102 Chapter 4 a b c Concentration (nM) TSPO occupancy (%) XBD173 HAB LAB 2 26.0 2.7 23 80.2 24.3 284 98.0 79.8 d e f g j i h k l XBD173 (nM) XBD173 (nM) XBD173 (nM) XBD173 (nM) XBD173 (nM) XBD173 (nM) Human TSPO module Mouse TSPO module Human random module Mouse random module XBD173 XBD173 VEH veh untr XBD173 veh untr Figure 8. TSPO ligand XBD-173 modulates classical pro-inflammatory myeloid cell function in mouse but not human myeloid cells. a-c The specific TSPO ligand XBD-173 reduces LPS-induced cytokine secretion in mouse BV2 microglia (a,b) and primary bone-marrow derived macrophages (c; BMDM, XBD = 10nM). (a P = 0.0007, F = 9.646, df = 5, n = 3, padj(100) = 0.014, padj(1000) = 0.003; b P = 0.0008, F = 9.282, df = 5, n = 3, padj(1000) = 0.006; c P = 0.005, n = 6). d-g XBD-173 does not reduce LPS-induced cytokine secretion by human primary monocyte-derived macrophages derived from rs6971 AA individuals (high affinity binders, HAB), rs6971 TT individuals (low affinity binders, LAB) (d,e) or by hiPSC derived microglia-like cells (f,g) (d HAB: P = 0.8333, K-W = 1.4624, df = 4, n = 6; LAB: P = 0.141, K-W = 5.8624, df = 4, n = 6; e HAB: P = 0.09999, K-W = 7.7796, df = 4, n = 6, LAB: P = 0.2097, F = 0.68, df = 4, n = 6; f P = 0.057, n = 7, XBD = 200nM; g P = 0.423, n = 7, XBD = 200nM). h,i XBD-173 enhances phagocytosis in mouse BMDM (h) but not human monocytes (i) (h P < 0.0001, F = 12.07, df = 4, n = 5; i P = 0.1728, K-W = 6.376, df = 4, n = 5). j-k TSPO gene co-expression modules from naïve and pro-inflammatory primary macrophages in mouse and human. Gene ontology biological processes for the mouse TSPO module is enriched in classical proinflammatory pathways (j) and the human TSPO module is enriched for bioenergetic pathways (k). 3 genes overlap between mouse and human TSPO modules (l, left panel), compared to 2 genes overlapping between human and mouse random modules of the same size (l, right panel). Statistical significance in a,b,d,e and i,j was determined by one way ANOVA or Kruskal-Wallis test when not normally distributed and by a two-tailed unpaired t-test or Mann-Whitney U-test when not normally distributed in c,f and g. Box and whiskers mark the 25th to 75th percentiles and min to max values, respectively, with the median indicated. Discussion Microglial activation accompanies and is a major contributor to neurodegenerative and neuroinflammatory diseases1,4-6,76. A better understanding of microglial activation in combination with a technique that could reliably quantify activated microglia in the human brain would have broad utility to monitor disease progression as well as response to therapy. TSPO PET has been applied bymany with this objective9,10. Herewe have tested thewidely held assumption that TSPO cellular expression increases upon microglial activation. We examined in vitro data from isolated myeloid cells across 6 species, multiple sequence alignment of the TSPO promoter region across 34 species, and ex vivo neuropathological and scRNAseq data from human neuroinflammatory and neurodegenerative diseases, with relevant marmoset and young and aged mouse models. We show that TSPO expression increases in mouse and rat microglia when they are activated by a range of stimuli, but that this phenomenon is
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