Xiangyuan Liu

AG490 inhibits NFATc1 expression and STAT3 activation during RANKL induced osteoclastogenesis.

Commonly, JAK/STAT relays cytokine signals for cell activation and proliferation, and recent studies have shown that the elevated expression of JAK/STAT is associated with the immune rejection of allografts and the inflammatory processes of autoimmune disease. However, the role which JAK2/STAT3 signaling plays in the receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis is unknown. In this study, we investigated the effects of AG490, specific JAK2 inhibitor, on osteoclast differentiation in vitro. AG490 significantly inhibited osteoclastogenesis in murine osteoclast precursor cell line RAW264.7 induced by RANKL. AG490 suppressed cell proliferation and delayed the G1 to S cell cycle transition. Furthermore, AG490 also suppressed the expression of nuclear factor of activated T cells (NFAT) c1 but not c-Fos in RAW264.7. Subsequently, we investigated various intracellular signaling components associated with osteoclastogenesis. AG490 had no effects on RANKL-induced activation of Akt, ERK1/2. Interestingly, AG490 partly inhibited RANKL-induced phosphorylation of Ser(727) in STAT3. Additionally, down-regulation of STAT3 using siRNA resulted in suppression of TRAP, RANK and NFATc1 expression. In conclusion, we demonstrated that AG490 inhibited RANKL-induced osteoclastogenesis by suppressing NFATc1 production and cell proliferation via the STAT3 pathway. These results suggest that inhibition of JAK2 may be useful for the treatment of bone diseases characterized by excessive osteoclastogenesis.

Interleukin-21 induces migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone erosion. Fibroblast‐like synoviocytes (FLS) play a pivotal role in RA pathogenesis through aggressive migration and matrix invasion, and certain proinflammatory cytokines may affect synoviocyte invasion. Whether interleukin (IL)‐21 influences this process remains controversial. Here, we evaluated the potential regulatory effect of IL‐21 on the migration, invasion and matrix metalloproteinase (MMP) expression in RA‐FLS. We found that IL‐21 promoted the migration, invasion and MMP (MMP‐2, MMP‐3, MMP‐9, MMP‐13) production in RA‐FLS. Moreover, IL‐21 induced activation of the phosphoinositide 3‐kinase (PI3K), signal transducer and activator of transcription‐3 (STAT‐3) and extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2) pathways, and blockage of these pathways [PI3K/protein kinase B (AKT) inhibitor LY294002, STAT‐3 inhibitor STA‐21 and ERK1/2 inhibitor PD98059] attenuated IL‐21‐induced migration and secretion of MMP‐3 and MMP‐9. In conclusion, our results suggest that IL‐21 promotes migration and invasion of RA‐FLS. Therefore, therapeutic strategies targeting IL‐21 might be effective for the treatment of RA.

RANKL downregulates cell surface CXCR6 expression through JAK2/STAT3 signaling pathway during osteoclastogenesis

The receptor activator of nuclear factor-κB ligand (RANKL), as a member of the tumor necrosis factor (TNF) family, plays an essential role in osteoclast differentiation and function. Chemokines and their receptors have recently been shown to play critical roles in osteoclastogenesis, however, whether CXCL16-CXCR6 plays role in RANKL-mediated osteoclastogenesis is unknown. In this study, we first reported that RANKL decreased CXCR6 in a dose-dependent manner, which may be through deactivation of Akt and STAT3 signaling induced by CXCL16. Interestingly, RANKL-mediated CXCR6 reduction may be associated to the activation of STAT3 by phosphorylation. When STAT3 activation was blocked by JAK2/STAT3 inhibitor AG490, RANKL failed to shut down CXCR6 expression during osteoclastogenesis. However, CXCL16 alone did not augment RANKL-mediated osteoclast differentiation and did not alter RANKL-receptor RANK mRNA expression. These results demonstrate that reduction of CXCL16-CXCR6 is critical in RANKL-mediated osteoclastogenesis, which is mainly through the activation of JAK2/STAT3 signaling. CXCL16-CXCR6 axis may become a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis and osteoporosis.

CXCL16 upregulates RANKL expression in rheumatoid arthritis synovial fibroblasts through the JAK2/STAT3 and p38/MAPK signaling pathway.

ObjectiveTo explore the influence of chemokine, CXCL16, on the expression of the receptor activator nuclear factor κB ligand (RANKL) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLS).MethodsThe expression of CXCL16/CXCR6 and RANKL in RA or osteoarthritis (OA) patient synovia was examined by Western blot and immunohistochemistry. The serum concentration of CXCL16 and RANKL was measured by enzyme-linked immunosorbent assay (ELISA). RA-FLS were treated with recombinant CXCL16, and RANKL mRNA and protein were measured using PCR, Western blot and ELISA.ResultsThe synovial expression of CXCL16, CXCR6, and RANKL was higher in RA patients than in patients with OA. The serum CXCL16 and RANKL levels were higher in RA patients compared with OA patients and healthy controls. CXCL16 correlated with erythrocyte sedimentation rate, C reactive protein, disease activity, serum rheumatoid factor, and RANKL. RA-FLS treated with CXCL16 showed markedly increased expression of RANKL. When STAT3 or p38 activation was blocked by an inhibitor, CXCL16 failed to upregulate RANKL expression. In contrast, inhibiting the Akt or Erk pathway did not achieve the same effect.ConclusionsCXCL16 upregulates RANKL expression in RA-FLS and these effects are mainly mediated by the JAK2/STAT3 and p38/MAPK signaling pathways.

