The Role Of Secretory GRP78 In Psoriasis

BackgroundPsoriasis is a common chronic inflammatory skin disease.Heredity,infection,immune disorder and other factors are related to the occurrence of the disease.The global prevalence rate of psoriasis is about 2-3%.The typical clinical manifestations of psoriasis are scattered red plaques with clear boundaries,covered with silver-white scales,and easy to occur repeatedly.The typical histopathological feature of psoriasis is the excessive proliferation of keratinocyte,and the epidermis is obviously thickened with hyperkeratosis or incomplete keratosis.Because angiogenic factors and other factors promote the proliferation of micro vessels in the lesion,the epidermis capillaries proliferate and dilate,the granular layer becomes thinner or disappears,and the spinous layer thickens,accompanied by superficial langerhans cells in the epidermis,CD8+T cell infiltration and neutrophil aggregation.The papilla of the dermis is extended,and the infiltration of dendritic cells and a variety of T cells such as CD4+Th cells could be seen in the superficial and middle layers of the dermis.The treatment of psoriasis brings great psychological and economic burden to patients.GRP78(glucose regulated protein 78)also known as immunoglobulin heavy chain binding protein(Bip),or heat shock protein A5(HSPA5),widely exists in the nucleus,endoplasmic reticulum,intracellular and capsule of cells.Some studies have shown that under stress,GRP78 can be secreted to play a role in the regression of inflammation in autoimmune diseases,such as rheumatoid arthritis.Our previous studies also found that sGRP78 can induce the formation of a group of CD19hiB cells.These cells highly express PD-L1,FasL and secrete a large amount of IL-10.sGRP78 can induce DCs apoptosis and down-regulate CD80,CD86 and MHCII molecules on their surface.Furthermore,sGRP78 inhibits the production of LPS-induced inflammatory cytokines,such as TNF-α and etc.The initiation and development of psoriatic dermatitis depend on the classical positive circulation of IL-23/IL-17,which can be summarized as that under the action of pathogenic microorganisms,KCs produce PAMPs and DAMPs and cytokines to act on plasmacytoid DCs(pDCs),which secrete IFN-α/β and a large amount of IL-23.pDCs prime T cells to differentiate into IL-17 secreting cells,such as Th17 and γδT17 cells etc.These cells in turn act on KCs to promote the occurrence and development of psoriatic dermatitis.Some studies reported that the expression of GRP78 in the undifferentiated keratinocytes near the basal layer in psoriatic skin was down-regulated.Given the anti-inflammatory role of sGRP78,in this study,we explore its role in the pathogeneisis and development of psoriatic dermatitis.Objective1.To detected the expression level of GRP78 in psoriatic lesion in IMQ-induced dermatitis.2.To explore the effect of sGRP78 on IMQ-induced psoriatic dermatitis.3.To explore the potential mechanisms underlying the allevation of sGRP78 on IMQ-induced psoriatic dermatitis.Methods1.Preparation of recombinant mouse sGRP78The pGEX-4T1-GST-sGRP78 plasmid preserved in the laboratory was transferred into BL21 escherichia coli.GST-GRP78 fusion protein was purified using Glutathione Spin Columns.GRP78 was obtained by thrombin cleavage and filtered by 50KD ultrafiltration tube.Endotoxins were removed by a Pierce High-capacity Endotoxin Removal Resin,and the final endotoxin concentration of protein samples was<10 EU/mg.2.Establishment of psoriasis model in mice6-week-old C57BL/6 female mice were randomly divided into control group,model group and experimental group.The animals in model group and experimental group were smeared with imiquimod(IMQ)ointment on the back every day,followed by intraperitoneal injection of GRP78 to animals in experimental group.Control animals was intraperitoneally injected with the same dose of saline every day.Before administration,each mouse was scored according to the psoriatic lesion score standard.The experiment was terminated at the seventh day.The eyeball blood,skin of the lesion,draining lymph nodes and spleen were taken for the follow-up experiment.3.Detection of the immune status in psoriatic dermatitis modelsFlow cytometry was used to detect the immune phenotypes of dendritic cells,macrophages,monocytes,neutrophils and all kinds of T cells in skin