Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

Summary

We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.

Abstract

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages.

We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

Introduction

The health benefits of fatty acids, such as oleic, linoleic and linolenic acids have been and continue to be documented. For example, oleic acid helps facilitate penetration of lipophilic drugs in the body^1,2, reduces coronary heart disease by 24% when substituted for saturated fatty acids³, and is used to treat metabolic diseases such as Adrenoleukodystrophy⁴ which is an X-linked genetic disorder of fatty acid metabolism. While a necessary precursor for arachidonic acid in mammals, linoleic acid (unlike oleic acid) is not synthesized by the body and must be obtained through outside sources such as by flax seed consumption.⁵ Studies show several beneficial health effects of linoleic acid such as: anti-aging properties for the skin;⁶ anti-inflammatory properties;⁷ reduced proliferation of colorectal and prostate carcinoma cells;⁸ and the ability to fight obesity and promotion of cardiovascular health.⁹ Linolenic acid plays a role in reducing periodontal inflammation,¹⁰ and modulating thromboxane and prostacyclin biosynthesis.¹¹

Arpita¹² studied the influence of bile fatty acids and cholesterol on V. cholerae's expression of virulence factors and motility. Yamasaki¹³ indicated that methanol extract from red chili peppers, and other naturally extracted compounds, can potentially decrease cholera toxin production. It is conceivable to consider the use of food products that are rich in the above fatty acids (such as flax seeds) in the prevention and alleviation of infectious disease such as cholera through providing mucosal immunity. We conducted investigations to solubilize fatty acids and to determine, using cell proliferation assays, the maximum concentration of fatty acids that human intestinal epithelial cells can tolerate without detrimental effects on cell viability. We hypothesized that oleic, linoleic and linolenic acids provide a beneficial effect on cell viability at lower concentrations, but that at higher concentrations they will be toxic to the cells. We also solubilized the cholera toxin and determined the maximum concentration of cholera toxin that BALB/C mouse macrophages can tolerate without a significant decrease in cell viability. We hypothesize a toxic effect of cholera toxin on cell viability even at very low level. The method of solubilizing a cholera toxin and using it to determine the maximum amount of the toxin that the cells can tolerate without a significant decrease in survivability provides an advantage for future studies. For example, a combination of the above methodologies can be used to determine whether fatty acids provide cells with mucosal immunity against cholera infections. To the best of our knowledge, this rational and methodology has not been explored.

We discuss how our preliminary data can be used in later investigations to determine if oleic, linoleic and linolenic acids provide cells with mucosal immunity against cholera infection.

Protocol

1. Tissue Culture

1. Use Mus musculus macrophages (BALB/c mice) for cholera toxin determinations. Initially culture all of M. musculus cells following vendor's instructions.
2. Propagate BALB/c mice cells in Dulbecco's Modified Eagle's Medium with L-glutamine completed with 10% fetal bovine serum, and 1% antibiotic/antimycotic or RPMI 1640 base media completed with 10% fetal bovine serum, 5% L-glutamine, and 1% antibiotic/antimycotic reagent.
3. Grow cells in 75 cm² corning flasks with vented caps at 37 °C, 95% air and 5% CO₂.
4. Bring up human intestinal epithelial cells within 24 hr of delivery as per manufacturer's instructions for the determination of fatty acid determinations.
5. Propagate the human intestinal epithelia cells in 75 cm² corning flasks with vented caps in HybriCare media completed with 10% FBS and 30 ng/ ml human EGF at 37 °C. Do not use Antibiotic/antimycotic reagents as per manufacturer's instructions.
6. Split all cells (1:3) at approximately 70% confluency.
7. Freeze and bring up all cells throughout the study using standard freezing protocols.
8. Note: For larger scale concentration determination of fatty acids, use the faster growing, easier to maintain, mouse macrophages initially. For fine scale fatty acid concentration determination, use human epithelial cells. Use mouse macrophages for all cholera toxin treatments (see discussion).

