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

Heni Susilowati; Sidna Artanto; Heribertus Dedy Kusuma Yulianto; Wihaskoro Sosroseno; Suryani Hutomo

doi:10.12688/f1000research.19327.2

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Research Article

Revised

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

[version 2; peer review: 2 approved]

Heni Susilowati ¹, Sidna Artanto², Heribertus Dedy Kusuma Yulianto³, Wihaskoro Sosroseno⁴, Suryani Hutomo⁵

Heni Susilowati ¹, Sidna Artanto², [...] Heribertus Dedy Kusuma Yulianto³, Wihaskoro Sosroseno⁴, Suryani Hutomo⁵

PUBLISHED 17 Dec 2019

Author details Author details

¹ Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, Yogyakarta, 55281, Indonesia
² Department of Microbiology, Faculty of Veteriner, Universitas Gadjah Mada, Sleman, Yogyakarta, 55281, Indonesia
³ Department of Dental Biomedical Science, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, Yogyakarta, 55281, Indonesia
⁴ Faculty of Dentistry, AIMST University, Bedong, Kedah, 08100, Malaysia
⁵ Department of Microbiology, Faculty of Medicine, Dutawacana Christian University, Yogyakarta, 55225, Indonesia

Heni Susilowati
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Original Draft Preparation

Sidna Artanto
Roles: Investigation, Methodology, Resources

Heribertus Dedy Kusuma Yulianto
Roles: Data Curation, Investigation, Software, Writing – Original Draft Preparation

Wihaskoro Sosroseno
Roles: Software, Visualization, Writing – Review & Editing

Suryani Hutomo
Roles: Investigation, Writing – Original Draft Preparation

OPEN PEER REVIEW

REVIEWER STATUS

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

Corresponding author: Heni Susilowati

Competing interests: No competing interests were disclosed.

Grant information: This study was supported by a research grant (no. 1048/UN1-P.III/LT/DIT-LIT/2016) from the Ministry of Research, Technology and Higher Education, the Indonesian Government.

Copyright: © 2019 Susilowati H et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Susilowati H, Artanto S, Yulianto HDK et al. The protective effects of antigen-specific IgY on pyocyanin-treated human lymphoma Raji cells [version 2; peer review: 2 approved]. F1000Research 2019, 8:1008 (https://doi.org/10.12688/f1000research.19327.2) First published: 04 Jul 2019, 8:1008 (https://doi.org/10.12688/f1000research.19327.1) Latest published: 17 Dec 2019, 8:1008 (https://doi.org/10.12688/f1000research.19327.2)

Revised Amendments from Version 1

Major differences of the new and previous version are listed below.
1. We replaced Figure 4A, B, and C as our response to Dr. Madhavan regarding magnification of micrograph.
2. We have already added two new references published in 2019 to the reference list. These are papers published by Li et al, 2019 and Ruffin & Brochiero, 2019.
3. We added new sentences in Discussion section line 21 as response to Dr. Boy Bachtiar comment about possible mechanism of PCN-induced cell death inhibition by IgY.
4. We have already added new data of quantitative results of acridine orange/ethidium bromide staining. Data is available in https://doi.org/10.6084/m9.figshare.8115701.v1 as figure 4D
5. We have already added new explanation to accommodate rational reason of the use of pyocyanin. The explanation is added to line 13 in Discussion section, and added new references No.23 and 24
6. According to reviewer comment to the reference no. 16 that PCN is not having a role in the bacterial invasion to the host cells we have revised the manuscript, precisely on the 3^rd line of the discussion section.
7. Our suggestion for the next study were listed in the end of Discussion section.

See the authors' detailed response to the review by Boy M. Bachtiar

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 treatments^5,6. The presence of P. aeruginosa in dental pulp and periapical lesions may cause failure of endodontic treatments^7,8. 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 functions^14,15 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 pathogen^18–20. 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 egg 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.

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).

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). The results showed that anti-PCN IgY did increase the survivability of PCN-exposed cells from 24.3% up to 72.6% at concentration at 55.49 μg/ml (Figure 4D). Statistically, significant differences existed between cell only and PCN-treated or PCN-IgY-treated cells. The survivability of cells treated by PCN was significantly lower than that treated with IgY prior to PCN stimulation (p<0.05). Raw cell viability counts, along with other raw results and images, are available as Underlying data²³.

