The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

Podsumowanie

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

Streszczenie

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1β and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.

Wprowadzenie

The intestine represents a dynamic system that acts as a barrier for commensal microorganisms, fights against invading pathogens, and regulates the microbial composition¹. The intestinal epithelial cells, consisting of enterocytes, goblet cells, Paneth cells and enteroendocrine cells, are the major cell populations that provide host defense responses against intestinal microbiota. The goblet cells produce mucins that create a demilitarized zone on the top of the epithelial layer². The Paneth cells and enterocytes produce antimicrobial peptides, cytokines, and reactive oxygen and nitrogen species that constitute antimicrobial host defense responses and contribute to shaping the intestinal microbial composition^3,4. In addition to epithelial cells, the immune cells including macrophages, dendritic cells, neutrophils, natural killer cells, lymphocytes, and innate lymphoid cells in the lamina propria and submucosa play a critical role in intestinal antimicrobial host defense responses by producing cytokines, chemokines, and other mediators^5-7. In order to understand how the mucosal immune system regulates microbiota and provides protection against microbial infection, it is important to consider the complex interaction of the heterogeneous cell populations of the gut. However, an in vitro model that encompasses all of the features of the intestine is not available. Therefore, molecular studies on host-pathogen interaction in the intestine are highly challenging.

Over the past few years, several model systems that mimic aspects of the intestinal mucosa have been developed for investigating the pathophysiological processes involved in inflammatory bowel diseases (IBD) and other gastrointestinal disorders^8-14. Immortalized intestinal epithelial cell lines are often used to study epithelial cell specific responses. However, because of differential gene expression and function in immortalized cells, the data obtained from using those cells do not often match with those observed in in vivo studies. Intestinal crypt organoid culture has recently emerged as a potential tool for assessing the response of the intestinal epithelium to different stimuli¹³. In this system, crypt stem cells are allowed to grow and develop a 3D organoid structure. While the organoid culture system is very useful for studying many aspects of the intestinal epithelium, it does not mimic the complex interaction of immune cells, epithelial cells and microbial products. The ex vivo culture of the intestinal tissue offers a better representation of in vivo host defense responses. In this method, a part of the intestine is cultured in a cell culture plate with appropriate media allowing the different types of cell populations in the intestine to be metabolically active for at least 48 h. Thus, an ex vivo culture of the organ can be used to measure the expression of antimicrobial genes and the host defense responses of the intestine to a particular stimulus.

Investigators have been using the ex vivo organ culture system to study host defense responses against microbial infection in the intestine^15-21. We recently adopted the organ culture system to study the role of the inflammasome in antimicrobial host defense responses in mouse colons²². The inflammasome is a molecular platform for the activation of caspase-1, which is required for the production of matured IL-1β and IL-18. We showed that IL-1β and IL-18 induce antimicrobial peptides which effectively kill commensal pathobionts such as E. coli. This observation was consistent with increased E. coli burden in inflammasome-defective mouse colons²². This system therefore can be used to study the role of pattern recognition receptors (PRRs) and other innate immune molecules in intestinal antimicrobial host defense responses as well as pathogenesis of intestinal disorders such as inflammatory bowel disease (IBD) and colorectal cancer (CRC). There are more than 200 IBD susceptibility genes, and mutations in many of these genes are associated with altered microbial composition in the gut. It is of great clinical significance to determine the precise mechanism through which the IBD-susceptibility genes regulate gut microbiota. The overall goal of this method is to introduce a basic protocol of ex vivo colon organ culture and demonstrate how this culture method can be used to study antimicrobial host defense responses of the intestine.

Protokół

All experiments described here were performed using 6-8 weeks old male wild-type (C57BL6/J) mice maintained in a specific pathogen free (SPF) facility at the Animal Resource Center (ARC), UT Southwestern Medical Center. All studies were approved by the Institutional Animal Care and Use Committee (IACUC) and were conducted in accordance with the IACUC guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

1. Collection and Preparation of the Colon

1. Euthanize mice with CO₂ asphyxiation followed by cervical dislocation.
2. Spray mice with 70% ethanol and pin limbs to a pinning board keeping the mouse dorsal side down on the board.
3. Using pre-sterilized dissecting scissors and forceps, make a mid-line incision in the peritoneum of the abdomen. Open the abdomen by folding the peritoneum with forceps.
4. Remove the intestine from the abdominal cavity with dissecting forceps and scissors. Separate the colon from the small intestine by cutting at the bottom of the cecum and the other end at the rectum.
5. Place the colon in a sterile petri dish containing ice-cold PBS. Flush the contents of the lumen of the colon with ice-cold PBS using a 20 mL syringe holding a 20 G needle or an oral gavage needle until the stool within the lumen of the colon is completely removed.
6. Cut the colon with scissors longitudinally. Wash the colon by shaking vigorously in ice-cold PBS in a sterile Petri dish.
7. Cut the colon tissue into pieces approximately 1 cm long using a sterile scalpel or scissors.
8. Record the weight of the colon pieces.
   NOTE: All the steps in the Section 1 can be performed either inside or outside of a biological safety cabinet. Once colons are ready for culture, all the steps must be performed inside a biological safety cabinet to maintain sterility.

