Wild-type 6-to-8-week-old female C57BL/6 mice were purchased form SAMTAKO BIO KOREA (Gyeonggi-do, Korea) and TLR2-, MyD88-, and TRIF-knockout (KO) mice on a C57BL/6 background were kindly provided by Dr. S. Akira (Osaka University, Japan). All animal procedures were approved by the Institutional Animal Care and Use Committee of Chungnam National University. Primary BMDMs were isolated from femurs of mice and then differentiated for 5~7 days in DMEM (Life Technologies BRL) containing macrophage colony stimulating factor (M-CSF, 25 ng/ml) (R&D systems, Minneapolis, MN, USA), 4 mM glutamine, and 10% FBS, as described previously (16).

All reagents and chemicals were purchased from Sigma-Aldrich except those noted below, which were obtained from the indicated suppliers. Specific Abs against phospho-ERK1/2 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), and phospho-JNK (Thr183/Tyr185) were purchased from Cell signaling Technology (Cell signaling, Beverly, MA, USA). Specific inhibitors of MAPKs [U0126 (MEK inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor)], the antioxidant N-acetyl-_L-cysteine (NAC), diphenyleneiodonium (DPI) as an inhibitor of NADPH oxidase, BAY11-7082 (Bay), and caffeic acid phenethyl ester (CAPE) were purchased from Calbiochem (San Diego, CA, USA). Anti-β-actin (I-19) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) was added to cultures at a concentration of 0.1% (v/v) as a solvent control.

M. scrofulaceum ATCC and clinical strains (KMRC 200-30113, -30114, -30115, -30116) were obtained from the Korean Institute of Tuberculosis (Osong, Chungbuk, Korea), maintained in Ogawa medium, and inoculated in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI, USA) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) (BD Pharmingen, San Diego, CA, USA), 0.5% glycerol, and 0.05% Tween 80 (Sigma-Aldrich) at 37℃. Inocula were prepared from the mid-exponential-growth phase cultures. Bacteria were washed four times with cold PBS and then concentrated or diluted as needed. The number of colony-forming units (CFU) per milliliter was determined by plating on 7H10 agar supplemented with OADC at 37℃. For the in vitro experiments, macrophage infections were performed as described previously (16). Briefly, cells were infected with mycobacteria at different MOIs and incubated for 4 h at 37℃ in a 5% CO₂ atmosphere. After allowing time for phagocytosis, cells were washed four times with fresh PBS to remove extracellular bacteria and then incubated with complete DMEM without antibiotics. As controls, cultures of uninfected macrophages were maintained under the same conditions. For the in vivo experiments, a bacterial inoculum was prepared by thawing a frozen aliquot of a mycobacterium. Mice were intravenously injected with a suspension of 5×10⁷ or 5×10⁸ CFU bacteria per mouse in a final volume of 100µl. Mice were euthanized at 0, 3, 7, 15, and 30 days after infection and the bacterial loads in the infected organs were determined by plating serial 10-fold dilutions of tissue homogenates on Middlebrook 7H10 medium supplemented with OADC. The plates were incubated for 4~6 weeks at 37℃. Control mice were injected with sterile saline.

Colony morphology was evaluated on 7H10 agar plates by microscopic observation. For detection of the cord formation, bacteria were cultured in 7H9 under stationary phase conditioned broth at 37℃ for 7 days. Culture suspensions were spun onto glass slides using a cytocentrifuge and the bacteria were heat-fixed and then stained with crystal violet dye. Bacteria were examined at ×40 or ×100 magnification using a light microscope (Olympus, Japan) and photographed.

BMDMs or mice were infected as indicated in the figures and processed for analysis by RT-PCR, Western blot, and sandwich ELISA as described previously (16). Briefly, total RNA was isolated from BMDMs or lungs of mice using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Then, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR were performed. The sequences of the primers used are as follows: TNF-α (forward) 5'-CGGACTCCGCAAAGTCTAAG-3', (reverse) 5'-ACGGCATGGATCTCAAAGAC-3', IL-6 (forward) 5'-GGAAATTGGGGTAGGAAGGA-3', (reverse) 5'-CCGGAGAGGAGACTTCACAG-3', IL-12p40 (forward) 5'-GGA AGCACGGCAGCAGAATA-3', (reverse) 5'-AACTTGAGGGAGAAGTAGGAATGG-3', β-actin (forward) 5'-TCATGAAGTGTGACGTTGACATCCGT-3', (reverse) 5'-CCTAGAAGCATTTGCGGTGCACGATG-3'. PCR was performed as follows: For proinflammatory cytokine PCR, 30 cycles of denaturation at 95℃ for 1 min, annealing at 58℃ for 1 min, and extension at 72℃ for 1 min, with a final extension at 72℃ for 10 min. Real-time PCR was performed using SYBR Green PCR Master Mix (Molecular Probes, Carlsbad, CA, USA), and transcript levels were quantified using an ABI 7900 Sequence Detection System (Applied Biosystems). The mean value of triplicate reactions was normalized against the mean value for β-actin.

