Cell culture and reagents. The human monocyte THP-1 (ATCC TIB-202) cell line; the human colon epithelial cell lines HT-29, HCT-116 and SW 620; and the mouse macrophage RAW 264.7 cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). HT-29, HCT-116, SW 620 and RAW 264.7 cells were cultured in complete growth media (CGM) using Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose, L-glutamine and sodium pyruvate (Cellgro, Corning, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologics, Lawrenceville, GA) and 1% penicillin-streptomycin (Cellgro, Corning, NY). THP-1 cells were grown in Roswell Park Memorial Institute (RPMI) medium with L-glutamine (Cellgro, Corning, NY) supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin. T84 cells were grown in complete growth medium, which is a 1:1 Dulbecco’s Modified Eagle’s Medium (DMEM) and Ham’s F12 medium with 2.5 mM L-glutamine and supplemented with 5% heat-inactivated fetal bovine serum (FBS). All cells were maintained in an incubator set at 37°C and 5% CO2 and allowed to grow to 80-90% confluency before being sub-cultured or used in experiments. Purity of DMA was confirmed by gas chromatography-mass spectroscopy. All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) or VWR (Bridgeport, NJ).
MTT assay for cell viability. Cell viability assays were performed as previously described (Pekson et al. 2016). A solution of 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) was prepared in phosphate buffered saline (PBS) at a concentration of 5mg/ml. HCT-116, SW620, and HT-29 cells were seeded at 18,000 cells/well whereas THP-1 cells were seeded at 30,000 cells/well in 96-well plates and incubated at 37°C and 5% CO2 overnight. The next day, cells were washed with phosphate buffered saline (PBS), pre-treated with different concentrations of DMA (0.01, 0.1, 1, 10, 20, 30, 40, 50 and 100 mM) in CGM for 2 h and then treated with LPS (Escherichia coli 026:B6) (Sigma, St. Louis, MO) at 1 µg/ml and human-tumor necrosis factor alpha (TNF-α) (R and D systems, Minneapolis, MN) at 40 ng mL−1 for 24 h. At the end of 24 h of treatment, 20 µl of MTT solution (5 mg/ml) (Alfa Aesar, Ward Hill, MA) was added to each well at a final concentration of 0.5 mg/ml. After incubation for 2 h at 37°C and 5% CO2, media was aspirated and dimethyl sulfoxide (DMSO) (100 µl/well) (BDH, Randor, PA) was added to dissolve formed purple formazan crystals. The plates were shaken on an orbital microplate shaker for 10 min to ensure complete solubilization of the crystals and the absorbance of the resulting purple solution was measured at 570 nm using an Opsys MR microplate reader (Dynex Technologies, Chantilly, VA).
Enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA was used to determine the levels of various cytokines and chemokines such as TNF-α, Interleukin (IL)-6, IL-1β, IL-8, monocyte chemoattractant protein (MCP), granulocyte-macrophage colony-stimulating factor (GM-CSF)-1 and IL-10 and high mobility group box 1 protein (HMGB1) in cell culture supernatants. Ready-SET-Go! sandwich ELISA kits (eBioscience, San Diego, CA) for various human cytokines and chemokines were used as per manufacturers’ protocols. First, the optimal sample dilution for each target cytokine was determined to ensure that the sample absorbance readings fall within the range of their respective standard curves. Standard curves were produced by serially diluting lyophilized or recombinant standards, provided with the kits, according to the manufacturers’ instructions.
