RESUMEN
Wnt5a signalling plays pathological roles in synovial inflammation and bone destruction. In the present study, we designed four human Wnt5a-based DNA recombinants and detected their effects on immunogenicity and anti-rheumatism in a collagen-induced arthritis (CIA) model. Histomorphometry and micro-CT scanning showed that the phWnt5a-NL was superior to other recombinants because it resulted in decreased severity of arthritis, histopathological scores of synovial inflammation and bone erosion in CIA mice. In addition, ELISA and TRAP staining showed that the phWnt5a-NL-immunized CIA mice had reductions in the serum concentrations of the rheumatoid-associated cytokines IL-1ß and RANKL and in osteoclastogenesis. Furthermore, flow cytometry showed that the phWnt5a-NL treatment increased the percentage of Treg cells. Finally, western blotting analysis showed that the phWnt5a-NL-immunization interrupted ß-catenin and JNK expression in osteoclast precursors derived from the CIA mice. The results suggest that depleting the carboxy-terminus in hWnt5a-based DNA recombinants may be beneficial for the treatment of chronic inflammatory disorders involving bone resorption.
Asunto(s)
Artritis Experimental/inmunología , Inmunización/métodos , Proteínas Recombinantes/inmunología , Proteína Wnt-5a/inmunología , Animales , Artritis Experimental/diagnóstico por imagen , Artritis Experimental/patología , Citocinas/sangre , Citocinas/inmunología , Humanos , Interleucina-1beta/sangre , Interleucina-1beta/inmunología , Proteínas Quinasas JNK Activadas por Mitógenos/inmunología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Osteoclastos/citología , Osteoclastos/inmunología , Osteoclastos/metabolismo , Osteogénesis/inmunología , Proteínas Recombinantes/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Microtomografía por Rayos X/métodosRESUMEN
The contributions of aryl hydrocarbon receptor (Ahr) to the pathogenesis of rheumatoid arthritis (RA), particularly bone loss, have not been clearly explored. The imbalance between osteoblasts and osteoclasts is a major reason for bone loss. The dysfunction of osteoblasts, which are derived from mesenchymal stem cells (MSCs), induced bone erosion occurs earlier and is characterized as more insidious. Here, we showed that the nuclear expression and translocation of Ahr were both significantly increased in MSCs from collagen-induced arthritis (CIA) mice. The enhanced Ahr suppressed the mRNA levels of osteoblastic markers including Alkaline phosphatase (Alp) and Runt-related transcription factor 2 (Runx2) in the differentiation of MSCs to osteoblasts in CIA. The 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated activation of Ahr dose-dependently suppressed the expression of osteoblastic markers. In addition, the expression of ß-catenin was reduced in CIA MSCs compared with control, and the TCDD-mediated activation of the Ahr significantly inhibited ß-catenin expression. The Wnt3a-induced the activation of Wnt/ß-catenin pathway partly rescued the osteogenesis decline induced by TCDD. Taken together, these results indicate that activated Ahr plays a negative role in CIA MSCs osteogenesis, possibly by suppressing the expression of ß-catenin.
Asunto(s)
Artritis Experimental/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Receptores de Hidrocarburo de Aril/metabolismo , beta Catenina/metabolismo , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Artritis Experimental/patología , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos DBA , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Dibenzodioxinas Policloradas/farmacología , Vía de Señalización WntRESUMEN
Dickkopf-1 (DKK1), a secretory inhibitor of canonical Wnt signaling, plays a critical role in certain bone loss diseases. Studies have shown that serum levels of DKK1 are significantly higher in rheumatoid arthritis (RA) patients and are correlated with the severity of the disease, which indicates the possibility that bone erosion in RA may be inhibited by neutralizing the biological activity of DKK1. In this study, we selected a panel of twelve peptides using the software DNASTAR 7.1 and screened high affinity and immunogenicity epitopes in vitro and in vivo assays. Furthermore, we optimized four B cell epitopes to design a novel DKK1 multiepitope DNA vaccine and evaluated its bone protective effects in collagen-induced arthritis (CIA), a mouse model of RA. High level expression of the designed vaccine was measured in supernatant of COS7 cells. In addition, intramuscular immunization of BALB/c mice with this vaccine was also highly expressed and sufficient to induce the production of long-term IgG, which neutralized natural DKK1 in vivo. Importantly, this vaccine significantly attenuated bone erosion in CIA mice compared with positive control mice. These results provide evidence for the development of a DNA vaccine targeted against DKK1 to attenuate bone erosion.
Asunto(s)
Artritis Experimental/terapia , Resorción Ósea/terapia , Epítopos de Linfocito B , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Vacunas de ADN/farmacología , Animales , Artritis Experimental/genética , Artritis Experimental/metabolismo , Artritis Experimental/patología , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/patología , Células COS , Chlorocebus aethiops , Femenino , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Endogámicos BALB C , Vacunas de ADN/genéticaRESUMEN
Ahr activation is known to be associated with synovitis and exacerbated rheumatoid arthritis (RA), but its contributions to bone loss have not been completely elucidated. Osteoblast proliferation and differentiation are abnormal at the erosion site in RA. Here, we reported that the expression of Ahr was increased in the hind paws' bone upon collagen-induced arthritis (CIA) in mice, and the levels of Ahr were negatively correlated with bone mineral density (BMD). In addition, immunofluorescent staining showed that the high expression of Ahr was mainly localized in osteoblasts from the CIA mice compared to normal controls. Moreover, the luciferase intensity of Ahr in the nucleus increased by 12.5% in CIA osteoblasts compared to that in normal controls. In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activation of the Ahr inhibited pre-osteoblast MC3T3-E1 cellular proliferation and differentiation in a dose-dependent manner. Interestingly, the levels of alkaline phosphatase (ALP) mRNA expression in the osteoblasts of CIA mice were reduced compared to normal controls. In contrast, decreased ALP expression by activated Ahr was completely reversed after pretreatment with an Ahr inhibitor (CH-223191) in MC3T3-E1 cell lines and primary osteoblasts on day 5. Our data further showed that activation of Ahr promoted the phosphorylation of ERK after 5days. Moreover, Ahr-dependent activation of the ERK signaling pathway decreased the levels of proliferation cells and inhibited ALP activity in MC3T3-E1 cells. These results demonstrated that the high expression of Ahr may suppress osteoblast proliferation and differentiation through activation of the ERK signaling pathway, further enabling bone erosion in CIA mice.