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.

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 acridine orange/ethidium bromide. Green or orange fluorescence stained cells are viable and dead cells, respectively. The cell survivability was increased on the cells treated with IgY antibody prior to PCN exposure (D).

Discussion

The results of the present study showed that PCN does induce cell death in Raji cells as also seen in our previous report Susilowati. Similarly, other also demonstrated that PCN plays an important role in P. aeruginosa pulmonary infection through the induction of neutrophil cell death, which involves the release of reactive oxygen species and the activation of mitochondrial acid sphingomyelinase¹⁶. 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 pathogen 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 immunization^17–19, suggesting that P. aeruginosa-specific IgY antibodies may inhibit cellular inflammatory responses induced by this pathogen. Pyocyanin is secreted by P. aeruginosa when the nutrition for the bacterium is limited. As important virulent factor, PCN plays a key role in bacterial infection because its ability to cross cell membrane which then causing disturbance in cellular electron transport and innate immune response²⁴. Since PCN functions to kill host cells²⁵ it is possible to inhibit cellular and tissue damage by blocking PCN using the anti PCN-antibody IgY. 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. Zhao et al. demonstrated the activation of caspase-3 in hepatoma HepG2 cell death mechanism induced by PCN²⁶. Other has shown that PCN stimulates NK cell death via increased intracellular calcium levels and mitochondrial disruption²⁷, suggesting that cell death induced by PCN may utilize different apoptotic signal pathways, perhaps, in a cell type-dependent fashion. 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 infections^1–4 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 flagellin²⁸. 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 colonization^18,19, 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, IgY antibodies derived from PCN-immunized chicken were able to inhibit PCN-induced Raji cell death, suggesting that PCN-specific IgY antibodies may be protective against PCN-induced Raji cell death. Future studies need to clarify the mechanisms involved in inhibiting the death of Raji cells induced by PCN. Furthermore, it is also necessary to study the effect of IgY antibodies derived from PCN-immunized chicken on the in vivo model of P. aeruginosa infection..

Data availability

Figshare: Cytotoxicity of PCN.xlsx. https://doi.org/10.6084/m9.figshare.8115701.v6²³.

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)
Fig 4D Cell survivability (raw image used for Figure 4D)
Acridine orange-ethidium bromide results (raw cell viability data)
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).

Acknowledgements

We also express our gratitude to Ericko Tedy Hananto, who has helped with full responsibility during antibody preparation.

