Immunomodulatory effects of Rhipicephalus haemaphysaloides serpin RHS2 on host immune responses

Abstract

Background: Rhipicephalus haemaphysaloides is a widespread tick species in China and other South East Asian countries, where it is the vector of many pathogens. The objective of this study was to study the role of serpin (serine protease inhibitor) during the tick-host interaction.

Methods: The differentiation of bone marrow-derived dendritic cells (BMDC) was induced in vitro, and the effect of RHS2 on the maturation of DCs was evaluated. The effects of RHS2 on T cell activation and cytotoxic T lymphocytes' (CTLs) activity were analyzed by flow cytometry. Antibody subtypes after immunization of mice with RHS2 and OVA were determined.

Results: RHS2 can inhibit the differentiation of bone marrow-derived cells into DCs and promote their differentiation into macrophages. RHS2 can inhibit the maturation of DCs and the expression of CD80, CD86 and MHCII. The number of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells secreting IFN-γ, IL-2 and TNF-α was decreased, and the number of CD3⁺CD4⁺ T cells secreting IL-4 was increased, indicating that RHS2 can inhibit the activation of CD4 T cells and CD8 T cells, leading to inhibition of Th1 immune response. RHS2 inhibits the elimination of target cells by cytotoxic T lymphocytes. After immunization of mice with RHS2 and OVA, serum IgG2b was significantly reduced and IgM was increased.

Conclusions: The results show that RHS2 has an inhibitory effect on the host immune response. Ticks have evolved various ways to circumvent adaptive immunity. Their serpin inhibits BMDC differentiation to reduce immune responses.

Keywords: BMDC; Immmunomodulatory; Rhipicephalus haemaphysaloides; Serpin; Tick.

Fig. 1

Differentiation of BMDC in the presence or absence of RHS2. a Recombinant RHS2 purified from a GST affinity column (GST-RHS2 and after cleavage of the GST tag, detected by Western blot). b, c Cultured cells were labeled with the designated monoclonal antibodies (mAbs) and analyzed by two-color flow cytometry. Results are shown as the mean percentage ± standard error of the mean (SEM) of CD45⁺ CD11c⁺ cells from duplicate wells. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 compared to cells cultured only with GM-CSF + IL-4 (control group). Data shown are representative of two experiments. d CD45⁺ CD11c⁺ and e CD45⁺ CD11b⁺ F4/80⁺, the results are shown as the mean percentage ± standard error of the mean (SEM)

Fig. 2

The effects of RHS2 on ERK, STAT3 and p38 phosphorylation. a Western blotting was performed to analyze the phosphorylation levels of ERK, STAT3 and p38 in DC differentiation, which was significantly higher in GM-CSF/IL-4 + RHS2 compared to GM-CSF/IL-4. b–d Gel-Pro analyzer 4 was used to detect gray value and the results were defined as normalized gray value of various experiments in reference to the control group. Each experiment was performed independently three times. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

RHS2 inhibited the maturation of BMDC. a Expression of co-stimulatory molecules incubated with LPS or with RHS2 after 36 hours using flow cytometry, MFI levels are shown from three independent experiments. b The percentage of co-stimulatory and MHC-II molecule expression on CD45+ CD11c+ cells. Each experiment was performed independently three times. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; bars represent the mean of three independent experiments ± SD

Fig. 4

Production of IL-2, IFN-γ and TNF-α-from CD4⁺ and CD8⁺ T cells. a Co-cultured cells stained with anti-CD3⁺ and CD4⁺ markers were analyzed by flow cytometry to evaluate levels of IL-2, IL-4, IFN-γ and TNF-α. The percentage of IL-2-, IL-4-, IFN-γ- and TNF-α-producing cells was determined in live CD4⁺ cells. b Co-cultured cells stained with anti- CD3⁺ and CD8⁺ markers were analyzed by flow cytometry to evaluate levels of IL-2, IFN-γ and TNF-α. The percentage of IL-2-, IFN-γ- and TNF-α-producing cells was determined in live CD8⁺ cells. The data are expressed as the mean percentages of production cytokines in CD4⁺ and CD8⁺ cells from triplicate wells plus SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Cytotoxicity assay. OVA-CD8-activated CD8⁺ cytotoxic lymphocytes were used as effector cells, whereas the OVA-expressing B16F10 cells were used as target cells. Triton X-100 was added to the positive control group. The data are presented as the percent specific lysis of the target cells in a 2 h RHS2-stimulate assay. Each point represents the mean of triplicate cultures. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

Effect of RHS2 on serum anti-OVA antibody in mice immunized subcutaneously. The serum was collected 10 days after the third immunization for antibody subtype and total antibody titer evaluation. a Anti-OVA antibody IgG, IgG1, IgG2a, IgG2b, IgG3, IgM and IgE were measured by ELISA. b IgG2b/IgG1 ratio. Each data point represents the mean antibody titer ± SEM with n = 5. *P < 0.05, **P < 0.01