RAW 264.7 mouse monocyte macrophages and A549 type II alveolar cells, purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified incubator with 5% CO₂ and sub-cultured every 2–3 days.

The UA, N^G-monomethyl-L-arginine (L-NMMA), and concanavalin A (Con A) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Throughout the present study, the cells (1×10⁵ cells/ml) were treated with 10 µg/ml UA and 5 mM L-NMMA for 6 h following M. tuberculosis infection, following which they were incubated for the specified durations. Immediately prior to infection or treatment, the medium were replaced with serum-free DMEM. In the case of the splenocytes, the cells (1×10⁶ cells/ml) were co-treated with 5 µg/ml Con A and 10 µg/ml UA for the specified durations.

Female Wistar rats (190–220 g; 8-weeks old; n=8) were purchased from Nara Biotech, Ltd. (Pyeongtaek-si, Korea). The animals were housed in a solid-bottomed cage at 23±2°C, maintained under a 12 h light-dark cycle and fed standard rodent chow (Purina Rodent Chow; Purina Co., Ltd., Seoul, Korea) and water ad libitum. The present study was performed according to the guidelines of the United States National Institutes of Health, and was approved by the ethics committee of Soonchunhyang University (Asan, Korea; SCH14-0031).

Primary splenocytes were collected from the rats following sacrifice by cervical dislocation, and aseptic collection of the spleen was performed. Each spleen was immersed in RPMI 1640 culture medium supplemented with 10% (v/v) heat-inactivated FBS and a 1% (v/v) antibiotic/antimycotic cocktail, comprising 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were passed through a cell strainer (BD Biosciences, Durham, NC, USA) to form a single cell suspension. Erythrocytes were excluded from the resulting cell suspension using lysis buffer (Life Technologies, Seoul, Korea), containing 0.15 M NH₄Cl, 10 mM KHCO₃ and 0.1 mM Na₂EDTA (pH 7.3). The cells were washed once with phosphate-buffered saline (PBS) and cultured at a density of 10⁶ cells/ml. Cell viability was determined according to the exclusion of Trypan blue (Sigma-Aldrich). The cells were maintained under standard culture conditions at 37°C.

The M. tuberculosis H37Rv strain was purchased from ATCC and was grown in Middlebrook 7H11 agar (Becton Dickinson, Sparks, MD, USA) or Ogawa medium for ~3 weeks. Isolated colonies were inoculated in Middlebrook 7H9 broth (Becton Dickinson) in am incubator with agitation for 15 days. To avoid clumping, the bacterial suspension was vortexed vigorously with glass beads, and then passed through an 8-µm filter to form a single cell suspension. The suspension was allowed to stand for several minutes to settle and two-thirds of the clear upper portion of suspension was used for quantification at 600 nm using the UVmini-1240 spectrophotometer (Shimadzu, Kyoto, Japan) adjusted with McFarland standards. A 0.5 McFarland standard was prepared by mixing 0.05 ml of 1.175% barium chloride dihydrate with 9.95 ml of 1% sulfuric acid. Subsequently, 10 µl of the suspension was inoculated in Middlebrook 7H11 agar or Ogawa medium at 37°C in an atmosphere of 5% CO₂ for 3–4 weeks to quantify the bacterial number in colony forming units (cfu). Following count determination, the bacterial suspension was aliquoted and stored at −76°C as a single volume

For in vitro infection, the RAW 264.7 (2×10⁵ cells) and A549 (2×10⁵ cells) cells were grown in six-well plates overnight and infected with M. tuberculosis H37Rv at a 1:10 ratio for 3 h in the aforementioned cell culture conditions. The cells were washed with warm PBS three to five times to remove extracellular bacteria. To confirm successful invasion of the bacteria, the final wash medium and cell extract (100 µl), following cell dissolution with 0.1% Triton X-100 (Sigma-Aldrich), were inoculated in Middlebrook 7H11 agar for colony counting, Middlebrook 7H9 broth for a resazurin assay (20) and in a Bactec MGIT system (Becton Dickinson) for the determination of time to detection (TTD) (21), as described previously (22). Prior to proceeding, it was confirmed that the bacteria present in the final wash medium were absent or few in number, compared with the high numbers of viable bacteria in the extracted cell suspension, which confirmed successful bacterial invasion.