Faculty Opinions recommended

References

1. Lamas Ferreiro JL, Álvarez Otero J, González González L, et al.: Pseudomonas aeruginosa urinary tract infections in hospitalized patients: Mortality and prognostic factors. PLoS One. 2017; 12(5): e0178178. PubMed Abstract | Publisher Full Text | Free Full Text
2. Tang X, Xiao M, Zhuo C, et al.: Multi-level analysis of bacteria isolated from inpatients in respiratory departments in China. J Thorac Dis. 2018; 10(5): 2666–2675. PubMed Abstract | Publisher Full Text | Free Full Text
3. Hattemer A, Hauser A, Diaz M, et al.: Bacterial and clinical characteristics of health care- and community-acquired bloodstream infections due to Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2013; 57(8): 3969–3975. PubMed Abstract | Publisher Full Text | Free Full Text
4. Caldas AR, Brandao M, Paula FS, et al.: Hypertrophic cranial pachymeningitis and skull base osteomyelitis by Pseudomonas aeruginosa: case report and review of the literature. J Clin Med Res. 2012; 4(2): 138–144. PubMed Abstract | Publisher Full Text | Free Full Text
5. Gowan JR, Deretic V: Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev. 1996; 60(3): 539–574. PubMed Abstract | Free Full Text
6. Juan C, Peña C, Oliver A: Host and Pathogen Biomarkers for Severe Pseudomonas aeruginosa Infections. J Infect Dis. 2017; 215(Suppl 1): S44–S51. PubMed Abstract | Publisher Full Text
7. Nord CE, Sjöberg L, Wadström T: Pseudomonas aeruginosa in oral infections. Acta Odontol Scand. 1972; 30(3): 371–381. PubMed Abstract | Publisher Full Text
8. Barnett F, Axelrod P, Tronstad L, et al.: Ciprofloxacin treatment of periapical Pseudomonas aeruginosa infection. Endod Dent Traumatol. 1988; 4(3): 132–137. PubMed Abstract | Publisher Full Text
9. Moradali MF, Ghods S, Rehm, BH: Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence. Front Cell Infect Microbiol. 2017; 7: 39. PubMed Abstract | Publisher Full Text | Free Full Text
10. Dietrich LE, Price-Whelan A, Petersen A, et al.: The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Mol Microbiol. 2006; 61(5): 1308–1321. PubMed Abstract | Publisher Full Text
11. Baron SS, Rowe JJ: Antibiotic action of pyocyanin. Antimicrob Agents Chemother. 1981; 20(6): 814–820. PubMed Abstract | Publisher Full Text | Free Full Text
12. O’Malley YQ, Reszka KJ, Rasmussen GT, et al.: The Pseudomonas secretory product pyocyanin inhibits catalase activity in human lung epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2003; 285(5): L1077–1086. PubMed Abstract | Publisher Full Text
13. Hall S, McDermott C, Anoopkumar-Dukie S, et al.: Cellular Effects of Pyocyanin, a Secreted Virulence Factor of Pseudomonas aeruginosa. Toxins (Basel). 2016; 8(8): pii: E236. PubMed Abstract | Publisher Full Text | Free Full Text
14. Ulmer AJ, Pryjma J, Tarnok Z, et al.: Inhibitory and stimulatory effects of Pseudomonas aeruginosa pyocyanine on human T and B lymphocytes and human monocytes. Infect Immun. 1990; 58(3): 808–815. PubMed Abstract | Free Full Text
15. Bianchi SM, Prince LR, McPhillips K, et al.: Impairment of apoptotic cell engulfment by pyocyanin, a toxic metabolite of Pseudomonas aeruginosa. Am J Respir Crit Care Med. 2008; 177(1): 35–43. PubMed Abstract | Publisher Full Text
16. Managò A, Becker KA, Carpinteiro A, et al.: Pseudomonas aeruginosa pyocyanin induces neutrophil death via mitochondrial reactive oxygen species and mitochondrial acid sphingomyelinase. Antioxid Redox Signal. 2015; 22(13): 1097–1110. PubMed Abstract | Publisher Full Text | Free Full Text
17. Döring G, Meisner C, Stern M, et al.: A double-blind randomized placebo-controlled phase III study of a Pseudomonas aeruginosa flagella vaccine in cystic fibrosis patients. Proc Natl Acad Sci U S A. 2007; 104(26): 11020–11025. PubMed Abstract | Publisher Full Text | Free Full Text
18. Kollberg H, Carlander D, Olesen H, et al.: Oral administration of specific yolk antibodies (IgY) may prevent Pseudomonas aeruginosa infections in patients with cystic fibrosis: a phase I feasibility study. Pediatr Pulmonol. 2003; 35(6): 433–440. PubMed Abstract | Publisher Full Text
19. Nilsson E, Larsson A, Olesen HV, et al.: Good effect of IgY against Pseudomonas aeruginosa infections in cystic fibrosis patients. Pediatr Pulmonol. 2008; 43(9): 892–9. PubMed Abstract | Publisher Full Text
20. Thomsen K, Christophersen L, Bjarnsholt T, et al.: Anti-Pseudomonas aeruginosa IgY Antibodies Induce Specific Bacterial Aggregation and Internalization in Human Polymorphonuclear Neutrophils. Infect Immun. 2015; 83(7): 2686–2693. PubMed Abstract | Publisher Full Text | Free Full Text
21. Susilowati H, Hutomo S, Siwi DP, et al.: Caspase-3-dependent cell death in B lymphocyte caused by Pseudomonas aeruginosa pyocyanin. J Dent Indonesia. 2015; 22(2): 51–57. Publisher Full Text
22. York JJ, Fahey KJ: Diagnosis of infectious laryngotracheitis using a monoclonal antibody ELISA. Avian Pathol. 1988; 17(1): 173–182. PubMed Abstract | Publisher Full Text
23. Susilowati H: Cytotoxicity of PCN.xlsx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8115701.v6
24. Rada B, Leto TL: Pyocyanin effects on respiratory epithelium: relevance in Pseudomonas aeruginosa airway infections. Trends Microbiol. 2013; 21(2): 73–81. PubMed Abstract | Publisher Full Text | Free Full Text
25. Ruffin M, Brochiero E: Repair Process Impairment by Pseudomonas aeruginosa in Epithelial Tissues: Major Features and Potential Therapeutic Avenues. Front Cell Infect Microbiol. 2019; 9: 182. PubMed Abstract | Publisher Full Text | Free Full Text
26. Zhao J, Wu Y, Alfred AT, et al.: Anticancer effects of pyocyanin on HepG2 human hepatoma cells. Lett Appl Microbiol. 2014; 58(6): 541–548. PubMed Abstract | Publisher Full Text
27. Li T, Huang X, Yuan Z, et al.: Pyocyanin induces NK92 cell apoptosis via mitochondrial damage and elevated intracellular Ca². Innate Immun. 2019; 25(1): 3–12. PubMed Abstract | Publisher Full Text | Free Full Text
28. Hurley MN, Cámara M, Smyth AR: Novel approaches to the treatment of Pseudomonas aeruginosa infections in cystic fibrosis. Eur Respir J. 2012; 40(4): 1014–1023. PubMed Abstract | Publisher Full Text | Free Full Text

Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 04 Jul 2019

Author details Author details

Sidna Artanto
Roles: Investigation, Methodology, Resources

Heribertus Dedy Kusuma Yulianto
Roles: Data Curation, Investigation, Software, Writing – Original Draft Preparation

Wihaskoro Sosroseno
Roles: Software, Visualization, Writing – Review & Editing

Suryani Hutomo
Roles: Investigation, Writing – Original Draft Preparation

Competing interests

No competing interests were disclosed.

Grant information

This study was supported by a research grant (no. 1048/UN1-P.III/LT/DIT-LIT/2016) from the Ministry of Research, Technology and Higher Education, the Indonesian Government.

Article Versions (2)

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Published: 17 Dec 2019, 8:1008

https://doi.org/10.12688/f1000research.19327.2

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https://doi.org/10.12688/f1000research.19327.1

© 2019 Susilowati H et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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 2; peer review: 2 approved]. F1000Research 2019, 8:1008 (https://doi.org/10.12688/f1000research.19327.2)

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Reviewer Report 20 Dec 2019

Boy M. Bachtiar, Department of Oral Biology and Oral Science Research Center, Faculty of Dentistry, Universitas Indonesia, Jakarta, Indonesia

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https://doi.org/10.5256/f1000research.23846.r57905

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Author Response 13 Jan 2020

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

13 Jan 2020

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Dear Dr. Boy M Bachtiar,

We express our high appreciation for the review, critical analysis, and suggestions given for the improvement of our manuscript. We hope this research report will be ... Continue reading Dear Dr. Boy M Bachtiar,

We express our high appreciation for the review, critical analysis, and suggestions given for the improvement of our manuscript. We hope this research report will be useful for the development of science in the health field.
Dear Dr. Boy M Bachtiar,

We express our high appreciation for the review, critical analysis, and suggestions given for the improvement of our manuscript. We hope this research report will be useful for the development of science in the health field.
Competing Interests: We declare that in the past and at present we have never had a conflict of interest with anyone regarding the process in our manuscript peer review. Close
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Author Response 13 Jan 2020

Heni Susilowati, Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, 55281, Indonesia

13 Jan 2020

Author Response

Dear Dr. Boy M Bachtiar,

We express our high appreciation for the review, critical analysis, and suggestions given for the improvement of our manuscript. We hope this research report will be ... Continue reading Dear Dr. Boy M Bachtiar,

We express our high appreciation for the review, critical analysis, and suggestions given for the improvement of our manuscript. We hope this research report will be useful for the development of science in the health field.
Dear Dr. Boy M Bachtiar,

We express our high appreciation for the review, critical analysis, and suggestions given for the improvement of our manuscript. We hope this research report will be useful for the development of science in the health field.
Competing Interests: We declare that in the past and at present we have never had a conflict of interest with anyone regarding the process in our manuscript peer review. Close
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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

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https://doi.org/10.5256/f1000research.21186.r52110

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

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 this.
Why isn't there a positive control? I can only see results with negative control. Please provide a justification for not including a positive control.
Best to also include other articles from year 2018-2019.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Immunogenetics, Antimicrobial resistance, Molecular Microbiology.

