Int J Clin Exp Pathol 2016;9(8):8406-8414 www.ijcep.com /ISSN:1936-2625/IJCEP0025163 Original Article The role of TNF-α and IFN-γ in the formation of osteoclasts and bone absorption in bone tuberculosis Zhong Li1, Housen Jiang1, Xuedong Yang1, Lin Shi1, Junhua Liu2, Xiaoqi Zhang3 1Hand and Foot Bone Surgery of Weifang City People’s Hospital, Weifang 261041, Shandong, China; 2Medicine of Shou Guang City People’s Hospital, Weifang 262700, Shandong, China; 3The Second People’s Hospital Tuberculo- sis of Weifang City, Weifang 261041, Shandong, China Received January 28, 2016; Accepted May 20, 2016; Epub August 1, 2016; Published August 15, 2016 Abstract: Bone-joint tuberculosis is one lytic bone lesion caused by Tubercle bacilli. Tumor necrosis factor (TNF)-α interferon (IFN)-γ can mediate bone formation and osteoclasts. This study aimed to investigate the effect of these factors in the effect of Tubercle bacilli on osteoclast formation and bone absorption. Bone marrow mononuclear cells were separated and generated for osteoblast-osteoclast co-culture system. The effect of Mt sonicate on os- teoclast formation and bone absorption was observed, with the correlation analysis between TNF-α and IFN-γ con- centration. Real-time PCR was employed to detect mRNA expression of receptor activator NF-κB ligand (RANKL) and osteoprotegerin (OPG) in osteoblasts. Immunofluorescence was used to quantify NFATc1 protein level. Antibody against TNF-α and IFN-γ was applied for observing bone absorption. Mt sonicate significantly enhance osteoclast formation and bone absorption and facilitate secretion of TNF-α and IFN-γ. It can also elevate mRNA expression of RANKL and OPG in osteoblast and decrease NFATc1 in osteoclast. The block of TNF-α or IFN-γ significantly weaken Mt sonicate-induced osteoclast formation and bone absorption, facilitate OPG expression, and decrease RNAKL and NFATc1 expression. Tubercle bacilli induced release of TNF-α and IFN-γ is important for bone absorption. TNF-α and IFN-γ interfere the interaction between osteoclast and osteoblast, causing bone destruction, via the modulation on the expression of OPG and RANKL in osteoblast. Keywords: Tuberculosis, bone-joint, inflammatory cytokine, TNF-α, IFN-γ Introduction Host immune function plays important roles on TB growth. Once infected with TB, body will initi- Tuberculosis is one severe infectious disease ate related immune reaction involving multiple caused by Tubercle bacilli (TB) [1]. Osteoarticular immune cells and inflammatory factors to man- tuberculosis (OAT) can be spread to bone and age infection [3, 4]. With the invasion of TB, joint tissues by TB, causing bone destruction innate immune cells including macrophage, NK and absorption featured with inflammatory infil- cells and dendritic cells are rapidly activated to tration. OAT has atypical asymptotic and inva- release multiple inflammatory factors such as sive manifestation, as lesion is featured with bone destruction and lysis, which has persis- interferon (IFN)-γ, tumor necrosis factor (TNF)-α, tence and slow inhalation [2]. The healthy mor- and interleukin (IL)-1, inducing protective infla- phology and normal bone volume depend on mmatory response for exerting immune clear- the dynamic balance between osteoblast and ance [5]. The over-production of protective anti- osteoclast. When certain pathological factor inflammatory factors, however, can interfere makes the enhancement of osteoclast function with the immune clearance against TB. In OAT beyond the compensatory mechanism of osteo- lesion featured with tuberculous granuloma, blast, bone absorption and destruction will both protective and pathological abnormality occur. There are multiple studies regarding the immune response co-exists, but having large mechanism of destruction by pulmonary tuber- difference regarding induced immune cells and culosis. However, the detailed mechanism of inflammatory factors [6]. Generally speaking, bone destruction during OAT is still unclear. protective immunity is mainly achieved via mac- Bone tuberculosis and cytokines rophage activation by CD4 Th cells via IFN-γ, with 10 mL α-MEM basic medium. After cen- while pathological abnormality reaction is trifugation at 1 200 rpm for 5 min, the superna- induced by delayed hypersensitive T cells via tant was discarded. α-MEM medium containing TNF-α and IL-1 [6]. Various inflammatory factors 10% FBS and 1% streptomycin/penicillin was involving in anti-TB immunity, also play impor- added to re-suspend cell spheres, which were tant roles in mediating osteoblast and osteo- then incubated at 37°C chamber with 5% CO2 clast functions. Multiple studies revealed that perfusion. With changing medium every three both TNF-α and IFN-γ could regulate differentia- days, few fibroblast-like attachment cells can tion and biological functions of osteoblast