OTII (OVA specific T cell receptor transgenic) C57BL/6 and control C57BL/6 mice were bred and maintained at the Pennsylvania State University (University Park, PA). The mice were fed synthetic diets made in the laboratory as described previously [26–28]. In one experimental design the mice were either control fed or fed diets that included 2% powdered mushrooms for 4 weeks (8–10 mice per group). In a second design mice were fed control, 1% or 2% WB or oyster mushrooms for 1 week prior to, and continuing through the DSS treatment (4–8 mice per group). Controls included additional mice that were fed the same diet but were not treated with DSS (3–4 mice per group). Experimental procedures were approved by the Office of Research Protection, Institutional Animal Care and Use Committee at the Pennsylvania State University.
Commercially available and commonly consumed mushrooms were used for this study. Agaricus bisporous or common name WB, brown Agaricus bisporous or common name crimini, Grifola frondosa or common name maitake, Lentnula edodes or common name shiitake, and Pleurotus eryngii common name king oyster mushrooms were obtained from Modern Mushroom Farm, Inc. (Toughkenamon, PA). The whole mushrooms were freeze-dried and ground into a fine powder. For the in vitro experiments, 10 mg of mushroom powder was suspended in 1 ml dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO) and sonicated (Branson Ultrasonic Corporation, Danbury, CT) for 8 min at room temperature. The crude unfiltered extracts were used for the experiments below. Mushroom extracts were tested for LPS levels (Limulus Ameobocyte Lysate assay, Cambrex, Walkersville, MD). There was a low level of LPS contamination of the extracts (3 ng/ml in WB and 3.2 pg/ml or less in the other extracts). The final concentration of LPS from the WB extracts added to the cultures was 30 pg/ml LPS. This concentration of LPS was well below the level of LPS required to stimulate cells in vitro. Although commercially produced according to standardized methods, there are likely to be important differences between mushrooms grown in different areas and under different conditions. Thus, freeze-dried mushrooms and extracts will be provided upon request.
RAW 264.7 cells were obtained from ATCC (Manassas, VA) and 106 cells per well were cultured in the presence of 100 μg of mushroom extracts or equal concentrations of DMSO (0.1 μl/ml) alone for 72 h in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum (FCS), 100 U/L penicillin, 100 mg/L streptomycin and 4 mmol/L glutamine (Invitrogen, Carlsbad, CA). Viability of the cultures after the 72 h was checked by trypan blue exclusion and was not different between the cells that received mushroom extract, DMSO or just media.
BMDM were obtained as previously described . BMDM from five C57BL/6 mice were pooled and cultured for 7 d at 37°C in the presence of recombinant macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN), in cold DMEM medium containing 25 mmol/L HEPES, 2 mmol/L glutamine, 5 × 10-5 mol/L 2-mercaptoethanol, 100 U/L penicillin and 100 mg/L streptomycin, supplemented with 100 ml/L FCS. More than 90% of the cells had macrophage morphology (large cells with irregular outlines, abundant cytoplasm and oval or indented nucleus) as determined by viewing cytospin preparations and by flow cytometry (F480 and CD11b positive). After 7 d in culture, the BMDM were washed with phosphate-buffered saline (PBS) and cells were cultured (106 cell/well) in the presence of mushroom extracts (100 μg/ml) alone, LPS (0.5 μg/ml, Sigma) alone, or mushroom extracts plus LPS for 72 h. Cell viability was checked at 72 h (trypan blue exclusion) and found not to be different among the different treatment groups.
Splenocyte suspensions from OT II mice were prepared and placed (106 cell/well) in complete RPMI 1640 (Sigma) medium-supplemented with 100 ml/L FCS, mmol/L HEPES, 2 mmol/L glutamine, 100 U/L penicillin and 100 mg/L streptomycin (Invitrogen, Gibco). Splenocytes in 24-well culture plates (Becton Dickinson Labware, Franklin Lakes, NJ) were cultured in the presence of crimini, shiitake, or oyster (100 μg/ml) and OVA (1 mg/ml, Sigma) or OVA alone for 72 h. In a separate series of experiments OT II splenocytes were cultured with WB (100 μg/ml) and OVA (1 mg/ml, Sigma) stimulation or OVA alone for 72 h. Cell viability was checked at 72 h (trypan blue exclusion) and found not to be different among the different treatment groups.
Splenocytes suspensions from C67BL/6 mice were prepared and 106 cells/well were placed in complete RPMI 1640. Splenocytes were cultured with LPS, 0.5 μg/ml), Con A (10 μg/ml) or media for 72 h when supernatants were collected for the detection of cytokines by enzyme-linked immunosorbent assay (ELISA). Cell viability was checked at 72 h (trypan blue exclusion) and found not to be different among the different treatment groups.
Mice were administered 3.5% DSS (MW = 40 kDa; ICN Biomedicals, Solon, OH) dissolved in filter-purified and sterilized water ad libitum for 5 d followed by a return to water for the remainder of the experiment (10 d following the start of DSS treatment).
The gross colonic blood scoring system previously described by Siegmund et al was used. The entire colon from cecum to anus was removed and the length was measured and reported as colonic length as described .
The distal colon was weighed and the same amount of tissue was cut open and washed in 1 × PBS containing penicillin (100 U/ml) and streptomycin (100 mg/ml). Tissue was then scrapped in 1 ml PBS using a razor blade. The colon tissue scrapings were centrifuged at 10,000 g at 4°C for 10 min. Cytokine concentrations in the supernatant of the colonic homogenate were measured by ELISA.
Parts of the stomach, intestine, kidney, and liver were saved in 10% formalin and sent to the Pennsylvania State Diagnostic Laboratories (University Park, PA) for staining (hematoxilyn and eosin) and evaluation of the tissue sections. The distal colon were removed from mice treated with DSS and histological analysis was performed blinded for pathology associated with DSS treatment as described .
ELISA kits (Pharmingen, San Diego, CA) were used to determine the levels of IFN-γ, IL-10, IL-1β, TNF-α, IL-4 and IL-12 in the supernatants, serum and colonic homogenates. The limits of detection were 125 pg/ml IFN-γ, 31.25 pg/ml IL-10, 31.25 pg/ml IL-1β, 31.25 pg/ml TNF-α, 31 pg/ml IL-4 and 125 pg/ml IL-12 p70.
Statistical analyses were performed using PRISM software (GraphPad Software, San Diego, CA) using ANOVAs and data that were not normally distributed were transformed prior to ANOVA. P values of 0.05 or less were considered statistically significant.