Mycobacterium tuberculosis infection increases the number of osteoclasts and inhibits osteoclast apoptosis by regulating TNF‑α‑mediated osteoclast autophagy
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- Published online on: June 18, 2020 https://doi.org/10.3892/etm.2020.8903
- Pages: 1889-1898
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Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Osteoarticular tuberculosis is a chronic inflammatory disease characterized by Mycobacterium tuberculosis (M.tb) infection that occurs through the respiratory tract and spreads through the blood to the bones and joints (1). This disease is characterized by enhanced bone and joint absorption and bone destruction (1). Osteoarticular tuberculosis causes limb deformity and nerve compression, greatly affecting the quality of life of patients and imposing a global health and socio-economic burden (2). Clinically, osteoarticular tuberculosis accounts for ~10% of all extrapulmonary tuberculosis cases, which is second only to pleural and lymphatic tuberculosis (3). Antituberculosis drugs have been widely used since the mid-20th century to effectively control this infection (4); however, to the best of our knowledge, the pathogenesis of osteoarticular tuberculosis has not been widely studied. In recent years, drug-resistant tuberculosis, particularly multidrug-resistant tuberculosis, has emerged globally (5). Additionally, an increase in longer chemotherapy cycles and drug side effects have presented major challenges to effectively control osteoarticular tuberculosis (6,7). Therefore, the pathogenic mechanism of osteoarticular tuberculosis must be determined to develop new and effective prevention and treatment strategies.
The earliest study of the pathogenesis of osteoarticular tuberculosis was reported in 1997, which suggested that heat shock protein Cpn10 in M.tb can promote chemotaxis of osteoclasts and locally infiltrate lesions, while inhibiting proliferation of osteoblast precursors and increasing bone resorption. Furthermore, histopathological analysis of samples from patients with osteoarticular tuberculosis showed that local abnormal activation of osteoclasts lead to bone destruction (8). In 2015 a study conducted using an osteoarticular tuberculosis rabbit animal model confirmed that the number of osteoclasts in the damaged spine increased, while the number of osteoblasts decreased (9). Therefore, increased numbers and activation of osteoclasts after M.tb infection are key factors leading to the pathogenesis of osteoarticular tuberculosis.
Autophagy is a basic metabolic process in cells and involves the degradation of intracellular proteins and invading pathogens by lysosomal pathways to maintain cell survival. This is the most primitive innate immune mechanism used by eukaryotic cells to clear invading pathogens (10,11). In a rheumatoid arthritis model, tumor necrosis factor (TNF)-α caused inflammation of the synovial membrane in the joint, increased expression of the autophagy pathway molecules autophagy-related protein (Atg)7 and Beclin1 in osteoclasts and promoted autophagy (12). A previous study demonstrated that TNF-α regulates autophagy and affects osteoclast production (13). Autophagy is also an innate immune mechanism by which macrophages control M.tb infection (14). Macrophage autophagy is activated through the action of type 1 T helper (Th1) cytokines such as interferon-γ, leading to degradation of phagocytic M.tb via the lysosomal pathway, preventing the proliferation of M.tb in macrophages and avoiding cell necrosis (15). However, whether Th1 cells and their cytokine networks activated by M.tb infection in patients with osteoarticular tuberculosis regulate and maintain the autophagy of infected osteoclasts remains to be elucidated.
In the present study, clinical specimens were evaluated and cell experiments were conducted. TNF-α activated autophagy and inhibited apoptosis of osteoclasts infected with M.tb, thereby maintaining cell homeostasis. The results have practical value for guiding the prevention and treatment of osteoarticular tuberculosis.
Materials and methods
Patients and samples
Patients were divided into two groups: The osteoarticular tuberculosis group and osteoarthritis (OA) group. The selection criteria for the osteoarticular tuberculosis group were as follows: Hospitalized patients from the First Hospital of Wuhan (Wuhan, China) were diagnosed as having osteoarticular tuberculosis according to their medical history, clinical symptoms, signs, imaging findings, related laboratory tests, and pathological examination. Lesions with necrotic bone and the synovium were collected as specimens, and 30 cases were included (14 males and 16 females; age range 61-70 years). The selection criteria for the OA group were as follows: Hospitalized cases from the First Hospital of Wuhan were diagnosed as OA according to their medical history, clinical symptoms, signs, imaging findings and relevant laboratory tests. Additionally, patients with surgical indications requiring joint replacement were included. Bone and synovial tissue removed during the operation were collected as specimens from a total of 25 cases (11 males and 14 females; age range 63-72 years). All 55 specimens were collected from January 2017 to December 2018. All experimental protocols were approved by the Clinical Research Ethics Committee of Wuhan First Hospital, Tongji Medical College, Huazhong University of Science and Technology. All patients or their parents provided informed consent.
