Regulation of microglial TMEM119 and P2RY12 immunoreactivity in multiple sclerosis white and grey matter lesions is dependent on their inflammatory environment

Abstract

Multiple Sclerosis (MS) is the most common cause of acquired neurological disability in young adults, pathologically characterized by leukocyte infiltration of the central nervous system, demyelination of the white and grey matter, and subsequent axonal loss. Microglia are proposed to play a role in MS lesion formation, however previous literature has not been able to distinguish infiltrated macrophages from microglia. Therefore, in this study we utilize the microglia-specific, homeostatic markers TMEM119 and P2RY12 to characterize their immunoreactivity in MS grey matter lesions in comparison to white matter lesions. Furthermore, we assessed the immunological status of the white and grey matter lesions, as well as the responsivity of human white and grey matter derived microglia to inflammatory mediators. We are the first to show that white and grey matter lesions in post-mortem human material differ in their immunoreactivity for the homeostatic microglia-specific markers TMEM119 and P2RY12. In particular, whereas immunoreactivity for TMEM119 and P2RY12 is decreased in the center of WMLs, immunoreactivity for both markers is not altered in GMLs. Based on data from post-mortem human microglia cultures, treated with IL-4 or IFNγ+LPS and on counts of CD3⁺ or CD20⁺ lymphocytes in lesions, we show that downregulation of TMEM119 and P2RY12 immunoreactivity in MS lesions corresponds with the presence of lymphocytes and lymphocyte-derived cytokines within the parenchyma but not in the meninges. Furthermore, the presence of TMEM119⁺ and partly P2RY12⁺ microglia in pre-active lesions as well as in the rim of active white and grey matter lesions, in addition to TMEM119⁺ and P2RY12⁺ rod-like microglia in subpial grey matter lesions suggest that blocking the entrance of lymphocytes into the CNS of MS patients may not interfere with all possible effects of TMEM119⁺ and P2RY12⁺ microglia in both white and grey matter MS lesions.

Keywords: Cortical lesions; Demyelination; Homeostatic microglia; Multiple sclerosis; Subpial lesions.

Fig. 1

Representative images of lesion types used in this study. Lesions are characterized by loss of PLP staining and amount of MHC-II+ cells. A large amount of of MHC-II+ cells can be observed in the demyelinated area (a) in active WMLs (b). Chronic-active demyelinated WMLs (c) feature a ‘rim’ of MHC-II cells (d) which is also visible in demyelinated active GMLs (e, f). Subpial demyelinated (g) GMLs hardly show MHC-II+ cells (h). Scalebar (a-h) = 200 μm. Dashed lines indicate the edge of the lesion

Fig. 2

Representative images of MHC-II (a, b, c), Iba-1 (d, e, f), TMEM119 (g, h, i) and P2RY12 (j, k, l) immunoreactivity in normal appearing matter and in the demyelinated center of active WMLs and chronic-active WMLs and in pre-active lesions (m, n, o, p). Scalebars (a-l and m-p) = 50 μm

Fig. 3

Representative images of MHC-II (a, b, c), Iba-1 (d, e, f), TMEM119 (g, h, i) and P2RY12 (i, k,l) immunoreactivity in NAGM, active subpial GMLs and non-active subpial GMLs. Arrows indicate rod-shaped microglia visible in subpial GMLs in Iba-1+ cells (f), TMEM119 + cells (i) and P2RY12+ cells (l). Scalebar = 50 μm

Fig. 4

Boxplot of semi-automatic quantification of the of the DAB stained area as percentage of the ROI. in the demyelinated center of lesions compared to normal appearing matter. Boxplots represent the mean, the 1st and 4th quartile and the minimum and maximum value. Post-hoc testing was done between WM groups and between GM groups. N = 15 for NAWM, N = 10 for active WML, N = 7 for chronic-active WML, N = 5 for leuko WML, N = 16 for NAGM, N = 8 for subpial GML, N = 5 for leuko GML. # = p = 0.07, ^* = p < 0.05, ** = p < 0.01, *** = p < 0.001

Fig. 5

Representative images of immunoreactivity for MHC-II (a, b, c), Iba-1 (d, e, f), TMEM119 (g, h, i) and P2RY11 (j, k, l) along the rim of various lesion types. Scalebar (a-l) = 50 μm

Fig. 6

Graphs of TMEM119 and P2RY12 mRNA levels in cultured primary human microglia derived from WM enriched areas or GM enriched areas treated with IFNγ+LPS or with IL-4 compared to untreated WM microglia. mRNA levels from GM derived cells are represented in the grey-coloured box. Data are presented as individual patient-derived microglia measurements and means (bars). N = 10 for all WM-derived microglia conditions, N = 7 for IL-4 treated GM-derived microglia N = 8 for IFNγ+LPS treated GM-derived microglia and N = 9 for untreated GM-derived microglia. * = p < 0.05

Fig. 7

Representative images of IFNγ and IL-4 immunoreactivity in active (a, b) and chronic-active WMLs (c, d), an active subpial GML (e, f) and subpial GMLs (g, h). Scalebar (a-h) = 50 μm

Fig. 8

Representative images of CD3, CD20, TMEM119 and P2RY12 immunoreactivity in subpial GMLs showing meningeal infiltration and in subpial GMLs without meningeal infiltration. In subpial lesions with infiltration of CD3+ cells (a) and CD20+ cells (b) show immunoreactivity for TMEM119 (c) and P2RY12 (d). In subpial GMLs without infiltration of CD3+ (e) and CD20+ (f), immunoreactivity for TMEM119 (g) and P2RY12 (h) is similar. Scalebar (a-h) = 100 μm. Inserts show magnifications of the area of interest. Scalebar inserts = 50 μm