were analyzed. (M) Total pathological score for sections from apical and medial parts of the ceca of two mice (indicated by the asterisk and dagger, respectively). (N) H&E-stained thin section of apical and medial parts of the cecum of the same mouse (asterisk). L, intestinal lumen; e, edema; de = desquamation; PMN inf., PMN infiltration. Scale bar = 200 µm.


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  		TABLE 3. Comparison of disease parameters for GF and StrSPF mice: statistical analysis of disease parameters from Fig. 5a


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  		TABLE 4. Comparison of disease parameters for mice shown in Fig. 5 receiving PBS or SB161 and for mice receiving SB161 or wild-type strain SL1344a

In some GF mice (Fig. 5E and 6M), the pathological scores of wild-type Salmonella serovar Typhimurium-infected tissues diverged significantly with respect to mucosal edema depending on the part of the cecum which was used for analysis (Fig. 6M). At the apical part of the cecum, where the cecal patch is located, no mucosal edema was present in some of the mice, in contrast to the medial part of the cecum (Fig. 6N). This phenomenon was not observed in cecal sections of StrSPF mice (unpublished observations). This indicated that there were local alterations in the severity of disease in the cecum of GF mice.

In GF and StrSPF mice we observed colonization of mLN by 24 h p.i. and colonization of mLN, livers, and spleens by 48 h p.i. (Fig. 3 and 5). At 48 h p.i. wild-type strain SL1344 and SB161 colonized the mLN of StrSPF mice slightly more efficiently than they colonized the mLN of GF mice (P < 0.05) (Fig. 5B). In the livers and spleens of GF mice, SB161 and wild-type strain SL1344 were present at slightly higher levels than they were in StrSPF mice. However, most of the differences were not statistically significant (Fig. 5C and D). Overall, the course of systemic Salmonella serovar Typhimurium infection was similar in GF and StrSPF animals at days 1 and 2 p.i.

Notably, GF mice infected with wild-type Salmonella serovar Typhimurium showed external signs of sickness (ruffled fur, hunched back) at day 2 p.i., while GF mice infected with SB161 or mock infected with PBS and all StrSPF mice appeared to be healthy. Considering the moderate level of systemic infection (102 to 104 CFU per organ) (Fig. 5C and D), this might indicate that GF mice are hyperreactive toward some virulence function of the SPI-1 TTSS.

In summary, GF and StrSPF mice exhibited only subtle differences in cecal colonization or pathology upon infection with both wild-type Salmonella serovar Typhimurium and SB161. The systemic infections were also quite similar, and the SPI-1 type III secretion system was required for colitis in both animal models. This suggested that Salmonella serovar Typhimurium colitis in StrSPF and GF mice is ruled by the same pathogenetic mechanisms.

GF mice fail to regenerate cecal epithelium upon infection with wild-type Salmonella serovar Typhimurium. While several parameters of Salmonella serovar Typhimurium colitis were similar in StrSPF and GF mice, there was a clear difference in the integrity of the cecal epithelium. The epithelium of GF mice infected with wild-type Salmonella serovar Typhimurium was ulcerated and showed only a few signs of regeneration (Fig. 4O). In StrSPF mice colitis was accompanied by marked regeneration of epithelial cells, resulting in crypt and villus elongation (Fig. 4F). To analyze this in more detail, we performed immunofluorescence studies of serial cecal sections of PBS-, SB161-, and wild-type Salmonella serovar Typhimurium-infected StrSPF and GF mice from the experiment shown in Fig. 3. Epithelial cells were stained with a polyclonal rabbit anti-cytokeratin antibody, and regeneration was studied using an antibody directed against Ki-67, an antigen expressed in the nuclei of proliferating cells (16).

In StrSPF and GF mice infected with SB161 or mock infected with PBS, the epithelial cell layer overlying the lamina propria was intact (Fig. 7D, E, M, and N), and proliferating cells were detected in a region close to the bottom of the crypts (Fig. 7G, H, P, and Q), where the proliferative zone is located. The epithelial layer in wild-type Salmonella serovar Typhimurium-infected StrSPF mice was also intact (Fig. 7F). Here, crypt elongation was clearly visible. Strong proliferation of epithelial cells was also reflected by the distribution of Ki-67+ nuclei along large parts of the crypt and also in the tips (Fig. 7I). In contrast, intact crypts were only rarely observed in GF mice infected with wild-type Salmonella serovar Typhimurium, and the mucosa was almost entirely replaced by infiltrating cells and connective tissue. Cytokeratin-positive epithelial cells, including the rare Ki-67-positive proliferating cells (probably enterocytes), were present only as a thin disordered layer on top of the mucosa (Fig. 7O and R).



