Effects Of VSTM1-V2on Regulation Of Inflammatory Response

The innate immunity plays a vital role in host defence, most important effectivecells in innate immune system include neutrophils, monocytes and macrophages. Thesecells are recruited at sites of inflammation immidiately upon infection, and recognize,phagocyte and eliminate pathogens, as well as recruite other immune cells by secretingpro-inflammatory mediators, subsequently activate the adaptive immunity. However theelimination of pathogen is followed by excessive immune response and severe damageof tissue, so far as to death due to fatal septic shock. Immune system relay on multiplemechanism regulate these intensive immune response, one of the most importantmechanism is the inhibition of activition and the balance of immune response by meansof the expression of inhibitoray receptors.Inhibitoray receptors are negative regulatoror that wide expressed protein ininmmune cells, these cells transduct inhibitory signals and inhibit activition of cells orincrease the threshold of cell activation through cytoplasmic immunoreceptortyrosine-based inhibitory motifs (ITIMs) upon ligand binding. These kind of cells canregulated the activaty of immune cells and control immune response by phagocytes,migration and apoptosis of cells and production of cytokines. There are almost300potential ITIMs-contained genes in human genome, most of these genes have no clearlyunderstand of functions except for only1/5of them, V-set and transmembrane domaincontaining1(VSTM1) is the right unknown functional gene.Up to now there are a few research about VSTM1. We analysed the expressionprofile and DNA sequences and found there are at least five spliceosome, includingVSTM1-V1～V5. The VSTM1-V1and VSTM1-V2is the main form of expression.VSTM1-V1is broad expressed in nomal tissues and cells, but VSTM1-V2is mainly expressed in immune system. VSTM1-V1is a typeⅠ membrance protein, and alsocalled signal inhibitory receptor on leukocytes-1(SIRL-1). It is a potential innateinhibitor to immune response because of the ITIMs in its cytoplasmic tail. VSTM1-V2is a classical secretary glycoprotein and is similar to VSTM1-V1, but has notransmembrance domain, and they are same in the region of ligand-binding domain, soit is considered that they are competitive to each other. It is known that VSTM1-V1hasan inhibitoray function to macrophages, however VSTM1-V2can promote the secretionof IL-17A by CD4+T cells and promote primary CD4+T cells differentiation to Th17cells. Furthermore it is increased in serum of rheumatoid arthritis patients, and it islikely to close relate to initiate or develop of autoimmune disease. Untill now thereseach of VSTM1-V2were focus on the regulation of adapte immunity, and there is noabout innate immunity regulation.[Objective] The fuction of VSTM1-V2in innate immunity particularly inanti-infection immunity by the research of inflammation related cells in vitro and themodel of acute inflammatory mice in vivo.[Methods] Human umbilical vein endothelial cells (HUVECs) and humanmonocyte lines THP-1cells were incubated in vitro, then they were treated bylipopolysaccharide (LPS) or Tumor necrosis factor (TNF)-α. Cells were divide intodifferent groups, including PBS group, TNF-α/LPS group, VSTM1-V2with differentconcentration group, TNF-α/LPS+VSTM1-V2group. The effect of VSTM1-V2oncell proliferation were measured by MTT assay. The effect of VSTM1-V2on cellmagration were assessed by wound healing assay. The MPO activity and NO levelswere measured in supernatant of cultured HUVECs and THP-1cells. RT-PCR wereperformed to check the mRNA transcription of VSTM1-V1and VSTM1-V2inHUVECs, the pro-inflammatory cytokines (TNF-α、IL-1β、IL-6), chemokines (IL-8、MCP-1、CXCL2) and cell surfaced adhesion molecules (VCAM-1) were also checkedby RT-PCR. The expression of inflammitory mediators by HUVECs or THP-1cellsthrough ELISA. Western blotting was used to detect the expression of VCAM-1, IL-6and