and be observed. Cells were further incubated until osteoclast via multiple pathways [7]. Whether reaching complete fusion (P0), 0.05% trypsin TNF-α and IFN-γ plays a role in OAT caused by was used to digest cells for passage. When P1 TB, is still unclear. generation cell reaches confluence of 60%~80%, osteoblast induction differentiation Materials and methods medium was used for changing every three days in 14-day induction differentiation incuba- Reagent and materials tion. The basic formation of osteoblast can be Low-glucose DMEM, α-MEM, FBS and osteo- observed by Alizarin red S staining. Osteoblasts blast induction differentiation medium (Gibco, were further passed until P2 generation for fur- US); Anti-tartaric acid phosphatase (TRAP) ther experiments. staining kit (Beyotime, China); ELISA kits for human TNF-α and IFN-γ (eBioscience, US); Osteoclast induction and Mt sonicate process- Human recombinant M-CSF (Peprotech, US); ing TB strain H37rv (Pharmaceutical Biological Double volumes of α-MEM medium were used Products Analysis Institute, China); PCR primer to dilute bone marrow, and were centrifuged at (Sangon, China); Fluorescent quantitative PCR 1 200 rpm for 5 min. Supernatant was discard- kit (Toyobo, Japan); Rabbit anti-human NFATc1 ed, with the addition of α-MEM medium con- monoclonal antibody (Abcam, US); Rabbit anti- taining 10% FBS, 20 ng/mL M-CSF and 1% TNF-α and mouse anti-IFN-γ body (R&D, US). streptomycin/penicillin to re-suspend cells. Processing of TB After adjusting 1 × 107/mL concentration, cells were seeded into 24-well plate with or without Mycobacterium tuberculosis H37rv suspen- cortical bone (0.5 mL each). Mt sonicate treat- sions were lysed under intermittent ultrasound ment groups at different concentrations (0 μg/ at 4°C for 60 min. Bacterial mixture was then mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL and 100 centrifuged at 2 500 rpm for 60 min, followed μg/mL) were prepared for changing medium by filtration through 0.22 μm filter, aliquoted, every 72 h in 8 consecutive days. In addition, to and frozen to prepare lyophilized powder, which better observe the effect of TNF-α and IFN-γ on were kept at 4°C for further use. osteoblast and osteoclast functions under Mt sonicate treatment, we further replenished Preparing calf cortical bone blocker groups including anti-TNF-α (50 ng/mL) Fresh cortical bones from calf limbs were cut and/or anti-IFN-γ (100 ng/mL), in parallel to into bone fraction (50 mm × 50 mm) and were control group using 100 μg/mL Mt sonicate, to smoothing. After fixation in 5% glutaraldehyde test related indexes. for 2 h, bone fraction was cut into sclerite, Establishment of co-culture system which was dehydrated in gradient ethanol. 0.25 mol/L ammonia was used to wash sclerite for Low-glucose DMEM medium containing 10% three times. After γ-irradiation for sterilization, FBS was added into the upper chamber of sclerite was immersed in 1000 U/mL strepto- Transwell for inoculation of induced P2 osteo- mycin/penicillin (in PBS) and was kept at 4°C blast. Transwell chambers were then placed for storage. into 24-well chamber containing osteoclasts Induction of osteoblast after treating using different concentrations of Mt sonicate. After 72 h incubation, culture 5 mL bone marrow samples were drawn from medium in the lower chamber was collected to ilium bone grafting patients, and were mixed quantify TNF-α and IFN-γ using ELISA approach. 8407 Int J Clin Exp Pathol 2016;9(8):8406-8414 Bone tuberculosis and cytokines Table 1. TNF-α, IFN-γ, osteoclast counting and bone absorption under Mt sonicate (N = 10) Bone absorption Bone absorption area Group Osteoclast TNF-α (pg/mL) IFN-γ (pg/mL) lacunae (μm2) 0 μg/mL 2.61±0.91 2.45±0.87 92.13±8.62 97.56±10.46 84.31±12.45 0.1 μg/mL 4.23±1.02 4.04±1.11 98.72±10.17 104.31±11.34 88.43±11.87 1 μg/mL 9.56±2.67a 7.67±2.33a 325.67±36.95a 228.78±21.66a 138.56±15.75a 10 μg/mL 21.54±4.53a 19.45±3.88a 3617.56±77.59a 356.73±31.82a 257.55±30.64a 100 μg/mL 65.25±9.21a 62.64±10.43a 15293.73±291.48a 768.26±41.45a 448.24±39.61a Note: a, P < 0.05 compared to 0 μg/mL group. Observation of bone absorptive lacunae and ddH2O were added. Reaction conditions were: osteoclast 95°C denature for 5 min, followed by 40 cycles each containing 95°C for 15 sec, and 60°C for Cells in 24-well plate without cortical bone were 1 min. Data were collected from ABI ViiA7 fluo- incubated for 8 days. Culture medium was dis- rescent quantitative PCR cycler, and were ana- carded, followed by the staining by TRAP kit. lyzed by 2ΔΔCt method. Under inverted microscope, those large cells with more than three nuclear with TRAP-positive Western blotting staining were identified as osteoclasts. Five fields were randomly selected under 10X objec- Nuclear proteins were extracted, separated in tive to count the number of osteoclasts.