Isolation and culture of human osteoclasts
The standards for healthy volunteers were as follows: Males aged 20-30 years, no metabolic bone disease according to blood and urine analysis, normal liver and kidney function, no hereditary, blood, or infectious diseases and no general infections, such as colds, within the past 1 month. Samples were collected at Wuhan First Hospital from January 2017 to December 2018. Informed consent was provided by the volunteers prior to elbow vein blood collection. Healthy male volunteers were selected, and 80-100 ml of whole blood was collected from the venous blood. Human monocytes were separated by density gradient centrifugation. DMEM (Gibco; Thermo Fisher Scientific, Inc.) was mixed with equal volumes of heparin-anticoagulated whole blood. Diatrizoate was added to a 50 ml centrifuge tube, and the mixture was slowly added along the tube wall to ensure a clear interface between the two samples, which were then centrifuged at 362 x g at 4˚C for 30 min. The obtained mononuclear cells were placed in two 25-mm petri dishes and cultured in 5% CO2 at 37˚C for 24 h, before being washed three times with DMEM. Unattached cells were eluted, and the remaining adherent cells were considered as mononuclear cells. Confluent cells were resuspended in DMEM containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 mg/ml streptomycin, 100 U/ml penicillin, 30 ng/ml macrophage colony-stimulating factor (PeproTech, Inc.) and 40 ng/ml receptor activator of NF-κB ligand (PeproTech, Inc.). Cells were then incubated at 37˚C in a humidified atmosphere with 95% (v/v) air and 5% (v/v) CO2. The culture medium was replaced every three days for 21 days.
Tartrate-resistant acid phosphatase (TRAP) staining
Patient-derived osteoclast droplets were placed on a loading slide to prepare a cell smear. The slide was allowed to dry naturally, and TRAP (Beijing Solarbio Science & Technology Co., Ltd.) fixative was added to fix the cells at 4˚C for 60 sec. After washing the slides with water and slight drying, the samples were placed in TRAP incubation solution. Slides were then incubated at 37˚C for 60 min in the dark, and then washed with water. Cells were stained with hematoxylin for 5 min or methyl green solution for 3 min at 37˚C. The samples were washed with water, dried, and examined using a light microscope at 40X objective.
M.tb infection and treatment
A homogenizer seal was first weighed. A pipette tip was used to scrape the bacteria (provided by Professor Fan Xionglin, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology) from the plate and the homogenizer was sterilized and sealed, weighing the homogenizer. The dry weight of the bacteria was calculated, and 1 ml of complete DMEM was added to the sample. The osteoclasts were ground and the supernatant was collected by centrifugation at 362 x g for 5 min at 4˚C. A total of 1 ml supernatant was added to the autophagy gene knockout type M.tb H37Rv (M.tb H37RvΔeis) and wild-type M.tb H37Rv (M.tb H37Rv WT) cultured complete DMEM and then mixed well. Osteoclasts not infected with M.tb were used as a blank control group. Following infection, cells were incubated at 37˚C for 24 h and osteoclasts were centrifuged at 362 x g at 4˚C for 5 min and washed twice with PBS.
For TNF-α treated experiments, infected cells were treated with 40 ng/ml TNF-α (cat. no. 1217202; Dakewe Biotech Co., Ltd.) for 24 h or pre-treated with 10 µM 3-methyladenine (3-MA) (cat. no. M9281; Sigma-Aldrich, Merch KGaA) before TNF-α administration.
RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was isolated from the harvested cells and tissues using TRIzol™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The RNA was denatured for 5 min at 70˚C and placed on ice for 5 min. Denatured RNA was added to a mixture of MMLV-RT, MMLV-RT buffer, horseradish peroxidase (HRP) RNA-RNA interaction/RNase inhibitor and dNTPs and incubated for 60 min at 42˚C. The mixture was inactivated by heating at 95˚C for 5 min. qPCR was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.) on a 7900HT thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the PCR: Initial denaturation at 50˚C for 2 min; 40 cycles of 95˚C for 10 min, 95˚C for 30 sec and 60˚C for 30 sec; and a final extension step at 60˚C for 30 sec. A total of 1 µg cDNA and 0.4 µl of primer were used for the qPCR, Relative gene expression levels were calculated using the 2-ΔΔCq method (16). GAPDH was used as an endogenous control to normalize the level of each mRNA. The sequences of the primers used are shown in Table I. All experiments were performed at least in triplicate.