Interleukin-21 promotes osteoclastogenesis in RAW264.7 cells through the PI3K/AKT signaling pathway independently of RANKL

Cytokines play a key role in the bone destruction of rheumatoid arthritis (RA). Interleukin-21 (IL-21) promotes osteoclastogenesis in RA in a receptor activator of nuclear factor-κB ligand (RANKL)-dependent way. Whether IL-21 is capable of promoting osteoclastogenesis directly in the absence of RANKL remains unknown. In the present study, we examined the osteoclastogenic activity of IL-21 in RAW264.7 cells in the absence of RANKL. We found that IL-21 enhanced osteoclastogenesis and this was demonstrated by increased numbers of tartrate-resistant acid phosphatase (TRAP)-positive stained, multinucleated cells compared with the negative control. Western blot analysis and immunocytochemistry showed the positive expression of calcitonin receptor (CTR) in the IL-21 group. RT-PCR and RT-qPCR also verified the increased mRNA expression of CTR and cathepsin K in the IL-21 group compared with the negative control. The scanning electronic microscope images showed a few resorption pits on the bone slices cultured with IL-21. The phosphoinositide 3-kinase (PI3K)/AKT pathway inhibitor LY294002 significantly suppressed IL-21-induced osteoclastogenesis. Taken together, these findings suggest that IL-21 has direct osteoclastogenic potential independently of RANKL. IL-21 may promote osteoclastogenesis through the PI3K/AKT signaling pathway. Therapy targeting IL-21 may be of value in preventing bone erosions in patients with RA.

Soluble TAM receptor tyrosine kinases in rheumatoid arthritis: correlation with disease activity and bone destruction

The TAM receptor tyrosine kinases (TAM RTK) are a subfamily of receptor tyrosine kinases, the role of which in autoimmune diseases such as systemic lupus erythematosus has been well explored, while their functions in rheumatoid arthritis (RA) remain largely unknown. In this study, we investigated the role of soluble TAM receptor tyrosine kinases (sAxl/sMer/sTyro3) in patients with RA. A total of 306 RA patients, 100 osteoarthritis (OA) patients and 120 healthy controls (HCs) were enrolled into this study. The serum concentrations of sAxl/sMer/sTyro3 were measured by enzyme‐linked immunosorbent assay (ELISA), then the associations between sAxl/sMer/sTyro3 levels and clinical features of RA patients were analysed. We also investigated whether sTyro3 could promote osteoclast differentiation in vitro in RA patients. The results showed that compared with healthy controls (HCs), sTyro3 levels in the serum of RA patients were elevated remarkably and sMer levels were decreased significantly, whereas there was no difference between HCs and RA patients on sAxl levels. The sTyro3 levels were correlated weakly but positively with white blood cells (WBC), immunoglobulin (Ig)M, rheumatoid factor (RF), swollen joint counts, tender joint counts, total sharp scores and joint erosion scores. Conversely, there were no significant correlations between sMer levels and the above indices. Moreover, RA patients with high disease activity also showed higher sTyro3 levels. In‐vitro osteoclast differentiation assay showed further that tartrate‐resistant acid phosphatase (TRAP)+ osteoclasts were increased significantly in the presence of sTyro3. Collectively, our study indicated that serum sTyro3 levels were elevated in RA patients and correlated positively with disease activity and bone destruction, which may serve as an important participant in RA pathogenesis.

Mimotope mimicking epidermal growth factor receptor alleviates mononuclear cell infiltration in exocrine glands induced by muscarinic acetylcholine 3 receptor.

The muscarinic type 3 receptor (M3R) plays a pivotal role in the pathogenesis of Sjögrens syndrome (SS). Characterization of the crosstalk between M3R and EGFR has been investigated in some human malignancies. In the current study, we sought to investigate whether EGFR mimic immunization could alleviate the abnormal immune responses in an experimental SS-like model triggered by M3R peptides. After immunization with the combination of mimotope and M3R peptide, the active immunization targeting EGFR induced by the mimotope could reduce the marked infiltration of mononuclear cells, the high titer of antibodies against M3R and the accumulation of crucial pro-inflammatory cytokines in mice immunized with M3R peptide. Mechanistic analysis showed that mimotope immunization could alleviate the autoimmune response through inhibiting mitochondrion-mediated anti-apoptosis and up-regulating the FAS apoptosis pathway. These results may help to clarify the role of M3R in the pathogenesis of SS and suggested that transactivation of the EGFR signaling pathway may help M3R activate the autoimmune response in the pathogenesis of SS.