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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

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https://doi.org/10.5256/f1000research.21186.r50799

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, and it was further examined in vitro experiment to mimic the naturally occurring of bacterium-host cell interaction. In my opinion, it is necessary to explain (in the discussion session), when the toxin (PCN) is secreted and injected into host cells. For example, after cell contact (adherence phase).

Specific commons:

Methods:

In the experiment, PCN was added into the cultured cell in the presence or absence of anti-PCN IgY (acted as control). This means, in the absence of the IgY, the toxin can enter the host cell (might be in adherence phase) due to the presence of a receptor, then induced the intracellular pathway leading to human B cell (Raji Cells) death (21). If this is the case, please explain it in the discussion session.

Results:

In order to exclude chicken serum as the other IgY source, please change “the immunized chickens” to “egg of immunized chickens”.
In this study, they used a microscope (fluorescence microscope?) to compare the Raji cells number between groups tested, qualitatively, thus the obtained results was not quantitative.

Discussion:

”anti-PCN IgY antibodies specific ………….to this virulence”. I suggest changing this virulence with this pathogen.
“the present results are supported by the fact that antigen-specific IgY antibodies did prevent P. aeruginosa infection…….., suggesting that P. aeruginosa-specific IgY antibodies may inhibit cellular inflammatory responses induced by this pathogen”. Since the IgY used in this study was specifically bind to the secreted PCN, not to bacterium’s whole cell, the rationale behind using this antibody is need to be explored.
According to the reference (16), the toxin is not having a role in the bacterial invasion into the host cell, but it involves in the mechanisms by which the bacterium kills the host cell tested (neutrophils).
“Furthermore, antigen-specific IgY antibodies were……, suggesting that PCN-specific IgY antibodies may be protective against PCN-induced Raji cell death”. Not clear, what (cell surface) antigen/s that the authors assumed to bind specifically to the IgY, in addition to the secreted toxin (PCN).
Based on this study, please suggest, what should be done in future studies.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Oral microbiology, host agent interaction

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Version 2

VERSION 2 PUBLISHED 04 Jul 2019

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Version 2 (revision) 17 Dec 19	read
Version 1 04 Jul 19	read	read

Boy M. Bachtiar, Universitas Indonesia, Jakarta, Indonesia
Priya Madhavan, Taylor’s University, Subang Jaya, Malaysia

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Reviewer Report

23 Views

20 Dec 2019 | for Version 2

Boy M. Bachtiar, Department of Oral Biology and Oral Science Research Center, Faculty of Dentistry, Universitas Indonesia, Jakarta, Indonesia

23 Views Cite this report Responses(1)

Approved

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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18 Views

27 Aug 2019 | for Version 1

Priya Madhavan, School of Medicine, Faculty of Health and Medical Sciences, Taylor’s University, Subang Jaya, Malaysia

18 Views Cite this report Responses(0)

Approved

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 this.
Why isn't there a positive control? I can only see results with negative control. Please provide a justification for not including a positive control.
Best to also include other articles from year 2018-2019.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Immunogenetics, Antimicrobial resistance, Molecular Microbiology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report

21 Views

16 Jul 2019 | for Version 1

Boy M. Bachtiar, Department of Oral Biology and Oral Science Research Center, Faculty of Dentistry, Universitas Indonesia, Jakarta, Indonesia

21 Views Cite this report Responses(0)

Approved With Reservations

In the experiment, PCN was added into the cultured cell in the presence or absence of anti-PCN IgY (acted as control). This means, in the absence of the IgY, the toxin can enter the host cell (might be in adherence phase) due to the presence of a receptor, then induced the intracellular pathway leading to human B cell (Raji Cells) death (21). If this is the case, please explain it in the discussion session.

Results:

In order to exclude chicken serum as the other IgY source, please change “the immunized chickens” to “egg of immunized chickens”.
In this study, they used a microscope (fluorescence microscope?) to compare the Raji cells number between groups tested, qualitatively, thus the obtained results was not quantitative.

