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Research Article

The protective effects of antigen-specific IgY on pyocyanin-treated human lymphoma Raji cells

[version 1; peer review: 1 approved, 1 approved with reservations]
PUBLISHED 04 Jul 2019
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Abstract

Background: Pyocyanin (PCN), a highly pathogenic pigment produced by Pseudomonas aeruginosa, induces caspase 3-dependent human B cell (Raji cells) death. The aim of the present study, therefore, was to assess whether antigen-specific IgY antibodies may be protective on PCN-induced Raji cell death.
Methods: Chickens were subcutaneously immunized with Freund's complete adjuvant containing PCN, and then given two boosted immunizations.  Anti-PCN IgY antibodies were purified from egg yolk and detected using an agar gel precipitation test (AGPT) and ELISA. Protective effects of antigen-specific IgY on Raji cells were tested using a cell viability assay.
Results: AGPT results showed the formation of strong immune complex precipitates, whilst ELISA further confirmed the presence of IgY antibodies specific to PCN at significant concentration. Further experiments showed that anti-PCN IgY antibodies significantly increased PCN-treated Raji cell viability in a dose-dependent fashion (p<0.05).
Conclusions: The results of the present study suggest that anti-PCN IgY antibodies may be protective on PCN-induced Raji cell death.

Keywords

Pseudomonas aeruginosa, pyocyanin, IgY, protective effect

Introduction

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, is found in the environment with a broad spectrum of habitats and is responsible for severe nosocomial infections in the urinary tract1, the respiratory tract2, the vascular system3 and the central nervous system4. It is known for one of the most common pathogens infecting patients with cystic fibrosis, leading to increase its morbidity and mortality due to the resisting abilities of this pathogen to against antibiotic treatments5,6. The presence of P. aeruginosa in dental pulp and periapical lesions may cause failure of endodontic treatments7,8. In the initial stage of infection, P. aeruginosa releases various virulent mediators, such as elastases, proteases, exotoxin A, and pyocyanin (PCN), after which chronic infection and persistent bacterial colonization at the P. aeruginosa-infected sites would be established9. PCN, a blue redox-active secondary metabolite and a member of tricyclic phenazine family, is known to function as a gene controller during the stationary growth phase10, an antibiotic11, an electron transfer facilitator12, and a potent mammary cell-damaging virulence factor13. Reports indicate that PCN inhibits B cell, T cell and macrophage functions14,15 and induces neutrophil apoptosis16, suggesting that PCN suppresses both innate and antigen-specific adaptive immune response.

The existence of multidrug-resistant (MDR) P. aeruginosa leads to the development of alternative treatment strategies to eradicate an established chronic P. aeruginosa infection. Of these treatments, both active and passive immunotherapies have been reported. Active immunization with P. aeruginosa-derived flagella in cystic fibrosis patients resulted in increased serum antigen-specific IgG antibodies and reduced number of P. aeruginosa strains, suggesting the reduction of P. aeruginosa infection risk in cystic fibrosis patients by active vaccination17. Passive immunization with egg yolk immunoglobulin (IgY) specific to P. aeruginosa in patients with cystic fibrosis prevented bacterial colonization and infection, perhaps by acting as an opsonin, which in turn enhanced neutrophil phagocytosis to this pathogen1820. A recent study showed that PCN induces caspase 3-dependent human B cell (Raji Cells) death21. The aim of the present study, therefore, was to determine whether antigen-specific IgY antibodies may prevent PCN-induced Raji cell death.

Methods

IgY preparation and purification

PCN (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO at a concentration of 1 mg/ml. Five Leghorn chickens aged 3 months were subcutaneously immunized with 500 μl of Freund's complete adjuvant (Sigma-Aldrich) containing 100 μg of PCN in the back of the neck. Two weeks later, a booster was given by injecting 500 μl incomplete Freund adjuvant containing 40 μg PCN as above and the same immunization regime was repeated two weeks later. Eggs were collected one week after the last immunization and IgY was isolated by using Pierce® Chicken IgY Purification Kit (Thermo Fisher Scientific Pierche Biotechnology, Rockford, USA) according to the manufacturer. The presence of anti-PCN IgY antibodies was detected using the agarose gel precipitation test (AGPT) as previously reported22 and its concentration was assessed using the Chicken IgY ELISA Kit (Elabscience Biotechnology Co., Ltd, USA). The AGPT test was performed three times, each of 4 isolates from the first and second IgY purification results. The ELISA was then performed on two IgY batches.

Cell cultures

Raji cells, a human B cell line, obtained from central university laboratory LPPT, Universitas Gadjah Mada, Yogyakarta, Indonesia, were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 UI/ml of penicillin-streptomycin, and 250 μl/ml of amphotericin B and then incubated in 5% CO2 humidity. All materials for culture medium were purchased from Sigma-Aldrich. The cells were cultured in 96-well plates and five replicates were carried out for assays.

