The Recombinant E g.P29-Mediated miR-126a-5p Promotes the Differentiation of Mouse Naive CD4+ T Cells via DLK1-Mediated Notch1 Signal Pathway

Abstract

Cystic echinococcosis (CE) is a zoonotic parasitic disease spread worldwide caused by Echinococcus granulosus (Eg), which sometimes causes serious damage; however, in many cases, people are not aware that they are infected. A number of recombinant vaccines based on Eg are used to evaluate their effectiveness against the infection. Our previous report showed that recombinant Eg.P29 (rEg.P29) has a marvelous immunoprotection and can induce Th1 immune response. Furthermore, data of miRNA microarray in mice spleen CD4⁺ T cells showed that miR-126a-5p was significantly elevated 1 week after immunization by using rEg.P29. Therefore, in this perspective, we discussed the role of miR-126a-5p in the differentiation of naive CD4⁺ T cells into Th1/Th2 under rEg.P29 immunization and determined the mechanisms associated with delta-like 1 homolog (DLK1) and Notch1 signaling pathway. One week after P29 immunization of mice, we found that miR-126a-5p was significantly increased and DLK1 expression was decreased, while Notch1 pathway activation was enhanced and Th1 response was significantly stronger. The identical conclusion was obtained by overexpression of mmu-miR-126a-5p in primary naive CD4⁺ T cells in mice. Intriguingly, mmu-miR-126a-5p was significantly raised in serum from mice infected with protoscolex in the early stages of infection and markedly declined in the late stages of infection, while has-miR-126-5p expression was dramatically reduced in serum from CE patients. Taken together, we show that miR-126a-5p functions as a positive regulator of Notch1-mediated differentiation of CD4⁺ T cells into Th1 through downregulating DLK1 in vivo and in vitro. Hsa-miR-126-5p is potentially a very promising diagnostic biomarker for CE.

Keywords: CD4+ T cells; Cystic Echinococcocosis; DLK1; Notch1; Th1; Th2; miR-126a-5p; rEg.P29.

Figure 1

Upregulation of miR-126a-5p expression in splenic CD4+ T cells 1 week after rEg.P29 immunization in mice. (A) rEg.P29 protein in SDS-PAGE: M, protein markers; line 1, Escherichia coli lysates; line 2, induction of E. coli lysates with IPTG; line 3, purified rEg.P29. (B) Flow assay of IFN-g and IL-4 expression levels in splenic CD4+ T cells 1 week after rEg.P29 immunization. (C) Statistical graph of flow analysis of IFN-g and IL-4. (D) Ratio of splenic CD4+T cells to CD8+ T cells 1 week after rEg.P29 immunization. (E) Expression levels of IFN-g, T-bet, IL-4, and GATA-3 in splenic CD4+ T cells after 1 week of rEg.P29 immunization by qRT-PCR. (F) Flow sorting of CD4+ T cells, CD8+ T cells, and B cells from spleen lymphocytes. (G) Expression of miR-126a-5p in CD4+ T cells, CD8+ T cells, and B cells was detected by qRT-PCR. **P<0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Figure 2

miR-126a-5p promotes the differentiation of naive CD4⁺ T cells into Th1. (A) The purification rate of naive CD4⁺ T cells from lymphocytes sorted by magnetic beads with flow cytometry. (B) Expression of IFN-γ, T-bet, IL-4, and GATA-3 in naive CD4⁺ T cells transfected with miR-126a-5p mimics or miR-126a-5p inhibitor was examined by qRT-PCR. (C) Flow cytometry determination of activation indicators CD25 and CD69 after transfection with miR-126a-5p mimics or miR-126a-5p inhibitor in naive CD4⁺ T cells. (D) CFSE assay of the proliferation levels in naive CD4⁺ T cells after transfection with miR-126a-5p mimics or miR-126a-5p inhibitor. (E) Flow cytometry measurement of IFN-γ and IL-4 expression after transfection with miR-126a-5p mimics or miR-126a-5p inhibitor in naive CD4⁺ T cells. (F) ELISA detection of IFN-γ, TFN-α, IL-2, IL-4, IL-5, and IL-10 expression in culture supernatants after transfection with miR-126a-5p mimics or miR-126a-5p inhibitor in naive CD4⁺ T cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

miR-126a-5p regulates the Notch1 signaling pathway by targeting DLK1. (A) Predicted binding site of miR-126a-5p on DLK1. (B) The luciferase activity of mir-126a-5p binding to DLK1 was measured by dual luciferase reporter gene. (C) DLK1 mRNA expression after transfection with miR-126a-5p mimics or miR-126a-5p inhibitor in naive CD4⁺ T cells determined by qRT-PCR. (D) DLK1 protein expression after transfection with miR-126a-5p mimics or miR-126a-5p inhibitor in naive CD4⁺ T cells by Western blot. (E) Notch1 and HES1 mRNA expression after transfection with miR-126a-5p mimics or miR-126a-5p inhibitor in naive CD4⁺ T cells determined by qRT-PCR. (F) The protein expression of Notch1, N1ICD, DLK1, HES1, and HES5 after transfection with miR-126a-5p mimics or miR-126a-5p inhibitor in naive CD4⁺ T cells by Western blot. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

