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

Introduction

Neurotropic pathogens encompass a wide range of parasitic organisms, from viruses to protozoan parasites, and are the causative agents of debilitating conditions affecting the central nervous system (CNS), often resulting in life-long impairments and death if left untreated. To date, most of these infections are studied using murine models of infection. Although these infection models often recapitulate the clinical outcomes observed in humans, there are serious ethical and biological implications associated with in vivo host-pathogen interaction studies. More recently, the generation of organoids developed in vitro from human stem cells have provided novel insights into developmental biology, and their potential application as alternative models to study host-pathogen interactions is starting to be recognised. These models offer an opportunity to interrogate human tissues that are difficult to access, such as CNS tissue. Indeed, human brain organoids comprising the diversity of cell types representative of the complex neuroepithelium are an increasingly attractive model system to interrogate how human nervous cells respond to infection. Currently, in vitro brain organoid systems are being used to study infections from ZIKA and SARS-CoV-2,^1–4 and have proven insightful for understanding other parasitic infections, including toxoplasmosis and malaria.^5,6 However, these in vitro systems have not been used to explore the pathogenesis of human African trypanosomiasis, a parasitic infection traditionally known for its devastating neurological effects.^7–10

Here, we explored whether human brain organoids can be used to model host-trypanosome interactions in vitro. Using bulk RNA sequencing, we observed that the human cortical brain organoids transcriptionally respond to the human pathogen T. brucei gambiense by upregulating gene pathways associated with innate immune functions, amongst others. Some of the upregulated genes are proposed to have antimicrobial properties, suggesting that human brain organoids are able to sense and respond to pathogens in the absence of innate immune cells (e.g., microglia). Using this novel in vitro system, we estimate a direct reduction of ~47% of animals required to achieve similar conclusions, and ~20% of animals used as donors to generate infectious parasites. The methods and results presented here have the potential to open new research avenues for the adoption of human brain organoids to model host-pathogen interactions with important implications for the 3Rs principles—replacement, refinement, and reduction.

Methods

Trypanosoma brucei gambiense - human brain organoids co-culture system

• 1. A total of 10⁵ parasites at log-phase of growth were co-cultured with the brain organoids on 12 well plates (Final ratio of 1 organoid:10⁵ parasites per well) for a period of 24 or 72 hours in HMI-9 media diluted 50:50 with CORD media at 37°C and 5% CO₂ in round-bottomed 96-well plates. These two time points were chosen to mimic acute (24 hours) and chronic (72 hours) responses and we determined that parasites grew well under these conditions, at least during the first 72 hours in culture (Figure 1B).

• 2. In parallel, organoids cultured in HMI-9 media diluted 50:50 with CORD media at 37°C and 5% CO₂ but without parasites were also seeded in round-bottomed 96-well plates and were included as controls to assess the effect of diluted media on the organoids transcriptome. As controls, we included organoids kept in 50:50 HMI-9:CORD organoid media alone.

• 3. After 24 or 72 hours, the organoids were gently washed 5 times with 1X PBS (Gibco) and wide bore pipette tips to remove parasites. Then, some organoids were fixed in 4% PFA for 24 hours at room temperature and preserved as paraffin-embedded blocks for immunohistochemistry analysis. The rest of the organoids were processed for bulk transcriptomics.

Figure 1. Bulk transcriptomics analysis of human cortical brain organoids co-cultured with T. brucei gambiense.

A) Schematic representation of the experimental design developed for this study. B) Growth curve assay for T. b. gambiense in HMI-9 alone (teal) or diluted 50:50 with CORD media (magenta). The arrow indicates a dilution step to bring the parasite cultures down to 10² parasites/ml. Data shown as mean ± standard deviation from three independent experiments. C) Representative H&E staining and MAP 2 Immunohistochemistry from naïve (left) and T. brucei gambiense-infected (right) organoid after 72 hours of in vitro co-culture. Scale bar = 50 μm. D) Principal component analysis of the samples including the bulk RNA sequencing analysis. Volcano plot of differentially expressed genes between untreated organoids and after (E) 24 hours and (F) 72 hours in culture with T. brucei gambiense. Dotted line represents the significance (-0.5 < Log₂FC > 0.5 and p adjusted value < 0.05). Pathway analysis of the genes dysregulated at (G) 24 hours and (H) 72 hours in culture with T. brucei gambiense. The adjusted p value (Q value) for each of the enriched pathways is included. MAP 2, microtubule associated protein 2.