For the NF-κB luciferase assay, BMDMs were transduced with NF-κB p65 luciferase reporter adenovirus [20 plaque-forming units (PFU)/cell] constructs. After 36 h, the culture medium was changed to new medium and then cells were infected with MS strains (MOI=1, 5, 10). After 4 h, the cells were harvested in lysis buffer and luciferase activity was measured by a dual luciferase assay (Promega, Madison, WI, USA) as recommended by the manufacturer.

NF-κB p65 translocation was detected by immunofluorescence microscopy analysis as described previously (9). Briefly, BMDMs differentiated on coverslips were infected with MS strains at 37℃ for 6 h, fixed with 4% (w/v) paraformaldehyde in PBS for 10 min at RT, permeabilized with 0.25% (v/v) Triton X-100 in PBS for 15 min at RT, and then blocked with 3% bovine serum albumin (BSA) in PBS for 1 h. Cells were immunostained with rabbit anti-mouse NF-κB p65 (1:400) for 2 h and the staining was detected using Alexa Fluor 488-conjugated anti-rabbit immunoglobulin (Ig) G (1:400, Molecular Probes, Eugene, OR, USA) for 1 h at RT. Nuclei were stained by incubation with 1µg/ml 4',6'-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). After mounting, fluorescence images were acquired with a confocal laser scanning microscope (DP ver. 1.2.108, Olympus).

For immunoblot analysis, primary Abs were diluted 1:1,000. Membranes were developed using a chemiluminescent reagent (ECL; Pharmacia-Amersham, Freiburg, Germany) (17). In sandwich ELISA, the levels of TNF-α, IL-6, and IL-12p40 in cell culture supernatants were analyzed using DuoSet antibody pairs (BD Pharmingen), according to the manufacturer's protocol.

ROS production was measured by DHE assay, as previously described (8). Briefly, BMDMs were infected with MS strains (MOI=5, 10) for 30 min and washed three times with phosphate-buffered saline (PBS) to remove extracellular bacteria. The cells were incubated with 2µM DHE dye to detect superoxide for 30 min at 37℃ in 5% CO₂ and then fixed with 4% paraformaldehyde in PBS at room temperature (RT) followed by flow cytometry using a FACSCanto II flow cytometer (Becton Dickinson, San Jose, CA, USA) and FlowJo software (Tree Star, Ashland, OR, USA).

Mice were sacrificed at 0, 3, 7, 15, and 30 days after infection with MS strains and lung tissue was collected from individual mice. Lung tissues were fixed in 10% formalin and then dehydrated, paraffin-embedded, and sectioned at 5µm. Sections of lung tissues were stained with haematoxylin and eosin for histopathological analysis. Morphometric image analysis was performed at ×100 or ×200 magnification. For immunohistochemical staining of COX-2, paraffin-embedded lung sections were stained using rabbit anti-mouse polyclonal COX-2 antibody (Abcam, Cambridge, UK), biotin-conjugated rabbit anti-mouse IgG, and a peroxidase-conjugated streptavidin antibody. Then, visualization was performed with 3,3-diaminobenzidine as the peroxidase substrate (Sigma-Aldrich).

The data were obtained as the mean (±standard deviation) value from independent experiments on 3~5 mice per group. The statistical analyses were performed using Student's t-test with Bonferroni correction or analysis of variance in addition to two-way ANOVA using GraphPad Prism version 5 (GraphPad software Inc, San Diego, CA, USA). p<0.05 was considered significant.

Previous studies demonstrated that the morphotype of mycobacteria is associated with virulence (18,19). To identify the correlation between morphotype and host immune responses of M. scrofulaceum, we prepared various strains of M. scrofulaceum including ATCC 19991 (ATCC), clinically isolated KMRC 00200-30113 (113S), MS KMRC 00200-30114 (114R), KMRC 00200-30115 (115S), and KMRC 00200-30116 (116S). Microscopic images indicated that only colonies of the 114R strain had a rough morphology similar to that of the M. tuberculosis virulent strain (H37RV) (Fig. 1A). We next examined whether the colony morphology affects the production of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12p40. We infected bone marrow-derived macrophages (BMDMs) with the ATCC, 113S, or 114R strains of M. scrofulaceum and examined the mRNA and protein expression of proinflammatory cytokines. As shown in Fig. 1B, the cells infected with 114R showed higher mRNA expression of TNF, IL-6, and IL-12p40 than those infected with ATCC or 113S at both 3 h and 6 h of infection. Consistent with these findings, protein expression of proinflammatory cytokines was highly increased in cells infected with 114R compared with those infected with ATCC, 113S, 115S, 116S in concentration- and time-dependent manners (Fig. 1C). These findings indicate that M. scrofulaceum-mediated proinflammatory responses depend on colony morphotype.