DLK1 modulates naive CD4⁺ T-cell differentiation through the Notch1 signaling pathway. (A) Notch1 and HES1 mRNA expression after transfection with OE-DLK1 or DLK1 siRNA in naive CD4⁺ T cells determined by qRT-PCR. (B) The protein expression of Notch1, N1ICD, DLK1, HES1, and HES5 after transfection with OE-DLK1 or DLK1 siRNA in naive CD4⁺ T cells by Western blot. (C) qRT-PCR detection of mRNA expression of IFN-γ, T-bet, IL-4, and GATA-3 after transfection with OE-DLK1 or DLK1 siRNA in naive CD4⁺ T cells. (D) Expression of IFN-γ and IL-4 after transfection with OE-DLK1 or DLK1 siRNA in naive CD4⁺ T cells determined by flow cytometry. (E) The expression of IFN-γ, TNF-α, IL-2, IL-4, IL-5, and IL-10 after transfection with OE-DLK1 or DLK1 siRNA in naive CD4⁺ T cells measured by ELISA. (F) qRT-PCR detection of mRNA expression of IFN-γ, T-bet, IL-4, and GATA-3 after transfection with DAPT in naive CD4⁺ T cells. (G) Expression of IFN-γ and IL-4 after transfection with DAPT in naive CD4⁺ T cells determined by flow cytometry. (H) The expression of IFN-γ, TNF-α, IL-2, IL-4, IL-5, and IL-10 after transfection with DAPT in naive CD4⁺ T cells measured by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

miR-126a-5p and DLK1 together can rescue the naive CD4⁺ T-cell differentiation induced by their separate actions. (A) The mRNA expression of IFN-γ, T-bet, IL-4, and GATA-3 after transfection with miR-126a-5p mimics+OE-DLK1 or miR-126a-5p inhibitor+DLK1 siRNA in naive CD4⁺ T cells performed by qRT-PCR. (B) The expression of IFN-γ and IL-4 after transfection with miR-126a-5p mimics+OE-DLK1 or miR-126a-5p inhibitor+DLK1 siRNA in naive CD4⁺ T cells conducted by flow cytometry. (C) The expression of IFN-γ, TNF-α, IL-2, IL-4, IL-5, and IL-10 after transfection with miR-126a-5p mimics+OE-DLK1 or miR-126a-5p inhibitor+DLK1 siRNA in naive CD4⁺ T cells measured by ELISA. (D) Notch1 and HES1 mRNA expression after transfection with miR-126a-5p mimics+OE-DLK1 or miR-126a-5p inhibitor+DLK1 siRNA in naive CD4⁺ T cells determined by qRT-PCR. (E) The protein expression of Notch1, N1ICD, DLK1, HES1, and HES5 after transfection with miR-126a-5p mimics+OE-DLK1 or miR-126a-5p inhibitor+DLK1 siRNA in naive CD4⁺ T cells by Western blot. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Figure 6

Reduced DLK1 expression and enhanced Notch1 signaling pathway in spleen CD4⁺ T cells from rEg.P29-immunized mice. (A) Identification of DLK1, Notch1, and HES1 mRNA expression in naive CD4⁺ T cells after rEg.P29 immunization in mice 1 week after qRT-PCR. (B) Western blot detection of DLK1, Notch1, N1ICD, HES1, and HES5 protein expression in CD4⁺ T cells 1 week after rEg.P29 immunization in mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7

Peripheral blood serum miR-126a-5p was elevated in the early stages of infection with Echinococcus granulosus and declined in the late stages of infection. (A) Examination of miR-126a-5p expression in peripheral blood serum of rEg.P29-immunized mice after 1 and 24 weeks by qRT-PCR. (B) Anatomical view of protoscolex intraperitoneally infected mice and PBS control mice after 24 weeks. (C) Protoscolex isolated from the cyst of a CE patient, magnification 20 × 10. (D) Analysis of peripheral blood serum miR-126a-5p expression in protoscolex intraperitoneally infected mice after 1 and 24 weeks by qRT-PCR. (E) Determination of serum miR-126a-5p expression in the peripheral blood of CE patients (n = 12) and healthy controls (n = 16) by qRT-PCR. *P < 0.05, **P < 0.01,***P < 0.001; ns, not significant.