Pythium insidiosum-antigen enhances neutrophil-mediated killing of zoospores

Abstract

Pythium insidiosum-antigen (PIA) immunotherapy has been used to treat human pythiosis. This study compared PIA-stimulated and unstimulated neutrophils on zoospore viability of P. insidiosum strains. We cultured and collected zoospores of 6 P. insidiosum strains, CBS 777.84, ATCC 58643, ATCC 90586, PEC1, PC10, and CBS 101039. PIA concentrations of 0.01, 0.1, 1, and 10 µg/ml were prepared and were used to stimulate neutrophils isolated from healthy volunteers. The MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay was used to calculate the zoospore viability of P. insidiosum strains. Neutrophils stimulated with 0.01 or 0.1 µg/ml PIA, or both, showed a significant reduction in the viability of zoospores of CBS 777.84, ATCC 58643, CBS 101039, PC10, and PEC1 strains. Furthermore, 1 µg/ml PIA-induced neutrophils elicited a significant decrease in the viability of zoospores of ATCC 58643, CBS 101039, and PC10 strains. However, a higher dose of PIA (10 µg/ml) did not demonstrate superiority in reducing the zoospore viability of all six strains. Our findings suggest that PIA immunotherapy improves the zoospore-killing activity of neutrophils, and neutrophils might be involved in the forefront mechanism responsible for the beneficial effects of PIA immunotherapy.

Introduction

Human pythiosis, caused by the pathogenic oomycete Pythium insidiosum, is an underdiagnosed and difficult-to-treat infectious disease with high morbidity and mortality¹. Human pythiosis is clinically classified into four groups: vascular pythiosis, ocular pythiosis, cutaneous or subcutaneous pythiosis, and disseminated pythiosis². Most pythiosis cases are reported in tropical, subtropical, and temperate areas, with temperatures between 28 and 37 °C, which is optimal for P. insidiosum growth³. The motile flagellate zoospores of P. insidiosum attach to the damaged tissue and cause infection by invading the host tissue through germination into hyphae that can start a new colonization⁴. The P. insidiosum-antigen (PIA) immunotherapy, commonly administered in combination with surgery and antimicrobials, has shown promising results in treating different forms of human pythiosis^5–10.

During pythiosis, antigens released by P. insidiosum hyphae trigger a T-helper 2 immune response through degranulation of eosinophils and mast cells around the hyphae, responsible for the extensive tissue damage in the host¹¹. A postulated mechanism of action of PIA immunotherapy is the recruitment of cytotoxic T lymphocytes to kill P. insidiosum by switching T-helper 2 lymphocytes to T-helper 1 lymphocytes⁹. Although PIA has proved beneficial in human pythiosis, there is limited research into how it empowers a patient’s immune system to eliminate P. insidiosum.

The PIA vaccine used in our study is derived from culture filtrate antigen (CFA) and soluble proteins prepared from P. insidiosum strain¹². β-glucans are immunostimulants and major constituents of the cell wall of P. insidiosum¹³. Dectin-1 is a major pattern recognition receptor for β-glucans, expressed by various leukocytes, including neutrophils^14,15. Recent evidence suggests that healthy human neutrophils can kill P. insidiosum zoospores and stimulate neutrophil extracellular traps (NETs) against P. insidiosum zoospores¹⁶. Recent data have also indicated that P. insidiosum zoospores induce interleukin 8 (IL-8) mRNA expression¹⁷. IL-8 recruits and activates neutrophils at the site of infection, where they eliminate pathogens¹⁸. Therefore, understanding PIA immunotherapy in neutrophil activation and its response to P. insidiosum is of great scientific and clinical interest.

We postulated that neutrophils stimulated with PIA may show increased anti-P. insidiosum response compared to the unstimulated neutrophils. Thus, we compared PIA-stimulated neutrophils and unstimulated neutrophils on zoospore viability in six P. insidiosum strains using MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay.

