ABSTRACT
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the most important infectious diseases worldwide. Mtb and its culture filtrates or sonic extracts induce apoptosis in macrophages. However, there is a little known about Mtb components that modulate apoptosis and their regulating mechanism. We identified Rv0753c protein with apoptotic potential through searching the biologic active proteins from the multidimensional fractions of Mtb culture filtrate. Here, we investigated the apoptotic effects of Rv0753c on RAW264.7 cells. The recombinant Rv0753c induced RAW264.7 cells apoptosis in a caspase-9-dependent manner. Dissipation of the mitochondrial transmembrane potential (ΔΨ m ), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with Rv0753c. Enhanced reactive oxygen species (ROS) production was required for Rv0753c-mediated apoptosis. Furthermore, ROS-mediated JNK activation was major signaling pathway for Rv0753c-induced apoptosis. Moreover, Rv0753c-mediated apoptosis is dependent on TLR4. Altogether, these results suggest that Rv0753c induce apoptosis through ROS-JNK signaling pathway in RAW264.7 cells.
ABSTRACT
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the most important infectious diseases worldwide. Mtb and its culture filtrates or sonic extracts induce apoptosis in macrophages. However, there is a little known about Mtb components that modulate apoptosis and their regulating mechanism. We identified Rv0753c protein with apoptotic potential through searching the biologic active proteins from the multidimensional fractions of Mtb culture filtrate. Here, we investigated the apoptotic effects of Rv0753c on RAW264.7 cells. The recombinant Rv0753c induced RAW264.7 cells apoptosis in a caspase-9-dependent manner. Dissipation of the mitochondrial transmembrane potential (ΔΨ m ), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with Rv0753c. Enhanced reactive oxygen species (ROS) production was required for Rv0753c-mediated apoptosis. Furthermore, ROS-mediated JNK activation was major signaling pathway for Rv0753c-induced apoptosis. Moreover, Rv0753c-mediated apoptosis is dependent on TLR4. Altogether, these results suggest that Rv0753c induce apoptosis through ROS-JNK signaling pathway in RAW264.7 cells.
ABSTRACT
B lymphocytes are produced from hematopoietic stem cells (HSCs) through the highly ordered process of B lymphopoiesis, which is regulated by a complex network of cytokines, chemokines and cell adhesion molecules derived from the hematopoietic niche. Primary osteoblasts function as an osteoblastic niche (OBN) that supports in vitro B lymphopoiesis. However, there are significant limitations to the use of primary osteoblasts, including their relative scarcity and the consistency and efficiency of the limited purification and proliferation of these cells. Thus, development of a stable osteoblast cell line that can function as a biomimetic or artificial OBN is necessary. In this study, we developed a stable osteoblastic cell line, designated OBN4, which functions as an osteoblast-based artificial niche that supports in vitro B lymphopoiesis. We demonstrated that the production of a B220⁺ cell population from Lineage⁻ (Lin⁻) Sca-1⁺ c-Kit⁺ hematopoietic stem and progenitor cells (HSPCs) was increased ~1.7-fold by OBN4 cells relative to production by primary osteoblasts and OP9 cells in coculture experiments. Consistently, OBN4 cells exhibited the highest production of B220⁺ IgM⁺ cell populations (6.7±0.6–13.6±0.6%) in an IL-7- and stromal cell-derived factor 1-dependent manner, with higher production than primary osteoblasts (3.7±0.5–6.4±0.6%) and OP9 cells (1.8±0.6–3.9±0.5%). In addition, the production of B220⁺ IgM⁺ IgD⁺ cell populations was significantly enhanced by OBN4 cells (15.4±1.1–18.9±3.2%) relative to production by primary osteoblasts (9.5±0.6–14.6±1.6%) and OP9 cells (9.1±0.5–10.3±1.8%). We conclude that OBN4 cells support in vitro B lymphopoiesis of Lin⁻ Sca-1⁺ c-Kit⁺ HSPCs more efficiently than primary osteoblasts or OP9 stromal cells.