The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Summary

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

Abstract

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.

The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

Introduction

Infection by enteric bacterial pathogens triggers gastrointestinal (GI) inflammation, as well as intestinal pathology and pathophysiology, including diarrhea and intestinal epithelial barrier dysfunction. The virulence mechanisms by which bacterial pathogens colonize the GI tract of their hosts, as well as specific host responses that defend against such infections are poorly understood, however recent advances in the modeling of enteric bacterial infections have begun to aid our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). The IBDs Crohn's Disease (CD) and UC are complex diseases of unknown etiology, characterized by chronic intestinal inflammation and tissue damage. Many mouse models of intestinal inflammation exist, from spontaneous inflammation in genetically modified strains, such as IL10 -/- mice, to chemical challenges with compounds, such as dextran sodium sulfate (DSS) and dinitrobenzene sulfonic acid (DNBS)¹. It has been hypothesized that the maladaptive chronic inflammatory responses present in IBD patients develop in genetically susceptible individuals upon abnormal exposure of the intestinal mucosal immune system to enteric bacteria², therefore the study of models of infectious colitis also offers significant potential for defining potentially pathogenic host responses to enteric bacteria. Citrobacter rodentium induced colitis is one of the rare models of infectious colitis that has been well characterized^1,3, allowing for the analysis of host responses to enteric bacteria and further understanding of potential mechanisms of IBD pathogenesis; an essential step in developing novel preventative and therapeutic treatments.

C. rodentium is a gram negative attaching and effacing (A/E), murine specific bacterial pathogen that is closely related to the important human pathogens enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC)^3-8. The family of A/E pathogens intimately attach to the apical host cell membrane of the cecal and colonic epithelium, forming a non-invasive pedestal-like structure on the host cell. Oral challenge with C. rodentium of 10⁸-10⁹ organisms produces a robust model of infectious colitis characterized by colonic hyperplasia or elongation of the crypts, mononuclear immune cell infiltration and goblet cell depletion^3,4. The initial site of colonization, a few hours after challenge, is at the cecal patch, followed by progression to the distal colon 2 to 3 days after infection³. In immunocompetent mouse strains, clearance of the pathogen is achieved 3 to 4 weeks after infection^1,3,4. However, many genetically modified strains, i.e. gene deficient or knockout (-/-) mice, have been found to display increased susceptibility to infection resulting in exaggerated damage and/or chronic infection and inflammation^9-14. Use of this infectious colitis model in these knockout strains, many lacking innate signaling proteins, has been indispensible in revealing several host proteins integral to resolution of intestinal infection and inflammation.

Protocol

1. Preparation of Citrobacter rodentium Inoculum and Oral Gavage of Mice

1. Prepare and autoclave Luria Bertani broth (LB).
2. Obtain viable C. rodentium from a frozen glycerol stock and streak onto LB agar plate using a sterile inoculating loop or pipette tip. Incubate at 37 °C overnight. Inoculate 3 ml of sterile LB broth in a falcon culture tube with colonies from the LB plate using an inoculating loop or pipette tip. Preparation of the inoculum should be done using aseptic techniques.
3. Incubate the C. rodentium culture aerobically at 37 °C overnight in a benchtop incubation shaker at 200 rpm. The inoculated LB broth should appear cloudy after overnight culture.
4. Use a bulb tipped gastric gavage needle attached to a 1 ml syringe to gavage each mouse with 100 μl of the overnight C. rodentium culture. Gently scruff the mouse, by firmly grasping the loose skin over its neck and back with thumb and fingers. Pull back the animal's head with your index finger to immobilize the head and straighten the esophagus for insertion of the gavage needle.
5. Maintain the mouse in an upright position and direct the bulb tip of the gavage needle along the side of its mouth and over its tongue. Gently pass the needle along the roof of the mouth and advance it down the esophagus. If there is any resistance felt during this procedure, remove the gavage needle and re-insert it.
6. Slowly inject 100 μl of the bacterial inoculum and gently remove the gavage needle. Monitor the breathing rate and behavior of the mouse after returning it to its cage.

Notes:

• In steps 2 and 3, also prepare a falcon culture tube using aseptic techniques with 3 ml of sterile LB broth to be cultured overnight to ensure that there is no bacterial contamination of the broth itself. The LB broth should appear clear after overnight culture.
• Gently agitate the culture within the falcon tube prior to loading syringes for gavage so that an equal dose of bacteria is delivered to each mouse, in step 4.
• Overnight culture should be discarded if it has been inoculated for over 24 hr.
• All C. rodentium infections and housing of infected animals should be carried out in a Biosafety Level 2 facility.

