This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.
In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3′-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms’ intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.
Intestinal permeability is considered as one of the main barriers related to the intestinal microbiota and mucosal immunity and is likely to be affected by several factors, such as gut microbiota modifications, epithelial impairment, or mucus layer alterations1. Recent papers have reported effective protocols to measure the intestinal permeability of cultured human intestinal cells by analyzing the fluorescence flux rates across the intestinal cell layer2, but fewer research papers present a suitable procedure for measuring the gut permeability in nematodes, particularly in C. elegans, by using FITC-dextran staining.
There are two representative protocols for measuring the gut permeability in C. elegans using Nile red3 and erioglaucine disodium (or the Smurf assay)4,5. In this protocol, we used FITC-dextran (average molecular weight 10,000), which has a much higher molecular weight than Nile red (MW = 318.37) and erioglaucine disodium (MW = 792.85). FITC-dextran is more similar than Nile red or erioglaucine disodium dyes to actual macromolecular nutrients such as carbohydrates, which are absorbed through the intestinal layer. The intestinal permeability of C. elegans fed with erioglaucine disodium (blue Smurf dye) can be easily evaluated without fluorescence microscopy. However, in the Smurf assay, quantitative analysis of intestinal permeability is difficult due to the lack of standardization and should be evaluated manually4,5. In the case of the Nile red assay, Nile red also stains lipid droplets in cells, which may interfere with the exact determination of gut permeability in C. elegans6. The present protocols enable rapid and precise quantitative analysis of intestinal permeability in C. elegans treated with various intestinal bacteria and chemicals while avoiding unspecific lipid staining.
C. elegans is a typical model in biological fields due to its affordable price, easy manipulation, limited animal ethics issues, and short lifespan, which is beneficial for rapid experimentation7. In particular, after the entire C. elegans genome was published, nearly 40% of genes in the C. elegans genome were found to be orthologous to genes that cause human diseases8. Moreover, the transparent body allows observation inside the organism, which is advantageous for researching cellular events and for fluorescence applications in cell biology, for example, stem cell staining with DAPI or immunohistochemistry9. C. elegans is often used as an experimental animal to study the interaction between the gut microbiota and the host; in addition, C. elegans is used to screen health-promoting probiotic bacteria10,11,12 as well as dietary chemicals promoting intestinal health13,14.
Pseudomonas aeruginosa and Enterococcus faecalis are well-known gut bacteria that negatively affect the gastrointestinal system, especially the colonic epithelial cells of the intestinal tract15,16. Therefore, measuring the gut permeability triggered by these bacteria is necessary for the screening and development of new drugs that can recover and reduce the damage caused by bacterial inflammation and infection. In this protocol, we tested the effects of these intestinal bacteria on the intestinal permeability of C. elegans.
We also report an optimized protocol for testing chemicals on the intestinal permeability of C. elegans. For this purpose, we used 3,3′-diindolylmethane (DIM) as a model chemical because DIM is a bioactive metabolite compound derived from indole-3-carbinol, which is present in Brassica food plants, and has been reported to have therapeutic effects on IBD in mice17,18. In addition, we recently discovered that DIM improves intestinal permeability dysfunction in both cultured human intestinal cells as well as the model nematode C. elegans19.
In this study, we used three different experimental conditions. First, we measured the effects of the different bacteria, P. aeruginosa and E. faecalis, on intestinal permeability (Figure 1). Second, we measured the effects of live and heat-inactivated P. aeruginosa on intestinal permeability (Figure 2). Third, we measured the effects of DIM (a model chemical) on the intestinal permeability of C. elegans fed with P. aeruginosa (Figure 3).
The objective of this study was to develop optimized protocols that measure the intestinal permeability of C. elegans, which is changed by treatment with various intestinal bacteria as well as with chemicals.
