Introduction
Interleukin-6 (IL-6) is a key cytokine upregulated by infection in pregnant and non-pregnant individuals. IL-6 can signal through two signalling pathways – cis and trans. Trans signalling is associated with a pathogenic response[1], and implicated in peripheral disease[2,3]. However, we lack evidence about trans signalling in human brain. As IL-6 can cross human placenta, and mediates induction of behavioural phenotypes in animal maternal immune activation models, differential mechanisms of signalling may hold relevance for aetiology of neurodevelopmental disorders. Microglia express both membrane-bound IL-6 receptor (IL-6R), and signal transducer gp130[4], enabling response through both pathways. We sought to explore whether microglia exhibit differential phenotypes when either cis- or trans IL-6 signalling is activated.
Methods
Human iPSC-derived macrophage precursors were differentiated into microglia-like cells as described previously [4]. On day 14, N=4 neurotypical donors were exposed to i) IL-6, ii) IL-6 and soluble gp130, (cis signalling), or iii) hyper IL-6, a fusion construct of IL-6/soluble IL-6Ra, (trans signalling), (or iv) vehicle,) for 3 and 24 hours. Transcriptomic changes were assessed via bulk RNA sequencing using the DESeq2 package in R (version 4.4.2). Pooled media samples from 24hr-treated microglia were incubated with proteome profiler array membranes, to provide a qualitative read-out of cytokine secretion. In addition, a FACS-based measure of phagocytosis of E. coli particles was performed.
Results
Microglia exhibited a robust transcriptional response to IL-6 (>2200 DEGs, 5% FDR), unaffected by blockade of trans signalling, at 3hrs. In contrast, trans signalling led to differential expression of 15 genes at 3hrs. Chiefly among these was STAT3, indicative of canonical IL-6 activation, which was increased to a similar level in all IL-6-treatment conditions. These patterns were conserved at 24hrs, though DEGs were relatively lower (~1300) in IL-6 and cis-treated cells. GO enrichment analysis identified generic inflammation-related pathways, including regulation of inflammatory response, and cellular response to interleukin-6, after IL-6 and trans-treatment, respectively. Most DEGs produced by trans signalling were also altered in IL-6 and cis-treated microglia. Uniquely, the gene IFI16 was upregulated by trans signalling only at both 3 and 24hrs. Of 34 cytokines measured, 10 were detectable, and seven, including MIP-1a, RANTES, MCP-1, and CCL1, were increased by IL-6 and cis-treatment. Trans-treated microglia upregulated MCP-1 and CCL1 alone. At 3 hours, two of four cell lines exhibited higher mean fluorescence intensity (MFI) in all IL-6-treated conditions, compared with vehicle, indicative of increased phagocytic behaviour. At 24 hours, MFI in the remaining two lines was consistently increased across IL-6-treatments, whilst the other two lines displayed more stochastic patterns. Given variability over
time, group-wise changes were not statistically significant (χ2(3)>1.72, p >0.32), and suggest high donor-driven variability.
Conclusions
Results suggest microglia exhibit a muted transcriptional response to IL-6 trans signalling, compared to cis signalling. This occurs despite indications of a standard cellular response to IL-6, including cytokine secretion and transcriptional activation of relevant pathways. Future studies will examine the role of genes, particularly IFI16, uniquely upregulated by IL-6 trans signalling. Further studies to determine functional consequences of these pathways on cell motility and morphology are currently underway.