Western blotting
Osteoclasts were rinsed 2-3 times with TBS. An appropriate volume of RIPA buffer (Biosharp) was added to plates and flasks for 3-5 min followed by incubation in an ice bath for 30 min. The supernatant was collected as a total protein solution. Total, cytoplasmic, and mitochondrial proteins were extracted using corresponding kits (Beyotime Institute of Biotechnology). Protein concentration was determined using a BCA protein assay kit. A total of 20 µg/lane extracted proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). After blocking in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) at 37˚C for 2 h, the blots were incubated with anti-Atg7 (1:500; cat. no. Ag27914; ProteinTech Group, Inc.), anti-Beclin1 (1:500; cat. no. 11306-1-AP; ProteinTech Group, Inc.), anti-Bax (1:1,000; cat. no. 50599-2-Ig; ProteinTech Group, Inc.), anti-Bcl-2 (1:1,000; cat. no. 10927-1-AP; ProteinTech Group, Inc.), anti-cytochrome C (1:500; cat. no. 12245-1-AP; ProteinTech Group, Inc.), anti-cleaved-caspase3 (1:1,000; 19677-1-AP; ProteinTech Group, Inc.), anti-microtubule-associated protein 1A/1B light chain 3A (LC-3; 1:500; cat. no. 18725-1-AP; ProteinTech Group, Inc.), anti-β-actin (1:1,000; cat. no. TDY051; Beijing TDY Biotech Co., Ltd.) and anti-voltage-dependent anion channel (1:3,000; cat. no. sc-32063; Santa Cruz Biotechnology; Dallas, TX, USA), overnight at 4˚C. β-actin served as an internal control. The membranes were then washed three times with TBS-T on a bleaching shaker at room temperature for 5 min. HRP-labeled goat anti-mouse antibody (1:3,000; cat. no. BL002A; Biosharp) diluted with TBS-T was incubated with the membranes for 30 min at room temperature. The membranes were washed three times with TBS-T on a decolorizing shaker at room temperature for 5 min. A total of 1 ml Electrochemiluminescence A (ECLA) (cat. no. BL520A; Biosharp) and electrochemiluminescence B (ECLB) (cat. no. BL520A; Biosharp) reagents were mixed in a centrifuge tube. The protein side of the PVDF membrane was brought into full contact with the mixed solution. After 1-2 min, the residual liquid was removed and the membrane was incubated in the dark. Protein expression was quantified using ImageJ v1.46 (National Institutes of Health). All experiments were performed at least in triplicate.
ELISA
The level of TNF-α in culture supernatant was measured using ELISA kits (cat. no. E-EL-H0109c; Elabscience) according to the manufacturer's instructions. Optical density was detected by a microplate reader at a wavelength of 450 nm.
TUNEL
DNA fragmentation was detected using an In Situ Cell Death Detection kit (Roche Diagnostics). After fixation with 4% paraformaldehyde at 37˚C for 1 h, the cells were incubated with 3% H2O2 and 0.1% Triton X-100 for 10 min and washed with PBS three times following each step. In accordance with standard protocols, the cells were stained with TUNEL inspection fluid (1:100) and DAPI at 37˚C for 1 h. The slides were mounted on a mounting solution containing an anti-fluorescent quencher (Fluoromont-G; cat. no. 0100-01; SouthernBiotech). Three fields of view on each slide were randomly chosen for observation with a fluorescence microscope.
Transmission electron microscopy
Medium was first decanted before cells were fixed with 2.5% glutaraldehyde (Sinopharm Chemical Reagent Co., Ltd.) at 4˚C for 15 min. Cells were subsequently collected by centrifugation at 362 x g and 37˚C and stored at 4˚C. Cells were rinsed three times with 0.1 M phosphate buffer (Sinopharm Chemical Reagent Co., Ltd.) for 15 min, followed by dehydration in a 30, 50, 70, 80, 85, 90 and 100% ethanol gradient for 15-20 min in each alcohol solution. The penetrant was composed of epoxy resin (Sinopharm Chemical Reagent Co., Ltd.) and acetone (Sinopharm Chemical Reagent Co., Ltd.). The infiltrated sample was placed in an embedding plate, before addition of embedding epoxy resin. The samples were embedded with epoxy resin (Hubei Xinkang Pharmaceutical Chemical Co., Ltd.) in a 60˚C incubator for 48 h. An ultramicrotome was used to slice the sections to 80-100 nm thickness. Uranyl acetate was added to the sections and incubated at room temperature for 15 min. The sections were dried overnight at room temperature and observed under an electron microscope.