Discussion:

”anti-PCN IgY antibodies specific ………….to this virulence”. I suggest changing this virulence with this pathogen.
“the present results are supported by the fact that antigen-specific IgY antibodies did prevent P. aeruginosa infection…….., suggesting that P. aeruginosa-specific IgY antibodies may inhibit cellular inflammatory responses induced by this pathogen”. Since the IgY used in this study was specifically bind to the secreted PCN, not to bacterium’s whole cell, the rationale behind using this antibody is need to be explored.
According to the reference (16), the toxin is not having a role in the bacterial invasion into the host cell, but it involves in the mechanisms by which the bacterium kills the host cell tested (neutrophils).
“Furthermore, antigen-specific IgY antibodies were……, suggesting that PCN-specific IgY antibodies may be protective against PCN-induced Raji cell death”. Not clear, what (cell surface) antigen/s that the authors assumed to bind specifically to the IgY, in addition to the secreted toxin (PCN).
Based on this study, please suggest, what should be done in future studies.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Oral microbiology, host agent interaction

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Responses (0)

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

[1] 1. Lamas Ferreiro JL, Álvarez Otero J, González González L, et al.: Pseudomonas aeruginosa urinary tract infections in hospitalized patients: Mortality and prognostic factors. PLoS One. 2017; 12(5): e0178178. PubMed Abstract | Publisher Full Text | Free Full Text

[2] 2. Tang X, Xiao M, Zhuo C, et al.: Multi-level analysis of bacteria isolated from inpatients in respiratory departments in China. J Thorac Dis. 2018; 10(5): 2666–2675. PubMed Abstract | Publisher Full Text | Free Full Text

[3] 3. Hattemer A, Hauser A, Diaz M, et al.: Bacterial and clinical characteristics of health care- and community-acquired bloodstream infections due to Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2013; 57(8): 3969–3975. PubMed Abstract | Publisher Full Text | Free Full Text

[4] 4. Caldas AR, Brandao M, Paula FS, et al.: Hypertrophic cranial pachymeningitis and skull base osteomyelitis by Pseudomonas aeruginosa: case report and review of the literature. J Clin Med Res. 2012; 4(2): 138–144. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Gowan JR, Deretic V: Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev. 1996; 60(3): 539–574. PubMed Abstract | Free Full Text

[6] 6. Juan C, Peña C, Oliver A: Host and Pathogen Biomarkers for Severe Pseudomonas aeruginosa Infections. J Infect Dis. 2017; 215(Suppl 1): S44–S51. PubMed Abstract | Publisher Full Text

[7] 7. Nord CE, Sjöberg L, Wadström T: Pseudomonas aeruginosa in oral infections. Acta Odontol Scand. 1972; 30(3): 371–381. PubMed Abstract | Publisher Full Text

[8] 8. Barnett F, Axelrod P, Tronstad L, et al.: Ciprofloxacin treatment of periapical Pseudomonas aeruginosa infection. Endod Dent Traumatol. 1988; 4(3): 132–137. PubMed Abstract | Publisher Full Text

[9] 9. Moradali MF, Ghods S, Rehm, BH: Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence. Front Cell Infect Microbiol. 2017; 7: 39. PubMed Abstract | Publisher Full Text | Free Full Text

[10] 10. Dietrich LE, Price-Whelan A, Petersen A, et al.: The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Mol Microbiol. 2006; 61(5): 1308–1321. PubMed Abstract | Publisher Full Text

[11] 11. Baron SS, Rowe JJ: Antibiotic action of pyocyanin. Antimicrob Agents Chemother. 1981; 20(6): 814–820. PubMed Abstract | Publisher Full Text | Free Full Text

[12] 12. O’Malley YQ, Reszka KJ, Rasmussen GT, et al.: The Pseudomonas secretory product pyocyanin inhibits catalase activity in human lung epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2003; 285(5): L1077–1086. PubMed Abstract | Publisher Full Text

[13] 13. Hall S, McDermott C, Anoopkumar-Dukie S, et al.: Cellular Effects of Pyocyanin, a Secreted Virulence Factor of Pseudomonas aeruginosa. Toxins (Basel). 2016; 8(8): pii: E236. PubMed Abstract | Publisher Full Text | Free Full Text

[14] 14. Ulmer AJ, Pryjma J, Tarnok Z, et al.: Inhibitory and stimulatory effects of Pseudomonas aeruginosa pyocyanine on human T and B lymphocytes and human monocytes. Infect Immun. 1990; 58(3): 808–815. PubMed Abstract | Free Full Text