2. Colon Organ Culture

1. Place a cell strainer (100 μm) on a 6-well cell culture plate. Transfer all of the colon pieces collected from one mouse into the cell strainer.
2. Add 5 mL DMEM/F12 medium containing 5% FBS, Penicillin-Streptomycin (1x), and Gentamycin (20 µg/ml). Completely cover the colon pieces with the media.
3. Incubate for 2 hr at 37 °C in an incubator with 5% CO₂ and 95% air.
4. Lift the cell strainer and aspirate the media.
5. Place the cell strainer back on the well and add 5 ml fresh media without any antibiotics. Lift the cell strainer and aspirate the media.
6. Repeat the above washing step (Step 2.5) twice more (total 3x) to remove all of the residual antibiotics.
7. Transfer the colon pieces from a single cell strainer into a single well of a sterile 12-well cell culture plate.
8. Add DMEM/F12 medium containing 5% FBS (without any antibiotics). Adjust the volume of the medium with the weight of the colon pieces, e.g., 1 ml medium for 100 mg of tissue. Incubate for 12 h at 37 °C in an incubator injecting 5% CO₂ and 95% air.
9. Collect the culture supernatant in a sterile 1.5 ml tube.
10. Centrifuge at 12,000 x g at 4 °C for 5 min. Separate the supernatant into a new 1.5 mL tube for use in the bacteria killing assay and/or other immune assays. The supernatant can be stored at -80 °C until the assay.
11. Collect the colon pieces in a tube for RNA isolation.

3. E. coli Killing Assay

1. Inoculate E. coli in 5 mL Luria-Bertani (LB) broth in a 15 ml tube and incubate at 37 °C with shaking at 200 rpm overnight. Keep the cap of the culture tube slightly loose.
2. Centrifuge the bacterial culture tube at 1,200 x g for 10 min at 4 °C. Remove the supernatant and resuspend the bacterial pellet into 5 ml ice-cold PBS.
3. Transfer 1 mL of the bacterial suspension into a cuvette and measure OD at 600 nm. Use PBS as a blank.
4. Calculate the colony forming unit (cfu) using a predetermined standard curve. Here, assume that 1 OD = 2 x 10⁹ cfu/ml (approximately).
5. Dilute the bacterial suspension to make the stock suspension 1 x 10⁵ cfu/mL.
6. Transfer the colon organ culture supernatant (500 µl/well) collected in Section 2.10 into duplicate wells of a 24-well cell culture plate.
7. Add 10 µL E. coli culture (1,000 cfu) into one well containing 500 µL colon organ culture supernatant. Leave the other well without any E. coli inoculation to confirm that the colon organ culture supernatant does not contain any contamination.
8. Incubate the same number of bacteria (1,000 cfu) in the culture media (DMEM/F12 plus 5% FBS) without any antibiotics as a control.
9. Incubate at 37 °C for 1 h.
10. Place 50 µL of each sample drop-wise on MacConkey agar plates. Incubate the MacConkey agar plates at 37 °C overnight.
11. Count the number of colonies and calculate the cfu/ml.

4. The Effect of Extrinsic and Intrinsic Factors on Colonic Antimicrobial Host Defense Responses

NOTE: The antimicrobial killing assay as described here can be adopted to examine the effect of pathogen associated molecular patterns (PAMPs) and cytokines on bactericidal activity of organ culture supernatant. An example of such experiment using IL-1β and IL-18 is described below.