The cell viability assay performed in the present study was based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) to formazan crystals by the mitochondrial dehydrogenase enzyme (23). In brief, the cells (1×10⁵ cells/ml) were seeded overnight to achieve ~80% confluence and were treated with 10 µg/ml UA for 0, 6, 12, 24, 48 and 72 h. At the determined time, 20 µl of 5 mg/ml MTT reagent was added. Following 4 h incubation at 37°C, the medium were aspirated, and 100 µl of dimethylsulfoxide (Samchun Pure Chemical Co., Ltd., Pyeongtaek, Korea) was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a Victor™ X3 multilabel reader (Perkin Elmer, Inc., Waltham, MA, USA).

Splenocyte viability was determined using an MTS-based assay (24). In brief, the cells were treated for the indicated times periods, and the detection reagent was prepared using MTS and phenazine methosulfate (Sigma-Aldrich) at a ratio of 20:1, which was added at a 1:5 ratio of reagent mixture to cell culture. The absorbance was detected at 492 nm using the Victor™ X3 multilabel reader.

The cells were infected and/or treated for the indicated time periods, following which the total RNA was extracted using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) and quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA) at 260 nm. The assay used RNA at a 260:280 nm ratio of 1.8–2.0, indicating high purity. Subsequently, cDNA was prepared using 1,000 ng of the total RNA using Oligo dT₁₅ Primer (Maxime™ RT PreMix kit; Intron Biotechnology, Inc., Seongnam, Korea) in a Veriti^® 96-Well Thermal Cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). Subsequently, qPCR was performed to amplify the cDNA using an iQ SYBR Green Supermix kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's protocol, in a CFX96TM real-time PCR detection system (Bio-Rad Laboratories, Inc.). The temperature cycle followed for qPCR was as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec, 42°C for 10 sec and 72°C for 20 sec. A dissociation curve was acquired to ensure the specificity of the PCR product in every PCR assay. The assay results were normalized to the endogenous control gene, glyceraldehyde 3-phosphate dehydrogenase. The primers used were purchased from Bioneer Corporation (Seoul, Korea) and are listed in Table I, with the exception of human interleukin 1-β (IL-1β), and interleukin 6 (IL-6), which were also purchased from Bioneer Corporation (Seoul, Korea; cat. nos. N-1058 and N-1063, respectively). Relative quantification was obtained using the comparative threshold cycle (∆∆Cq) method. A CFX96™ real-time PCR detection system (Bio-Rad Laboratories, Inc.) with a default calculation system was used.

NO release was measured by detecting the concentration of nitrite produced, using the Griess reagent system (Promega Corporation, Madison, WI, USA). Briefly, nitrite standards were prepared, ranging between 100 and 1.56 µM (100, 50, 25, 12.5, 6.25, 3.13 and 1.56 µM) by serial 2-fold dilutions. The cell-free supernatants were obtained by centrifugation at 2,000 × g for 1 min at room temperature. The nitrite standards and cell-free supernatants from the infected and/or treated samples (50 µl) were added to a 96-well tissue culture plate in triplicate. Subsequently, 50 µl sulphanilamide solution was added to each well, and incubated for 10 min at room temperature in the dark, followed by the addition of 50 µl N-1-napthylethylenediamine dihydrochloride. The absorbance was measured at 540 nm using the Victor™ X3 multilabel reader.

Data are presented as the mean ± standard deviation. At least three individual experiments were performed. Statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA), Differences between groups were analyzed using one-way analysis of variance, followed by the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Prior to the experiments, it was necessary to evaluate the effect of UA on cell viability. Cell viability was determined using an MTT assay for the RAW 264.7 and A549 cells (Fig. 1A), and using an MTS assay for the rat splenocytes (Fig. 1B). Measurements over time established that significant cytotoxicity was evident, beginning at 24 h, in the two cell lines. At 72 h, 10 µg/ml UA resulted in cytotoxicity towards the RAW 264.7 cells, which was almost 11% higher than that in the A549 cells (Fig. 1A). By contrast, the splenocytes showed less cytotoxicity under similar treatment conditions between 24 and 72 h, compared with the RAW 264.7 and A549 cells. No significant difference in splenocyte viability was found at any time point, with the exception of 72 h (Fig. 1B), at which the cell viability was decreased by almost 35%, which was marginal, compared with the other two cell types at 72 h (Fig. 1B).