Active immunization with Tocilizumab mimotopes induces specific immune responses

BackgroundTocilizumab is a humanized monoclonal antibody showing high-affinity binding to both soluble interleukin-6 receptor (sIL-6R) and membrane bound IL-6R (mIL-6R), thereby preventing pro-inflammatory effects of IL-6. However, therapeutic antibodies still have practical limitations. To overcome these limitations, we generated Tocilizumab specific epitope mimics by using the phage display technology and tested whether the peptide mimics could induce similar humoral responses in mice immunized with the peptides.ResultsSeven phage mimics were obtained by using phage display peptide library. Four phage mimics (YHTTDKLFYMMR, YSAYEFEYILSS, KTMSAEEFDNWL and LTSHTYRSQADT) were shown to mimic Tocilizumab epitope using immunoassays. The mimotopes were conjugated to immunogenic carrier proteins and used to intraperitoneally immunize BALB/c mice. Sera from the mimotopes immunized mice not only showed specific binding to recombinant IL-6R, but can also IL-6R expressed in Hela, U-937, Jurkat cell lines and in fibroblast-like synoviocytes from patients with RA (FLS-RA). Furthermore, sera from mice immunized with mimotopes-KLH conjugate could reduce the level of phosphorylated- signal transducers and activator of transcription (STAT3), STAT3, phosphorylated- extracellular signal-regulated kinase (Erk) 1/2 and Erk1/2 in HeLa and Jurkat cells. Antibody-dependent cellular cytotoxicity (ADCC) assay showed that antibodies induced by mimotopes-KLH conjugate could elicit specific lysis in Hela and U-937 cells.ConclusionsFrom phage display library, we successfully isolated four Tocilizumab mimotopes which induced specific humoral and cellular reponses in vitro and in vivo.

A Case Report of X-Linked Hyperimmunoglobulin M Syndrome with Lipoma Arborescens of Knees

The X-linked hyperimmunoglobulin M syndrome (HIGM), caused by mutations in the CD40LG gene, is a kind of primary immunodeficiency disease (PID). Patients with X-linked HIGM are susceptible to infection as well as autoimmune diseases. Lipoma arborescens (LA) is a rare benign tumor, of which the pathogenesis mechanism has not been clearly understood. We report a case of HIGM combined with LA in a 22-year-old male patient. A new deletion mutation of CD40LG gene was detected in this case. The possible relationship between HIGM and LA was also discussed.

Autoantibodies against interferon-γ reduce the frequency of pulmonary fibrosis and concentration of C-reactive protein in patients with primary Sjögren's syndrome

Briefly, 96-well ELISA plates (MaxiSorp; Thermo Fisher Scientific, Shanghai, China) were coated by incubation overnight at 4 ° C with 1 μ g/ml rIFNγ (Sigma, MO, USA). The plates were then washed, blocked and incubated with 1:500 dilutions of plasma samples from the patients or controls for 2 h at room temperature. The plates were again thoroughly washed. Horseradish peroxidase (HRP) – conjugated Fc-specific IgG fractions from polyclonal goat antiserum against human IgG (Abcam, Hongkong, China) were added to a final concentration of 0.5 μ g/ml. The plates were incubated for 1 h at room temperature and then washed. Bound antibodies were detected with 2,2 ′ azinobis[3-ethylbenzothiazoline-6-sulfonicacid]-diammonium salt (ABTS) (Sigma, MO, USA) as the substrate. Absorbance was read at a wavelength of 405 nm (optical density [OD] 405 nm) with an ELISA reader (Thermo Fisher Scientific, Shanghai, China). The presence of anti-IFNγ aAbs in the sera of patients with pSS is shown in Figure 1. The median absorbance value at 405 nm of the anti-IFNγ antibodies was 0.0098 in the healthy controls and 0.1500 in pSS. The cutoff values (the mean plus two standard deviations of healthy sera) for positivity for the anti-IFNγ aAbs were 0.1656. The titers of the anti-IFNγ aAbs were significantly higher in the sera of patients with pSS compared with those in the healthy controls ( * * * , P 0.0001). The prevalence of the anti-IFNγ aAbs in patients with SS was 44.8%. The concentration of CRP in the sera of patients with anti-IFNγ aAbs was lower than that in the patients without aAbs ( P 0.0341). The frequency of pulmonary fibrosis was lower in the patients with antiIFNγ aAbs than in the patients without aAbs ( P 0.0172) (Table 1). No significant correlation was observed between the titer of aAbs against IFNγ and clinical manifestations and laboratory parameters (data not shown). The presence of aAbs against cytokines has been reported in various autoimmune diseases. Neutralizing aAbs against interleukin-1 alpha (IL-1 α ) have been demonstrated in the sera from patients with rheumatoid arthritis (RA) [11], and Mod Rheumatol, 2015; 25(2): 325–327