2. Fatty Acid and Cholera Toxin Treatments

1. Transfer oleic, linoleic and linolenic acids from company-provided glass ampules to sterilized glass vials.
2. Transfer each fatty acid to a separate sterilized Eppendorf tube and dissolve it first in 100% ethanol at a dilution of 1:6, and then in RPMI 1640 incomplete media for a final concentration of 10 μg/μl.
3. Vortex each solution and transfer it to glass vials for storage in the freezer (-20 °C).
4. Plate cells at 2,500 cells/well in 96 well tissue culture plates.
5. Add the appropriate complete media to bring the total well volume to 200 μl for treatment of cells with fatty acids in preparation for MTT assays.
6. After a 24 hr proliferation period, remove the media and replace it with fresh media.
7. Add the appropriate concentrations of each fatty acid to be tested to the wells (see results). Add complete media to bring the total well volumes to 200 μl.
8. Incubate the treated plates for 24 hr before beginning the MTT assay.
9. Dissolve the cholera toxin in PBS at a concentration of 1 mg/ml and aliquot the solution in cryovials and store the cryovials at 2-8 °C.
10. For cell treatments, dilute the toxin 1:100 in either sterilized PBS (pH 7.4) or incomplete DMEM for a final working concentration of 1 ng/μl.
11. To treat cells with cholera toxin in preparation for MTT assays, plate cells as described above.
12. After a 24 hr proliferation period, add the appropriate concentrations of cholera toxin (for our concentrations, see results) to be tested to the wells following the procedures used in fatty acid applications (above).
13. Incubate the treated plates for 24 hr before beginning the MTT assay.
14. As a positive (positive c throughout the paper) and negative control (negative c), for both fatty acid and cholera toxin treatments, incubate cells with complete medium and 70% ethanol, respectively.
15. For all samples and treatments use a minimum of three replications (n=3), with more replications for positive controls (n=6 in this study).

3. MTT Assay

1. Make a MTT stock solution to a concentration of 2.5 mg/ml.
2. Remove the solution in each well of the 96 well plate to be tested and replace it with 200 μl of fresh complete media solution.
3. Add 10 μl of the MTT solution to each well.
4. Incubate the plate at 37 °C for 3-4 hr.
5. Discard the media solution and add 100 μl of 0.04 M HCl in isopropanol to each well.
6. Incubate the plate at room temperature for five minutes.
7. Transfer the solution from each well to a new centrifuge tube.
8. Centrifuge at 20,000 x g, at room temperature, for 1 min, or until a pellet was formed.
9. Transfer between 20-40 μl of each sample to a microplate reader.
10. Read absorbance at 570 nm using a spectrophotometer.

Results

Determination of the Optimum Concentration of Fatty Acids

The optimum concentration for fatty acids is defined as the maximum concentration at which cell growth is comparable to or exceeds that of control cells, with relatively low variability in results. To determine the optimum concentration of oleic, linoleic and linolenic acids cells were initially treated with varying concentrations of each fatty acid in large increments and later with smaller increments. Figure 1 shows the ...

Discussion

Suggestion of Concentration of Fatty Acids and Cholera Toxin

While the exact mechanism of how fatty acids enhance mucosal immunity is unknown, several studies have attempted to investigate their beneficial effects. Our study aims to provide methodology to determine the maximum concentration of fatty acids that cells can tolerate as well as the maximum concentration of cholera toxin that cells can tolerate without a significant influence on cell survival.

To determ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cells/Reagent
Mus musculus macrophages	ATCC	ATCC RAW 264.7
Dulbecco's Modified Eagle's Medium	ATCC	30-2002
L-glutamine	ATCC	30-2115
Fetal bovine serum	Bio-west	S0250
Antibiotic/antimycotic	Hyclone	SV3007901
Human intestinal epithelial cells	ATCC	ATTC CCL-241
HybriCare media	ATCC	46-X
Oleic Acid	Sigma-Aldrich	O1008
Linoleic Acid	Sigma-Aldrich	L-1376
Linolenic Acid	Sigma-Aldrich	L-2376
Cholera toxin	Sigma-Aldrich	C8052
Equipment
BD Falcon 96-Well Cell Culture Plates	BD Biosciences	351172
Spectrophotometer with Dynex Revelations 4.22 software	Dynex	91000101