Statistical analysis
The results are presented as the mean ± SD. Statistical analysis was performed using SPSS 20.0 software (IBM Corp.). Two groups were compared using an independent sample t-test, and three or more groups were compared by one-way ANOVA followed by Dunnett's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.
Results
Autophagy is enhanced in osteoarticular tuberculosis tissues
The number of osteoclasts in 30 osteoarticular tuberculosis pathological tissues and 25 osteoarthritis pathological tissues was detected by TRAP staining. As shown in Fig. 1A, the osteoclast content was markedly increased in osteoarticular tuberculosis pathological tissues compared with OA samples. Autophagic activity during osteoarticular tuberculosis was subsequently investigated. RT-qPCR was performed to detect Atg7 and Beclin1 mRNA levels in osteoarticular tuberculosis tissues. The results showed that Atg7 and Beclin1 mRNA levels were significantly increased in osteoarticular tuberculosis samples compared with OA samples (Fig. 1B and C). Additionally, the expression of Atg7, Beclin1 and LC3-II/I protein were investigated by western blotting. The results showed that the protein levels of Atg7, Beclin1 and LC3-II/I were increased in osteoarticular tuberculosis pathological tissue samples compared with OA samples (Fig. 1D). Furthermore, transmission electron microscopy was conducted to observe autophagy. As shown in Fig. 1E, increased accumulation of autophagosomes was observed in osteoclasts from tuberculosis lesions, as indicated by yellow arrows. The results indicated that autophagy was enhanced in osteoarticular tuberculosis compared with OA samples.
Autophagy induction reduces the toxicity of M.tb to osteoclasts
Osteoclasts were transfected with M.tb H37Rv∆eis or M.tb H37Rv and mRNA expression of Atg7 and Beclin1 was investigated by RT-qPCR. As shown in Fig. 2A and B, M.tb H37Rv∆eis increased the mRNA expression levels of Atg7 and Beclin1 compared with the M.tb H37Rv infection group and control group. Western blot analysis was performed to detect Atg7, Beclin1, and LC3-II/I protein levels in osteoclasts. The results showed that the expression levels of these proteins were increased in the M.tb H37Rv∆eis infection group compared with the control group (Fig. 2C).
The role of autophagy in regulating apoptosis of osteoclasts infected with M.tb. was studied using M.tb H37Rv∆eis- and M.tb H37Rv-infected osteoclasts. RT-qPCR was performed to detect the apoptotic markers Bax, Bcl-2, and cleaved-caspase 3 mRNA levels in osteoclasts. Bax and cleaved-caspase 3 mRNA levels were significantly reduced in the H37Rv∆eis-infected group compared with controls and H37Rv-infected samples, whereas Bcl-2 mRNA levels significantly increased (Fig. 2D-F). Similarly, M.tb H37Rv∆eis infection decreased the expression of cleaved-caspase 3 and Bax protein levels and increased the expression of anti-apoptotic Bcl-2 protein levels compared with controls and H37Rv-infected samples (Fig. 2G). In addition, attenuated translocation of mitochondrial cytochrome c to the cytoplasm was detected by western blot analysis (Fig. 2H). TUNEL analysis further confirmed that autophagy reduced osteoclast apoptosis (Fig. 2I).
TNF-α enhances autophagic activity of osteoclasts infected with M.tb
TNF-α plays an important role in the development of tuberculosis (17). ELISA was performed to detect the secretion of TNF-α from osteoclasts infected with M.tb. As shown in Fig. 3A, M.tb infection significantly increased the levels of TNF-α compared with controls. Subsequently, the effect of TNF-α on osteoclasts infected with M.tb was tested. Osteoclasts infected with M.tb were treated with TNF-α (40 ng/ml) for 24 h. Untreated osteoclasts infected with M.tb were used as controls. RT-qPCR, western blotting and transmission electron microscopy were performed to determine autophagy activation. The results demonstrated that mRNA levels of Atg7 and Beclin1 were significantly upregulated in osteoclasts infected with M.tb and treated with TNF-α compared with corresponding cells that were not treated with TNF-α (Fig. 3B and C). Western blotting results showed that protein expression levels of Atg7, Beclin1 and LC3-II/I increased following TNF-α treatment of osteoblasts infected with M.tb (Fig. 3D). Furthermore, increased accumulation of autophagosomes was observed in infected osteoclasts treated with TNF-α as indicated by the arrows (Fig. 3E). The results indicated that TNF-α enhanced the autophagy of osteoclasts infected with M.tb.