lesions,lymph nodes and spleen.Flow CBA,and RT-PCR were used to detect the expression of cytokines.4.In vitro experimentsBone marrow cells from C57BL/6 mice(6-and 8-week-old)were isolated and induced by IL-4 and GM-CSF for 6 days to generate mature DCs.The bone marrow-derived DCs were divided into four groups:control group,sGRP78 group,IMQ group and IMQ+sGRP78 group.Media,IMQ,SGRP78 and IMQ+SGRP78 were added,respectively.After 6 days,the cells were collected and cultured superintendents were used to detect the expression of CD80,CD86 and MHCII on the cell surface.The expression of related cytokines was detected by flow cytometry(CBA).DCs induced by sGRP78 were co-incubated with T cells for 72 h and T cell phenotypes were detected.Results1.Expression of GRP78 was downregulated in IMQ-induced psoriatic dermatitis modelsCompared with the control group,IMQ treatment decreased the expression of GRP78 in lesional skin of psoriatic mice.2.sGRP78 relieve IMQ-induced psoriatic dermatitis in mice IMQ-induced psoriasis model group began to show skin thickening,erythema,scales and bleeding on the third day of the experiment.With the progress of the experiment,the skin lesions were aggravated,which was the most serious before the termination of the experiment.Similarly,compared with the control group,the IMQ+sGRP78 group also had a certain degree of skin thickening and mild erythema,scales and bleeding.However,compared with IMQ group,the skin thickened and the degree of erythema,scales and bleeding in IMQ+sGRP78 group were lighter.3.Effect of sGRP78 on the release of cytokines in IMQ-induced psoriatic lesionRT-PCR results showed that the expression of IL-17A,IL-17F,IL-17C,IL-22,IL-23,TNF-α,IFN-α and IL-1β in psoriatic lesion of IMQ group were significantly higher than that of control group.Compared with the IMQ group,the expression of other inflammatory factors except IL-17C and TGF-β was significantly decreased in the IMQ+Sgrp78 group,while the expression of IL-10 was significantly increased.4.The effect of sGRP78 on DC cellsIn psoriatic mice model,IMQ treatment up-regulated the frequencies of DCs,promoted their activation,and increased the expression of proinflammatory cytokines such as IL-23 in lesion-derived DCs,while sGRP78 treatment down-regulated DCs frequencies and quantities.Meantime,sGRP78 can also down-regulate the proportion of neutrophils.5.GRP78 down-regulated the proportions and numbers of IL-17 secreting T cellsIMQ treatment significantly increased the frequencies of Th17 and γδT17 cells in peripheral blood and in draining lymph nodes.sGRP78 supplement down-regulated the proportions and numbers of above IL-17 secreting T cells,and increased the proportion of Tregs in lymph nodes.6.GRP78-conditioned BMDCs had anti-inflammatory effectThe expression of CD80,CD86 and MHCII were all up-regulated on the surface of IMQ-treated BMDCs.However,GRP78 reversed such IMQ-induced upregulation.RT-PCR detection confirmed that the RNA expression of IL-23 and IL-1β in BMDCs were increased after IMQ treatment,while decreased in sGRP78 treatment group.Meanwhile,less T cells co-incubated with sGRP78-conditioned DCs differentiated into Th17 cells than those with IMQ-conditioned DCs.ConclusionIn vivo and in vitro experiments suggested that sGRP78 can partially alleviate psoriatic dermatitis induced by IMQ.This may be due to the fact that sGRP78 inhibits the activation of DC cells induced by IMQ,down-regulates their expression of proinflammatory cytokines such as IL-1β,IL-23 and IL-17A/IL-17F,decreases the frequencies of IL-17 secreting T cells.Hence,the mechanism underlying how GRP78 alleviates IMQ-induced Psoriasis-like lesions is summarized as following:1.sGRP78 down-regulates the expression of proinflammatory cytokines such as IL-17A,IL-17F,IL-17C,IL-22,IL-23,TNF-α,IFN-α and IL-1β in IMQ-induced lesional tissues,and up-regulates the expression of anti-inflammatory cytokines IL-10.2.sGRP78 down-regulates the proportion of DCs and induces the formation of tolerogenic DCs.3.sGRP78 down-regulated the proportion of Th17 and γδT17 cells cells in IMQ-induced psoriasis-like lesions in mice,and up-regulated the proportion of Treg cells.In view of the classic KCs-DCs-IL-23-Th17-IL-17-KCs axis about the occurrence and development of psoriasis,sGRP78 plays a comprehensive role in inhibiting the progression of psoriatic dermatitis.