101 TSPO in neurodegeneration Figure 7. TSPO is increased in mouse but not human pro-inflammatory activated and disease-associated microglia. a-c Boxplots and dotplots showing the significantly elevated expression of Tspo in mouse models of pro-inflammatory activation using LPS (GSE115571), of acute EAE (GSE130119) and of AD (GSE98969). The percentage of cells that express Tspo in mouse microglia is relatively low, but it is considerably increased after LPS treatment, in the EAE model and in the DAM cells. d-f TSPO is not significantly upregulated in microglia-like cells from the CSF of AD (GSE134578) and MS (GSE138266) patients. The percentage of cells that express a given gene corresponds to the size of the dot, whereas the average expression corresponds to the fill colour of the dot. TSPO is mechanistically linked to classical pro-inflammatory myeloid cell function in mice but not humans. Having demonstrated species-specific differences in TSPO expression and regulation, we then sought to examine TSPO function in mouse and human myeloid cells. We first examined the effect of pharmacological modulation of the classical microglial pro-inflammatory phenotype using the high affinity TSPO ligand, XBD173. Consistent with the literature11-13, we found that in primary mouse macrophages and the BV2 mouse microglial cell line, XBD173 reduced LPS induced release of proinflammatory cytokines (Fig. 8a,b,c). However, in primary human macrophages and in human induced pluripotent stem cell (hIPSC) derived microglia, XBD173 had no impact on the release of these cytokines, even at high concentrations associated with 98% TSPO binding site occupancy (Fig. 8d,e,f,g). We found similar results for zymosan phagocytosis. Primary mouse microglia demonstrated a dose dependant increase in phagocytosis upon exposure to XBD173 (Fig. 8h). However, we sawno increase in phagocytosis in primary human macrophages upon XBD173 exposure (Fig. 8i). XBD173 is metabolised by CYP3A4, which is expressed in myeloid cells. We therefore used LC-MSMS to quantify XBD173 in the supernatant in order to test the hypothesis that the lack of drug effect on human myeloid cells was due to depletion of XBD173. The measured concentration of XBD173 in the supernatant at the end of the assay was no different to the planned concentration (Fig. S5), excluding the possibility that XBD173 metabolism explained the lack of effect. To understand if TSPO is associated with divergent functional modules in mouse and human we then used weighted gene co-expression network analysis to examine the genes whose expression are correlated with TSPO in mouse and human myeloid cells. To construct the gene co-expression networks, we used four publicly available and one in-house RNA-seq data from human (n = 47) and five publicly available mouse (n = 35) datasets of myeloid cells treated with LPS or LPS and IFNγ. In mouse myeloid cells, the gene ontology biological processes associated with the TSPO network related to classical pro-inflammatory functions such as responses to type 1 and 2 interferons, viruses and regulation of cytokine production (Fig. 8j, Supplementary File 1). However, in human myeloid cells, the processes associated with the TSPO co-expression network related to bioenergetic functions such as ATP hydrolysis, respiratory chain complex assembly, and proton transport (Fig. 8k, Supplementary File 1). In mouse myeloid cells there was no overlap in the genes that TSPO is co-expressed with, relative to human (Fig. 8l).
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