Curcumin was purchased from Sigma-Aldrich (St. Louis, MO, USA), was dissolved in dimethylsulfoxide (DMSO), and stored at −20°C. The final concentration of DMSO in all experiments was <0.1%. The JNK inhibitor SP600125 and the p38 inhibitor SB203580 were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Cell culture reagents, medium, fetal bovine serum (FBS), L-glutamine, and antibiotics were purchased from Thermo Fisher Scientific (Gibco^®; Waltham, MA, USA). The antibodies targeting total extracellular signal-regulated kinase (ERK), phospho-ERK (Thr202/Tyr204), total JNK, phospho-JNK (Thr183/Tyr185), total p38, phospho-p38 (Thr180/Tyr182), TLR2 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA).

The M. tuberculosis strain H37Rv was obtained from the American Type Culture Collection (ATCC 5618; Manassas, VA, USA) in lyophilized form, was reconstituted and used as previously described (20). M. tuberculosis 19-kDa lipoprotein (P19) was purified as previously described (21). Briefly, cell-wall fractions were obtained by sonication of resuspended M. tuberculosis H37Rv in ice-cold water (20 kHz, 5 cycles, 5 min each). Forty micrograms of protein were dissolved in a reducing sample buffer that contained 0.05 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 1% glycerol, 10% 2-mercaptoethanol, and 0.5 mM/ml Tris-HCl (pH 6.8), were heated for 5 min at 95°C and were subjected to 12% SDS-polyacrylamide gel electrophoresis. Next, proteins were transferred onto a nitrocellulose membrane and stained with Ponceau Red to identify the 19-kDa band; this band was also identified using a rabbit monoclonal antibody IT-19 targeting the 19-kDA M. tuberculosis antigen (TB Research Material and Vaccine Testing Contract, Colarado State University, Colarado, USA). Then the band was excised, solubilized in DMSO and precipitated with a carbonate/bicarbonate sodium buffer (0.05 M, pH 9.6). The pellet was rinsed thrice with phosphate-buffered saline (PBS, pH 7.4) and stored at −20°C. The concentration of the protein was determined with the Bradford method (Bio-Rad, Hercules, USA).

The WBC 264-9C macrophage line (ATCC HB-8902) was cultivated in RPMI-1640 medium supplemented with 15% FBS, 10 mM HEPES (2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid), 2 mM L-glutamine, and 50 μg/ml gentamicin, at 37°C in a humidified incubator containing 5% CO₂. For P19 treatment, WBC 264-9C cells were placed in 12-well plates with glass coverslips at a density of 5×10⁵ cells/ml for 24 h. Cells were then washed and incubated for an additional 18 h in the medium supplemented with 0.1% FBS. The macrophage monolayers in the tissue culture plates were washed with pre-warmed RPMI-1640 medium, which was then replaced with 1 ml of RPMI-1640 medium supplemented with 10 mM HEPES and 0.4% human serum albumin. The cells in the controls groups were cultured in the same RPMI-1640 medium and environmental conditions mentioned above, but were treated with 10% DMSO vehicles later.

The effect of P19 and curcumin on the viability of macrophages were evaluated using the MTT assay. Briefly, WBC 264-9C macrophages (1×10⁴ cells/well) were seeded into a 96-well culture plate. Following adherence, the cells were treated with various concentrations of P19 (5, 10 or 20 μg/ml) or curcumin (10, 20, 40 and 80 μM), JNK inhibitor SP600125 (30 μM) or p38 inhibitor SB203580P38 (20 μM), and incubated at 37°C for various time periods. Then the medium was removed, and cells were incubated with 100 μl (0.5 mg/ml) of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich) for 4 h at 37°C. The formazan crystals were solubilized with DMSO, and the absorbance was measured at 490 nm with a microplate reader (Tecan Trading AG, Männedorf, Switzerland).

The concentrations of IL-6 and tumor necrosis factor-α (TNF-α) in the supernatant of the cell cultures were determined by using Invitrogen™ enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific) for the corresponding human proteins following the manufacturer’s instructions.

Following treatment with P19 and/or curcumin, macrophages were rinsed with pre-warmed PBS and lysed in an ice-cold extraction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1 mM dithiothreitol, and a protease inhibitor cocktail). The homogenate was incubated on ice for 20 min and centrifuged at 13,000 × g for 20 min at 4°C. Next, the supernatant was collected, and the concentration of the protein in the supernatant was quantified using the Bradford method. The whole-cell lysate from the macrophages was subjected to 12% SDS-PAGE, and subsequently blotted onto a nitrocellulose membrane. The membrane was then incubated with the specific antibodies, using GAPDH as an internal control. Quantification of the protein bands was performed by densitometry using the QuantityOne software (Bio-Rad).