Numerous studies have reported that mycobacteria and their antigenic components activate intracellular signaling cascade through innate recognition of TLRs (9,12,16,17,20). To determine the roles of TLR2 in mycobacteria-induced production of proinflammatory cytokines in macrophages, BMDMs from TLR2 wild type (WT) and knockout (KO) mice were stimulated with ATCC, 113S, or 114R strains of M. scrofulaceum in an MOI-dependent manner. As shown in Fig. 2A, the mycobacteria-induced production of TNF, IL-6, and IL-12p40 was absolutely inhibited in BMDMs from TLR2 KO mice. Because the adaptor proteins MyD88 and TRIF are essential for signal transduction of TLR (21), we further examined whether the production of these three strain-induced cytokines was dependent on either MyD88 or TRIF. BMDMs from MyD88 KO mice but not from TRIF KO mice showed a significant decrease of TNF, IL-6, and IL-12p40 levels after infection with each of the strains (Fig. 2B). These results suggest that the TLR2/MyD88 signaling pathway is important for host inflammatory responses in M. scrofulaceum infection.

As a consequence of cross-talk between pattern-recognition receptors (PRRs) and mycobacteria, inflammatory responses are mainly initiated by a conserved signaling pathway involving NF-κB activation (12). We therefore examined NF-κB luciferase activity and nuclear translocation in BMDMs infected with the ATCC, 113S, or 114R strains of M. scrofulaceum. As shown in Fig. 3A, 114R-infected cells showed higher NF-κB luciferase activity than cells infected with ATCC or 113S in a dose-dependent manner. NF-κB nuclear translocation is followed by phosphorylation of the IκB kinase (IKK) complex and the inhibitor of NF-κB proteins (IκB)-α, which is critical for the transcriptional activation of target genes including proinflammatory cytokines (22). We found that 114R infection resulted in a greater enhancement of nuclear translocation of p65 (Fig. 3B) than ATCC or 113S infection. In addition, pretreatment with NF-κB specific inhibitors including Bay 11-7085 (BAY) and caffeic acid phenethyl ester (CAPE) significantly inhibited the mRNA expression of 114R-induced proinflammatory cytokines such as TNF-α and IL-6 (Fig. 3C). These results suggest that 114R activated NF-κB signaling more strongly than ATCC or 113S to produce proinflammatory cytokines.

We previously found that mycobacterial infection induced phosphorylation of MAPK such as p38, JNK, and ERK1/2, resulting in production of inflammatory cytokines (16,20). To determine whether M. scrofulaceum-induced inflammatory responses were dependent on MAPK signaling, we monitored MAPK activation in macrophages. Although these three strains induced phosphorylation of p38, SAPK/JNK and ERK1/2, the 114R strain especially induced rapid and strong activation within 30 min (Fig. 4A and B). We next investigated the role of MAPK signaling in proinflammatory cytokine production induced by the 114R strain. As shown in Fig. 4C, pretreatment with specific inhibitors of JNK (SP600125), p38 (SB203580), or MEK (U0126) caused significant inhibition of 114R-induced TNF-α, IL-6, and IL-12p40 production in a concentration-dependent manner (Fig. 4C). Together, these results suggest that the 114R strain strongly induces the production of proinflammatory cytokines through enhanced activation of MAPK signaling compared to ATCC or 113S in macrophages.

Previous studies proposed that Gp91phox/NADPH oxidase (NOX) 2 is critical for regulation of TLR-mediated inflammatory responses and host innate immune responses against M. tuberculosis (15) and M. massiliense (20). We first investigated whether M. scrofulaceum induced intracellular superoxide production in BMDMs using the oxidative fluorescent dye dihydroethidium (DHE). Flow cytometric analysis (Fig. 5A) showed that 114R infection, but not that of ATCC or 113S, strongly induced superoxide generation at MOI 5 and 10. We further examined whether 114R-induced ROS generation was essential for the production of proinflammatory cytokines in macrophages infected with the three strains. As shown in Fig. 5B, pretreatment with the general ROS scavenger NAC and NADPH oxidase inhibitor DPI significantly attenuated the production of TNF-α in a concentration-dependent manner. These data suggest that 114R activated ROS generation more strongly than ATCC or 113S, which resulted in hyperactivation of inflammatory responses.

Mycobacterial infection is known as a major source of granulomatous reactions in humans and in animal models (23). We first investigated whether infection by M. scrofulaceum ATCC, 113S, and 114R strains induced granuloma formation in a murine model. At day 7, 114R-infected mice showed a stronger granulomatous response and infiltration of immune cells (Fig. 6A) than ATCC- or 113S-infected mice. In comparison to animals infected with the ATCC or 113S strain, 114R-infected mice showed significantly higher bacterial loads at day 7 post infection (Fig. 6D).

During M. tuberculosis infection, inflammatory responses are closely associated with granulomatous reactions (24). We thus examined the inflammatory responses in mice infected with the three strains. As shown in Fig. 6C, expression of cyclooxygenase-2, a key enzyme for activation of the inflammatory cascade, was significantly higher in the lungs of 114R-infected mice than in those of ATCC- or 113S-infected mice. We further analyzed the mRNA expression of proinflammatory cytokines in mouse lungs infected with the three strains. mRNA expression of TNF-α, IL-6, and IL-12p40 was higher in the lungs of 114R-infected mice than in those of ATCC or 113S-infected mice (Fig. 6B). Together, these data indicate that 114R infection induces strong inflammatory responses that finally result in enhanced granulomatous reactions.