TNF-α inhibits apoptosis of osteoclasts infected with M.tb by activating autophagy
To understand whether TNF-α affects the apoptosis of osteoclasts infected with M.tb, M.tb H37Rv∆eis or M.tb H37Rv was transfected into human osteoclasts before the cells were stimulated with TNF-α. Following osteoclast treatment with TNF-α (40 ng/ml) for 24 h, mRNA and protein expression of apoptosis-associated factors were quantified by RT-qPCR and western blotting. The mRNA and protein expression of pro-apoptotic factors (cleaved-caspase 3 and Bax) in osteoclasts infected with M.tb significantly decreased after treatment with TNF-α, whereas the mRNA and protein expression of anti-apoptotic Bcl-2 was significantly increased compared with the respective TNF-α-untreated groups (Fig. 4A-D). In addition, western blot analysis detected attenuated translocation of mitochondrial cytochrome c in TNF-α-treated groups when compared with corresponding TNF-α-untreated groups (Fig. 4E). Additionally, TUNEL staining showed that TNF-α treatment decreased the apoptosis of osteoclasts infected with M.tb (Fig. 4F).
To investigate the beneficial effects of TNF-α on the autophagy of osteoclasts infected with M.tb, cells were pre-treated with the autophagy inhibitor 3-MA before TNF-α administration. As shown in Fig. S1, 3-MA treatment decreased the protein levels of Atg7, Beclin1 and LC3-II/I compared with the corresponding 3-MA-untreated groups. Pre-treatment with 3-MA significantly attenuated the effects of TNF-α on the expression of apoptosis-related factors compared with the respective 3-MA-untreated groups (Fig. 4G-K).
Discussion
Recent studies have shown that autophagy plays an important role in diverse biological and pathological processes, including cell proliferation, differentiation, apoptosis, and carcinogenesis; however, little is known about the role of autophagy in the development of osteoarticular tuberculosis (18-21). The present results demonstrated that the number of osteoclasts increased and autophagy was enhanced in the lesions of patients with osteoarticular tuberculosis compared with patients with OA. Activation of autophagy inhibited the apoptosis of osteoclasts infected with M.tb and increased the expression levels of TNF-α. Furthermore, TNF-α enhanced the autophagic activity of osteoclasts infected with M.tb and inhibited cell apoptosis.
Osteoclasts are the only cells in the body that perform bone resorption and are closely related to normal bone metabolism and various diseases (22). The pathological changes in vertebrae destruction in spinal tuberculosis are closely related to abnormal activation and proliferation of osteoclasts caused by M.tb (23). The synergistic effect of osteoclasts and osteoblasts is crucial in bone formation and development. Once the balance between osteoclasts and osteoblasts is disrupted, abnormal bone diseases such as bone and joint tuberculosis can occur (24). Autophagy plays an important role in the formation, apoptosis and function of osteoclasts as an important mechanism of action to maintain cell homeostasis (25). Both osteoclast formation and bone resorption require the involvement of Atg5, Atg7, Atg4B and LC3. For example, Atg5 and Atg7 promote bone resorption in vivo and in vitro, mainly through the actin loop of osteoclasts as a target for lysozymes (26). Atg5, Atg7, Atg4B and LC3 are important components and regulatory molecules in autophagy, suggesting an important role for autophagy in osteoclast formation (19). LC3 is also involved in osteoclast differentiation, which is modified by Atg4B to block the absorption activity and expression of cathepsin K and inhibit the degradation of collagen in the bone matrix (27). The present results showed that tuberculosis infection induced autophagy, which inhibited the apoptosis of osteoclasts, further demonstrating a key role of autophagy in maintaining the osteoclast steady state.
