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

Introduction

Pythium insidiosum, an oomycete, is a causative agent of a life-threatening human pythiosis marked by high morbidity and mortality rates.¹ Pythiosis cases are found in Thailand, India, Malaysia, China, Japan, Australia, New Zealand, Spain, Israel, Columbia, Brazil, Costa Rica, and the United States.² The clinical presentations of human pythiosis are classified as cutaneous/subcutaneous, ocular, vascular, and disseminated pythiosis.³ P. insidiosum produces motile flagellate zoospores, which are attracted to damaged tissues and cause infection.⁴ Significant challenges in the clinical management of human pythiosis include an underdiagnosis of the disease, a lack of standard treatment, and a poor patient prognosis.¹

Vascular pythiosis is characterized by chronic non-healing skin lesions, arterial insufficiency syndrome, gangrenous ulcer, and iliac or abdominal aortic aneurysm.² Patients with vascular pythiosis have been associated with hematological disease and thalassemia.⁵ Itraconazole, terbinafine, and Pythium vaccine combination therapy have successfully managed vascular pythiosis.^3,5,6 Successful treatment of vascular pythiosis relies on the surgical removal of the infected arteries, aneurysms, and surrounding tissues.^6,7 Despite the surgical intervention, the mortality rate of patients with vascular pythiosis is high (36%-40%), and better control strategies for vascular pythiosis are required.^1,3

Immunotherapy, which targets or modulates the body’s immune system and induces the body’s defenses to eliminate pathogens, holds promise for vascular pythiosis cases.⁸ Over the decades, immunotherapeutic P. insidiosum-antigen (PIA) has shown its ability to activate immune responses and treat pythiosis in humans and animals.^9–11 It has been speculated that the success of PIA immunotherapy in pythiosis is due to the switch from a T helper lymphocyte type 2 (Th2) to a T helper lymphocyte type 1 (Th1) response.¹² Administration of PIA, as a part of the treatment in vascular pythiosis, the enzyme-linked immunosorbent assay (ELISA) value of P. insidiosum-specific antibody greater than 8 correlated with the patient survival with adequate host immune response to PIA in vascular pythiosis.¹³ The application of immunotherapy in treating vascular pythiosis can be a distinctive approach to harnessing the power of the patient’s immune system. To achieve this objective, we must thoroughly understand the mechanisms of immune clearance of P. insidiosum in vascular pythiosis.

Polymorphonuclear neutrophils (PMN) are essential innate immune cells in the human body and provide the first-line defense against infections.¹⁴ Neutrophils generally serve as potent innate immune cells against pathogenic microbes by exhibiting phagocytic activity, releasing antimicrobial granules, and forming neutrophil extracellular traps (NETs).¹⁵ Interleukin-8 (IL-8) level is elevated during P. insidiosum infection.^16,17 Human neutrophils are activated by IL-8 through G-protein-coupled CXC chemokine receptors, CXCR1 and CXCR2, as part of the inflammatory response.¹⁸ A recent study showed that neutrophils could kill P. insidiosum zoospores, generate NETs, and have high levels of double-stranded DNA (dsDNA) production, providing insight into immune response in pythiosis.¹⁹

On the basis that neutrophils enhance the immune response in pythiosis, we hypothesized that the decreasing trend of serum IL-8, involved in neutrophil activation, is demonstrated in PIA-treated recovered cases. Here, we monitored the IL-8 level, assuming, “Is there any elevation of IL-8 levels in a blood sample of vascular pythiosis patient as reported in a patient with ocular pythiosis^16,17?” Also, we hypothesized that in ex vivo, PIA-stimulated neutrophils enhance the anti-P. insidiosum activity compared to unstimulated neutrophils. The present study evaluated the serum IL-8 level in pre- and post-PIA-treated vascular pythiosis patients. Our primary objective in this study was to investigate the efficacy of neutrophil function induced by PIA against P. insidiosum strains. We characterized the effect of PIA-stimulated neutrophils on the viability of zoospores using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, visualized NET formation via microscopy, and measured the levels of dsDNA using PicoGreen dsDNA quantitation assay, using neutrophils from healthy volunteers and P. insidiosum strains from vascular pythiosis patients.