Results

We optimized the MTT assay and validated it in the laboratory to assess zoospore viability in the PIA-stimulated neutrophils. Neutrophils were treated with PIA concentrations of 0.01, 0.1, 1, and 10 µg/ml to demonstrate their killing effect against the zoospores of the six P. insidiosum strains. The key findings of MTT assay across the six P. insidiosum strains are summarized in Table 1 and compared in Fig. 1.

Table 1.

Effect of PIA-stimulated neutrophils on the zoospore viability of 6 P. insidiosum strains.

P. insidiosum strain	Zoospore viability inference
CBS 777.84	PIA-stimulated neutrophils significantly reduced the number of viable zoospores
ATCC 58643	Neutrophils significantly increased zoospore killing after treatment with PIA
ATCC 90586	PIA-stimulated neutrophils were not efficient in killing zoospores compared to unstimulated neutrophils
PEC1	Neutrophils treated with low doses of PIA were effective in killing zoospores
PC10	PIA-treated neutrophils exhibited significantly higher zoospore killing
CBS 101039	PIA was effective in inducing neutrophils to kill zoospores

Fig. 1.

MTT viability assay results performed on zoospores of (a) CBS 777.84, (b) ATCC 58643, (c) ATCC 90586, (d) PEC1, (e) PC10, and (f) CBS 101039 with neutrophils stimulated with various concentrations of PIA. * indicates a comparison between PIA-stimulated and unstimulated neutrophils. Error bars denote ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

CBS 777.84

We observed that neutrophils stimulated with PIA concentrations of 0.01, 0.1, 1, and 10 µg/ml significantly reduced the number of zoospores compared with the control (P < 0.01 for 0.01 and 0.1 µg/ml PIA; P < 0.05 for 1 and 10 µg/ml PIA) (Fig. 1a). Treatment of neutrophils with 0.01 and 0.1 µg/ml of PIA significantly reduced the number of zoospores compared with the untreated neutrophils (P < 0.05). Meanwhile, neutrophils stimulated with 1 and 10 µg/ml PIA did not significantly reduce the viability of zoospores compared to the untreated neutrophils (P > 0.05). Also, untreated neutrophils did not significantly reduce the viability of zoospores compared to the control (P > 0.05).

ATCC 58643

Neutrophils treated with all concentrations of PIA and untreated neutrophils significantly reduced the viability of zoospores compared to the control (P < 0.01 for untreated neutrophils and 1 µg/ml PIA; P < 0.001 for 0.01, 0.1, and 10 µg/ml PIA) (Fig. 1b). Neutrophils treated with the PIA concentrations of 0.01, 0.1, and 1 µg/ml showed a significant reduction in viable zoospores compared to the untreated neutrophils (P < 0.05 for 0.01 and 1 µg/ml PIA; P < 0.01 for 0.1 µg/ml PIA). However, neutrophils treated with 10 µg/ml PIA did not significantly reduce the viability of zoospores compared to the untreated neutrophils (P > 0.05).

ATCC 90586

Both PIA-treated and untreated neutrophils significantly decreased the viability of zoospores compared to the control (P < 0.01) (Fig. 1c). Neutrophils stimulated with PIA did not exhibit superior zoospore killing compared to the unstimulated neutrophils (P > 0.05).

PEC1

Neutrophils stimulated with all concentrations of PIA significantly reduced the viability of zoospores compared to control (P < 0.01 for 0.01 and 0.1 µg/ml of PIA; P < 0.5 for 1 and 10 µg/ml PIA) (Fig. 1d). Neutrophils treated with 0.01 and 0.1 µg/ml of PIA showed a significant decrease in the viability of zoospores compared to the untreated neutrophils (P < 0.01). However, neutrophils treated with 1 and 10 µg/ml PIA did not significantly reduce the viability of zoospores compared to the untreated neutrophils (P > 0.05). Also, untreated neutrophils did not significantly reduce zoospore viability compared to the control (P > 0.05).

