The Immunosuppressive Function Of A Tick Salivary Gland Protein,DsCystatin,identified From Dermacentor Ticks And Its Potential Application In Inflammatory Disease Models

Ticks are blood-feeding arthropods and transmit numerous human pathogens;ticks secrete immunosuppressant molecules which can inhibit host inflammatory and immune responses and provide survival advantages to pathogens at tick feeding sites on mammalian hosts.Mammalian Cathepsins play important roles in innate and adaptive immune responses,including the physiological process of apoptosis;antigen processing and presentation.Cystatins are natural inhibitors for Cathepsins,and widely expressed in animals and other species,and they are evolutionally conserved for sequences and functions.In this study,we characterized a novel tick secreted protein,DsCystatin,which belong to the Cystatin protein family,from Dermacentor silvarum of China.The DsCystatin gene encodes 128 amino acids that contain a signal peptide of 21 amino acids.DsCystatin inhibited enzymatic activity of human Cathepsin L and B in a dose dependent manner.GST pull down assay suggested that DsCystatin can directly interactions with human Cathepsins L and B.DsCystatin impaired antigen presentation process in mouse bone marrow derived dentritic cells(BMDCs).DsCystatin suppressed inflammatory cytokines such as IL1?,IFN?,TNF? and IL6 production at mRNA and protein levels from mouse bone marrow derived macrophage(BMDMs)that stimulated with LPS or Borrelia burgdorferi.DsCystatin inhibited the activation of bone marrow derived macrophage and dendritic cells by downregulating the surface expression of CD80 and CD86.DsCystatin was found to inhibit the expression of inflammatory cytokine at mRNA level,and the activation of NF?B is the key transcriptional factor that regulates proinflammatory cytokine expression.We further showed that DsCystatin can inhibit LPS or Borrelia burgdorferi induced activating of NF?B.We used Luciferase reporter assay was performed in 293T-TLR4 cells to explore the potential role of DsCystatin in TLR4 pathway.We found that DsCystatin inhibited MYD88 and TRAF6 induced NF?B activating.DsCystatin decreased the p50 expression in bone marrow derived macrophage.In Raw264.7 cells,DsCystatin inhibited the expression and phosphorylation of IkB?.In Raw264.7 and 293T-TLR4 cells,DsCystatin inhibited LPS induced TRAF6 and TAK1 expression.Finally,we tested the role of DsCystatin in mouse inflammatory diseases models.DsCystatin can relief the joint inflammation induced by Complete Freund's adjuvant.DsCystatin also suppressed Borrelia burdorferi induced joint swelling and antibody production.These data suggested that DsCystatin is a novel immunosuppressive protein and can be potentially used in the treatment of inflammatory diseases.