Cytokines are critical in the development and regulation of immune responses. Several phytochemicals are involved in the immunomodulatory activities of the immune system and, thus, are key in immunosuppression. The expression levels of genes encoding TNF-α, IL-6 and IL1-β in different cells was evaluated following infection and/or treatment. The M. tuberculosis-infected RAW 264.7 cells showed a significant induction of the mRNA expression of TNF-α at 12 and 24 h (Fig. 2A). This induction was significantly reduced by treatment of the RAW 264.7 cells with UA. In the A549 cells, the induction of TNF-α by infection, and the reduction by UA were not significant (Fig. 2B). UA treatment alone reduced the mNRA levels of TNF-α in the RAW 264.7 cells at 12 h, however, the expression returned to a basal level at 24 h. In the A549 cells, UA did not induce significant changes at any time point (Fig. 2A and B). In the splenocytes, the Con A mitogen was used to investigate the immunoregulatory activity of UA. Con A increased the mRNA expression of TNF-α by ~2-fold at 12 h, which increased to 3-fold at 24 h. At both time points, UA significantly reduced the mRNA expression levels of TNF-α, compared with the control (Fig. 2C).

IL-6 acts as pro-inflammatory, as well as an anti-inflammatory, cytokine. In the RAW 264.7 cells, infection induced the mRNA expression of IL-6 at 24 h, and was significantly reduced by UA treatment. Of note, the reduction was even lower than the basal level. Treatment with UA alone had no significant effect on the mRNA expression of IL-6 (Fig. 3A). In the A549 cells, infection and/or treatment showed no significant change at 12 h, however, at 24 h the mRNA expression of IL-6 increased almost 5-fold. UA significantly reduced this induced expression (Fig. 3B). No significant changes in the mRNA expression of IL-6 mRNA were observed in the Con A and/or UA-treated splenocytes at 12 h. However, at 24 h, the expression of IL-6 was significantly induced when treated with Con A, and UA successfully reduced this induced expression (Fig. 3C).

IL-1β is a pro-inflammatory cytokine, which was found to be significantly induced by M. tuberculosis H37Rv infection in RAW 264.7 cells (Fig. 4A) and A549 cells (Fig. 4B) at 12 and 24 h. Their induction was effectively reduced by UA treatment, however, in the RAW 264.7 cells, the reduction was below the basal level at 12 h, compared with the control (Fig. 4A and B). Compared with the RAW 264.7 and A549 cells, no significant changes were observed in the splenocytes treated with Con A and/or UA at 12 h. However, at 24 h, Con A induced the expression of IL-1β, whereas UA had no significant effect (Fig. 4C).

The iNOS and COX-2 genes are usually induced by inflammation. To evaluate the effect of UA on their expression levels, the present study infected RAW 264.7 cells with M. tuberculosis H37Rv and/or treated the cells with UA. Infection induced the expression of the two genes significantly at 12 and 24 h. These levels of expression were significantly suppressed by UA at both time points. The gene expression of iNOS and COX-2 were suppressed below the basal level at 24 h. No significant changes in the gene expression of iNOS or COX-2 were observed in the cells treated with UA only (Fig. 5A and B).

The inflammatory response was induced in the splenocytes by treatment with the Con A mitogen. At 24 h, the expression of the iNOS gene was induced almost 5-fold, compared with the control, however, UA suppressed the expression by ~2-fold. No significant changes in the expression of iNOS were evident at 12 h in the samples treated with Con A and/or UA (Fig. 5C). However, at 12 and 24 h, the expression of COX-2 was induced significantly by Con A treatment. UA treatment reduced this induced expression significantly at 24 h, but not at 12 h (Fig. 5D). Treatment of the splenocytes with UA alone did not significantly alter the gene expression levels of iNOS or COX-2 from the basal level (Fig. 5C and D).

NO is a signaling molecule, which is important in modulating the release of various inflammatory mediators from a wide range of cells (25). Infection of RAW 264.7 (Fig. 6A) and A549 (Fig. 6B) cells with M. tuberculosis H37Rv produced a significant release of NO into the culture medium at 24 and 48 h, respectively. The alveolar epithelial A549 cells did not appear to release NO prior to 48 h. In addition, treatment with UA or the NO synthase inhibitor significantly reduced the release of NO in the two cell types at these time points. Of note, UA and the NO synthase inhibitor also induced NO release in the two cell type, although without statistical significance (Fig. 6A and B).