| Ticks are ectoparasites which maintain their life by sucking lots of blood and could spread multiple pathogens during blood-sucking,and they severely endanger the health and life of human and animals.Bioactive molecules produced from tick contribute to evading the host immune response.These bioactive molecules contain multiple proteases and protease inhibitors.Among the numerous protease inhibitors,cysteine protease inhibitors named Cystatins are reversible inhibitors,which could inhibit the activity of cysteine protease.Cystatins are distributed in almost all organisms and are involved in various biological processes.The intense research on ticks bioactive molecular contributes to elucidating the interaction and interrelationship of ticks-pathogen-host.In this study,two Cystatins named RHcyst-1 and RHcys-2 from Rhipicephalus haemaphysaloides were used as the study subjects.The flowing three aspects were mainly studied.Firstly,the effect of RHcyst-1 and RHcyst-2 on the differentiation and maturation of DCs.Two Cystatins isolated from the tick were successfully constructed and efficiently expressed in this study.In order to test the effect of RHcyst-1 and RHcyst-2 on the differentiation of DCs,the bone marrow monocyte was induced with GMCSF/IL-4 with or without RHcyst-1 and RHcyst-2 treatment,respectively.The cells were collected on the third and sixth day and the ratio of DCs was evaluated by flow cytometry.The results showed that the ratio of DCs was significantly inhibited at different time points in the RHcyst-1 treatment group,while the ratio of DCs in the RHcyst-2 treatment group was lower than the control group on the sixth day,but not on the third day.On the basis,possible mechanism of RHcyst-1 regulating bone marrow monocytes differentiated into DCs was studied.The results showed that DCs differentiation at different time points,transcriptional levels of p38,ERK and STAT were significantly changed.The phosphorylation of p38 increased significantly after RHcyst-1 treatment on the third day,and the opposite result on the sixth day;but the phosphorylation of ERK was reduced on the sixth day,and there was no difference on the third day.The phosphorylation of STAT3 and STAT5 was suppressed on the third day but increased on the sixth day.These results suggested that RHcyst-1 may affect the transcription and phosphorylation of p38 MAPK and STAT pathway during DCs differentiation.In order to test the effect of two Cystatins on the maturation of DCs,flow cytometry was used to evaluate the effect of them on DCs maturation.The results revealed that the protein level of CD40+,CD80+,CD86+ and MHC-II in RHcyst-1 or RHcyst-2 treatmen group has no significant changes compared to the control group,respectively.These results suggest that RHcys-1 and RHcyst-2 have no significant effect on the maturation of DCs.Secondly,the effect of RHcyst-1 and RHcyst-2 on T cell activation and antibody response.To explore the role of two Cystatins in immune response stage,DCs with OVA activation and T-cell coculture assay was performed to test the effect of RHcyst-1 and RHcyst-2 on the levels of cytokines.The results showed that T cells co-cultured with DCs treated with RHcyst-1 produced significantly less TNFα,IFNγ and IL-2 than the control group,while only the expression of TNFα and IL-2 but not IFNγ was significantly inhibited with RHcyst-2 treatment.The effect of RHcyst-1 and RHcyst-2 on humoral immune responses were studied after co-immunization with OVA.The results showed that the antibody subtype and antibody titer were not changed between the Cystatins treatment and the control groups,respectively.And there was no difference in the ratio of IgG2a/IgG1 b.These results revealed that RHcyst-1 and RHcyst-2 have no effect on the humoral response and could not affect the imbalance of Th1 and Th2.In order to determine the effect of RHcyst-1 and RHcyst-2 on CLT response,the release of lactic dehydrogenase(LDH)was tested.The results showed that RHcyst-1 and RHcyst-2 have no affect on CTL response.Thirdly,the preliminary study of antitumor activity of RHcyst-1.According to the high efficiency of RHcyst-1 protease inhibitor activity and the high expression of protease in tumor microenvironment,our study target tumor as the preliminary study object in RHcyst-1 application.A549,HeLa,SKOV-3 and B16-F10 were used in vitro.Proliferation activity was evaluated using CCK-8,and migration and invasion were determined by scratch wound and Transwell invasion assays,respectively.Results showed that RHcyst-1 significantly inhibited the proliferation,migration,and invasion of all four different tumor cells but not normal cells in vitro.In addition,a mouse B16-F10 tumor model was used to evaluate the antitumor activity of RHcyst-1 in vivo.The results showed that RHcyst-1 treatment inhibited tumor growth and improved survival.A decrease and a increase in MDSCs levels were observed in PBMCs and in the spleen,respectively,after RHcyst-1 application.But no obvious difference in the levels of CD3+CD8+ and CD3+CD4+ T cells was observed between the RHcyst-1 treatment and the control,both in PBMCs and spleens.RHcyst-1 promote CD8+ T cells infiltration to tumor tissue by immunohistochemical detection.To determine specific binding,active uptake,or direct action of RHcyst-1 on the extracellular environment,Western blotting and IFA were used to test the location of RHcyst-1.The results showed that RHcyst-1 could be detected only in the cell supernatants but not in the cell lysis solution and cell surface,which suggested that RHcyst-1 exerted antitumor effects may act on the extracellular environment but not on active uptake or specific binding.In order to test the effect of RHcyst-1 entering the tumor cells,RHcyst-1 was overexpressed by transfection on B16-F10 cells.The results showed that mRNA expression of different cathepsins was supressed after RHcyst-1 transfection.And RHcyst-1 intratumor injection differentially regulated the mRNA expression of different cathepsins.In summary,we found that the type 1 Cystatin RHcyst-1 isolated from the tick was involved in the regulation of the host immune response,and involved in multiple stages of immune response,which indicated the pluripotency of Cystatins isolated from tick.However,the type 2 Cystatin RHcyst-2 has less effect on the host immune response.The results of RHcyst-1 antitumor activity indicated that the multi-function natural bioactive molecule isolated from tick has a potential application prospect in the biological agents. |
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