Surface proteins of Propionibacterium freudenreichii MJ2 inhibit RANKL-induced osteoclast differentiation by lipocalin-2 upregulation and lipocalin-2-mediated NFATc1 inhibition.

Osteoclasts degrade bone and osteoclast differentiation has been implicated in bone destruction in rheumatoid arthritis. The dairy bacterium Propionibacterium freudenreichii MJ2 (MJ2) isolated from raw milk inhibits osteoclast differentiation and ameliorates collagen-induced arthritis. This study aimed to investigate the inhibitory effect of the surface proteins of MJ2 on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and explain the underlying mechanism. The murine macrophage cell line RAW 264.7 was used to study the inhibition of osteoclast differentiation. The surface proteins significantly inhibited RANKL-induced osteoclast differentiation in a protein concentration-dependent manner by inhibiting the expression of genes and proteins related to osteoclast differentiation. RNA microarray analysis showed that the surface proteins significantly upregulated lipocalin-2 (lcn2) expression. In addition, they downregulated c-fos and NFATc1 and inhibited the expression of NFATc1-downstream genes Atp6v0d2, Calcr, and Ctsk. siRNA silencing of lcn2 decreased the extent of surface protein inhibition on osteoclast differentiation, suggesting that lcn2 plays an important role in the inhibition of RANKL-induced osteoclast differentiation. In conclusion, surface proteins of MJ2 show inhibitory effects on RANKL-induced osteoclast differentiation by upregulating lcn2 expression, in turn downregulating NFATc1, leading to the inhibition of NFATc1-downstream osteoclastogenesis-related gene expression.

Propionibacterium freudenreichii Inhibits RANKL-Induced Osteoclast Differentiation and Ameliorates Rheumatoid Arthritis in Collagen-Induced Arthritis Mice.

Osteoclast differentiation is crucial for bone absorption, and osteoclasts are involved in bone destruction in rheumatoid arthritis (RA). Dairy Propionibacterium freudenreichii is used as a cheese starter and possesses prebiotic and postbiotic properties. It is known to stimulate the growth of bifidobacteria and produces valuable metabolites, such as vitamin B12 and propionic acid. However, limited information is available on the beneficial effects of P. freudenreichii on human disease. Herein, we aimed to investigate the inhibitory effect of P. freudenreichii MJ2 (MJ2) isolated from raw milk on osteoclast differentiation and evaluate the improvement in RA. The murine macrophage cell line, RAW 264.7, and a collagen-induced arthritis (CIA) mouse model were used to perform in vitro and in vivo studies, respectively. Heat-killed P. freudenreichii MJ2 (hkMJ2)-treated cells significantly inhibited RANKL-induced osteoclast differentiation and TRAP activity. HkMJ2-treated cells exhibited significantly decreased expression of genes and proteins related to RANKL-induced osteoclast differentiation. MJ2 administration decreased the arthritic score in the CIA mouse model. Live and dead MJ2 inhibited bone loss and afforded protection against bone erosion and joint damage in CIA mice. MJ2 decreased the levels of collagen-specific antibodies and inflammatory cytokines and the expression of osteoclast differentiation-related genes and proteins in CIA mice. Interestingly, live and dead MJ2 showed similar RA improvement effects in CIA mice. In conclusion, P. freudenreichii MJ2 inhibited osteoclast differentiation by inhibiting the NF-κB signaling pathway and ameliorated CIA.