Materials and methods

Population

We recruited 18 years and older patients with vascular pythiosis from the Mycology Unit, King Chulalongkorn Memorial Hospital, from January 2010 to July 2016, a total of 6.5 years period in this study based on the inclusion criteria as follows:

1. Patients were diagnosed with vascular pythiosis with at least two of the following criteria²⁰:

   1. Successful isolation of P. insidiosum and zoospore production

   2. Positive polymerase chain reaction (PCR) and sequencing by internal transcribed spacer (ITS) gene and cytochrome oxidase II (COX2) regions either from the isolates or directly from the clinical specimens

   3. Positive P. insidiosum specific IgG antibody (Pi-Ab) by established in-house ELISA

2. Patients received a combination therapy of surgery, systemic antifungal agents: systemic itraconazole (Sporal®), systemic terbinafine (Lamisil®), and immunotherapy using PIA, prepared from P. insidiosum standard strain classified as clade 1,²¹ according to the research treatment protocol for at least 1 year period for a total 7 doses according to PIA schedule generated by King Chulalongkorn Memorial Hospital. In brief, the first 2 mg/mL PIA vaccine was administered subcutaneously after the definitive diagnosis was established. Subsequent booster doses were administered every 2 weeks for 1.5 months, followed by 3 doses of PIA vaccine at 3, 6, and 12 months.

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 participants provided informed consent to participate in the study.

Serum samples

Based on the PIA-treated patient monitoring protocol generated by KCMH, whole blood samples of patients were collected before the PIA immunotherapy course initiation and at each patient visit for vaccination. All samples were collected before PIA injection for Pi-Ab examination. The leftover sera of patients selected above were used in this study. Based on the final clinical outcome of patients 1 year after the first vaccination, the sera were classified into 2 groups: the survival group and the deceased group. We collected basic clinical data of patients of recruited sera, including underlying disease, surgeries, and clinical status after each visit (stable or pain at the surgical site).

IL-8 assay

To measure the level of IL-8, 1 ml of sera stocked at −80°C was thawed and measured by a human cytokine platinum kit (eBiosciences, USA) based on the principle of direct ELISA technique according to the manufacturer’s protocol. Briefly, the 96-microwell plate was coated with IL-8 specific antibody and incubated overnight at 4°C. Then the reaction was washed twice by soaking approximately 400 µl washing buffer per well for 10–15 seconds before removing all buffer. 100 μl of standard dilution was added to the standard wells, while 100 μl of each serum sample was added to the test wells in duplicate. Then, the 50 µl of the biotin-conjugate solution was added to all wells and incubated for 2 h at 25°C. The washing was performed before adding 100 µl of diluted streptavidin-HRP to all wells, including the blank ones. After that, the plate was incubated at 25°C for 1 h, followed by the washing step. 100 μl of TMB substrate solution was added to all wells, and the plate was incubated at 25°C for 10 min in the dark. Finally, the stop solution was added, and the color development was detected immediately by the ELISA reader at 620 nm. The levels of IL-8 were determined by comparing the OD₆₂₀ with the standard curve and interpreted in the concentration unit of picogram/milliliter (pg/ml).

