73 Activated microglia do not upregulate translocator protein shift across lesion type. In active MS lesions microglia and macrophages predominantly express all three commonly used markers CD68, IBA-1 and HLA-DR suggesting that these cells have the capacity to scavenge, present antigens as well as phagocytose debris from their environment35-38. In control and NAWM tissue, microglia and macrophages predominantly expressed IBA-1 and CD68 but not HLA-DR, indicating that HLA-DR expression arises during activation of microglia in active MS lesions, corroborating previous studies19. Recently, P2ry12 and TMEM119 have been identified as markers of homeostatic microglia20,39,40, some of which express TSPO11. Microglia expressing TMEM119 also express CD68 in control tissue as well as in early MS lesions19 indicating that resident microglia (expressing P2ry12 and TMEM119) express activation markers the balance of which is likely regulated by inflammatory cytokines in their environment41. As expected, we show that TMEM119 and P2ry12 expression is constitutive in regions where microglia are homeostatic (control, NAWM) and lost in areas where microglia are activated (active lesions and the rim of chronic active lesions). We also show the colocalization of P2ry12 and HLA-DR, suggesting that these cells are either transitioning from their homeostatic status towards a more inflammatory profile or vice versa, from active to homeostatic cells. That almost all TSPO+ cells across a range of white matter regions were microglia, and that – as expected - the phenotype in control and NAWM was homeostatic, and the phenotype in active lesions and the rim of chronic active lesions was activated, allowed for examination of the relationship between activation and TSPO expression. We analysed the percentage of TSPO+ pixels in TSPO+ cells (n=12,225 cells) and compared regions containing homeostatic microglia with regions containing activated microglia. There was no difference in pixel count per cell between these regions. This implies that although TSPO+ cells in active lesion areas are activated microglia/macrophages, their TSPO expression does not increase on a per cell basis relative to microglia/macrophages from homeostatic regions. Additionally, there was no correlation between the morphology and the amount of TSPO expression in microglia and macrophages. These findings are consistent with previous reports from our own group and others showing a lack of increase in TSPO expression in activated myeloid cells8,42, and co-localisation of TSPO with markers of both pro-inflammatory and reparative phenotypes in the MS brain11. In support of our studies, a recent study showed that single cell RNA-seq of microglia in Alzheimer’s disease reported comparable TSPO mRNA levels across all microglial sub-types suggesting TSPO PET to be a good proxy for total microglia count10. Taken together, these findings support generalisation of the hypothesis that when human myeloid cells adopt a pro-inflammatory phenotype, TSPO expression itself does not increase, and any change in signal likely reflects an increase in myeloid cell density rather than a phenotypic shift. The data also highlights the limitations of extrapolating from rodents to humans when examining TSPO biology.
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