46 Chapter 2 inflammatory (CD40) and anti-inflammatory (mannose receptor, CD206) activated microglia and macrophages also were used61,62. Microglia/macrophages that express both CD40 and CD206 are described as having an intermediate phenotype. No significant differences were found in proportions of TMEM119+ microglia expressing TSPO across the lesion sub-types (Fig. 2A-D). As reported previously56, P2RY12+ cells expressing TSPO were substantially reduced in active lesions (Fig. 2E-H). Numbers of macrophages and microglia showing an intermediate phenotype, and expressing TSPO were very low in control cases and were not detected in the NAWM in multiple sclerosis (Fig. 2I,J). The density of microglia/macrophages with the intermediate phenotype represented 18% of the total TSPO+ cell population, in active lesions and in the rims of chronic active lesions (Fig. 2J). By contrast, few TSPO+ microglia/ macrophages with the intermediate phenotype were present in the centres of chronic active lesions or in inactive lesions, (Fig. 2J). Cells that were solely expressing the pro-inflammatory marker CD40 or the anti-inflammatory mannose receptor CD206 always expressed TSPO, but were present in very low numbers in multiple sclerosis lesions (<1% of total TSPO+ cells, data not shown). Thus, TSPO expression was not specifically associated with either solely pro- or anti-inflammatory microglia/macrophage phenotype based on these markers. CON WM NAWM Active CA rim CA centre Inactive 0 100 200 300 400 TSPO+CD40+MR+ cells / mm2 ** ** ** ** CON WM NAWM Active CA rim CA centre Inactive 0 100 200 300 400 TSPO+P2ry1 + cells / mm2 * * *** **** J L TSPO/CD20 CON WM NAWM Active CA rim CA centre Inactive 0 100 200 300 400 TSPO+TMEM119+ cells / mm2 D H TSPO/CD206/CD40 I TSPO/P2RY12 G TSPO/TMEM119 C TSPO/CD3 K NAWM NAWM TSPO/TMEM119 TSPO/P2RY12 TSPO/P2RY12 A E F B TSPO/TMEM119 Figure 2. TSPO expression in microglial phenotypes and lymphocytes. Resident microglia expressing TSPO in an active and chronic active lesion and the periplaque white matter (A,B) as well as in NAWM (C). Resident microglia did not show any significant difference in TSPO expression compared to control or NAWM (D). Overview of expression of the homeostatic marker P2RY12 with TSPO in an active and chronic active lesion and the periplaque white matter (E,F), as well as in NAWM (G). A loss in homeostatic microglia expressing TSPO was found in active and chronic active lesions stages (H). Both TSPO+ and TSPO- microglia were found expressing TMEM119 or P2RY12 in multiple sclerosis lesions (black arrowheads; inserts; C,G). In contrast, active and chronic active lesions showed an increase in CD206+CD40+ cells expressing TSPO (I,J). T-cells (CD3) showed low expression of TSPO in multiple sclerosis lesions (K) in contrast to B-cells (CD20) which showed strong localisation with TSPO (L). *=P<0.05; **=P<0.01; ***=P<0.001; ****=P<0.0001. Scale bars (A,B,E,F) 200 μm, (C,G,K,L) = 50 μm, (I) = 25 μm. Inserts are digitally zoomed in to 800x., NAWM = normal appearing white matter, A =active, CA = chronic active, IA = inactive.
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