PMN isolation

Peripheral blood was collected from healthy volunteers into a tube containing an anticoagulant. Blood samples were treated with Polymorphprep^TM (Axis-shield, Norway) at a 1:1 ratio.²² Blood was carefully layered with the pipette tip close onto Polymorphprep^TM. The tube was centrifuged at 1700 rpm for 30 min. After centrifugation, six separate layers were observed: plasma, peripheral blood mononuclear cells (PBMC), Polymorphprep^TM, neutrophils, more Polymorphprep^TM, and the red blood cells pellet. The top three layers of plasma, monocytes, and Polymorphprep^TM were discarded. The layer of neutrophils and all the Polymorphprep^TM beneath the neutrophils were collected in a centrifuge tube. The RPMI-1640 medium with 25 mM HEPES and 2 mM L-Glutamine supplemented with 10% fetal bovine serum was added to the neutrophil solution and centrifuged at 2000 rpm for 5 min to remove residual Polymorphprep^TM. After removing the supernatant, the red pellet was subjected to ammonium chloride lysis buffer (for lysis of residual erythrocytes) containing 13.4 mM KHCO₃, 155 mM NH₄Cl, 96.7 µM EDTA at a 9:1 ratio with the RPMI 1640 medium with 25 mM HEPES and 2 mM L-Glutamine supplemented with 10% fetal bovine serum. The lysis solution was mixed by inversion and centrifuged at 2000 rpm for 5 min. The supernatant was removed, and the 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. After this, neutrophils were counted using a hemocytometer and trypan blue.

P. insidiosum culture and zoospore production

From 50 vascular pythiosis cases mentioned above in the “population” section, only 3 P. insidiosum strains were successfully isolated. Isolation of Pythium strains from blood clot samples or vascular tissues is recommended because of the higher positive rate than the blood samples.²¹ However, in this study, we utilized the leftover blood samples from the patients submitted for detection of Pi-Ab, which resulted in isolating only three P. insidiosum strains. Those strains were classified as clade 2 P. insidiosum before being recruited in this study. All clade 2 strains were maintained on Sabouraud dextrose agar (SDA) at 37°C. P. insidiosum zoospores were developed with modifications as described by Mendoza et al.²³ The hyphae of each P. insidiosum strain were transferred to the Petri dish containing sterilized grass fragments (Axonopus compressus) placed on the surface of cornmeal agar (CMA). The cultures were incubated at 37°C for 48 hr. Subsequently, the grass fragments were transferred to a Petri dish containing an induction medium suggested by Chaiprasert et al.²⁴ and incubated at 37°C for 18–20 hr. The induction medium contained solution A (K₂HPO₄ 87.09 g, KH₂PO₄ 68.05 g, (NH₄)₂ HPO₄ 66.04 g, distilled water 500 ml) and solution B (MgCl₂.6 H₂O 25.42 g, CaCl₂.2 H₂O 18.38 g, distilled water 250 ml). The final composition of induction media was 500 µl of solution A, 100 µl of solution B, and 1000 ml of distilled water. The induction medium was collected and centrifuged for 10 min. Zoospores were washed by centrifugation with 1X phosphate-buffered saline (PBS). The zoospore concentration was determined by a hemocytometer and trypan blue.

PIA preparation

As described elsewhere,¹¹ PIA was prepared from clade 1 P. insidiosum strain and used as an inoculum in our study. A 2 mg/ml PIA stock solution was prepared with PCR-grade water and stored at −80°C. The PIA stock solution was further diluted to the required PIA concentrations of 0.01, 0.1, 1, and 10 µg/ml for experimental use.

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

The MTT assay was performed to assess the viability of zoospores, where the viable zoospores converted MTT into a purple-colored formazan product.²⁵ Freshly prepared neutrophils were added to zoospores (1 × 10⁵ cells) with effector to target ratio (E:T) 1:1 in a 96-well round-bottom plate. Neutrophils were treated with various PIA concentrations (0, 0.01, 0.1, 1, and 10 µg/ml). Untreated zoospores were used as controls. The plate was incubated for 2 hr at 37°C in the CO₂ incubator and then centrifuged for 10 min. The cell pellet was resuspended in 1X PBS and centrifuged for 10 min. Subsequently, the neutrophils were lysed in distilled water at room temperature for 5 min, followed by vigorous vortexing for approximately 5 sec to disperse the zoospores. The plate was centrifuged for 10 min, and the cell pellet was resuspended in 1X PBS, to which 5 µL of MTT solution was added. The plate was incubated for 2 hr at 37°C in the CO₂ incubator, and then 50 µL of sodium dodecyl sulfate- hydrochloric acid (SDS-HCl) was added to each well. The plate was again incubated for 2 hr at 37°C in the CO₂ incubator. Finally, the absorbance of the samples was measured on the plate reader at 570 nm. The following equation calculated the percentage of viability of zoospores²⁶:

Viability of zoospores (%) = [(A570 of zoospores incubated with PMNs or PIA-treated PMNs)/A570 of zoospores alone] x 100

NET imaging and quantification

Fluorescence microscopy was used for NETs visualization using DAPI (4′,6-diamidino-2-phenylindole), a blue-fluorescent DNA stain.^27,28 Freshly isolated neutrophils (2 × 10⁵ cells) were seeded in a 24-well flat-shaped bottom plate on glass coverslips coated with poly-L-lysine. Neutrophils were stimulated with various PIA concentrations (0, 0.01, 0.1, 1, and 10 µg/ml) and incubated for 1 hr (37°C, 5% CO₂). Heat-inactivated zoospores (E:T = 1:1) and Phorbol-12-myristate-13-acetate (PMA, positive control) were added and incubated for 2 hr (37°C, 5% CO₂). Unstimulated neutrophils were included as a control to check for spontaneous NETosis. NETosis was stopped with 1% formaldehyde fixation for 5 min; cells were permeabilized with 1X Tris-buffered saline (TBS) in 0.05% tween and incubated with a blocking solution of 1X TBS in 2% bovine serum albumin (BSA) for 30 min. Subsequently, cells were stained using DAPI. Cells were imaged with an inverted fluorescence microscope (Olympus IX81, Japan). Neutrophils releasing NETs were quantified by visual identification and counting of the NETs forming neutrophils per 100 neutrophils.

PicoGreen double-stranded deoxyribonucleic acid (dsDNA) quantitation assay

Neutrophils (2 × 10⁵ cells) were seeded in a 24-well flat-shaped bottom plate on glass coverslips, stimulated to undergo NETosis as described above, and incubated for 2 hr (37°C, 5% CO₂). The media was collected, and 0.1 M CaCl₂ and 50 U/ml micrococcal nuclease (Sigma-Aldrich, USA) were added for DNA fragmentation and digestion and incubated for 10 min at 37°C. Nuclease reactions were stopped by adding 0.5 M ethylenediaminetetraacetic acid (EDTA). dsDNA levels were quantified using a Quant-iT PicoGreen (Invitrogen, UK) and fluorometry (Varioskan Flash, Thermo Fisher Scientific, Finland). In brief, a standard curve was prepared with lambda-DNA standard on a 96-well flat-bottom plate. A working solution of Picogreen was prepared, mixed well with samples, and incubated in the dark for 5 min at room temperature. The plate was read using a fluorescence microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

Statistical analysis

All statistical evaluations were performed with IBM SPSS Statistics 28.0. Independent sample t-test (unpaired, two-tailed) analyzed the differences between the two groups. Error bars denote ±SD. p-values < .05 were considered to be statistically significant.

Results

Based on the inclusion criteria mentioned in the methods section, the sera from 50 vascular pythiosis cases and 10 healthy volunteers were examined in this research. For the vascular pythiosis cases, the survival group included 45 patients, whereas the deceased group included 5.

IL-8 level

A linear IL-8 pg/ml standard curve was generated, and the concentration of IL-8 in the samples was determined by comparing the OD of the samples to the standard curve. The serum level of IL-8 was significantly higher in patients with vascular pythiosis than the healthy subjects (p < .001) before PIA immunotherapy course initiation, as shown in Table 1. As we hypothesized, there was no statistically significant difference in the mean level of IL-8 between survived and deceased cases (p > .05) before PIA immunotherapy course initiation. There was about a 3-fold decrease in serum IL-8 level by 1.5 months and a 17-fold decrease in serum IL-8 level by 12 months among the surviving cases of vascular pythiosis.