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  		FIG.7. Epithelial regeneration in Salmonella serovar Typhimurium-infected GF and StrSPF mice. Serial thin sections of cryo-embedded cecal tissues of StrSPF mice (A and I) and GF mice (J and R) from the experiment described in the legend to Fig. 2 were stained as described in Materials and Methods. (A to C and J to L) H&E-stained thin sections (5 µm). Mice were mock infected with PBS (A and J) or infected with SB161 (B and K) or wild-type strain SL1344 (C and L). (D to F and M to O) Immunofluorescence analysis of the cecal epithelium. Thin sections (7 µm) were stained with DAPI, polyclonal rabbit anti-mouse cytokeratin, TRITC-phalloidin, and polyclonal anti-rabbit IgG-FITC antibodies as described in Materials and Methods. Mice were treated with PBS (D and M), SB161 (E and N), or wild-type strain SL1344 (F and O). (DNA, blue; actin, red; cytokeratin, green). (G to I and P to R) Regeneration of epithelial cells. Thin sections (7 µm) were stained with DAPI, polyclonal rabbit anti-mouse Ki-67, TRITC-phalloidin, and polyclonal anti-rabbit IgG-FITC antibodies as described in Materials and Methods. Mice were treated with PBS (G and P), SB161 (H and Q), or wild-type strain SL1344 (I and R). (DNA, blue; actin, red; Ki-67, green). L, intestinal lumen; e, submucosal edema. Scale bars = 50 µm.

In conclusion, epithelial integrity and proliferation were comparable in noninflamed, SB161-treated, and PBS-treated StrSPF and GF mice. Wild-type Salmonella serovar Typhimurium-induced inflammation triggered marked epithelial proliferation in StrSPF mice. In GF mice massive epithelial damage but only a little regeneration was observed. This is likely to account for the morphological difference between the cecal mucosa of StrSPF and GF mice upon infection with wild-type Salmonella serovar Typhimurium.


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DISCUSSION
 
Wild-type Salmonella serovar Typhimurium induces colitis in StrSPF and GF mice. Here, we compared the two animal models. Infection kinetics and most parameters of the intestinal inflammation were strikingly similar for GF and StrSPF mice. Most notably, in both GF and StrSPF mice, the SPI-1 TTSS was essential for induction of cecal inflammation.

GF and StrSPF mice both lack CR, but they differ in several aspects of gut physiology. StrSPF mice are associated with a "normal" microflora throughout their life until 24 h before infection. They have a fully developed gut-associated immune system, and the gut physiology matures in response to the colonization. In contrast, GF mice have never been in contact with a microflora and consequently have an "immature" gut physiology, including a reduced barrier function, altered mucus secretion, and no tolerance to bacterial products (21). Colonization of GF mice with a "normal" SPF flora was known to cause acute self-limited colitis (33). These observations suggested that GF mice might mount an inflammatory response to SB161, which lacks SPI-1-dependent protein secretion but produces ample amounts of microbe-associated molecular patterns (MAMP) (e.g., lipopolysaccharide, peptidoglycan, flagellin) and even colonizes the intestinal tissue, as indicated by the high number of SB161 cells present in the mLN. However, we found that this is not the case. Salmonella serovar Typhimurium requires the SPI-1 TTSS to "actively" induce colitis in GF mice. This is similar to the results obtained for StrSPF mice (2; this study) and suggests that the same pathogenetic mechanisms are responsible for Salmonella serovar Typhimurium colitis in StrSPF and GF mice. Physiological differences between GF and StrSPF mice (mucus, IgA production, M cells, developmental state of Peyer's patches) do not seem to be of great importance.

Exactly how the SPI-1 TTSS induces colitis is still a matter of debate. Effector proteins injected into intestinal cells via the SPI-1 TTSS could trigger signaling cascades which lead directly to the proinflammatory gene expression (14). Indeed, significantly elevated levels of various proinflammatory cytokines were detected in bovine ligated ileal loops infected with wild-type Salmonella serovar Typhimurium but not in loops infected with a mutant lacking the SPI-1 TTSS effector protein genes sipA, sopA, sopB, sopD, and sopE2 (2, 54). Alternatively, SPI-1-mediated cell invasion and penetration of the epithelial layer might simply increase Salmonella serovar Typhimurium loads in the mucosal tissue. This would lead to elevated extraluminal concentrations of bacterial products (MAMP). In this case, recognition of elevated MAMP levels by Toll-like receptors (45) would represent the primary trigger for inflammation. So far, it has not been clear which of these pathways is responsible for colitis triggered via the SPI-1 TTSS. A scenario including both mechanisms is also conceivable.

