Background The severe bone destruction during rheumatoid arthritis (RA) is mainly mediated by osteoclasts and thus, reducing osteoclast number is a keystep in ameliorating the outcome of the disease. Yersinia strains are secreting a broad number of effector proteins. The yersinia outer protein M (YopM) has the ability to translocate into host cells by two N-terminal alpha-helices and subsequently down-regulates inflammatory response in infected host tissues.
Objectives We investigated the effect of recombinant YopM on osteoclastogenesis in vitro and bone destruction in the hTNFtg mouse model in vivo.
Methods After cloning recombinant YopM into pET-24 b (+) expression vector, it was expressed in Bl21 cells and purified. The uptake of YopM into bone marrow macrophages (BMMs) and osteoclasts was analysed using Cy3-coupled YopM and laser scanning microscopy. The effect of YopM on osteoclastogenesis in an in vitro osteoclast formation assay was studied. The effects of YopM on the activation of ERK, p-38, AKT and NFkappaB signalling was determined by Western Blot analysis. With respect to a potential in vivo application of YopM, we analysed the distribution of YopM-Cy5 injected in the hindpaws of hTNFtg mice by fluorescence reflection imaging (FRI). Finally, we treated hTNFtg mice with YopM and an inactive deletion mutant of YopM. Clinical parameters were recorded and histomorphometric analyses of joint destruction were performed in hind paw sections.
Results In contrast to mature osteoclasts, YopM penetrated the cell membrane of BMMs in vitro and accumulated in the cells. The incubation of BMMs with YopM resulted in a strong reduction of osteoclast precursors and mature osteoclasts. We found that YopM reduced the activation of NF-kB by reducing the phosphorylation of IkB alpha. Phosphorylation of MAP kinases and AKT, however, was not altered by YopM. YopM-Cy5 injected into the hind paws was detectable by FRI during the monitored time of 48 houres. The treatment of hTNFtg mice with YopM showed a delayed onset of paw swelling and reduced bone destruction and inflammation in YopM treated hTNFtg mice in comparison to untreated hTNFtg mice.
Conclusions These results suggest that YopM has the potential to reduce inflammation and bone destruction in vivo. Therefore, the underlying mechanisms and functional properties of YopM may serve as basis for the development of novel strategies for the treatment of RA.
Disclosure of Interest None Declared