The Effects Of MPDL1-hIg On Collagen-Induced Arthritis

BackgroundProgrammed death 1(PD-1) is a CD28 family member. Activated T cells and B cell express PD-1. PD-1 delivers a coinhibitory signal upon binding to either of its two ligands, programmed death ligand 1 (PDL1) or programmed death ligand 2(PDL2). T cells and B cells play important roles in the pathogenesis of rheumatoid arthritis(RA). As an important negative regulator of the activation of T cells and B cell, PD-1 might be the target for the treatment of RA.mPDL1-hIg was the fusion protein composed of the extracellular domain of mouse PDL1 and human IgGFc and had biological effects of mouse PDL1. mPDLl-hIg could be easily detected and purified because it included human IgGFc. Moreover, human IgGFc of mPDLl-hlg might theoretically extend the half-live of mPDL1 of mPDLl-hIg, which could contribute to the effects of mPDLl of mPDL1-hIg in vivo.Collagen II (CII)-induced arthritis (CIA) is an experimental model of arthritis that has been used to dissect the pathogenesis of human RA.ObjectiveTo investigate the effects of mPDL1-hIg on CIA on the basis of successful construction of the vector expressing mPDLl-hIg and the obtainment of the cell strain stabily expressing mPDL1-hIg and mPDL1-hIg, which would provide new idea, new method and experimental evidence for the exploration of the treatment of RA.Methods①After activated erythrocyte-depleted splenocytes from normal mice interacted with mPDL1-hIg, they interacted with fluorescence labelling anti-human IgG antibody again. Flow cytometry detected the binding rate of activated erythrocyte-depleted splenocytes from normal mice and mPDL1-hIg. ②After erythrocyte-depleted splenocytes from DBA/1 J mice immunized twice with bovine collagen II(CII) were stained with CFSE, they were stimulated by denatured collagenⅡ(dCII). Flow cytometry analyzed the effects of mPDL1-hIg on cell proliferation in respose to bovine dCII and ELISA detected the effects of mPDL1-hIg on the production of IL-17 and IL-23 in respose to bovine dCII.③DBA/1 J mice were immunized twice with bovine CII to elicit CIA). The immunized mice were randomly divided and treated with the following regimens. Each group of mice received either control IgG(human IgG) or mPDL1-hIg. Mice were examined daily from day 0(The day of second immunization was designated as day 0).④Serum samples from the control IgG-treated or mPDL1-hIg-treated mice were collected at day 9 before CIA mice were killed. The expression of IL-17 and IL-23 in the serum was measured by ELISA.⑤The control IgG-treated or mPDL1-hIg-treated mice were killed at day 9. Mice's paws were removed, fixed, decalcified, embedded, sectioned, and stained with HE.⑦After erythrocyte-depleted splenocytes from CIA mice were stained with CFSE, they were stimulated by bovine dCII. Cell proliferation was analyzed by flow cytometry and the production of IL-17 and IL-23 in the culture supernatant were measured by ELISA.⑦Significant differences between experimental groups were analyzed by the Mann-Whitney U test. Values of p< 0.05 were considered to be significant.RESULTS①The results of flow cytometry indicated that activated erythrocyte-depleted splenocytes from normal mice and mPDL1-hIg could bind and the binding rate was about 43.62% in our experimental conditions.②Flow cytometry proved that mPDL1-hIg could have inhibitory effects on the proliferation of erythrocyte-depleted splenocytes from the CII-immunized mice in response to bovine dCII. And the results of ELISA showed that mPDL1-hIg could inhibit the production of IL-17 and IL-23 by erythrocyte-depleted splenocytes from the CII-immunized mice in response to bovine dCII.③The administration of mPDL1-hIg significantly decreased the mean arthritis score of CII-immunized mice, although the incidence of disease was not affected(The incidence of disease of each group is 6/7(86%)). The paw sections from the control IgG-treated mice and mPDL1-hIg-treated mice showed that the mPDLl-hIg treatment could ameliorate histopathological changes that are characteristic features of arthritis. These results demonstrated that the treatment with mPDL1-hlg could inhibit the disease severity of CIA, suggesting the involvement of the PD-1-PDL pathway in the pathogenesis.④The results of ELISA showed that the mPDLl-hlg treatment could reduced the expression of IL-17 and IL-23 in the serum of CIA mice.⑤Flow cytometry proved that the mPDLl-hlg treatment could have inhibitory effects on the proliferation of erythrocyte-depleted splenocytes from CIA mice in response to bovine dCII. And the results of ELISA showed that the mPDLl-hIg treatment could inhibit the production of IL-17 and IL-23 by erythrocyte-depleted splenocytes from CIA mice in response to bovine dCII. These results suggested that the mPDL1-hlg treatment might inhibit cell proliferation and the production of IL-17 and IL-23 in response to CII to ameliorated CIA.ConclusionmPDL1-hlg significantly ameliorated CIA as assessed by clinical arthritis score and histology in the joints. The expression of IL-17 and IL-23 in the serum was reduced by mPDLl-hIg treatment. The mPDLl-hlg treatment might inhibit cell proliferation and the production of IL-17 and IL-23 in response to CII to ameliorated CIA.