As per manufacturers’ protocols, clear flat-bottom Maxisorp 96-well plates (Thermo Fisher Scientific, Waltham, MA) were coated and incubated overnight at 4°C with capture antibody diluted in coating buffer for each analyte. The following day, each well was washed with washing buffer (1X PBS with 0.05% Tween 20) three times and then blocked for non-specific binding sites with assay buffer for one h. Samples were added to the wells in duplicate undiluted or diluted with assay buffer and incubated at for 2 h at room temperature. After the incubation period, each well was washed three times and the detection antibody diluted in assay buffer for the respective analytes was added and samples were incubated for one more h. Following the detection antibody incubation each well was washed thrice and avidin-HRP labeled secondary antibody diluted in assay buffer was added and incubated for another 30 min. Before proceeding to the last step, wells were washed five times and tetramethylbenzidine (TMB) substrate was added and the samples were incubated for 15 min, which resulted in a blue-colored solution. After 15 min., the reaction was concluded by adding a stop solution (1M H3PO4), turning the blue solution to yellow and the sample absorbance was measured at 450 nm using an Opsys MR microplate reader. The concentration of each analyte was interpolated using a second order polynomial equation created from the standard curve, using GraphPad Prism 6 software, and then the value was multiplied by the dilution factor used in the assay to calculate the concentration of the analyte in the original undiluted sample.
Protein immunoblotting. To study the mechanism of DMA’s effect on cytokine secretion, its effect on nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) expression in LPS stimulated THP-1 cells was examined. To perform the experiment, THP-1 cells were seeded at a density of 3 x 106 per T25 cm2 tissue culture flask and incubated overnight at 37°C and 5% CO2 using respective CGM. The next day, cells were washed with sterile PBS and pre-treated with different concentrations of DMA (0.1, 1 or 10 mM) or plain CGM for the untreated group, for 2 h. Then, LPS was added to all cells, except for the untreated group, at a final concentration of 1 µg/ml and cells were incubated for another 30 min. Whole cell lysates were prepared at the end of the respective treatment periods as described below.
Preparation of whole cell lysates. Whole cell lysates were prepared using a modified protocol as described by Abcam. Cells were scraped off using a cell scraper (Greiner Bio-one, Monroe, NC) and collected in a pre-chilled 5 ml snap cap centrifuge tube and then centrifuged at 14,000 x g for 2 min. The old medium was aspirated from the 5 ml tubes and cells were washed twice with ice-cold PBS. The washed pellets were again collected into 1.5 ml pre-chilled centrifuge tubes and were then centrifuged at 1000 x g for 5 min. The supernatants were removed without disturbing the cell pellets, which were washed with ice-cold PBS once again and centrifuged at the same settings as mentioned above. Again, the supernatants were removed without disturbing the cell pellets and the pellets were resuspended in 65 µl of radio immunoprecipitation assay (RIPA) lysis and extraction buffer (G-Biosciences, St. Louis, MO). The RIPA lysis buffer was freshly supplemented with EDTA-free protease inhibitor cocktail set III (Calbiochem, San Diego, CA) (1:200 dilution) and with phenylmethylsulfonylfluoride (PMSF) (Calbiochem, San Diego, CA) at a concentration of 100 mM in ethanol (1:100 dilution). The cell pellets were thoroughly mixed with the prepared lysis buffer and were kept on ice with vortexing for 5 sec every 10 min., for a total of 30 min. The lysed cells were centrifuged at 14,000 x g for 20 min. at 4°C (Eppendorf 5424R, Hauppauge, NY). The whole cell lysates were collected in labelled 0.65 ml microcentrifuge tubes and stored at -80°C until further analysis.
Automated capillary western blot analysis (WES Simple Western). WES, an automated capillary-based electrophoresis system (ProteinSimple, San Jose, CA) was used for performing the protein expression analysis. The total protein concentration of each experimental sample was calculated using Pierce bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, MA). The volumes of the lysates used for WES analysis were determined based on total protein concentrations, using a bovine serum albumin (BSA) (Thermo Fisher Scientific, Waltham, MA) standard curve. The final concentration of protein loaded into the WES plate is optimized to be 1µg/ml. All the reagents required for WES were prepared as per the manufacturer’s instructions. The provided 10X sample buffer was diluted using ultrapure water to 0.1X, which was used to dilute lysates. First, all standard pack reagents (DTT, 5X Fluorescent master mix and biotinylated ladder) were prepared. Then the lysates were mixed with prepared 5X fluorescent master mix in a 4:1 ratio to have the final concentrations of the lysates at 1 µg/µl. Finally, the lysates were heated at 95°C for 5 min.