Cell viability

PCN (Sigma-Aldrich) was initially dissolved in DMSO (Sigma-Aldrich) at a concentration of 1 mg/ml and then diluted in RPMI to a final concentration of 1 μg/ml, 10 μg/ml, 25 μg/ml, and 50 μg/ml. Raji cells at 2 × 104 cells incubated without the presence of PCN were used as a negative control. After exposure to various concentration of PCN then the cultures were incubated at 37°C for 24 hours. In the next experiments, the cells, at a concentration of 2 × 104 cells/well, were treated with 50 μg/ml PCN with or without the presence of various concentration (6.71 μg/ml, 13.42 μg/ml, 28.19 μg/ml, 55.49 μg/ml, 111.87 μg/ml, and 223.75 μg/ml) of anti-PCN IgY were cultured in 96-well plates and incubated for 16 hours. Cell survivability was assessed by MTT assay as described previously21. Experiments were carried out three times with 8 replicates in each group.

In order to assess cell viability, 5 × 104 cells/well were cultured on sterile coverslips in 24-well plates for 24 hours and then treated with PCN in the presence or absence of anti-PCN IgY (55.49 µg/ml) for 16 hours. Subsequently, the cells were stained with acrydine orange/ethidium bromide and viewed under Digital Carl Zeiss-Axioscope 40 (Carl Zeiss Vision, Oberkochen, Germany) by which viable and death cells appeared as green and orange/red, respectively.

Statistical analysis

The results of PCN cytotoxicity assay on Raji cells were analyzed by using one way analysis of variance followed by LSD test. Data obtained from the experiments on the effects of anti-PCN IgY on PCN-treated Raji cells was analysed by using one-way ANOVA followed by Tukey’s Test. Statistical analysis was calculated by using IBM SPSS Statistics Version 22 (SPSS Inc., IBM Corp., Chicago, IL).

Results

Following isolation and purification of IgY from the immunized chickens, PCN-IgY complexes were detected by using AGPT. As seen in Figure 1, clear lines of precipitates from two IgY batches in the agarose matrix indicated the presence of PCN-specific antibodies. A further assessment using ELISA demonstrated that the first batch gives high amount of specific IgY antibodies (8.95 μg/μl) than that one of the second (3.02 µg/µl) which were then used for the rest of experiments.

12b87e4d-34aa-4445-96c4-edf2b718475e_figure1.gif

Figure 1. Agarose gel precipitation test of pyocyanin (PCN)-IgY antibody complex batch I and II.

The presence of PCN-IgY antibody was detected through the presence of precipitates formed on agarose gel.

The results of this study showed that PCN at 1 mg/ml was toxic to the Raji cells. This cytotoxic effect of PCN on the cells was steadily increased in a dose dependent fashion (p<0.05) (Figure 2).

12b87e4d-34aa-4445-96c4-edf2b718475e_figure2.gif

Figure 2. The effects of pyocyanin (PCN) on Raji cell survivability.

After incubation with various concentration of PCN, Raji cell viabilities were assessed by MTT assay. PCN-treated Raji cell survivability was calculated against the control cells. *p<0.05.

Further experimentation demonstrated that anti-PCN IgY at concentrations of 28.19 μg/mL or higher was able to suppress the cytotoxic effect of PCN on Raji cells as compared with the negative control (p<0.05) (Figure 3). No significant differences between the cells treated with PCN and specific anti-PCN IgY antibodies at the concentration above 55 μg/ml were observed, however (p>0.05) (Figure 3). Microscopically, the number of viable cells treated with PCN-IgY complexes was much higher than those treated with PCN only (Figure 4). Raw cell viability counts, along with other raw results and images, are available as Underlying data23.

12b87e4d-34aa-4445-96c4-edf2b718475e_figure3.gif

Figure 3. The effects of IgY specific antibodies on pyocyanin (PCN)-treated Raji cell survivability.

PCN was incubated with various concentration of IgY antibodies. Raji cells were then incubated with the PCN-IgY mixtures. Viable cells were assessed by MTT and their percentage was calculated as in Figure 2. *p<0.05.

12b87e4d-34aa-4445-96c4-edf2b718475e_figure4.gif

Figure 4. Microscopic features of Raji cells treated with pyocyanin (PCN) or PCN-IgY antibody complexes.

Raji cells were incubated without PCN (A) and with PCN (B) or the mixture of PCN-IgY antibodies (C) and then stained with acrydine orange/ethidium bromide. Green or orange fluorescence stained cells are viable and dead cells, respectively.

Discussion

The results of the present study showed that PCN does induce cell death in Raji cells as also seen in our previous report21. Similarly, other also demonstrated that PCN of P. aeruginosa plays an important role in the invasion of host cells by inducing neutrophil cell death16. Therefore, efforts to inhibit excessive host cell damage induced by PCN are imminent.