Clearly, the SPI-1 TTSS plays an important role in initiating intestinal inflammation in GF mice (this study) and StrSPF mice (2, 18). Does it also contribute to systemic infection, as observed in oral infections in the murine typhoid model (15)? In a recent study it was shown that the SPI-1 TTSS strongly and significantly enhances invasion of the large intestinal epithelium and lamina propria of StrSPF mice (19) (Fig. 1). However, generally this does not lead to dramatically enhanced systemic infection. Colonization of the liver and spleen by wild-type Salmonella serovar Typhimurium seems to be only slightly enhanced compared to colonization by SB161. A significant difference is detected only in some experiments (e.g., spleen colonization at day 2 p.i.) (Fig. 5C, StrSPF mice) (P < 0.05). This is in line with our first observations (2). Thus, in StrSPF (and GF) mice, invasion of the intestinal epithelium does take place but does not seem to represent the rate-limiting step in reaching the mesenteric lymph nodes. Rather, the data are consistent with bacterial transport via M cells or dendritic cells (34, 38). Future research will be aimed at characterizing this pathway in more detail.

Pathology was somewhat aggravated (severe epithelial ulceration) in GF mice infected with wild-type Salmonella serovar Typhimurium compared to StrSPF mice. We found that this difference is associated with sluggish epithelial regeneration in GF mice. In contrast, Salmonella serovar Typhimurium colitis in StrSPF mice is accompanied by enhanced epithelial cell regeneration, resulting in crypt elongation. In uninfected GF mice the transit time of epithelial cells from intestinal crypts to villus tips is known to be longer than that in mice associated with an SPF microflora (40). Possibly this is attributable to the lack of Toll-like receptor signaling in GF mice (37). The low intrinsic epithelial cell turnover in GF mice might therefore explain the failure to respond adequately (by fast epithelial regeneration) to the insult imposed by wild-type Salmonella serovar Typhimurium.

Streptomycin can have several toxic side effects (28, 32). Therefore, whether intoxication of intestinal epithelial cells might contribute to Salmonella serovar Typhimurium colitis in StrSPF mice (i.e., by loosening tight junctions or disruption of the brush border) remained a matter of discussion. Here, we found that GF mice, which do not require streptomycin pretreatment, develop Salmonella serovar Typhimurium-induced colitis. This argues against the hypothesis that streptomycin-mediated intoxication of enterocytes plays a role in the streptomycin-pretreated mouse model.

In summary, this study shows that GF and StrSPF mice provide similar but not identical murine models for Salmonella serovar Typhimurium-induced colitis. In both model systems, the lack of CR allowed Salmonella serovar Typhimurium colonization of the murine large intestine at high levels and induction of SPI-1 TTSS-dependent colitis. This opens the door to profit from the advantages offered by each animal model. While streptomycin pretreatment allows workers to use a wide variety of knockout and transgenic mice obtained from SPF-certified facilities, GF mice have a different advantage: they guarantee that Salmonella serovar Typhimurium is the only bacterium present in the intestine and that there is no contamination by remains of the indigenous microflora. This might be advantageous for a range of experiments, including "Affymetrix chip-type" gene expression analyses. We expect that both animal models will contribute to elucidation of the molecular pathways of Salmonella serovar Typhimurium colitis.


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ACKNOWLEDGMENTS
 
We are grateful to Mathias Heikenwalder and Cosima Pelludat for comments and critical reading of the manuscript.

This work was supported by grant 3100A0-100175/1 to W.D.H. from the Swiss National Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Microbiology, ETH Zürich, Wolfgang-Paulistrasse 10, HCI G413, CH-8093 Zürich, Switzerland. Phone: 01-632-5143. Fax: 01-632-1129. E-mail: hardt{at}micro.biol.ethz.ch. Back

Editor: J. B. Bliska

{dagger} Present address: McMaster University, 1200 Main St. W., HSC 3N51, Hamilton, ON, L8N 3Z5 Canada. Back


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Infection and Immunity, June 2005, p. 3228-3241, Vol. 73, No. 6
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.6.3228-3241.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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