Published on April 25th, 2023
⏱ 11 min read
Cerebral malaria is the main cause of death in patients infected with Plasmodium falciparum. Artemisinin and its derivatives, such as artesunate (ATN), are the first-line treatment prescribed for severe malaria and display immunomodulatory properties (Mancuso et al, 2021).
In order to study the effects of artesunate in the brains of Plasmodium-infected hosts, Wang and colleagues have analysed the transcriptome in the brain of mice infected with P. berghei, a murine malaria species that also induces cerebral inflammation, and subsequently treated with ATN (Wang et al, 2022).
As expected, they observed the differential expression of various cytokines (in particular Tnf-α and Il-1β) upon infection and then treatment with ATN. Gene Set Enrichment Analysis (GSEA) indicated the dysregulation of pathways related to the JAK-STAT signaling pathway, apoptosis, and Toll-like receptor signaling pathway.
As the raw transcriptomic data is currently available on the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under the ID GSE162535, I decided to take a look at it using the Omics Playground Platform and, where possible, expand on the original findings by the authors.
The platform was run by intersecting the default choices of methods for differential gene expression analysis (limma, EdgeR and DEseq2), with cutoffs set at p<0.05 and logFC>0.5. For GSEA, the camera, fgsea and gsva methods were intersected with cut-offs set at p<0.05 and logFC>0.2.
The data shows a strong clustering based on infection and treatment, with the infected samples (“ECM” and “Artesunate”) forming a distinct cluster compared to control samples and being further separated by treatment on a UMAP plot (Fig. 1A).
Functional annotation of the clustered heatmap (Fig. 1B) highlights, among others, the upregulation (cluster S3) of various inflammatory pathways, apoptosis and the IL6-JAK-STAT3 pathway, based on the Hallmark gene set collection (Fig. 2A), and of the Toll-like receptor signaling pathways, based on the GO Terms collection (Fig 2B), following infection with P. berghei.
This replicates the main findings of the publication.
A useful information that can be gained from the platform regarding the nature of an experiment is obtained by comparing a selected expression signature against a collection of datasets collected from the GEO public database.
In this case, the gene expression signature generated by malaria infection against uninfected samples indicated a strong correlation with an experimental infection with P. berghei in mice (Fig. 3), which was part of a larger study on the pathogenesis of neuroinflammation (Torre et al, 2017).
Other pairwise comparisons with ATN-treated samples were still dominated by the P. berghei-induced signature, indicating the same dataset as the most correlated one (data not shown).
Differential gene expression analysis highlights, as expected, the upregulation of several cytokines and other immune-related genes following infection (Fig. 4). Additionally there was also a marked downregulation of three hemoglobin genes.
Treatment of infected cells with ATN resulted in the dampening of immune responses, as highlighted by the downregulation of several immunity-related genes, and the recovery of hemoglobin gene expression (Fig. 5).
GSEA was performed against all the collections available on the platform. Consistently with the gene expression analysis, infection resulted in the upregulation of various gene sets associated with inflammatory responses, particularly interferon activation (Fig. 6).
Apoptosis, JAK-STAT signaling and Toll-like receptor signaling pathways were also upregulated. This was accompanied by a loss of neuronal activity, as highlighted by the downregulation of glutamate receptor activity (Fig. 7).
Treatment with ATN effectively reversed these trends, dampening immune responses and restoring neuronal activity (Fig. 8).
The platform offers access to the L1000 drug connectivity map, a collection of more than a million expression profiles of 1000 genes from cell lines exposed to various drug concentrations, as well as individual gene silencing and overexpression experiments.
Individual gene manipulation comparison indicated that infection was positively correlated with the overexpression of various cytokines (Fig. 9).
When comparing the infection signature against those generated by various drugs, a strong positive correlation with topoisomerase inhibitors was observed (Fig. 10). As these result in cell apoptosis, it could reflect the pro-apoptotic properties of parasite hemozoin against neuronal cells (Eugenin et al, 2019) or a consequence of the expression of NF-κB induced by pro-inflammatory cytokines in the brain (Punsawad et al, 2013).
Furthermore, the platform indicated that dopamine receptor antagonists could be used to reverse the effects induced by ECM (Fig. 10). Since aberrant dopamine expression plays a critical role in cerebral malaria (Kumar and Babu, 2020), this could represent a plausible novel approach for treating cerebral malaria.
Comparing ATN-treated infected samples vs the control samples highlighted a weak positive correlation with the silencing of gene GABARAPL1 (Fig. 11). This gene is involved in autophagosome maturation (Chakrama et al, 2010) and since artemisinin and its derivatives have been known to induce apoptosis in cancer cells (Kiani et al, 2020), this was a counter-intuitive correlation.
However, a publication from 2018 suggested that artemisinin prevents neuronal cell apoptosis by activating the AKT pathway (Lin et al, 2018). Since activation of the AKT pathway inhibits expression of GABARAPL1 (Su et al, 2017), this could explain the correlation detected in the dataset.
Nonetheless, there is no indication of a downregulation in the expression of GABARAPL1 following ATN treatment, nor any negative correlation of ATN with known AKT inhibitors, which could have been used to imply an activating role (data not shown). It must be stressed that the available data is not suited to address this question, due to the presence of malaria parasites in ATN-treated samples.
Through the platform, the main findings of the original publication could be replicated. Thus I observed the upregulation of inflammatory responses and a loss of neuronal function in infected samples and a reversal of these trends upon treatment with ATN.
Thanks to access to the drug connectivity map database, further insights could also be gained from the dataset.
ATN treatment was positively correlated with gene expression profiles associated with topoisomerase inhibitors, known to induce apoptosis, one of the main modes of action of ATN. Meanwhile, the aberrant dopamine expression associated with cerebral malaria indicated the therapeutic potential of dopamine receptor antagonists, which has hitherto remained unexplored in this context.
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Axel Martinelli’s academic background lies in molecular biology and parasitology. He took his Ph.D. on the genetics of strain-specific immunity against malarial infections.
After his Ph.D. he undertook a Master degree in bioinformatics and specialised in the analysis of omics data. Over the course of his postdoctoral career he worked on genomics and transcriptomics studies in various protozoal parasites, as well as the helminth H. contortus.
He is currently Head of Biology at Bigomics Analytics.