Further results of the present study demonstrated that anti-PCN IgY antibodies specific to PCN significantly reduce the ability of this virulence to induce Raji cell death in a dose-dependent fashion. Whilst no previous studies showing prevention of PCN-induced cell death by antigen-specific IgY have yet been reported to our knowledge, the present results are supported by the fact that antigen-specific IgY antibodies did prevent P. aeruginosa infection in humans by both active and passive immunization1719, suggesting that P. aeruginosa-specific IgY antibodies may inhibit cellular inflammatory responses induced by this pathogen. Antigen-specific IgY antibodies also stimulated P. aeruginosa aggregation and increased human neutrophil phagocytic activities20. The exact mechanism by which antigen-specific IgY antibodies inhibited PCN-induced Raji cell death seen in the present study remains unclear, however. Our previous study indicated that PCN induced Raji cell death via a caspase 3-activation pathway21. It seems plausible, therefore, that PCN-IgY antibody complexes may fail to activate Raji cell-derived caspase 3 and hence, inhibit cell death. However, more studies are required to delineate this speculation.

The extrapolation of the results of the present study in the therapy of P. aeruginosa infection remains to be further investigated. P. aeruginosa with its multiple mechanisms for adaptation and survival is well known as one of the main pathogens that lead to increase nosocomial infections14 and morbidity and mortality in cystic fibrosis patients5. The difficulty to eradicate P. aeruginosa infection has been even more complicated by the presence of MDR P. aeruginosa, and hence alternative supplemental treatment approaches have been put forward based upon its bacterial virulent factors, such as exotoxin, lipopolysaccharide, and flagellin24. Immunotherapies using IgY specific to P. aeruginosa to delay initial infection and reduce both frequency of infection and development of chronic infection seem to be promising. Both passive and active immunization with antigen-specific IgY antibodies in humans resulted in inhibition of P. aeruginosa infection. For example, oral immunization with specific IgY antibodies in patients with cystic fibrosis led to the inhibition of P. aeruginosa colonization18,19, suggesting the usefulness of antigen-specific IgY antibodies an immunotherapy to prevent P. aeruginosa infection in patients with cystic fibrosis. Therefore, PCN-specific IgY antibodies used as an immunotherapy alongside with the common antibiotic treatments for P. aeruginosa infection are highly possible.

In conclusion, the present study showed that eggs from PCN-immunized chickens contain substantial amount of IgY antibodies that recognize PCN. Furthermore, antigen-specific IgY antibodies were able to inhibit PCN-induced Raji cell death, suggesting that PCN-specific IgY antibodies may be protective against PCN-induced Raji cell death.

Data availability

Figshare: Cytotoxicity of PCN.xlsx. https://doi.org/10.6084/m9.figshare.8115701.v123.

This project contains the following underlying data:

  • Cytotoxicity of PCN.xlsx (raw cell viability data following treatment with pyocyanin)

  • The effect of IgY on cell viability.xlsx (raw cell viability data following treatment with pyocyanin and IgY)

  • Fig 4A untreated cells.JPG (raw image used for Figure 4A)

  • Fig 4B PCN-treated cells.JPG (raw image used for Figure 4B)

  • Fig 4C PCN IgY-treated cells.JPG (raw image used for Figure 4C)

  • IMG-AGPT.jpg (raw image of agar gel precipitation test)

  • Elisa results Sept 1.xls (raw ELISA data)

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

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Susilowati H, Artanto S, Yulianto HDK et al. The protective effects of antigen-specific IgY on pyocyanin-treated human lymphoma Raji cells [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2019, 8:1008 (https://doi.org/10.12688/f1000research.19327.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
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Reviewer Report 27 Aug 2019
Priya Madhavan, School of Medicine, Faculty of Health and Medical Sciences, Taylor’s University, Subang Jaya, Malaysia 
Approved
VIEWS 18
Overall, the study design is of an acceptable quality. Immunotherapy using IgY specific to P. aeruginosa looks promising. Minor English editing and grammar check are required.
  • Magnification/Scale on Figure 4 is missing. Best to include
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Madhavan P. Reviewer Report For: The protective effects of antigen-specific IgY on pyocyanin-treated human lymphoma Raji cells [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2019, 8:1008 (https://doi.org/10.5256/f1000research.21186.r52110)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 16 Jul 2019
Boy M. Bachtiar, Department of Oral Biology and Oral Science Research Center, Faculty of Dentistry, Universitas Indonesia, Jakarta, Indonesia 
Approved with Reservations
VIEWS 21
General comments:

In this manuscript, the authors provide data account of an IgY-antibody that was purified from eggs of hens immunized with PCN secreted by P. aeruginosa. The specificity of PCN-IgY binding was determined by using AGPT, ... Continue reading
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HOW TO CITE THIS REPORT
Bachtiar BM. Reviewer Report For: The protective effects of antigen-specific IgY on pyocyanin-treated human lymphoma Raji cells [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2019, 8:1008 (https://doi.org/10.5256/f1000research.21186.r50799)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

Comments on this article Comments (0)

Version 2
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Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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