Paper Critique: Aspergilla’s Fumigatus Anti-inflammation

Paper Critique

Mark S Gresnigt, Silvia Bozza, Katharina L. Becker, Leo A B Joosten, Shahla Abdollahi-Roodsaz, Wim B van der Berg, Charles A. Dinarello, Mihai G Netea, Thierry Fontaine, Antonella De Luca, Silvia Moretti, Luigina Romani, Jean-Paul Latge, Frank L. van de Veerdonk.

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A Polysaccharide Virulence Factor from Aspergillus fumigatus Elicits Anti-inflammatory Effects through Induction of Interleukin-1 Receptor Antagonist

Introduction

The study performed by Mark S. Gresnigt et al. (2013) reconnoitred the possible techniques to extract and test whether a component of the cell wall of the fungus; aspergilla’s fumigatus have anti-inflammatory effects. The component used to test this theory use in vitro (biological molecules outside their normal habitat) PBMC (peripheral blood mononucleated cells) such as monocytes, macrophages and lymphocyte stimulation assays. The component from the cell wall of the fungus is Galactosaminoglycan or GAG, this component possess certain anti-inflammatory properties.

Glycosaminoglycan’s produce anti-inflammatory processes in humans by blocking T-helper 1 and 17 of the immune system which is involved in inflammatory responses. Glycosaminoglycans achieve this by blocking Interleukin 1 involved in pro-inflammatory processes during infections. This induces an antagonist called interleukin 1 receptor antagonist which binds to the receptor as a competitive inhibitor binds in enzyme complex substrates in order to block or dampen the normal response of interleukin 1.

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Methods

Firstly Glycosaminoglycan’s isolated from A.fumigatus was collected and purified, the glycosaminoglycan’s go through a phase of lyophilised which is a preservation technique involving dehydration. The aforementioned lyophilised glycosaminoglycan’s are then suspended in hydrochloric acid, to avoid contamination. The glycosaminoglycan’s were incubated in polymixin B which is usually used as an antibiotic for gram negative bacteria.

An isolate of A. fumigatus V05-27 was used for stimulation, conidia (fungal spores) and hyphae (a filamentous segment of fungus) was extracted and exposed to high temperatures, this process is called heat killing. Recombinant human interleukin 1beta, 23, 12, and 18 were purchased from R&D systems. The above mentioned interleukins are involved in inflammation. Recombinant human IL-Ra was also purchased. The PBMC were isolated, counted and adjusted, and placed on a 96 well round-bottom plate. The PBMC’s were pre stimulated with Glycosaminoglycan’s, culture medium and heat killed A. fumigatus conidia, IL1beta, IL23 or IL12/IL18 were added. All of these assays are performed in the presence of anti-IL-Ra antibody. The plates were incubated at 37 degrees, 5% CO2 for 24 hours for 7 days and serum were continuously added, after incubation the plates are collected, stored at -20 degrees and the cytokine content is measured.

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An ELISA was used to check the amount of cytokines. Mice were collected and breed under non-specific pathogen conditions, strains from aspergillum were obtained and injected in mice that were anaesthetized with isofluoran (an agent used for anaesthesia). The mice were treated with glycosaminoglycan. From days 1 to 3 post injection fungal levels were monitored. Histological samples were used to manage the fungal growth in the mice; the lungs were filled with a pyrogen (a fever causing agent) free saline fluid. The fluid was collected and centrifuged at 400 grams for 5 minutes; a Broncho alveolar Lavage used for diagnostic purposes was used to check the growth of the fungus. Stool samples and histological samples were taken, these method were carried out to check for IL1ra mRNA expression.

Fig 1 shows a direct Elisa test for a viral sample. In the paper cytokines need to be detected, this is done using a sandwich Elisa test to detect the concentration of cytokines and chemokines using capture antibodies. (Image from creative commons)

Results

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Fig 4 in the paper shows how the interleukin 1 receptor antagonist increases susceptibility to asperigillosis. The main result of the study showed that Glycosaminoglycan’s can control T-helper cytokine responses, this means that Glycosaminoglycan’s block the pro inflammatory t-helper production of cytokines. Another key result showed Glycosaminoglycan’s ability to produce an interleukin 1 receptor antagonist; Glycosaminoglycan’s achieve this by blocking the interleukin 1 pathway. The experiment showed Glycosaminoglycan’s induce the interleukin 1 receptor antagonist and the susceptibility to asperigillosis. As seen in fig 1 of the paper glycosaminoglycan blocks Aspergillums T-cell cytokine production. A study by Jane K. Relton et al. shows positive effects of the receptor antagonist. It was shown in rats that the receptor antagonist inhibits negative ischaemic effects and excitotoxic (a process were the neurons are damaged and killed) neuronal damage in rats. This study on the receptor antagonist shows that there are more functions of the IL1ra apart from just blocking inflammation. Another study on interleukin 1 receptor antagonist showed that there are behavioural effects on lipopolysaccharide in rats. The findings show that there are many possibilities on the functions of the receptor antagonist.

My opinion

The applications of this study in my opinion can be a breakthrough in science. Aspergillus fumigatus is a fungus that causes infection, the fungus goes through many strategies to evade the immune system mainly by stopping recognition of the pathogen. This study employs only a small part of a potentially fatal infection and uses it to benefit the immune system. However any conclusions made in the mice are not directly applicable to humans.

The statistics in the figures showed the differences in the stimulations using a Wilcoxon signed rank test and the data was considered significant, the test were done twice and presented as a mean or standard error of mean and a graph pad prism was used to organise, compare and combine all the statistical data. In my opinion the graphs were properly explained and allowed the reader to draw and confirm accurate results, the fact that two test were done and a mean taken shows that the data is more reliable. The signal transduction pathway which was activated by the polysaccharide is a very promising area for future research. New treatments can be developed using knowledge gained from the ILRa in fungal infections. Diseases caused by inflammatory processes such as bone and joint arthritis could be controlled with this antagonist. In conclusion the glycosaminoglycan can be recruited in treatment of interleukin 1 inflammatory disease. More research must be done or a follow up study should be carried out on the role and effects of glycosaminoglycan IL1Ra on inflammatory diseases.

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References

1. ‘Effects of interleukin=1 receptor antagonist o the behavioural effects of lipopolysaccharide in rat’, Rose-Marie Bluthe et al.
2. ‘Interleukin 1 receptor antagonist inhibits ischaemic and excitotoxic neuronal damage in rat’ Jane K. Relton et al.
3. “ELISA diagram” by Cavitri – Own work. Licensed under Creative Commons Attribution 3.0 via Wikimedia Commons