1. Preparation of P. aeruginosa PAO1 and Escherichia coli OP50 Culture
2. Preparation of Enterococcus faecalis KCTC 3206 Culture
3. Preparation of Heat-inactivated E. coli OP50 and Heat-inactivated P. aeruginosa PAO1 Cultures
4. Preparation of Nematode Growth Medium (NGM) Plates for Testing the Effects of Different Bacteria on the Intestinal Permeability of C. elegans
5. Preparation of NGM Plates for Testing the Effects of a Chemical (DIM) on the Intestinal Permeability of C. elegans Fed with P. aeruginosa
6. Preparation of Age-synchronized C. elegans
7. Treatment of Bacteria or DIM and FITC-dextran Feeding
8. Imaging C. elegans with the Operetta Imaging System and Determination of Intestinal Permeability by Measuring the FITC-dextran Fluorescence Uptake
NOTE: Fluorescent stereomicroscopy can be used for image analysis instead of the Operetta system.
9. Statistical Analysis of the FITC-dextran Fluorescence of C. elegans
After incubation with P. aeruginosa PAO1, C. elegans showed a significant increase in FITC-dextran fluorescence in the worm body compared to the fluorescence shown after incubation with the other two bacterial strains (Figure 1). The fluorescence intensities of worms fed with E. coli OP50, P. aeruginosa PAO1, and E. faecalis KCTC3206 were 100.0 ± 6.6, 369.7 ± 38.9, and 105.6 ± 10.6%, respectively. The data emphasize that P. aeruginosa caused more vital damage to the epithelial gut barrier, and therefore, the worms exhibited a dramatic increase in their intestinal permeability. Based on this result, FITC-dextran can easily penetrate through the intestinal layer, so P. aeruginosa was selected as a potential candidate pathogen for screening the effects of DIM. Although E. faecalis is a gut pathogen that can produce extracellular superoxide and hydrogen peroxide, which damage colonic epithelial cell DNA15, in some cases, E. faecalis is also known as a potential probiotic bacteria due to its ability to produce bacteriocins against some pathogens25,26. The functions of a probiotic include adherence to epithelial surfaces, persistence in the human gastrointestinal tract, immune stimulation and antagonistic activity against intestinal pathogens26. Therefore, the gut permeability of worms incubated with E. faecalis remained unchanged compared with that of the vehicle control worms. This result shows that the amount of the infection can be studied by the increase in the fluorescence intensity, and P. aeruginosa causes more intestinal permeability than other strains.
Figure 2 shows the difference between live and heat-inactivated P. aeruginosa PAO1 based on the intensity of FITC-dextran fluorescence in the worm body. Both the fluorescence images and statistical data indicate that the pathogen could not trigger any toxicity to nematodes after heat inactivation. P. aeruginosa can produce exotoxin A — a potent extracellular cytotoxin that is lethal for many animals27. Exotoxin A can be rapidly abolished by heating at 45 °C to 60 °C28. Therefore, heat-inactivated P. aeruginosa was unable to damage the permeability of the worms' intestinal epithelia. The supernatant of P. aeruginosa PAO1 culture significantly damaged intestinal permeability, and therefore, the culture supernatant instead of whole P. aeruginosa cells can be used to induce intestinal permeability dysfunction19. The supernatant contains endotoxins, exotoxin A, and lipopolysaccharides29, and these components are known to induce cell toxicity30,31. Therefore, these components of the supernatant may affect the intestinal permeability, although we did not check the direct effect of exotoxins and lipopolysaccharides on the intestinal permeability in C. elegans.
DIM cotreatment for 48 h significantly decreased the FITC-dextran fluorescence intensity inside the guts of worms compared with the P. aeruginosa single treatment (Figure 3C,D). Statistical analysis by one-way ANOVA and Tukey's multiple comparison test showed that after treatment with DIM, the mean fluorescence intensity was significantly decreased in comparison with the fluorescence intensity of the P. aeruginosa-only treatment. The fluorescence intensities of the worms treated with P. aeruginosa-only and P. aeruginosa plus DIM were 486.3 ± 41.7 and 414.2 ± 25.0%, respectively (Figure 3E). Based on this result, DIM can be considered a good natural product to cure intestinal permeability dysfunction caused by bacterial infections. This result indicated that DIM can attenuate cell inflammation in gut cells, which reduces the permeability of the intestine19. This result is similar to the results obtained in a mouse model, in which DIM showed a significant reduction in inflammation in the colon32.