[15] 15. Bianchi SM, Prince LR, McPhillips K, et al.: Impairment of apoptotic cell engulfment by pyocyanin, a toxic metabolite of Pseudomonas aeruginosa. Am J Respir Crit Care Med. 2008; 177(1): 35–43. PubMed Abstract | Publisher Full Text

[16] 16. Managò A, Becker KA, Carpinteiro A, et al.: Pseudomonas aeruginosa pyocyanin induces neutrophil death via mitochondrial reactive oxygen species and mitochondrial acid sphingomyelinase. Antioxid Redox Signal. 2015; 22(13): 1097–1110. PubMed Abstract | Publisher Full Text | Free Full Text

[17] 17. Döring G, Meisner C, Stern M, et al.: A double-blind randomized placebo-controlled phase III study of a Pseudomonas aeruginosa flagella vaccine in cystic fibrosis patients. Proc Natl Acad Sci U S A. 2007; 104(26): 11020–11025. PubMed Abstract | Publisher Full Text | Free Full Text

[18] 18. Kollberg H, Carlander D, Olesen H, et al.: Oral administration of specific yolk antibodies (IgY) may prevent Pseudomonas aeruginosa infections in patients with cystic fibrosis: a phase I feasibility study. Pediatr Pulmonol. 2003; 35(6): 433–440. PubMed Abstract | Publisher Full Text

[19] 19. Nilsson E, Larsson A, Olesen HV, et al.: Good effect of IgY against Pseudomonas aeruginosa infections in cystic fibrosis patients. Pediatr Pulmonol. 2008; 43(9): 892–9. PubMed Abstract | Publisher Full Text

[20] 20. Thomsen K, Christophersen L, Bjarnsholt T, et al.: Anti-Pseudomonas aeruginosa IgY Antibodies Induce Specific Bacterial Aggregation and Internalization in Human Polymorphonuclear Neutrophils. Infect Immun. 2015; 83(7): 2686–2693. PubMed Abstract | Publisher Full Text | Free Full Text

[21] 21. Susilowati H, Hutomo S, Siwi DP, et al.: Caspase-3-dependent cell death in B lymphocyte caused by Pseudomonas aeruginosa pyocyanin. J Dent Indonesia. 2015; 22(2): 51–57. Publisher Full Text

[22] 22. York JJ, Fahey KJ: Diagnosis of infectious laryngotracheitis using a monoclonal antibody ELISA. Avian Pathol. 1988; 17(1): 173–182. PubMed Abstract | Publisher Full Text

[23] 23. Susilowati H: Cytotoxicity of PCN.xlsx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.8115701.v6

[24] 24. Rada B, Leto TL: Pyocyanin effects on respiratory epithelium: relevance in Pseudomonas aeruginosa airway infections. Trends Microbiol. 2013; 21(2): 73–81. PubMed Abstract | Publisher Full Text | Free Full Text

[25] 25. Ruffin M, Brochiero E: Repair Process Impairment by Pseudomonas aeruginosa in Epithelial Tissues: Major Features and Potential Therapeutic Avenues. Front Cell Infect Microbiol. 2019; 9: 182. PubMed Abstract | Publisher Full Text | Free Full Text

[26] 26. Zhao J, Wu Y, Alfred AT, et al.: Anticancer effects of pyocyanin on HepG2 human hepatoma cells. Lett Appl Microbiol. 2014; 58(6): 541–548. PubMed Abstract | Publisher Full Text

[27] 27. Li T, Huang X, Yuan Z, et al.: Pyocyanin induces NK92 cell apoptosis via mitochondrial damage and elevated intracellular Ca². Innate Immun. 2019; 25(1): 3–12. PubMed Abstract | Publisher Full Text | Free Full Text

[28] 28. Hurley MN, Cámara M, Smyth AR: Novel approaches to the treatment of Pseudomonas aeruginosa infections in cystic fibrosis. Eur Respir J. 2012; 40(4): 1014–1023. PubMed Abstract | Publisher Full Text | Free Full Text

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

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Methods

IgY preparation and purification

Cell cultures

Cell viability

Statistical analysis

Results

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

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

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

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

Discussion

Data availability

Acknowledgements

References

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