1. Collect colons from mice as described in Section 1.
2. Place the colon on a sterile paper towel. Cut the colon longitudinally into three parts using scissors (Figure 2).
3. Wash each part of the colon in ice cold PBS as described in Section 1.
4. Cut each part of the colon into small pieces and weigh aseptically. Transfer the pieces of each part of the colon into three separate cell strainers (100 μm) placed on three wells of a 6-well cell culture plate (Figure 2).
5. Add 2 ml DMEM/F12 medium containing 5% FBS, Penicillin-Streptomycin (1x), and Gentamycin (20 µg/ml).
6. Incubate for 2 h at 37 °C in an incubator injecting 5% CO₂ and 95% air.
7. Wash the colon pieces as described in 2.4-2.6. Transfer the colon pieces of a single cell strainer into a single well of a sterile 12-well cell culture plate. The three wells containing three parts of the colon from a single mouse should be designated as untreated, IL-1β, and IL-18 (Figure 2).
8. Add DMEM/F12 medium containing 5% FBS (without any antibiotics). Adjust the volume of the medium with the weight of the colon pieces, e.g., 1 mL medium for 100 mg of tissue.
9. Stimulate the colon organ culture with IL-1β (20 ng/mL) or IL-18 (20 ng/mL) for 12 h. The untreated colon organ culture serves as control.
10. After 12 h incubation at 37 °C in an incubator injecting 5% CO₂ and 95% air, collect the culture supernatant in a sterile 1.5 mL tube.
11. Transfer 500 µL culture supernatant into duplicate wells of a 24-well plate.
12. Inoculate E. coli (1,000 cfu) in colon organ culture supernatant as described in Section 3. For each condition, inoculate E. coli in a single well. The other well containing same organ culture supernatant without E. coli will serve as a control for bacterial contamination.
13. Incubate the same amount of bacteria in culture media without any antibiotics as a control.
14. Incubate at 37 °C for 1 h.
15. Place 50 µL of each sample drop-wise on MacConkey agar plates. Incubate the MacConkey agar plates for overnight at 37 °C.
16. Count the number of colonies and calculate the cfu/mL (Figure 4).

5. Measurement of the Expression of Antimicrobial Genes

1. After overnight incubation (12 h) of colonic organ culture as described in Sections 2 and 4, wash the colon pieces with 2 ml ice-cold PBS (2x).
2. Collect the colon pieces into a 2 mL RNase/DNase free screw-cap tube. Place the tubes on ice.
3. Add 1 mL commercial Trizol reagent and lysing matrix beads into the tube.
4. Lyse the tissue using an automated tissue homogenizer.
5. Collect the tissue lysate into a new microcentrifuge tube.
6. Isolate RNA using standard protocol.
7. Measure RNA concentration.
8. Dilute RNA appropriately with deionized water and use 500 ng RNA to synthesize cDNA.
9. Use the cDNA for real-time RT-PCR analysis of targeted antimicrobial genes, cytokines, chemokines, and other genes of interest.

Wyniki

A representative picture of colons in organ culture is shown in Figure 1. The colon pieces in the culture remain metabolically and physiologically active. They respond efficiently to exogenous stimuli added to the culture media. A schematic work flow of the preparation of the colon tissue for ex vivo culture and stimulation with exogenous stimuli, e.g. IL-1β and IL-18, is shown in Figure 2. The representative data in Figure ...

Dyskusje

The intestinal epithelial cells are very sensitive in terms of their growth requirements and therefore difficult to culture. The epithelial cells isolated by EDTA treatment do not survive in conventional cell culture media such as DMEM⁸. Therefore, host-pathogen interaction studies using isolated crypt or primary epithelial cells are very challenging. Recently, Sato et al. described a crypt organoid culture system which is very promising and useful for studies related to intestinal pathophysiology...

Materiały

Name	Company	Catalog Number	Comments
Advanced DMEM/ F12	Life Technologies	12634-010
Dulbecco's phosphate buffered saline, modified, w/o Calcium chloride & Magnesium chloride	Sigma	5634
FBS, heat inactivated	Sigma	F4135
Penicillin-Streptomycin	Life Technologies	15070063
Gentamicin solution	Sigma	G1272
Mouse IL-1b recombinan	Reprokine	RKP10749
Mouse IL-18 recombinant	Reprokine	RKP70380
TRIzol Reagent	Thermo Fisher Scientific	15596018
Difco Luria-Bertani Broth	BD Bioscience	244620
BD Difco Dehydrated Culture Media: MacConkey Agar	Fisher Scientific	DF0075-17-1
NanoDrop 1000 Spectrophotometer	Thermo Scientific		Uded to measure RNA concentration
UV/Vis Spectrophotometer	BECKMAN	DU 530	Used to determine E. coli count
iScript RT Supermix, 100 rxns	Bio-Rad	1708841
iTaq Univer SYBR Green Supermix	Bio-Rad	1725125
Lysing Matrix S (1/8"), 2 ml Tube	MP Biomedicals	116925500	Used to homgenize colon organ for RNA isolation
FastPrep-24 5G System	Bio-Rad	116005500
100 mm x 15 mm Petri Dish	Falcon	5687
Plate 6 well ps TC CS100, Cellstar, 6w, tc, F-bottom (Flat), w/lid, sterile	Cellstar	5085
100 μm cell strainer	Falcon	5698
Sorvall Legend Micro 21R Centrifuge	Thermo Fisher Scientific
Sorvall ST40R Centrifuge	Thermo Fisher Scientific
Forma Scientific orbital shaker	Thermo Fisher Scientific