2. Measuring Colonic Epithelial Barrier Permeability in C. rodentium-infected Mice

1. Measure barrier integrity at a desired time point post-infection.
2. On the day of the assay, prepare enough 4 kDa fluorescein isothiocyanate (FITC)-dextran dissolved in phosphate buffered saline (PBS) to a concentration of 80 mg ml^-1 to gavage each mouse with 150 μl and to prepare the standard curve.
3. Scruff the mouse and gavage with 150 μl of FITC-dextran. Withdraw food from the cage at this time.
4. Anesthetize mice 4 hr after gavaging and collect as much blood as possible (~450 μl) through cardiac puncture. Add the blood to a final concentration of 3% acid-citrate dextrose in a microcentrifuge tube to deter coagulation. Euthanize the mice and collect colonic tissues in PBS for bacterial counts (steps 3.1 - 3.5) and in 10% formalin for tissue fixation and ultimately immunofluorescence staining (steps 4.1 - 4.8).
5. Spin the blood samples at 1,000 x g for 12 min in a centrifuge at 4 °C. Collect the serum and dilute to 1/10 and 1/100 in PBS. Add 100 μl of each sample at these two dilutions to a 96 well plate in replicates.
6. To prepare the standard curve, dilute the original 80 mg ml^-1 FITC-dextran used to gavage the mice in PBS to the following concentrations: 800, 400, 200, 100, 50, 25, 12.5, 6.25, and 0 μg/ml. Add 100 μl of each concentration to a 96 well plate in triplicate.
7. Quantify the fluorescence of each sample using a fluorometer at an excitation wavelength of 485 nm and 535 nm emission wavelength.
8. Analyze the raw data by plotting the standard curve. Input the raw data for each sample into the equation of the line generated by the standard curve to determine the concentration of FITC-dextran in each sample.

Notes:

• From step 4 onward, keep blood samples on ice and minimize exposure to light.
• When measuring epithelial barrier integrity a time point at, or before, 7 days post-infection is optimal, as severe tissue damage occurs after this point. Measuring barrier integrity prior to severe damage occurring allows you to determine maximal differences in permeability during C. rodentium infection between mouse strains.
• Ensure that uninfected control mice are also tested for epithelial barrier integrity.

3. Measurement of Bacterial Load in Tissues of C. rodentium-infected Mice

1. Add 1 ml sterile PBS and an autoclaved metal bead to a round bottomed 2 ml centrifuge tube using aseptic technique. Individual tissues collected from each animal will be placed in separate PBS tubes. Weigh the tubes prior to addition of the tissue.
2. Remove the cecum and colon from the mouse. Separate the cecum from the colon and push out the luminal contents using forceps. Add the contents to a PBS tube. After separating 0.5 cm sections from the distal colon and cecum for 10% formalin fixation, cut open the remaining colon and cecum longitudinally and wash with sterile PBS before placing tissues in tubes.
3. Weigh the PBS tubes with the tissue and then homogenize the samples using a bead beater for 6 min at 30 Hz.
4. Using aseptic techniques, add 180 μl sterile PBS per well to a 96 well plate. Add 20 μl of each homogenized sample to the first well in each row. Mix well and serially dilute, adding 20 μl from each previous well to obtain dilutions from 10^-1 (first well) to 10^-6. Plate 10 μl of each dilution from each sample in triplicate on a square bottom LB plate with a grid. Incubate overnight at 37 °C.
5. Count colony forming units (CFU), then average the 3 counts, and record the dilution at which each sample was counted. Multiply average CFU by 50, as 20 μl of the original 1 ml sample was used, and by the dilution factor at which the sample was counted resulting in CFU/ml. Finally, divide by the tissue weight to determine CFU/g.