Figure 1: The effects of different bacteria on the intestinal permeability of C. elegans. Microscopy images of the worms including bright-field, FITC fluorescence (green channel), and merged images. Microscopy images of worms from (A) E. coli OP50 without FITC-dextran feeding, (B) E. coli with FITC-dextran feeding, (C) P. aeruginosa PAO1 with FITC-dextran feeding, and (D) E. faecalis KCTC3206 with FITC-dextran feeding. Scale bar = 1 mm (white), and 200 µm (black). Age-synchronized L4 larvae were incubated for 48 h in NGM plates seeded with E. coli (A, B), P. aeruginosa (C), and E. faecalis (D). Then, the worms were transferred to plates containing FITC-dextran (B–D), except for the vehicle control (A). (E) The FITC fluorescence intensity of the different bacterial treatments. A higher percentage of FITC fluorescence indicated a higher gut permeability. Columns and error bars indicate the mean ± SD. ***P < 0.001 for significant difference from the vehicle control.###P < 0.001 for significant difference from the FITC-dextran-treated worms fed with P. aeruginosa PAO1 (ANOVA, n = 5). This graph is representative of two independent experiments. Please click here to view a larger version of this figure.
Figure 2: The effects of live and heat-inactivated P. aeruginosa PAO1 on the intestinal permeability of C. elegans. Microscopy images of worms from (A) live E. coli OP50 without FITC-dextran feeding, (B) live E. coli with FITC-dextran feeding, (C) live P. aeruginosa PAO1 with FITC-dextran feeding, and (D) heat-inactivated P. aeruginosa with FITC-dextran feeding. Scale bar = 1 mm (white), and 200 µm (black). Age-synchronized L4 larvae were incubated for 48 h in NGM plates seeded with live E. coli (A, B), live P. aeruginosa (C), and heat-inactivated P. aeruginosa (D). Then, the worms were transferred to plates containing FITC-dextran (B–D), except for the vehicle control (A). (E) FITC fluorescence intensity comparing live and heat-inactivated P. aeruginosa PAO1. Columns and error bars indicate the mean ± SD. ***P < 0.001 and **P < 0.01 for significant difference from the vehicle control.###P < 0.001 for significant difference from the FITC-dextran-treated worms fed with live P. aeruginosa PAO1 (ANOVA, n = 5). This graph is representative of two independent experiments. Please click here to view a larger version of this figure.
Figure 3: The effect of DIM on the intestinal permeability of C. elegans fed P. aeruginosa. Microscopy images of worms from (A) E. coli OP50 without FITC-dextran feeding, (B) E. coli with FITC-dextran feeding, (C) P. aeruginosa PAO1 with FITC-dextran feeding, and (D) P. aeruginosa and DIM (100 µM) cotreatment with FITC-dextran feeding. Scale bar = 1 mm (white), and 200 µm (black). Age-synchronized L4 larvae were incubated for 48 h in NGM plates seeded with live E. coli (A, B), live P. aeruginosa (C), live P. aeruginosa and DIM (D). Then, the worms were transferred to plates containing FITC-dextran (B–D), except for the vehicle control (A). (E) The FITC fluorescence intensity indicates that the gut permeability of C. elegans was affected by DIM. Columns and error bars indicate the mean ± SD. ***P < 0.001 for significant difference from the vehicle control.###P < 0.001 and ##P < 0.01 for significant difference from the FITC-dextran-treated worms fed with P. aeruginosa PAO1 (ANOVA, n = 5). This graph is representative of two independent experiments. Please click here to view a larger version of this figure.
By utilizing this new method for determining gut permeability in C. elegans, which combines automated fluorescence microscopy and quantitative image analysis, the differences caused by intestinal microorganisms or chemicals can be determined in vivo, specifically in the C. elegans intestine. This protocol is useful for gut permeability investigations and applicable to many tasks, such as reactive oxygen species (ROS) determination under stress conditions and morphological examinations, due to its convenience and easy manipulation. Moreover, this method can be used to determine the effects of therapeutic and preventive methods against multiple pathogens in C. elegans. In particular, it may be an efficient protocol to investigate the mechanisms of different bacterial strains, including both harmful and useful bacteria. Some pathogens and probiotic bacteria with similar structures exert different effects on the host33,34. This procedure can assist in evaluating which mechanism the bacteria are utilizing and where the targeted substrates can be secreted extracellularly or intracellularly. In addition, this method can also help to strengthen the hypothesis that heat treatment plays a vital role in pathogen inactivation.