The data were expressed as mean ± SEM from three independent experiments, and were analyzed using the SAS 8.2 software package (SAS Institute Inc., Cary, NC, USA). One-way analysis of variance (ANOVA) followed by Bonferroni correction was used to compare the means of three or more groups in the analyses of cell survival, cytokine production and protein expression. The phosphorylation levels of ERK, JNK and p38 were expressed as phosphorylated protein/total protein. P<0.05 was considered to indicate statistically significant differences.

The MTT assay was used to measure the viability of macrophages exposed to P19 or curcumin for 48 h (Fig. 1A). P19 inhibited the cell viability of WBC 264-9C macrophages in a concentration- and time-dependent manner (F_{concentration (3, 100)} = 55.0, P<0.0001; F_{time (4, 100)} = 18.6, P<0.0001; F_{interaction (12, 100)} = 4.87, P<0.0001). Post-hoc analysis revealed that P19 significantly inhibits macrophage viability (10 μg/ml inhibition rate, 32.4%, P<0.0001; 20 μg/ml inhibition rate, 61.5%, P<0.0001). Similarly, curcumin also inhibited macrophage viability (Fig. 1B) in an concentration- and time-dependent manner (F_{concentration (4, 125)} = 123, P<0.0001; F_{time (4, 125)} = 21.8, P<0.0001; F_{interaction (16, 125)} = 4.86, P<0.0001). In WBC 264-9C macrophages, high concentrations of curcumin resulted in 12.1–20.6% inhibition of cell viability (40 μM, P<0.0001 and 80 μM, P<0.0001, respectively). However, low concentrations of curcumin (10 and 20 μM) did not cause a significant inhibition of cell viability. Therefore, 20 μg/ml P19, and 10 and 20 μM curcumin were chosen for the following experiments.

Human macrophages were exposed to vehicle (saline) treatment as a control, or to 20 μg/ml P19 alone or combined with 10 or 20 μM curcumin for 48 h. As expected, P19 treatment alone markedly reduced the cell viability (58.7%, P<0.0001) (Fig. 2). Notably, the combined treatment with P19 and a low concentration of curcumin (10 μM) attenuated the inhibition of cell viability (32.2%, P<0.0001), compared to P19 treatment alone (Fig. 2). A higher concentration of curcumin (20 μM) further decreased (20.6%, P<0.0001) the inhibition in cell viability induced by P19.

ELISA was used to measure the IL-6 and TNF-α levels in the macrophages treated with 20 μg/ml P19 alone or combined with 10 or 20 μM curcumin for 48 h (Fig. 3). The IL-6 and TNF-α levels were not significantly affected by treatment with 20 μM curcumin alone. As expected, IL-6 and TNF-α levels were significantly elevated upon P19 treatment (P<0.0001), whereas the combination of 10 μM curcumin and P19 significantly decreased the P19-induced IL-6 and TNF-α production (IL-6, P<0.01; TNF-α, P<0.0001). Strikingly, treatment with 20 μM curcumin completely offset the P19-induced cytokine production.

To investigate the mechanism underlying the protective effects of curcumin against the P19-induced inflammatory responses, the expression of TLR2 and the phosphorylation of ERK, JNK and p38 were measured by western blot analysis (Fig. 4A). The expression of TLR2 did not significantly change following treatment with 20 μM curcumin (Fig. 4B), whereas treatment with 20 μg/ml P19 significantly increased the TLR2 level (P<0.05). The P19-induced increase in the TLR2 level was not affected by cotreatment with 10 or 20 μM curcumin.

As shown in Fig. 4, P19 decreased the phospho-ERK level (P<0.05), and increased the phospho-JNK (P<0.0001) and the phospho-p38 (P<0.0001) levels. Although treatment with 20 μM curcumin alone had no significant effect on the p38 basal level, its phosphorylation was significantly decreased relative to treatment with P19 upon treatment with 20 μM curcumin (P<0.05). Furthermore, the phosphorylation of JNK was decreased by both curcumin treatment alone (P<0.05) and when curcumin was combined with P19.

To determine the role of JNK and p38 phosphorylation in P19-induced inflammatory responses, we pre-treated the macrophages with 20 μM SP600125 (the JNK-specific inhibitor) or 30 μM SB203580 (the p38-specific inhibitor) for 1 h prior to treatment with 20 μM curcumin and 20 μg/ml P19. Cytokine production was determined by ELISA. Fig. 5 shows that treatment with the JNK inhibitor significantly decreased IL-6 (P<0.05) and TNF-α (P<0.01) levels, compared to the combination of curcumin and P19. IL-6 and TNF-α levels were slightly reduced after treatment with the p38 inhibitor, but the difference was not significant. These results demonstrated that activation of JNK, but not p38, may play a role in the protective effect of curcumin against P19-induced inflammatory responses.