PC10

Neutrophils stimulated with PIA concentrations of 0.01, 0.1, and 1 µg/ml showed a significant reduction in viable zoospores compared to the control (P < 0.5 for 0.01 µg/ml PIA; P < 0.01 for 0.1 and 1 µg/ml PIA) (Fig. 1e). Neutrophils stimulated with PIA concentrations of 0.01 and 1 µg/ml showed a significant decrease in viable zoospores compared to the unstimulated neutrophils (P < 0.05). Neutrophils stimulated with 10 µg/ml PIA and unstimulated neutrophils did not significantly reduce the viability of zoospores compared to control (P > 0.05).

CBS 101039

Neutrophils stimulated with 0.01, 0.1, and 1 µg/ml PIA showed significantly reduced zoospore viability compared to the unstimulated neutrophils and control (P < 0.01 for 0.01 and 1 µg/ml PIA; P < 0.001 for 0.1 µg/ml PIA) (Fig. 1f). We did not observe a significant reduction in the viability of zoospores with the neutrophils treated with 10 µg/ml PIA and untreated neutrophils compared to the control (P > 0.05).

Discussion

P. insidiosum-derived antigens have been used in immunotherapy to stimulate a protective response against human pythiosis^8,11. Neutrophils are the first innate immune defense line against invading microorganisms¹⁹. Neutrophils have displayed the killing of P. insidiosum through phagocytosis and the formation of NETs¹⁶. To continue unraveling the protective effect of neutrophils in human pythiosis, we investigated whether neutrophils stimulated with PIA show improved killing activity against P. insidiosum zoospores. In this study, we found that the neutrophils stimulated with PIA reduced the viability of P. insidiosum zoospores compared to the unstimulated neutrophils and that the zoospore killing efficacy of PIA-stimulated neutrophils is both strain-specific and dose-specific.

In the present study, we optimized the MTT assay with suitable concentrations of zoospores and incubation time because neutrophils are short-lived cells²⁰, and zoospores of P. insidiosum rapidly develop germ tube that elongates into a filament²¹. A previous study in our laboratory has shown that neutrophils in all six strains of P. insidiosum (CBS 777.84, ATCC 58643, ATCC 90586, PEC1, PC10, and CBS 101039) reduced zoospore viability in Trypan Blue staining assay¹⁶. In this study, PIA-unstimulated neutrophils did not reduce zoospore viability in CBS 777.84, PEC1, PC10, and CBS 101039 strains. Unstimulated neutrophils were only found to decrease the zoospore viability in ATCC 58643 and ATCC 90586 strains. The MTT analysis showed that neutrophils stimulated with PIA were superior to unstimulated neutrophils in anti-P. insidiosum activity (Fig. 1). However, the neutrophils stimulated with the highest concentration of PIA (10 µg/ml) used in our study did not exhibit superiority in reducing the zoospore viability of all six strains compared to unstimulated neutrophils. It can be argued that over-activating neutrophils with higher PIA concentrations might lead to decreased anti-P. insidiosum activity of neutrophils. Overall, the results have shown that neutrophils stimulated with lower concentrations of PIA (0.01, 0.1, and 1 µg/ml) used in our study achieve a higher zoospore killing of respective P. insidiosum strains compared to unstimulated neutrophils, except in ATCC 90586. Our study evaluated the viability results in individual strains because P. insidiosum isolates are from different hosts and geographical regions and are placed in separate clades^22,23.

The MTT tetrazolium assay has been widely adopted for determining cell viability due to the simplicity and sensitivity of this assay²⁴. Unavoidable cell death and the additional dissolving step necessary for measuring formazan absorbance may lead to a false estimation of cell number²⁵. Therefore, our findings must still be verified through multiple experiments in multiple strains in further studies to arrive at the same conclusions. However, we observed lower variability in our viability data, implying that fewer experimental repeats would be required with this MTT assay to obtain reliable results.

Previous studies in P. insidiosum have utilized different inoculum types, such as hyphal plug, hyphal suspension, and zoospores^26–28. In a study investigating the potential of dendritic cells (DCs) to induce cellular immunity against P. insidiosum, (1,3)(1,6)-β-glucan, PitiumVac^®, and heat-inactivated zoospores were used as antigens to pulse DCs²⁹. Although the hyphae is the morphological structure invading host tissues, using zoospores as inoculum in the experiments does not influence the anti-P. insidiosum activity results compared to results when the hyphae are used as an inoculum because β-glucan is present in hyphae and zoospores^13,21. P. insidiosum zoospores can be produced in the laboratory, however, generating zoospores for experimental studies is troublesome because of the difficulty in inducing zoosporogenesis, low abundance of zoospores, and longer generation time of zoospores²⁷.

