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Plasmodium falciparum and TNF-a Differentially Regulate Inflammatory and Barrier Integrity Pathways in Human Brain Endothelial Cells

ABSTRACT: Cerebral malaria is a severe complication of Plasmodium falciparum infection characterized by the loss of blood-brain barrier (BBB) integrity, which is associated with brain swelling and mortality in patients. P. falciparum-infected red blood cells and in!ammatory cytokines, like tumor necrosis factor alpha (TNF-a), have been implicated in the development of cerebral malaria, but it is still unclear how they contribute to the loss of BBB integrity. Here, a combination of transcriptomic analysis and cellular assays detecting changes in barrier integrity and endothelial activation were used to distinguish between the effects of P. falciparum and TNF-a on a human brain microvascular endothelial cell (HBMEC) line and in primary human brain microvascular endothelial cells. We observed that while TNF-a induced high levels of endothelial activation, it only caused a small increase in HBMEC permeability. Conversely, P. falciparum-infected red blood cells (iRBCLs) led to a strong increase in HBMEC permeability that was not mediated by cell death. Distinct transcriptomic pro"les of TNF-a and P. falciparum in HBMECs con"rm the differential effects of these stimuli, with the parasite preferentially inducing an endoplasmic reticulum stress response. Our results establish that there are fundamental differences in the responses induced by TNF-a and P. falciparum on brain endothelial cells and suggest that parasite-induced signaling is a major component driving the disruption of the BBB during cerebral malaria, proposing a potential target for much needed therapeutics.

ORGANISM(S): Homo sapiens

PROVIDER: GSE211439 | GEO | 2022/08/20

REPOSITORIES: GEO

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Project description:Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterised by the accumulation of infected erythrocytes in the microvasculature of the brain, due to parasite adhesins on the surface of infected erythrocytes binding to human receptors on microvascular endothelial cells. The parasite and host molecules involved in this interaction are unknown. We used the Human Brain Endothelial Cell line HBEC-5i to identify the malaria parasite ligands responsible for binding to human brain endothelial cells. Three P. falciparum strains (HB3, 3D7 and IT/FCR3) were selected for binding to HBEC5i and the whole transcriptome of selected and unselected parasites was analysed using a variant surface antigen-supplemented microarray chip. After selection, the only highly upregulated genes were a subset of group A-like var genes (HB3var3, 3D7_PFD0020c, ITvar7 and ITvar19), that showed 11 to >100-fold higher transcription levels in selected parasites. These genes are highly diverse in sequence, but do however show strong similarities in PfEMP1 architecture. Antibodies raised to the HB3var3 variant recognized the surface of infected erythrocytes and abolished the binding of infected erythrocytes to brain endothelial cells. The subset of Group A PfEMP1 variants identified here provides a new target for interventions to treat or prevent cerebral malaria. Unselected Plasmodium falciparum parasites (Uns) only poorly cytoadhere to human brain endothelial cells (HBEC-5i). Plasmodium falciparum HB3, 3D7 and IT/FCR3 strains were selected for binding to HBEC-5i (HB3-HBEC1, HB3-HBEC2, HB3-HBEC-TNF, 3D7-HBEC and IT-HBEC). HBEC-selected and unselected cultures were tightly synchronised before a timecourse experiment was performed. 6 samples, named time points 1 to 6, were taken every 8 hours. 12μg of RNA from the unselected parasites (HB3-Uns, 3D7-Uns or IT-Uns) at each of the 6 time points was combined together to form the reference pool. The pool and 12μg of each individual time point sample from both selected and unselectedparasites were then used for cDNA synthesis. For HB3-HBEC1 (pilot experiment), each HB3-HBEC1 time point (pilot experiment) was hybridised directly with HB3-Uns1 time points. For microarray hybridizations, each cDNA sample was coupled to Cy5 (red dye) while Cy3 (green dye) was added to the pool. Cy5-labelled time point samples were mixed with the same amount of Cy3-labelled pool sample. The solution was loaded on a microarray slide and hybridized for 14–16 h.

Project description:The blood brain barrier (BBB) acts both a physical as well as a metabolic barrier isolating the Central Nervous System (CNS) from systemic circulation, blocking intravascular toxin entry and promoting toxic substance elimination to blood stream. The BBB is formed by four main cellular elements: endothelial cells, astrocyte end-feet, microglial cells, and pericytes. There is strong evidence that astrocytes are able to regulate the phenotypes of endothelial cells by directly contacting or secreting a range of chemical agents, resulting in BBB permeability changes and so on. Conversely, endothelium-derived leukemia inhibitory factor (LIF) has been shown to induce astrocytic differentiation, from studies in normal condition of cell culture. However, in pathological conditions, the specific regulatory mechanism that endothelial cells exert on astrocytes is still less known. In our recently study, a transwell-based, endothelial-astrocyte co-culture model was established to investigate this regulatory relationship in bacterial infection. An extraintestinal pathogenic Escherichia. coli strain was used to infect human brain microvascular endothelial cells (hBMEC) planted in the upper chamber, astrocytes co-cultured in the bottom well were actived without direct contact with bacteria or endothelial cell. The transcription levels of some chemokines and cytokines such as TNF-α and CXCL10 were significantly upregulated. We hypothesized that the secreted proteins from endothelial cells is the main cause of astrocyte activation and the culture supernatant of hBMEC infected by E.coli was obtained to perform the mass spectrometry analysis. In summary, the proteomic identification results showed that 2,692 common proteins were identified in the two groups of control and infected hBMEC, including 140 differentially expressed proteins. The bioinformatics analysis indicated that differentially expressed proteins are mainly classified into structural molecular activity and constituent of ribosome. KEGG pathway enrichment analysis showed that many proteins are involved in focal adhesion as well as a bunch of signaling pathways, PI3K-Akt, Rap1, Ras, MAPK and NF-kappaB signaling pathways, for example. The present study laid an important foundation for the subsequent study of secreted proteins of hBMEC in bacterial infections.

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Plasmodium falciparum and TNF-a Differentially Regulate Inflammatory and Barrier Integrity Pathways in Human Brain Endothelial Cells

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