TNF-α can promote granuloma formation, envelop tubercle bacillus, limit pathogen spread and promote pathological changes caused by tuberculosis (28). High levels of TNF-α were detected in the serum and cerebrospinal fluid of patients with spinal tuberculosis (29). TNF-α, a pro-inflammatory factor, is key in the immunopathological response of tuberculosis. TNF-α can inhibit M.tb infection and accelerate bone destruction. It is closely related to the occurrence, development, severity, therapeutic effect and prognosis of the disease (30). Additionally, Mattos et al (31) reported that the levels of TNF-α were significantly lower after tuberculosis chemotherapy than before, demonstrating that TNF-α may play an important role in osteoarticular tuberculosis. Once M.tb enters the bone through the blood or lymphatic system, it activates mononuclear macrophages around the lesion to produce a large amount of TNF-α, which directly acts on osteoclast precursor cells in the bone marrow to form a large number of osteoclasts, causing bone destruction (32). Kobayashi et al (33) reported that TNF-α directly induced osteoclast precursor cells (mononuclear macrophages) in the bone marrow to differentiate into osteoclasts, which does not depend on any other route. TNF-α was reported to directly induce osteoclast formation in the bone marrow, as well as induce osteoblast apoptosis, reduce the proliferation and differentiation of osteoblasts, inhibit new bone formation and aggravate bone destruction; thus, TNF-α inhibitors are effective for reducing bone loss (34,35). Autophagy is activated by the proinflammatory cytokine TNF-α in the osteoclasts of patients with rheumatoid arthritis (13,36). The present study showed that TNF-α enhanced autophagic activity and inhibited apoptosis of osteoclasts infected with M.tb. Autophagy induced osteoclast differentiation and apoptosis and stimulated osteoclast-mediated bone resorption in vitro, thereby highlighting autophagy as a novel mediator of TNF-α induced bone resorption (37).
However, the present study had certain limitations. The study preliminarily explored the relationship between TNF-α and autophagy and did not determine the mechanism of TNF-α-activated autophagy. Subsequent studies should be performed on TNF-α-activated autophagy signaling pathways. The present study was limited to the cellular level, and no animal experiments were performed. The results of this experiment should be further verified in animal models.
In conclusion, to the best of our knowledge, this is the first study to show that autophagy is an important factor in maintaining the homeostasis of osteoclasts infected with M.tb. Additionally, TNF-α promoted the autophagy of mature osteoclasts infected with M.tb and inhibited the apoptosis of mature osteoclasts. These findings reveal a new osteoarticular tuberculosis-activated cytokine network through which autophagy may regulate bone metabolism and play an important role in the pathogenesis of osteoarticular tuberculosis. These results provide a foundation for the development of new drugs for treating osteoarticular tuberculosis based on TNF-α-mediated osteoclast autophagy.
Supplementary Material
Effect of 3-MA on autophagy protein expression. Protein expression levels of Atg7, Beclin1 and LC3-II/I in osteoclasts infected with M.tb, treated with TNF-α, and untreated or treated with 3-MA. M.tb H37RvΔeis, inhibition of autophagy gene knockout type M.tb H37Rv; M.tb H37Rv wild type M.tb H37Rv; TNF-α, tumor necrosis factor-α; Atg7, autophagy-related protein 7; 3-MA, 3-methyladenine; LC-3, microtubule-associated proteins 1A/1B light chain 3A; M.tb, Mycobacterium tuberculosis.
Acknowledgements
The authors would like to thank Dr Shuwen Jin from the College of Acupuncture of Orthopedics, Hubei University of Chinese Medicine, Wuhan, China for providing technical assistance. The authors would like to thank Professor Fan Xionglin, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China for providing the bacteria strain used in the study.
Funding
The current study is supported by grants from the Research Project of Hubei Province Health and Family Planning Commission (grant no. WJ2017Z022), Research Projects of Traditional Chinese Medicine of Hubei Health Commission (grant no. ZY2019F025), Wuhan Health and Family Planning Scientific Research Fund (grant nos. WZ18Q05 and WX19Q11) and the Funded Research Project of Wuhan First Hospital (grant no. 2019Y01).
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
WL contributed to study conception and design, data collection and analysis and manuscript writing. JZ and FN contributed to study conception and design and in critical revision and review of the manuscript. FFP, ZWW, MH and XLZ analyzed the data and reviewed the manuscript. LY and PFT performed the experiments. PX and JF analyzed and interpreted the data and contributed to final manuscript preparation. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Clinical Research Ethics Committee of Wuhan First Hospital, Tongji Medical College, Huazhong University of Science Technology. All patients or their parents provided informed consent.
Patient consent for publication
Patients provided consent for publication.
Competing interests
The authors declare that they have no competing interests.
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