This study has some limitations. Firstly, we collected blood and isolated neutrophils from healthy volunteers; however, neutropenia is reported to be associated with poor prognosis in patients with vascular pythiosis². Generalizing results from healthy people in our study is challenging because of the differences in patient characteristics among those infected with pythiosis and receiving PIA immunotherapy. Secondly, we did not evaluate and compare the viability of P. insidiosum zoospores with other viability assessment methods. Thus, our study can be considered a preliminary attempt, and large-scale research must be conducted to validate our findings.

This study is the first to show the zoospores-killing ability of neutrophils stimulated with PIA in P. insidiosum strains, CBS 777.84, ATCC 58643, ATCC 90586, PEC1, PC10, and CBS 101039, using MTT assay. In addition, we demonstrated that neutrophils stimulated with PIA are superior to the unstimulated neutrophils in killing zoospores of specific P. insidiosum strains. We, therefore, conclude that PIA immunotherapy in human pythiosis improves the immune response of the innate arm in protection against P. insidiosum. Further extensive studies must be performed to evaluate the immune mechanisms of PIA immunotherapy in different strains and clades of P. insidiosum for clinical translation.

Methods

Isolation of human neutrophils

We collected peripheral blood from healthy volunteers (n = 3), and blood was placed in an anticoagulant tube. Blood was processed within 15 min after withdrawal. The procedure involved density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to isolate high-purity neutrophils. Whole blood was treated with Polymorphprep™ (Axis-shield, Norway) in a 1:1 ratio. Blood was spun down at 1700 rpm for 30 min. The top three plasma layers, monocytes, and Polymorphprep™ were discarded after centrifugation. The supernatant containing neutrophils was transferred to a clean tube, and the cells were washed with RPMI-1640 medium with 25 mM HEPES and 2 mM L-Glutamine supplemented with 10% fetal bovine serum (centrifuged at 2000 rpm for 5 min). Subsequently, RBC Lysis Buffer was added to the cell pellet and mixed by inversion. The RBC Lysis Buffer contained 13.4 mM KHCO₃, 155 mM NH₄Cl, and 96.7 µM EDTA diluted to 1X working concentration with RPMI 1640 (containing 25 mM HEPES and 2 mM L-glutamine). Cells were spun down at 2000 rpm for 5 min. The cell pellet was gently resuspended in 1 ml of RPMI 1640 medium with 25 mM HEPES and 2 mM L-Glutamine supplemented with 10% fetal bovine serum. A small aliquot was diluted in Trypan Blue, and the unstained neutrophils were counted using a hemocytometer. Neutrophil viability was assessed by Trypan Blue dye exclusion assay, and neutrophils with viability of greater than 95% were used during the experiments.

This study was approved by the Institutional Review Board (IRB) of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (Certificate of Approval No. 0701/2023). All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained from each participant before beginning the experiments.

PIA preparation

For the experimental use in our study, PIA was prepared from CFA of clade 1 P. insidiosum strain with slight modifications from the method described previously¹². The P. insidiosum strain was initially incubated in Sabouraud Dextrose Broth at 37 °C. After 5 days of growth, the culture was inactivated with thimerosal (0.02%) and filtered. The retained mycelial mass was disrupted. The antigens of P. insidiosum were obtained by precipitating the supernatant mixture of culture and disrupted mycelial mass. The antigens were lyophilized and stored at -80 °C until use.

A 2 mg/ml PIA stock solution was prepared and stored at − 80 °C. PIA concentrations of 0.01, 0.1, 1, and 10 µg/ml were prepared from the stock solution in RPMI 1640 medium with 25 mM HEPES and 2 mM L-Glutamine supplemented with 10% fetal bovine serum for experimental use in our study right before use. Lipopolysaccharide (LPS) can trigger neutrophil functional responses, activating innate immunity³⁰. LPS was removed from the PIA preparation to prevent the interfering effect of LPS on the activation of neutrophils³¹.

