Abstract
Maternal immune activation (MIA) as a consequence of either infectious or non-infectious stimuli is an environmental risk factor associated with increased risk for psychiatric disorders with a putative neurodevelopmental origin in the offspring. At least in part, this association is driven by elevations in cytokines in the foetal brain downstream of the maternal immune response. Although this likely involves many such molecules, there is evidence to suggest that elevations in the levels of maternal and foetal interleukin (IL)-6 is specifically associated with higher risks for schizophrenia (SZ), bipolar disorder (BD), and autism spectrum condition (ASC) in the offspring. In this context, IL-6 appears to act as sensor, transducer, and effector. Consistent with this view, blocking IL-6 signalling in pregnant mice after induction of MIA prevents the development of behavioural phenotypes relevant for ASC and SZ. However, our understanding of the cellular and molecular mechanisms specific to human physiology that drive these relationships is limited, particularly in non-neuronal cells such as microglia that are implicated in the neurobiology of psychiatric disorders. In part this reflects a lack of access to human brain tissue at early developmental stages. To bridge this gap, we have developed a novel human induced pluripotent stem cell (iPSC)-derived model, to study the impact of IL-6 exposure on both neurons and microglia. By using hiPSC to retrain the donor's genetic background, we can explore connections between genetic risk and environmental factors that can be modelled in vitro, such as IL-6. This is crucial for studying underlying mechanisms, given the known interactions between genetic and environmental factors in the context of psychiatric disorders, which is currently challenging to achieve using mouse models.First, we characterized the effect of acute IL-6 exposure on iPSC-derived microglia-like cells (MGL) and cortical neural progenitor cells (NPC) in monoculture. Human forebrain NPCs did not respond to acute IL-6 exposure in monoculture due to the absence of IL-6Ra expression and sIL-6Ra secretion.
The addition of recombinant IL-6Ra however, enabled NPCs to respond to IL-6 in dose-dependent manner via trans-signalling as measured by phosphorylation of STAT3. By contrast, MGLs express IL-6Ra and secrete sIL-6Ra, hence acute IL-6 exposure resulted in rapid STAT3 phosphorylation and increased expression of genes downstream of STAT3. Transcriptomic analysis of MGLs following acute IL-6 exposure revealed overlapping changes between gene sets and pathways identified using post-mortem brain tissue of individuals with SZ, with no effect on the expression of risk genes for SZ as measured by GWAS. Live imaging showed increased MGL cytoplasm ruffling, consistent with observations in rodent MIA models. Finally, MGLs exhibited elevated levels of cytokines and chemokines, consistent with observations MIA rodent models such as MIP-1𝛼.
Having provided evidence for cell-specific responses to acute IL-6 exposure in vitro, we proceeded to evaluate how this interacts with a known genetic risk profile for psychiatric disorders. To achieve this, we used hiPSC cell lines donated by individuals with 22q.11 deletion syndrome (DS), one of the most penetrant genetic risk factors for ASC and SZ. To study how the MGL response to IL-6 might influence the development of cortical neurons, a trans-well culture system was developed that allows communication between these cell types without direct physical interaction, so the responses of each cell type may be assessed. Cytokine secretion and transcriptomic RNAseq analysis revealed a robust response by MGLs to IL-6 exposure and differed in 22q11.2DS cells which associated with the “regulation of vasculature development” consistent with the upregulation of VEGFA expression that was not noted in control cells, which is consistent with a dysfunctional endothelial cells and blood-brain barrier in 22q11.2DS. Separately, staining of mature cortical neurons, that were exposed to acute IL-6 during their NPC development stage in combination with microglia, showed a decrease in vGlut1 synaptic puncta in 22q11.2DS neurons but not in control cells which is consistent with data from post-mortem studies and in vivo PET imaging for reduced pre-synaptic proteins in SZ.
In summary, this thesis aimed to develop a human gene (22q11.2DS) by environment (IL-6) model to investigate the effects of acute IL-6 exposure on microglia and cortical neuron development. The findings enhance our understanding of NDD mechanisms by: (1) identifying that NPCs are unable to respond to IL-6 by cis-signalling; (2) the MGL transcriptome response overlaps with post-mortem SZ transcriptome; and (3) acute IL-6 reduces vGlut1 expression in mature neurons with the 22q11.2 deletion. The findings were validated against previously published data from both human and mouse MIA models, demonstrating cell-specific changes relevant to NDDs. Further investigation with additional cell types, such as endothelial cells, is necessary to study this complex interaction in a human iPSC model. Taken together, these results emphasize the importance of studying gene-environment interactions in a human-specific context using multiple cell types.
| Date of Award | 1 Nov 2023 |
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| Original language | English |
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| Supervisor | Anthony Vernon (Supervisor) & Deepak Srivastava (Supervisor) |