Results

African trypanosomes cause extensive neurological changes resulting in neuropsychiatric disorders and culminating in death if not treated adequately. 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. Importantly, we did not detect significant morphological or histological changes in the organoids exposed to the parasites based on the morphological aspects observed upon H&E staining and MAP2 staining (Figure 1C), suggesting that T. brucei gambiense does not seem to elicit tissue damage over a 72 hour culture period, at least not with the markers used in this study. 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 Log₂ 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. 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 inflammation¹² (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 inflammation¹⁵ (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. 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.

To gain a better understanding of the broad transcriptional responses triggered in the human brain organoids to T. b. gambiense infection, we performed Gene Ontology analysis on genes significantly dysregulated. After 24 hours of exposure to T. b. gambiense, the iPSC-derived human brain organoids upregulate genes associated with blood vessel and vasculature development, signalling, and chemotaxis, with a concomitant reduction in genes associated with response to hypoxia, defence response against viruses, and protein ubiquitination (Figure 1G). At 72 hours, the pathways overrepresented in the organoids transcriptome were associated with glial cell differentiation, positive regulation of CD8⁺ T cells, chemotaxis, and vascular and blood vessel differentiation, and a significant reduction of gene pathways associated with cell cycle progression, protein transport and proteasome-mediated protein degradation (Figure 1H). Taken together, these data suggest that T. b. gambiense trigger a broad innate-like immune response in the iPSC-derived human brain organoids accompanied by upregulation of genes involved in vascular development, immune chemotaxis, and cytokine-mediated immune signalling.

Discussion and outlook

In this study, we tested the possibility of using stem cell-derived human brain organoids as an in vitro system as an alternative mode to live animals to study host-trypanosome interactions, in line with the 3Rs principles. We firstly set up an in vitro co-culture system whereby iPSC-derived human brain organoids were co-cultured with the human pathogen T. b. gambiense and assessed the response of these organoids to the pathogen using histology and transcriptomics as a proxy for global responses to the pathogen. The data presented here demonstrate that iPSC-derived human brain organoids trigger a transcriptional programme associated with an innate-like immune response when exposed to T. brucei gambiense. Bulk transcriptomics has enabled us to identify that the brain organoids specifically respond to T. brucei gambiense in vitro by upregulating several genes with putative immune functions such as CXCL14, the alarmin IL33, the complement component C4B, as well as vasculogenesis and vascular repair such as VEGFC. CXCL14 is a potent antimicrobial cytokine secreted in response to inflammatory processes and is critical for human neutrophil recruitment,^14,16,17 which have been proposed as important players in controlling CNS infections.^18,19 Similarly, the upregulation of several genes critical for angiogenesis and development of vascular beds, including VEGFA, VEGFB, and VWF, suggests that they may potentially support vasculogenesis in the presence of this pathogen. All of these observations require further testing at the protein and functional level but provide an initial robust framework to dissect the relevance of these 3D culture systems to model brain-trypanosome interactions.

Our work provides an initial approach to explore the utility of complex 3D culture systems to study host-Trypanosoma interactions in vitro, adding African trypanosomes to the compendium of pathogens that have been tested to model host-pathogen interactions using brain organoids. However, there are many challenges and considerations that need to be addressed for the full implementation of these in vitro systems, with the aim of replacing animal models of infection. One of the critical hurdles is to generate fully mature organs in vitro, encompassing all the cell types typically identified in vivo, including microglia and vasculature cells,²⁰ which are likely to be the main drivers of and/or responders to infection. The incorporation of additional organoids (e.g., vascular or choroid plexus organoids¹) or inclusion of additional cell types (e.g., endothelial cells, microglia), referred to as “building blocks”,²¹ will support the development of mature cortical brain organoids that could faithfully recapitulate the immunological responses observed in vivo. Given these technical and biological limitations, we are unable to examine the role of these cell types using our in vitro host-pathogen culture system. Future work addressing these key challenges will improve the quality of these organoids to model CNS infections in vitro, facilitating the reduction and/or replacement of animals in research. Our work provides a wealth of data that can be further mined to design, refine, or implement in vitro experiments (e.g., using stem-cell derived astrocytes) as alternatives for in vivo work, and provides a foundation for future work in this area.

In summary, we delivered an initial proof-of-concept framework for future adoption of these in vitro systems for neuro-immunology research, motivated by the need to reduce and/or fully replace to use animals to study brain-pathogen interactions, in line with the 3Rs principles under the Animals (Scientific procedure) Act, 1986. Based on our estimations, with this model in place, animals used to study brain infections with African trypanosomes, typically considered to be moderate to severe procedures, would have been reduced by ~47%, with an additional ~27% reduction in the number of immunocompromised mice used as donors to generate infectious parasites.