Table 1. Serum levels of IL-8 in the different study groups.

Groups	IL-8 (pg/ml) before 1^st dose of PIA	IL-8 (pg/ml) before 2^nd dose of PIA	IL-8 (pg/ml) before 3^rd dose of PIA	IL-8 (pg/ml) before 4^th dose of PIA	IL-8 (pg/ml) before 5^th dose of PIA	IL-8 (pg/ml) before 6^th dose of PIA	IL-8 (pg/ml) before 7^th dose of PIA
Survived (N = 45)	2996.49 (189.15)	2936.18 (224.78)	1771.82 (187.83)	836.49 (104.13)	429.13 (53.44)	220.76 (52.49)	172.98 (33.95)
Deceased (N = 5)	3041.60 (180.70)	3170.00 (228.54)	2953.60 (198.04)	2410.00 (191.16)
Healthy (N = 10)	116.90 (13.59)

Values are expressed as mean (SD). IL: Interleukin; PIA: Pythium insidiosum antigen.

The treatment protocols and clinical patterns among the survived and deceased groups were similar, resulting in the minimal effect of the confounding factors in the IL-8 levels. All patients had thalassemia as an underlying disease. Definitive surgeries, defined by the achievement of negative surgical margins, were achieved in all patients. While the clinical status after surgery as “stable” was determined in the survival group, among five patients in the deceased group, the clinical symptoms of pain at the surgical site caused by the surgical wound infection were diagnosed (the residual pythiosis was found afterward) before death.

Cell viability analysis

As shown in Figure 1, neutrophils (PIA-stimulated and PIA-unstimulated) reduced zoospores viability compared to control. Neutrophils stimulated with 0.01 µg/ml PIA reduced zoospores viability significantly compared to PIA-unstimulated neutrophils for strain 1 and strain 3 (Figure 1a,c). In Figure 1b, neutrophils stimulated with 0.01, 0.1, 1, and 10 µg/ml PIA exhibited lower zoospore viability than PIA-unstimulated neutrophils for strain 2.

Figure 1. Cell viability results for strain 1 (a), strain 2 (b), and strain 3 (c). * indicates a comparison between control (RPMI+zoospores) and other groups; + indicates a comparison between unstimulated PMNs and PIA-stimulated PMNs. *p < .05, **p < .01, ***p < .001, ⁺p < .05, ⁺⁺p < .01.

Viability of zoospores co-cultured with PIA treated PMNs.

NET formation analysis

The images in Figure 2 show the NET formation with DAPI staining in PIA-stimulated and unstimulated neutrophils. Neutrophils (control) released minimal NETs, while unstimulated neutrophils incubated with zoospores released significantly higher numbers of NETs releasing neutrophils than the control for all strains (Figure 3a–c). Neutrophils stimulated with 1 µg/ml PIA showed a significantly higher number of NETs releasing neutrophils than unstimulated neutrophils for strain 3 (Figure 3c).

Figure 2. Representative microscopic images (40×) of NETs with DAPI staining of PIA-stimulated and unstimulated neutrophils. NETs were visualized for strains with neutrophils stimulated with 0, 0.01, 0.1, 1, and 10 µg/ml of PIA (a), negative control (b), and PMA (c).

NETs production (DAPI staining) of neutrophil treated with PIA.

Figure 3. Release of NETs from neutrophils. Quantification of NETosis percentage for strain 1 (a), strain 2 (b), and strain 3 (c). * indicates a comparison between control (RPMI+PMN) and other groups; + indicates a comparison between untreated PMNs and PIA-treated PMNs. *p < .05, **p < .01, ***p < .001, ⁺p < .05.

NETs production (Quantification of NETosis percentage) of neutrophil treated with PIA.

