Regulatory effect of two Trichinella spiralis serine protease inhibitors on the host's immune system

Abstract

Trichinella spiralis (T. spiralis) is widely distributed throughout the world and can cause serious zoonotic parasitic diseases. Serine protease inhibitors (SPIs) have unique enzyme inhibitory activity and occupy an important position in the interaction between parasites and hosts. In order to further understand the immunoprotective effect of SPIs on T. spiralis invasion in vivo, the Kazal and Serpin type SPI of T. spiralis (TsKaSPI and TsAdSPI) were mixed with Freund's adjuvant in equal volume to immunize mice. The results showed that the expression of IgG1 and IgG2a in serum, the proliferation of spleen cells, and the expression level of cytokines were all increased. The results of flow cytometry showed that the expression of CD4+CD25+Foxp3+ Tregs, CD8+CD28- T cells, CD19+CD5+CD1d^hi Bregs in spleen were also increased. Therefore, both TsKaSPI and TsAdSPI could induce strong humoral and cellular immune responses. And the results of adult reduction rate and pathological changes of intestine after adult invasion also indicated that both TsKaSPI and TsAdSPI could prevent T. spiralis from invading intestine. To explore the regulatory effects of TsKaSPI and TsAdSPI on the immune function of macrophage, the results of ELISA showed that the expression of cytokines in cell supernatant were increased. And the results of Western blot showed that both TsKaSPI and TsAdSPI could induce phosphorylation of JAK2 and STAT3 receptors, thereby affecting the signal transduction of macrophages. This experiment demonstrated that SPIs could act as effector molecules affecting the immune function of host when infected with T. spiralis.

Figure 1

Proliferation of spleen cells extracted from each group after ConA, RPMI-1640, recombinant protein stimulation in vitro. Data are shown as mean ± SD of 3 mice per group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus PBS group; ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001 versus HT-TsKaSPI group; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus FCA/FIA group; ^§P < 0.05, ^§§P < 0.01, ^§§§P < 0.001 for TsKaSPI vs TsAdSPI; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 for ConA, TsKaSPI/TsAdSPI vs RPMI-1640 in the same group; ^¥P < 0.05, ^¥¥P < 0.01, ^¥¥¥P < 0.001 for ConA vs TsKaSPI/TsAdSPI in the same group.

Figure 2

Demonstration of the gating strategy for the flow cytometric analysis of mouse CD4 CD25+Foxp3+ Treg (a), CD8+CD28− T cell (b), CD19+CD5+CD1d^hi Breg (c) from spleen. In this experiment a single cell suspension was prepared from the spleen of each group and stained with CD4 (FITC), CD25 (APC), Foxp3 (PE), CD8 (PE), CD28 (APC), CD19 (FITC), CD5 (APC), CD1d (PE) based on surface and intracellular staining protocols, respectively. Data were collected with FACSDiva flow cytometer and analyzed. Lymphocytes are identified by their scatter properties (FSC-A × SSC-A plot).

Figure 3

The percentage of CD4+ T lymphocytes in the total lymphocytes in the gate (a), the percentage of CD4+CD25+Foxp3+ Tregs in the total lymphocytes in the gate (b) and the percentage of CD4+ T lymphocytes in CD4+CD25+Foxp3+ Tregs (c) in spleens of five groups were showed in (A); the percentage of CD8+CD28− T cells in the total lymphocytes in the gate were showed in (B); the rate of CD4+/CD8+ T cells in spleens of five groups were showed in (C); and the percentage of CD19+ B lymphocytes in the total lymphocytes in the gate (a), the percentage of CD19+CD5+CD1d^hi+ Bregs in the total lymphocytes in the gate (b) and the percentage of CD19+ B lymphocytes in CD19+CD5+CD1d^hi Bregs (c) in spleens of five groups were showed in (D). Data are shown as mean ± SD of 3 mice per group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus PBS group; ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001 versus HT-TsKaSPI group; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus FCA/FIA group; ^§P < 0.05, ^§§P < 0.01, ^§§§P < 0.001 for TsKaSPI vs TsAdSPI.

Figure 4

Analyze changes of IgG subtypes: IgG1 and IgG2a. Data are shown as mean ± SD of 3 mice per group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus PBS group; ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001 versus HT-TsKaSPI group; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus FCA/FIA group; ^§P < 0.05, ^§§P < 0.01, ^§§§P < 0.001 for TsKaSPI vs TsAdSPI; ^☆P < 0.05, ^☆☆P < 0.01, ^☆☆☆P < 0.001 for IgG1 vs IgG2a in the same group.

Figure 5

The expression changes of IL-1β, IL-4, IL-6, IL-10, IL-12, IFN-γ, TNF-α and TGF-β were detected by ELISA. Data are shown as mean ± SD of 3 mice per group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus PBS group; ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001 versus HT-TsKaSPI group; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus FCA/FIA group; ^§P < 0.05, ^§§P < 0.01, ^§§§P < 0.001 for TsKaSPI vs TsAdSPI.

Figure 6

The number of adults detected and the adult reduction rate were showed in (A). Data are shown as mean ± SD of 3 mice per group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus PBS group; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus FCA/FIA group; ^§P < 0.05, ^§§P < 0.0, ^§§§P < 0.001 for TsKaSPI vs TsAdSPI; ^☆P < 0.05, ^☆☆P < 0.01, ^☆☆☆P < 0.001 for each group on the 3rd, 7th, 10th day vs the corresponding group on the 1st day; ^◇P < 0.05, ^◇◇P < 0.01, ^◇◇◇P < 0.001 for each group on the 7th, 10th day vs the corresponding group on the 3rd day; ^※P < 0.05, ^※※P < 0.01, ^※※※P < 0.001 for each group on the 10th day vs the corresponding group on the 7th day. Light micrograph of HE-stained colonic section were showed in (B) (a). Scale bar represents 200 μm. And the pathological score were showed in (B) (b). TsKaSPI group and TsAdSPI group showed significant improvement than the PBS group.

Figure 7

The expression changes of IL-1β, IL-4, IL-6, IL-10, IL-12, IFN-γ, TNF-α and TGF-β in the cell supernatant were showed in (A), and the representative gel of the Western Blot were shown in (B) (a), and the graph of the quantified band density were also shown in (B) (b). Data are shown as mean ± SD of 3 mice per group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus PBS group; ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001 versus HT-TsKaSPI group; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus FCA/FIA group; ^§P < 0.05, ^§§P < 0.01, ^§§§P < 0.001 for TsKaSPI vs TsAdSPI.

Figure 8

SDS-PAGE analysis of the expression products of before and after purification. M: Protein Marker; 1: Unpurified Protein; 2: Purified Protein.