signal transduction pathway proteins (JNK1and P65). The acute inflammatory infective models were established by means of endotracheal injection Klebsiellapneumoniae (Kp). VSTM1-V2protein were injected through tail vein, then serum andlung tissues were obtained at different time points. HE staining wre performed toobserve the effect of VSTM1-V2on acute pneumonia. The total RNA were isolated andthe real-time qPCR were performed to check the expresstion of cytokines (IL-6、IL-1β、TNF-α、CXCR2and MIP-2), the activity of MPO and iNOS was measured using lungtissue homogenate. Cytometric bead array (CBA) were used to check effects ofVSTM1-V2protein on serum cytokines (IL-6、MCP-1and IL-10).[Results](1) VSTM1-V1and VSTM1-V2genes were expressed in HUVECs, butthey are low expression. The VSTM1-V2expression were increased relative toVSTM1-V1in TNF-α treated HUVECs. The expression of VSTM1-V1increasedsignificantly; The expression prorotion of VSTM1-V1and VSTM1-V2had noobviously change by pre-treatment with VSTM1-V2followed by TNF-α.(2) HUVECsstaining showed that the increase of cells were reduced in VSTM1-V2treating groupcompare with PBS group. MTT assay also indicated VSTM1-V2can inhibitor HUVECsgrowth significantly.(3) Wound healing assay showed VSTM1-V2reduced themagrition increase by TNF-α. The increasing expression of adhesion molecule VCAM-1by TNF-α were also inhibited by VSTM1-V2by RT-PCR and Western blotting.(4) Themain cytokines were checked by RT-PCR and Western blotting, it was show that theexpression of pro-inflammatory cytokines and chemokines were increased by treatingwith TNF-α, however these increase were reduced by pre-treatment with VSTM1-V2.(5) The level of NO show, VSTM1-V2pre-treatment increased the NO level whichinduced by TNF-α.(6) NF-κB and MAPK pathways were check by western blotting, theincreasing expression of P65and JNK1induced by TNF-α were reduced bypre-treatment VSTM1-V2.(7) The model of acute inflammation by Kp injection wereestiblished. The acute lung injure were inhibited by VSTM1-V2in the slide of HEstaining. The activity of MPO and iNOS were reduced by using VSTM1-V2in lunghomogenate of acute lung inflammatory mice.(8) The mRNA level of cytokines such asIL-6、IL-1β、TNF-α、CXCR2�'�MIP-2were increased in Kp models, but they were decreased by injecting VSTM1-V2.(9) CBA were used to detect secretory cytokines inserum, the expression of TNF-α、IL-6and MCP-1were decreased in VSTM1-V2+Kpgroup compared with Kp group.(10) The opposite results were found in VSTM1-V2treated THP-1cells, there were a increasing reaction in VSTM1-V2treated THP-1, suchas it could promote the aggregation and adhison of THP-1, and improve the reaction ofTHP-1toward LPS. The increasing MPO activity and P65level in VSTM1-V2treatedTHP-1showed VSTM1-V2were a pro-inflammation protein, and the different reactionbetween endothelial cells and monocyte shoud be more clear by lots of work.[Conclusions](1) There are expression of VSTM1gene in HUVECs and thetranscription of VSTM1-V2was increased in TNF-α treated HUVECs which candecrease the activated threshold and promote the activity of HUVECs. VSTM1-V2canimprove the transcription of VSTM1-V1gene and increase cell threshold and inhibit theactivity of HUVECs.(2) VSTM1-V2can inhibit the proliferation of HUVECs in vitro,as well as inhibit the migration, adhesion and the production of cytokines in TNF-αinduced HUVECs. VSTM1-V2also inhibit the signal tranduction JNK1and P65whichinduced by TNF-α.(3) VSTM1-V2can inhibit the lung damage and the production ofpro-inflammatory cytokines in Kp indued acute pneumonia models.(4) VSTM1-V2hasa opposite function in THP-1, it can promote the proliferation, aggregation and theactivity of MPO in THP-1, as well as improve the activity of LPS indued THP-1. Thedifferent response of VSTM1-V2between HUVECs and THP-1might be due to thedifferent expression levels of the gene, the different distribution of ligands and thedifferent signal pathways.