Yang Jihyun, Park Ok-Jin, Kim Jiseon, Baik Jung Eun, Yun Cheol-Heui, Han Seung Hyun
Department of Oral Microbiology and Immunology, DRI and BK21 Plus Program, School of Dentistry, Seoul National University, Seoul, Republic of Korea.
Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea; Institute of Green Bio Science Technology, Seoul National University, Pyeongchang, Republic of Korea.
J Endod. 2016 Apr;42(4):570-4. doi: 10.1016/j.joen.2016.01.012. Epub 2016 Feb 23.
Enterococcus faecalis is associated with persistent endodontic infection and refractory apical periodontitis. Recently, we have shown that heat-killed E. faecalis attenuates osteoclast differentiation. Because lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria, we investigated the effect of LTA from E. faecalis (EfLTA) on osteoclast differentiation.
EfLTA was purified through organic solvent extraction, hydrophobic interaction column chromatography, and ion exchange column chromatography. Bone marrow cells from C57BL/6 or Toll-like receptor 2-deficient mice were incubated with macrophage colony-stimulating factor (M-CSF) for 5 days to generate macrophages (bone marrow-derived macrophages [BMMs]). The cells were differentiated into osteoclasts with M-CSF and receptor activator of NF-κB ligand (RANKL) in the presence or absence of EfLTA. The degree of osteoclast differentiation was determined by tartrate-resistant acid phosphatase staining. The expression of NFATc1 and c-Fos transcription factors was determined by Western blotting. A phagocytosis assay was performed by measuring the uptake of carboxyfluorescein diacetate succinimidyl ester-stained E. faecalis. An enzyme-linked immunosorbent assay was used to determine the amount of cytokines and chemokines.
When BMMs were treated with EfLTA, osteoclast differentiation was attenuated. EfLTA inhibited the RANKL-induced expression of NFATc1 and c-Fos. EfLTA inhibition of osteoclast differentiation was not observed in TLR2-deficient BMMs. In addition, EfLTA sustained the phagocytic capacity of BMMs even after the differentiation into osteoclasts, whereas it induced the expression of inflammatory cytokines and chemokines.
EfLTA inhibits the differentiation of macrophages into osteoclasts and thereby maintains the phagocytic and inflammatory capacities of macrophages, potentially contributing to refractory apical periodontitis.
粪肠球菌与持续性牙髓感染及难治性根尖周炎有关。最近，我们发现热灭活的粪肠球菌可减弱破骨细胞分化。由于脂磷壁酸（LTA）是革兰氏阳性菌的主要毒力因子，我们研究了粪肠球菌的LTA（EfLTA）对破骨细胞分化的影响。
通过有机溶剂萃取、疏水相互作用柱色谱和离子交换柱色谱纯化EfLTA。将C57BL/6或Toll样受体2缺陷小鼠的骨髓细胞与巨噬细胞集落刺激因子（M-CSF）孵育5天以生成巨噬细胞（骨髓来源的巨噬细胞[BMMs]）。在有或没有EfLTA的情况下，用M-CSF和核因子κB受体活化因子配体（RANKL）将细胞分化为破骨细胞。通过抗酒石酸酸性磷酸酶染色确定破骨细胞分化程度。通过蛋白质印迹法测定NFATc1和c-Fos转录因子的表达。通过测量羧基荧光素二乙酸琥珀酰亚胺酯染色的粪肠球菌的摄取进行吞噬试验。使用酶联免疫吸附测定法测定细胞因子和趋化因子的量。
当用EfLTA处理BMMs时，破骨细胞分化减弱。EfLTA抑制RANKL诱导的NFATc1和c-Fos表达。在TLR2缺陷的BMMs中未观察到EfLTA对破骨细胞分化的抑制作用。此外，即使在分化为破骨细胞后，EfLTA仍维持BMMs的吞噬能力，而它诱导炎性细胞因子和趋化因子的表达。
EfLTA抑制巨噬细胞向破骨细胞的分化，从而维持巨噬细胞的吞噬和炎症能力，可能导致难治性根尖周炎。
