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. 2022 Apr 4;15(1):121.
doi: 10.1186/s13071-022-05240-8.

Regulatory effects of a novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae on mice immune cells

Jing-Yun Xu  1 , XiaoBin Gu  1 , Yue Xie  1 , Ran He  1 , Jing Xu  1 , Lang Xiong  1 , XueRong Peng  2 , GuangYou Yang  3
Affiliations

Regulatory effects of a novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae on mice immune cells

Jing-Yun Xu et al. Parasit Vectors. .

Abstract

Background: The giant panda (Ailuropoda melanoleuca) is a well-known, rare and endangered species. Baylisascaris schroederi is a pathogenic ascarid. Infection with B. schroederi may cause death in giant pandas. At present, the immune evasion mechanism of B. schroederi is little known. Cysteine protease inhibitors (CPI) play important roles in the regulation of host immune responses against certain nematodes. In this study, we focused on the analysis of the regulation of B. schroederi migratory larvae CPI (rBsCPI-1) on mice immune cells.

Methods: First, the pattern recognition receptors on the surface of peripheral blood mononuclear cells (PBMCs) and the signal pathways that transduce extracellular signals into the nucleus activated by rBsCPI-1 were identified. Then, the regulatory effects of rBsCPI-1 on PBMCs physiological activities were detected. Finally, the effects of rBsCPI-1 on TLR signaling pathway activation and NF-κB phosphorylation in mice immunized with recombinant protein were analysed.

Results: The results suggested that rBsCPI-1 secreted by B. schroederi migratory larvae is mainly recognized by TLR2 and TLR4 on PBMCs. Extracellular signals are transduced into the nucleus through the MAPK and NF-κB signaling pathways, enhancing the phagocytosis, migration, and apoptosis of PBMCs; meanwhile, rBsCPI-1 induces high expression of NO. Thus, rBsCPI-1 plays a role in immune regulation. In addition, the high expression of negative regulatory factors also ensured that TLR activation is maintained at the optimal level.

Conclusions: rBsCPI-1 can transduce regulatory signals into immune cells by activating the TLR2/4-NF-κB/MAPK signaling pathway, having a certain regulatory effect on the physiological activities. Meanwhile, rBsCPI-1 can maintain the immune response in a balance by limiting the over-activation of the TLRs signaling pathway and thus contributes to B. schroederi immune evasion.

Keywords: Baylisascaris schroederi; Cysteine protease inhibitor; Immune evasion mechanism; PBMC; TLRs signal pathway.

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Conflict of interest statement

The authors declare that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Figures

Fig. 1
Fig. 1
Binding of recombinant cysteine protease inhibitor of Baylisascaris schroederi migratory larvae (rBsCPI-1) with peripheral blood mononuclear cells (PBMCs) as detected by enzyme-linked immunosorbent assay (ELISA). A Binding of PBMC proteins at various coating concentrations when incubated with 1 µg/mL rBsCPI-1. B Binding of 1 µg/mL PBMC proteins when incubated with different concentrations of rBsCPI-1. Data are shown as mean ± SD of 3 replicates per group
Fig. 2
Fig. 2
Recombinant cysteine protease inhibitor of Baylisascaris schroederi migratory larvae (rBsCPI-1) affects the proliferation of peripheral blood mononuclear cells (PBMCs). Proliferation of PBMCs treated with ConA along or combined with rBsCPI-1 were measured by Cell Counting Kit 8 (CCK-8) assay. The OD450 values were considered as the cell proliferation index. PBMCs used for all replicates of distinct treatments in each experimental repetition were derived from the same mouse. Data are shown as mean ± SD of 3 replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001 for other groups vs 0 µg/mL group; or other groups vs 0 h group
Fig. 3
Fig. 3
Recombinant cysteine protease inhibitor of Baylisascaris schroederi migratory larvae (rBsCPI-1) affects the relative expression of Toll-like receptor (Tlr) genes in peripheral blood mononuclear cells (PBMCs). Data are shown as mean ± SD of 3 replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group
Fig. 4
Fig. 4
Western blotting detected the activation of the MAPK and NF-κB signaling pathways. A representative Western blotting is shown in A, and the graph of the quantified band density is shown in B. Data are shown as mean ± SD of 3 replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; §P < 0.05, §§P < 0.01, §§§P < 0.001 versus pET-32a group
Fig. 5
Fig. 5
Quantitative real-time PCR (qRT-PCR) detected the relative expression of the negative regulators of the Toll-like receptor (TLR) signaling pathway. Data are shown as mean ± SD of 3 replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; §P < 0.05, §§P < 0.01, §§§P < 0.001 versus pET-32a group
Fig. 6
Fig. 6
Recombinant cysteine protease inhibitor of Baylisascaris schroederi migratory larvae (rBsCPI-1) affects the apoptosis of peripheral blood mononuclear cells (PBMCs). Apoptosis of PBMCs was determined by staining with annexin V and PI followed by flow cytometry (A). The total apoptosis rate presented is representative of three independent experiments (B). qRT-PCR detected the transcription levels of pro-apoptotic genes (Fas, Bax) and anti-apoptotic genes (Bcl-2, Bcl-xL) (C). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; §P < 0.05, §§P < 0.01, §§§P < 0.001 versus pET-32a group
Fig. 7
Fig. 7
Recombinant cysteine protease inhibitor of Baylisascaris schroederi migratory larvae (rBsCPI-1) increased the phagocytosis of peripheral blood mononuclear cells (PBMCs). Phagocytosis of PBMCs was determined by phagocytosed FITC-dextran using flow cytometry (A). The percentage of FITC+ cells presented is representative of three independent experiments (B). The relative expression of pro-inflammatory and anti-inflammatory cytokines is shown in (C). Data are shown as mean ± SD of 3 replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; §P < 0.05, §§P < 0.01, §§§P < 0.001 versus pET-32a group
Fig. 8
Fig. 8
Effects of recombinant cysteine protease inhibitor of Baylisascaris schroederi migratory larvae (rBsCPI-1) on peripheral blood mononuclear cell (PBMC) migration. Cells were incubated with PBS (control), pET-32a, and rBsCPI-1. Then, the random migration was determined using a Neubauer counting chamber; the migration index was calculated as number of cells in the lower chamber/2 × 105 cells (A). The relative expression of chemokines (Ccl2, Cxcl10) is shown in B. Data are shown as mean ± SD of 3 replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; §P < 0.05, §§P < 0.01, §§§P < 0.001 versus pET-32a group
Fig. 9
Fig. 9
Effects of recombinant cysteine protease inhibitor of Baylisascaris schroederi migratory larvae (rBsCPI-1) on NO production by peripheral blood mononuclear cells (PBMCs). The NO concentration (µmol/L) was calculated using a standard curve. Data are shown as mean ± SD of 3 replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; §P < 0.05, §§P < 0.01, §§§P < 0.001 versus pET-32a group
Fig. 10
Fig. 10
Analysis of the activation of the Toll-like receptor (TLR) signaling pathway in vivo. The relative expression of Tlr2, Tlr4, and the negative regulators of the TLR pathway are shown in A. A representative western blotting is shown in B, and the graph of the quantified band density is shown in C. Data are shown as mean ± SD of 3 replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; §P < 0.05, §§P < 0.01, §§§P < 0.001 versus pET-32a group
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