4. Histological Assessment and Immunofluorescence Staining of Infected Colon Tissues

1. Collect tissues as in step 3.2. Store tissues in formalin overnight, then wash with 70% ethanol. Embed tissues in paraffin and cut 5 μm sections for staining. Stain with hematoxylin & eosin for histological assessment and proceed to the following steps for immunofluorescence staining.
2. To deparaffinize tissues prior to staining, place slides in a coplin staining jar and put in 65 °C water bath for 10 min. Next, place slides in xylene for four washes of 2 min each. Slides should then be rehydrated with two 5-minute washes in 100% ethanol, followed by one 5 min wash in each of the following: 95% ethanol, 75% ethanol and dH₂0. These washes should be done in a fume hood to avoid exposure to harmful vapors.
3. Place slides in the coplin jar with pre-warmed sodium citrate buffer and then place into a steamer for 30 min, then let sit for 30 min. This process breaks the protein cross-links formed by formalin fixation retrieving potential antigenic sites.
4. Wash with PBS azide for 5 min, three times. Dry slides and mark area around tissue with a PAP pen to create a hydrophobic barrier, keeping the staining reagents localized on the tissue.
5. Block tissue for 1 hr at room temperature with blocking buffer (e.g. α-goat serum) in an immunostaining moisture chamber.
6. Dilute primary antibody to desired concentration using antibody dilution buffer. Pour off blocking buffer and add 50-100 μl of primary antibody to tissues. Incubate at 4 °C overnight or at room temperature for 2 hr.
7. Wash with PBS azide for 5 min, three times. Add 50-100 μl of secondary antibody diluted in antibody dilution buffer to the tissues and incubate at room temperature for 1 hr in the dark. Wash with dH₂O for 5 min, three times.
8. Dehydrate slides, add DAPI Prolong Gold mounting medium and apply a coverslip. View slides under a fluorescence microscope.

Notes:

• PBS azide can be substituted with freshly prepared PBS as an alternative to avoid bacterial growth in the wash medium from step 4 onward.

Results

During a standard infection experiment, mice are infected with approximately 2.5 x 10⁸ CFU through gavage of 100 μl overnight C. rodentium culture. Infection of C57BL/6 mice with C. rodentium results in modest and transient weight loss and diarrhea. Although a rare occurrence with C57BL/6 mice, animals may become ill and require euthanization. Therefore, mice should be monitored for degree of weight loss and symptoms of distress such as piloerect fur and hunched posture, to determine...

Discussion

Citrobacter rodentium infection provides a valuable model for the study of both infectious disease and colitis in mice. This unique model allows for the characterization of both host responses, as well as the pathogenic properties of bacteria. The steps outlined in this protocol detail the successful use of this model.

There are several critical steps in this protocol to keep in mind when inducing colitis and analyzing responses. First, the preparation of a fresh overnight ...

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Comments
Luria Broth	ABM	G247	Add 25 g of LB powder to 1L of water. Autoclave before using.
Square bottom plate with grid	Fisher	08-757-11A
Falcon culture tube	Sarstedt	62.515.006
Bulb tipped gastric gavage needle	Fine Science Tools	18060-20
1 ml syringe	BD Biosciences	309659
4 kDa FITC-dextran	Sigma	FD-4
Citric acid	Sigma	C7129
Sodium citrate	Fisher	S279-500
Dextrose	Fisher	D16.1
Acid citrate dextrose			20 mM ctiric acid, 110 mM sodium citrate, 5 mM dextrose
Black 96-well plate	Fisher	07-200-762
Metal beads (5 mm)	Qiagen	69989
10% formalin	Fisher	5F93-4
5 ml vial	DiaMed	STK3205
Hematoxylin	Fisher	H345-23
Eosin	Fisher	E511-100
Xylene	Fisher	HC700-1GAL
Tween 20	Sigma	P5927
Coplin staining jar	VWR	47751-792
Sodium citrate buffer			10 mM sodium citrate, 0.05% Tween 20, pH 6.0
Pap pen	Cedarlane	Mu22
Goat serum	Sigma	G902-3
Bovine Serum Albumin (BSA)	Fisher	BP1600-100
Triton X-100	Sigma	T8532
Sodium azide	Sigma	SZ002
Blocking buffer			2% goat serum, 1% BSA, 0.1% triton X-100, 0.05% Tween 20, 0.05% sodium azide, 0.01 M PBS, pH 7.2, mix & store at 4 °C.
Antibody dilution buffer			0.1% triton X-100, 0.1% BSA, 0.05% sodium azide, 0.04% EDTA
Blocking buffer & Antibody dilution buffer for tir			Same recipes as above, but without addition of detergents (triton X-100 and tween 20)
Prolong Gold Antifade Reagent with DAPI	Invitrogen	P-36931
Coverslips	Fisher	12.54SE
Benchtop incubation shaker	Barnstead Lab Line	Max Q4000
Fluorometer	Perkin Elmer	Victor2D
Refrigerated centrifuge	Beckman Coulter	Microfuge 22R
Steamer	Black & Decker
Fluorescence microscope	Zeiss	Axio Image.Z1