PicoGreen dsDNA quantitation assay analysis

The levels of dsDNA were significantly higher in the PIA-unstimulated neutrophils incubated with zoospores for all strains compared to controls (neutrophils only) (Figure 4a–c). For strain 1, neutrophils stimulated with 10 µg/ml PIA had significantly elevated levels of dsDNA compared to the control (Figure 4a). Correspondingly, levels of dsDNA were significantly higher with neutrophils stimulated with 1 µg/ml PIA for strain 2 (Figure 4b), and neutrophils stimulated with 0.01, 1, and 10 µg/ml PIA exhibited significantly higher levels of dsDNA for strain 3 compared to control (Figure 4c). The mean levels of dsDNA did not differ significantly between the PIA-stimulated neutrophils and unstimulated neutrophils for all strains.

Figure 4. Levels of extracellular dsDNA during NETosis for strain 1 (a), strain 2 (b), and strain 3 (c). * indicates a comparison between control (RPMI+PMN) and other groups; + indicates a comparison between PIA-treated PMNs and untreated PMNs. All figures demonstrate as mean ± SD, *p < .05, **p < .01, ***p < .001.

Extracellular dsDNA during NETosis of PMNs treated with PIA.

Discussion

P. insidiosum zoospores adhere to the skin lesions and germinate into hyphae stimulated by the host’s body temperature.²⁹ Vascular pythiosis is characterized by the invasion of hyphae within the arteries and surrounding tissues of the patient.² Immunotherapy with PIA remains one of the main treatment strategies in vascular pythiosis, in addition to wide surgical excision and antifungal drugs.^3,5,10 Our results showed that PIA-stimulated neutrophils enhance the anti-P. insidiosum activity from the cell viability data obtained from the MTT assay compared to unstimulated neutrophils. Based on the observations of NET quantification, PIA-stimulated neutrophils do not significantly increase NETosis compared to unstimulated neutrophils. The efficacy of PIA against P. insidiosum is dose-dependent and specific to strain and clade, helping to understand immune responses in vascular pythiosis.

Consistent with a previous study in ocular pythiosis caused by P. insidiosum,¹⁶ our study showed that IL-8 is elevated upon infection with P. insidiosum in vascular pythiosis. The persistently high levels of IL-8 after initiating PIA treatment indicated the case status of treatment failure. IL-8 is a chemotactic factor, and it recruits neutrophils to sites of infection.³⁰ It is believed that IL-8 increases intracellular Ca⁺² and triggers Ca⁺² signals in the neutrophil activation process.³¹ Neutrophils are likely activated by elevated IL-8 in P. insidiosum infection, thereby indicating the primary role of neutrophils in the defense against P. insidiosum. Our findings suggest that IL-8 could be used as a biomarker for monitoring the therapeutic effect of the PIA vaccine in vascular pythiosis patients. However, the impact of concomitant medications on IL-8 levels must be assessed by clinicians to justify when decreasing IL-8 levels as a treatment biomarker for PIA immunotherapy is suggested. In our study, itraconazole and terbinafine were used as a maintenance therapy for the patients during PIA immunotherapy, and both of these drugs do not decrease IL-8 levels.^32,33 Also, PIA treatment protocol, comorbidities, health behaviors, and severity of cases might affect the IL-8 levels in patients.^34,35 It necessitates the adjustment for IL-8 levels considering the confounding variables for a valid estimate of the PIA immunotherapy and IL-8 levels in further studies.

Neutrophils are the primary cellular mediators of the innate host defense against microorganisms.³⁶ The life span of PMNs is short, and they do not proliferate.³⁷ We optimized and adapted the MTT assay to measure the viability of P. insidiosum zoospores. Cell viability assays play an essential role in screening the response of cells against an agent in in vitro cell culture systems.³⁸ However, one must consider that the underlying mechanism of MTT assay has not been fully understood. MTT assay measures the integrated set of enzyme activities that are related in various ways to cell metabolism, resulting in over/underestimation of cell viability.³⁹ Regardless of the degree of variability in the MTT assay, we observed the assay produced replicable data with minimal discrepancy in multiple experiments. Our results highlighted the role of neutrophils in killing P. insidiosum zoospores. Lower doses of PIA appeared to be effective in stimulating neutrophils with high efficiency in killing P. insidiosum zoospores. The enzymatic activities observed in P. insidiosum strains include alkaline phosphatase, esterase, esterase lipase, lipase, acid phosphatase, urease, naphthol-AS-BI-phosphohydrolase, and β-glucosidase.^40,41 The biochemical characteristics differ among and within the P. insidiosum strains, which may explain the differences observed in the viability of zoospores with distinct strains and doses of PIA.⁴⁰