The prepared lysates, blocking reagent, primary antibodies, HRP-conjugated secondary antibodies, chemiluminescent substrate mix (Luminol:Peroxide mixture in 1:1 ratio) and wash buffer were dispensed into designated wells in an assay plate provided by the manufacturer and as per the manufacturer’s instructions. The assay plate, along with the respective capillary cartridge, was placed into the WES instrument (ProteinSimple, San Jose, CA), which carries out all further assay steps automatically using default settings. Anti-I𝜅Bα antibody(Cell Signaling technologies, Danvers, MA), diluted 1/50, was used as the primary antibody and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Cell Signaling Technologies, Danvers, MA), diluted 1/10,000, was used for the gel loading control. All primary antibodies were diluted in an antibody diluent provided by the manufacturer and the optimal dilution for each primary antibody was determined using Simple Western antibody database as a reference. In approximately 3 h, molecular weight and quantitative signals for target proteins were automatically reported by the Compass software (ProteinSimple, San Jose, CA). Protein expression was analyzed using ImageJ software (NIH, Bethesda, MD) and normalized to that of GAPDH.
Animals. Nine-week-old male C57Bl/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). All experimental protocols were approved by the Mount Sinai Medical Center Institutional Animal Care and Use Committee (IACUC). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Mice were euthanized by carbon dioxide asphyxiation.
In vivo studies on the effect of DMA on dextran sodium sulphate (DSS)-induced colitis. Animal studies were approved Colitis was induced with DSS. A total of ten male C57Bl/6 mice weighing between 20 and 28 g were given drinking water containing 2.5% DSS. Mice were then randomly assigned to two groups. Group I mice (n=5) were negative controls and were injected intraperitoneally (ip) with 0.2 mL of PBS once a day for nine days. Group II mice (n=5) were injected ip with 0.2 mL of 33% DMA (2.1 g/kg) once a day for four days. One sham mouse was allowed to drink plain drinking water and injected with 0.2 mL PBS once a day for nine days. Mice were euthanized on the ninth day of the experiment and necropsied. The intestines were removed in their entirety for histologic examination.
Histological evaluation and grading. Several sections of the distal portion of the colon from each mouse were fixed in formalin, paraffin embedded, sectioned at 4 µM and stained with hematoxylin and eosin. Sections were examined by a practicing anatomic pathologist, who was blinded to the experimental conditions (S.E.R.), and scored, following the method described by Laroui et al. . Each section was scored for severity of inflammation (0 = rare inflammatory cells in the lamina propria, 1 = increased inflammatory cells in the lamina propria, 2 = confluence of inflammatory cells extending into the submucosa, 3 = transmural inflammation); crypt injury (0 = intact crypts, 1 = loss of the basal one third of crypts, 2 = loss of the basal two thirds of crypts, 3 = loss of entire length of crypts, 4 = focal erosion of epithelial surface, 5 = confluent areas of erosion of the epithelium); and ulceration (0 = absence of ulceration, 1 = 1 or 2 foci of ulceration, 2 = 3 or 4 foci of ulceration, 3 = confluent ulceration). Scores were added to yield maximum histologic grade of 11. Sections were examined with a Nikon Eclipse 80i light microscope and images were captured with a Nikon Digital Sight camera (Nikon, Melville, NY).
Statistical analysis. All the data are represented as the mean +/- SEM of at least four independent experiments. The data were analyzed using GraphPad Prism 6 software (San Diego, CA, USA). The statistical significance among and between groups was tested using a one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison post hoc test for the cell viability, cytokine secretion, and immunoblotting protein analyses. The Kruskal Wallis test was used to analyze differences in histologic scores. The a priori significance value was P<.05.