Ex vivo observation of Pythium insidiosum-antigen treated neutrophils on three Pythium insidiosum strains isolated from vascular pythiosis patients

ABSTRACT

The mechanisms of Pythium insidiosum-antigen (PIA) immunotherapy activating a patient’s immune system are unknown. We evaluated the interleukin-8 (IL-8) serum levels during P. insidiosum infection and after vaccination with PIA in vascular pythiosis cases. Furthermore, we studied the anti-P. insidiosum activity of neutrophils stimulated with various concentrations of PIA ex vivo in 3 strains of P. insidiosum isolated from vascular pythiosis patients. IL-8 serum levels were evaluated using the ELISA technique. We assessed the effect of PIA-stimulated neutrophils on the viability of zoospores using MTT assay, visualized neutrophil extracellular trap (NET) formation via microscopy, and measured the levels of double-stranded DNA (dsDNA) using PicoGreen dsDNA quantitation assay in 3 strains of P. insidiosum isolated from vascular pythiosis patients. Serum levels of IL-8 gradually lowered from the early to the end phases of vaccination with PIA among the surviving group of vascular pythiosis cases. Neutrophils stimulated with 0.01 µg/ml PIA reduced zoospore viability significantly compared to PIA-unstimulated neutrophils for strain 1 and strain 3 (p < .05). Neutrophils stimulated with 0.01, 0.1, 1, and 10 µg/ml PIA exhibited significantly lower zoospore viability than PIA-unstimulated neutrophils for strain 2 (p < .05). IL-8 can be used as a biomarker for monitoring vascular pythiosis cases treated with the PIA vaccine. Also, anti-P. insidiosum activity of PIA-stimulated neutrophils was probably due to the disruption of cellular activity in zoospores rather than the mechanisms based on the formation of NETs.

KEYWORDS:

• MTT
• NET
• dsDNA
• zoospores
• PIA
• ELISA
• IL-8

Author’s contributions

Conception and design: AS, NP, and NW; drafting of the paper: SM; revising it critically for intellectual content: SM, NP, PT, and RP; final approval of the version to be published: SM, NP, PT, RP, and AC. All authors have read and agreed to the published version of the article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research project was supported by the Second Century Fund (C2F), Chulalongkorn University and Thailand Science Research and Innovation Fund, Chulalongkorn University [CU_FRB66_HEA663700096]. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.