NETosis is one of the mechanisms deployed by neutrophils to kill invading microorganisms.⁴² NETs entrap the pathogens, released as decondensed chromatin and associated granule products to the extracellular space from activated neutrophils.⁴³ The encysted P. insidiosum zoospore size is 8–10 µm, which is too large to be phagocytosed in vivo.^44,45 Neutrophils respond to microorganisms that are too large to phagocytose by releasing NETs.⁴⁶ The induction of NET formation due to the P. insidiosum zoospores is supported by a recent study of the in vitro function of human neutrophils against P. insidiosum.¹⁹ Our study utilized the optimum incubation period of 2 hr because NETosis is a slow process.⁴⁷ However, the signaling events leading to NETosis in pythiosis are not understood yet.

The extracellular dsDNA has been used as a surrogate biomarker for NETosis.⁴⁸ In our study, we found no significant differences in the levels of dsDNA between PIA-stimulated and unstimulated neutrophils. This raises the possibility that immune therapy with PIA in vascular pythiosis may not have therapeutic benefits based on the NETosis mechanism. Recent experimental evidence is consistent with our findings of PIA-unstimulated neutrophils producing higher levels of dsDNA when incubated with P. insidiosum zoospores.¹⁹ We used heat-inactivated zoospores for NET assay, which might render zoospores less virulent and compromise the ability of the neutrophils to elicit strong immune responses.

In all experiments regarding neutrophil function, we used the zoospore form of Pythium strains instead of the hyphae form in pythiosis patients. Given the number of countable zoospores, the result is consistent and reproducible. Regarding the property of neutrophil activation among zoospores and the hyphae form, based on the theory of Dectin-1 receptor recognizing beta-glucan,⁴⁹ it is not the primary concern because the beta-glucan is present in both forms of Pythium strain.

There are significant challenges to developing an effective vaccine for difficult-to-target P. insidiosum with public health implications.⁵⁰ Our study shows that a concept of personalized vaccine depending on the clinical strain P. insidiosum should be applied in vascular pythiosis because of the pathogen genetic variability.⁵¹ Our study did not investigate the ex vivo biochemical characteristics and their interplay with the immunological response, limiting the scope of PIA treatment in understanding its immunopathological mechanisms in pythiosis. The strain specificity of the immune response by PIA-stimulated neutrophils provides a basis for further comparative analysis to reflect the diversity among the P. insidiosum strains.⁵² Identifying the variations between P. insidiosum strains would give better insights into comparing the immune dynamics and extracting meaningful information about PIA immunotherapy in vascular pythiosis.

Overall, our study demonstrates the efficacy of PIA-stimulated neutrophils in killing P. insidiosum zoospores, specific to individual strains of P. insidiosum. However, further validation studies in multiple strains of P. insidiosum are necessary to effectively utilize our preliminary evidence of neutrophil involvement in PIA immunotherapy. Furthermore, comparative analyses of strains that do and do not respond to PIA immunotherapy and longitudinal studies of IL-8 and NETosis trends between successful and failed cases must be undertaken to prevent our results from being misleading. Although there has been greater emphasis on the pharmacotherapy of vascular pythiosis, the field of immunotherapy offers new hope for treating vascular pythiosis. A better understanding of the biochemical changes in P. insidiosum zoospores after treatment with PIA could lead to better therapeutic guidelines with the combined use of antifungals and strain-specific PIA in vascular pythiosis.

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.