However, there are also some difficulties during this procedure. First, the number of worms in each plate is important for statistically meaningful results and robust worm detection, so 70–90% confluency (at least 50 worms per well) is recommended. Accordingly, trial experiments should be performed to obtain suitable worms. Second, the plate properties are one of the important factors that affect the quality of images and intensity determination, particularly for the bottom material. In some advanced experiments, a glass bottom is the best option for the measurement of fluorescence due to its excellent optical quality. However, because of the high price, a polystyrene bottom is often used, although the quality is not as high as that of the glass bottom. Finally, the plate coating methods for C. elegans require optimization. In this experiment, fluorescent mounting medium was applied because it can preserve and enhance the labeling of tissue sections, but it is unable to fix the worms on the plate bottom properly.
This protocol has limitations; as C. elegans is a nematode, further experiments, such as evaluation in human intestinal cell monolayers and in mammals, should be performed. In this method, the phenotypic changes in C. elegans fed pathogenic bacteria and chemicals were determined. Therefore, the molecular and genetic mechanisms underlying the pathogenic or probiotic effects of intestinal bacteria as well as the therapeutic effects of chemicals should be further elucidated. Specific signaling pathways exerted by pathogenic bacterial infection and therapeutic effects of chemicals can be evaluated using both various mutant bacteria and mutant worms. In addition, further in-depth studies identifying the chemical components responsible for the pathogenic and probiotic effects of intestinal bacteria would be beneficial.
Here, we report an experimental protocol for measuring intestinal permeability in C. elegans treated with different bacteria and chemicals by using FITC-dextran feeding. We believe that these protocols will be helpful for basic biological research, such as studying the interaction between the intestinal microbiota and gut health, as well as for the development of probiotics and nutraceuticals for the prevention and treatment of intestinal health problems.
The authors have nothing to disclose.
This study was supported by a Korea Institute of Science and Technology intramural research grant (2E29563).
3,3’-diindolylmethane | Sigma | D9568 | |
90×15 mm Petri dishes | SPL Life Sciences, South Korea | 10090 | |
60×15 mm Petri dishes | SPL Life Sciences, South Korea | 10060 | |
Bactor Agar | Beckton Dickinson | REF. 214010 | |
Formaldehyde solution | Sigma | F1635 | |
Brain Heart Infusion (BHI) | Becton Dickinson | REF. 237500 | |
Caenorhabditis elegans N2 | Caenorhabditis Genetics Center (CGC) | Wild type | |
Cholesterol | Sigma | C3045 | |
Costa Assay Plate, 96 Well Black With Clear Flat Bottom Non-treated, No Lid Polystyrene | Corning Incorporated | REF. 3631 | |
Dimethyl sulfoxide | Sigma | D2650 | |
Enterococcus faecalis KCTC 3206 | Korean Collection for Type Culture | KCTC NO. 3206 | Falcutative anaerobic |
Escherichia coli OP50 | Caenorhabditis Genetics Center (CGC) | ||
Fluorescein isothiocyanate – dextran | Sigma | FD10S | |
Harmony software | PerkinElmer | verson 3.5 | |
Luria-Bertani LB medium | Merck | VM743185 626 1.10285.5000 | |
Magnesium sulfate heptahydrate | Fisher Bioreagents | BP2213-1 | |
Fluoromount aqueous mounting medium | Sigma | F4680 | |
Operetta CLS High-Content Analysis System | PerkinElmer | HH16000000 | |
Peptone | Merck | EMD 1.07213.1000 | |
Pseudomonas aeruginosa PA01 | Korean Collection for Type Culture | KCTC NO. 1637 | |
Sodium Chloride | Fisher Bioreagents | BP358-1 | |
Stereo Microscope | Nikon, Japan | SMZ800N | |
Yeast extract | Becton Dickinson | REF. 212750 |