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Excretory-secretory product of third-stage Gnathostoma spinigerum larvae induces apoptosis in human peripheral blood mononuclear cells

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Abstract

Human gnathostomiasis caused by third-stage Gnathostoma spinigerum larvae (G. spinigerum L3) is an important zoonotic disease in tropical areas of the world. The excretory-secretory products (ES) that are excreted by infective larva play a significant role in host immune evasion and tissue destruction. To investigate the poorly understood mechanisms of G. spinigerum L3 pathogenesis, we focused on the potential effect of ES on inducing apoptosis in human immune cells by using human peripheral blood mononuclear cells (PBMCs) as a model. Early and late apoptosis of PBMCs were assessed following the exposure of these cells to G. spinigerum L3 ES (0.1, 0.5, and 1.0 μg/ml) for 6–48 h. The apoptotic cells were identified by flow cytometric staining of PBMC with FITC-annexin V and propidium iodide. The expression of regulatory genes related to apoptosis mechanisms in ES-treated PBMCs was investigated using a Human Apoptosis RT2 Profiler™ PCR Array. The results showed significant levels of early phase apoptosis at 18 h and of late phase apoptosis at 24 h. We speculate that this apoptosis in PBMCs occurs via the extrinsic pathway. Apoptosis in the ES-induced PBMCs was observed as quickly as 90 min after exposure, and the highest effect was observed at 18–24 h. Furthermore, ES can trigger apoptosis lasting for 48 h. Our findings expand the understanding of one of the mechanisms involved, immune-evasive strategy mechanism used by G. spinigerum larvae during human gnathostomiasis.

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Acknowledgements

This study was supported by a Research Grant from the Faculty of Tropical Medicine, Mahidol University, Fiscal Year 2013–2014 for YM. It was also supported in part by Grant-in-Aids from the Ministry of Health, Labor, and Welfare of Japan (No. H20-Shinko-Ippan-015) to YE and from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Kiban B, Overseas, No. 20401050) to YE. The authors gratefully acknowledge the Mahidol-Oxford Tropical Medicine Research Unit (MORU) for providing access to the FACSCalibur flow cytometer and CellQuest software. We thank Mr. Paul R. Adams for English proofreading and Katie E. Oakley, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a later draft of this manuscript.

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Authors and Affiliations

  1. Parasitology Unit, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand

    Nareerat Viseshakul

  2. Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand

    Wilanee Dechkhajorn & Yaowapa Maneerat

  3. Center of Excellence for Antibody Research, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand

    Surachet Benjathummarak

  4. Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand

    Supaporn Nuamtanong

Authors
  1. Nareerat Viseshakul
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  2. Wilanee Dechkhajorn
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  3. Surachet Benjathummarak
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  4. Supaporn Nuamtanong
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  5. Yaowapa Maneerat
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Contributions

SN was responsible for the collection of G. spinigerum L3 cultures and preparation of ES. YM and WD were responsible for laboratory work, PBMC culture, and sample preparation for all experiments, and they carried out the FACS analysis. NV and SB contributed to the microarray experiment study design, performed the RNA extraction, and carried out the real-time PCR assays and analyses. WD was responsible for data analysis and statistical analysis. YM and NV participated in the study design, conceived the study, participated in its design and coordination, and contributed to writing the manuscript. All authors interpreted the results and have read and approved the final manuscript. The funding was granted to YM and YE.

Corresponding author

Correspondence to Yaowapa Maneerat.

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The authors declare that they have no competing interests.

Electronic supplementary material

Supplementary Fig. 1

Profile of gene expression and their functions in PBMC cultured with G. spinigerum L3 ES at the dose of 0.5 μg/ml for 30 min in comparison to those in medium alone. (JPG 908 kb)

Supplementary Fig. 2

Profile of gene expression and their functions in PBMC cultured with G. spinigerum L3 ES at the dose of 0.5 μg/ml for 60 min in comparison to those in medium alone. (JPG 920 kb)

Supplementary Fig. 3

Profile of gene expression and their functions in PBMC cultured with G. spinigerum L3 ES at the dose of 0.5 μg/ml for 90 min in comparison to those in medium alone. (JPG 935 kb)

Supplementary Fig. 4

Profile of gene expression and their functions in PBMC cultured with G. spinigerum L3 ES at the dose of 0.1 μg/ml for 3 h in comparison to those in medium alone. (JPG 928 kb)

Supplementary Fig. 5

Profile of gene expression and their functions in PBMC cultured with G. spinigerum L3 ES at the dose of 0.5 μg/ml for 3 h in comparison to those in medium alone. (JPG 924 kb)

Supplementary Table 1

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Viseshakul, N., Dechkhajorn, W., Benjathummarak, S. et al. Excretory-secretory product of third-stage Gnathostoma spinigerum larvae induces apoptosis in human peripheral blood mononuclear cells. Parasitol Res 116, 2783–2794 (2017). https://doi.org/10.1007/s00436-017-5589-5

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