P. insidiosum culture and zoospore production

The following P. insidiosum strains were studied: CBS 777.84 isolated from a Culex quinquefasciatus larva in India; ATCC 58,643 isolated from an equine with pythiosis in Costa Rica; ATCC 90586 isolated from human with pythiosis in Texas, USA; PEC1 isolated from water reservoir in Central Thailand; PC10 isolated from human with pythiosis in Northeastern Thailand; and CBS 101039 isolated from human with pythiosis in India^32,33. All P. insidiosum strains were cultured in Sabouraud Dextrose Agar at 37 °C. Production of P. insidiosum zoospores was performed with modifications of the method described by Mendoza et al.³⁴. Sterilized grass fragments (Axonopus compressus) were placed on the surface of cornmeal agar (CMA), and the hyphae of each P. insidiosum strain were laid down among the grass fragments and incubated at 37 °C for 48 h. The infected grass fragments were transferred to a Petri dish containing an induction medium (500 µL of solution A [K₂HPO₄ 87.09 g, KH₂PO₄ 68.05 g, (NH₄)₂HPO₄ 66.04 g, distilled water 500 ml], 100 µL of solution B [MgCl₂·6H₂O 25.42 g, CaCl₂·2H₂O 18.38 g, distilled water 250 ml], and 1,000 ml of distilled water) as suggested by Chaiprasert et al.³⁵ and incubated at 37 °C for 18–20 h. The supernatant induction medium was collected and centrifuged at 5000 rpm for 10 min. Zoospores pellet was washed with 1X PBS by centrifuging at 5000 rpm for 10 min. Zoospores were then counted using a hemocytometer.

Zoospore viability measurement using an MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay

The anti-P. insidiosum activity of PIA-stimulated neutrophils against P. insidiosum zoospores was assessed using a colorimetric MTT assay following the manufacturer’s recommendations (Thermo Fisher Scientific), and absorbance was measured at the wavelength of 570 nm using a spectrophotometer. The assay relies on reducing MTT, a water-soluble yellow tetrazolium component, into an insoluble purple-colored formazan crystal by the mitochondria of viable cells, which can be measured spectrophotometrically³⁶. Briefly, neutrophils were stimulated with various PIA concentrations (0, 0.01, 0.1, 1, and 10 µg/ml) and were seeded in a 96-well round-bottom plate (1 × 10⁵ cells/well). Neutrophils were then treated with a 1:1 ratio of neutrophils/zoospores. Cells were incubated for 2 h at 37 °C. The plate was centrifuged, and the cell pellet was washed two times with 1X PBS. The resulting cell pellet was resuspended in distilled water for neutrophil lysis at room temperature, followed by vigorous vortexing to disperse the zoospores. The plate was centrifuged again, and the cell pellet was resuspended in 1X PBS. After that, 5 µL of 12-mM MTT solution was added to the wells, followed by 2 h incubation at 37 °C. 50 µL of sodium dodecyl sulfate-hydrochloric acid (SDS-HCl) solution freshly prepared by adding 10 mL of 0.01 M HCl to one tube containing 1 gm of SDS were added to each well and incubated for another 2 h at 37 °C. Experiments were performed in triplicate. The zoospore viability percentage was obtained using the following equation³⁷:

Statistical analysis

Data was analyzed using IBM SPSS Statistics 28.0. The Shapiro–Wilk test was used to test the normality of the data. Statistical analysis was done using an independent sample t-test (unpaired, two-tailed). The results were considered significant with P-values < 0.05.

Author contributions

AS, NP, and NW conceived and designed the study. SM and AS acquired the data. SM drafted the article. SM, NP, PT, and RP revised the article critically for intellectual content. SM, NP, PT, RP, and AC contributed to the article’s final version. All authors read and approved the final version of the manuscript.

Data availability

The data supporting this study’s findings are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.