Modelling host-Trypanosoma brucei gambiense interactions in vitro using human induced pluripotent stem cell-derived cortical brain organoids

Abstract

Background: Sleeping sickness is caused by the extracellular parasite Trypanosoma brucei and is associated with neuroinflammation and neuropsychiatric disorders, including disruption of sleep/wake patterns, and is now recognised as a circadian disorder. Sleeping sickness is traditionally studied using murine models of infection due to the lack of alternative in vitro systems that fully recapitulate the cellular diversity and functionality of the human brain. The aim of this study is to develop a much-needed in vitro system that reduces and replaces live animals for the study of infections in the central nervous system, using sleeping sickness as a model infection. Methods: We developed a co-culture system using induced pluripotent stem cell (iPSC)-derived cortical human brain organoids and the human pathogen T. b. gambiense to model host-pathogen interactions in vitro. Upon co-culture, we analysed the transcriptional responses of the brain organoids to T. b. gambiense over two time points. Results: We detected broad transcriptional changes in brain organoids exposed to T. b. gambiense, mainly associated with innate immune responses, chemotaxis, and blood vessel differentiation compared to untreated organoids. Conclusions: Our co-culture system provides novel, more ethical avenues to study host-pathogen interactions in the brain as alternative models to experimental infections in mice. Future work is required to increase the complexity of the organoids (e.g., addition of microglia and vasculature). We envision that the adoption of organoid systems will be beneficial to researchers studying mechanisms of brain infection by protozoan parasites. Furthermore, organoid systems have the potential to be used to study other parasites that affect the brain, including neurocysticercosis, significantly reducing the number of animals undergoing moderate and/or severe protocols associated with the study of neuroinflammation and brain infections.

Modelling host-Trypanosoma brucei gambiense interactions in vitro using human induced pluripotent stem cell-derived cortical brain organoids

“…22,23 We also detected significant expression of the interleukin-17 receptor subunit A and D (IL17RA and IL17RD, respectively), interleukin-10 receptor subunit a (IL10RA), and the Interferon gamma receptor 1 (IFNGR1) (Table 1 and Table S2 in Underlying data), 22,23 indicating that these organoids are primed to sense and respond to T. brucei gambiense by activating IL-17, IL-10, and IFNγ signalling pathways. Furthermore, we also detected genes involved in angiogenesis and endothelial function, including the vascular endothelium growth factor subunit c (VEGFC), cadherin 5 (CDH5), the integrin associated protein CD47, and von Willebrand factor (VWF) (Table 1 and Table S2 in Underlying data), 22,23 suggesting that co-culture with T. b. gambiense also induces the expression of genes associated with vasculogenesis and vascular repair. Some of these genes showed a temporal expression dynamic, with some genes involved in immune sensing, recruitment and tissue repair (e.g., CXCL14, VWF, TLR4, IL4R) being exclusively detected after 72 hours of exposure to T. b. gambiense compared to naïve controls.…”

“…Principal component analysis demonstrates that at a transcriptional level, the samples segregate mainly based on infection status and experimental time point, but with limited transcriptional variation between samples harvested at 24 and 72 hours (Figure 1D). In these brain cortical organoids, we identified a total of 6,157 dysregulated genes at 24 hours (3,234 and 2,923 downregulated and upregulated genes, respectively) and 6,677 dysregulated genes at 72 hours (3,468 and 3,209 downregulated and upregulated genes, respectively) (Figure 1E and 1F, and Table S1 in Underlying data), 22,23 defined as genes with an adjusted p value < 0.05 and a Log 2 Fold change of -2< or >2. To obtain a broad overview of immune related pathways, we examined cytokine, chemokine, and immune receptors that were significantly dysregulated in brain organoids exposed to T. b. gambiense.…”

“…Although this disease is frequently modelled using experimental infections in mice, for ethical reasons around the use of animals in research, we were motivated to explore the utility of induced pluripotent stem cell (iPSC)-derived human cortical brain organoids to model brain-trypanosome interactions in vitro as alternatives to in vivo infections. Thus, using an in vitro co-culture system, we set out to characterise the transcriptional responses of the iPSC-derived human brain organoids to the human pathogen T. brucei gambiense (Figure 1A), 22,23 compared to organoids that were not incubated with the parasites. These time points were selected to evaluate early (24 hours) and late (72 hours) responses, in an attempt to gain as much insight as possible into the temporal dynamics associated with tissue responses to infection.…”

“…We detected several genes with canonical immune functions such as CD274, that encodes for Programme death ligand 1(PD-L1), the complement factor C4B, and the glial fibrillary acidic protein (GFAP), typically associated with gliosis during CNS inflammation 12 (Table 1 and Table S2 in Underlying data). 22,23 Additionally, we detected the expression of several interleukins and chemokines such as interleukin-34 (IL34) that promotes monocyte and macrophage survival and differentiation, 13 the chemokine CXCL14 involved in immune cell recruitment, 14 transforming growth factor beta 1 (TGFB1), and the alarmin IL33, which is a critical mediator of innate immune responses and inflammation 15 (Table 1 and Table S2 in Underlying data). 22,23 We also detected significant expression of the interleukin-17 receptor subunit A and D (IL17RA and IL17RD, respectively), interleukin-10 receptor subunit a (IL10RA), and the Interferon gamma receptor 1 (IFNGR1) (Table 1 and Table S2 in Underlying data), 22,23 indicating that these organoids are primed to sense and respond to T. brucei gambiense by activating IL-17, IL-10, and IFNγ signalling pathways.…”

“…22,23 Additionally, we detected the expression of several interleukins and chemokines such as interleukin-34 (IL34) that promotes monocyte and macrophage survival and differentiation, 13 the chemokine CXCL14 involved in immune cell recruitment, 14 transforming growth factor beta 1 (TGFB1), and the alarmin IL33, which is a critical mediator of innate immune responses and inflammation 15 (Table 1 and Table S2 in Underlying data). 22,23 We also detected significant expression of the interleukin-17 receptor subunit A and D (IL17RA and IL17RD, respectively), interleukin-10 receptor subunit a (IL10RA), and the Interferon gamma receptor 1 (IFNGR1) (Table 1 and Table S2 in Underlying data), 22,23 indicating that these organoids are primed to sense and respond to T. brucei gambiense by activating IL-17, IL-10, and IFNγ signalling pathways. Furthermore, we also detected genes involved in angiogenesis and endothelial function, including the vascular endothelium growth factor subunit c (VEGFC), cadherin 5 (CDH5), the integrin associated protein CD47, and von Willebrand factor (VWF) (Table 1 and Table S2 in Underlying data), 22,23 suggesting that co-culture with T. b. gambiense also induces the expression of genes associated with vasculogenesis and vascular repair.…”