In preactive WMLs which appeared in white matter that showed no demyelination (Fig. These findings correspond with previous observations that also showed microglial TMEM119 and P2RY12 immunoreactivity along the edgeof (chronic-)active WMLs [15, 17]. 2n), TMEM119 (Fig. https://doi.org/10.1016/S1474-4422(08)70191-1, Kutzelnigg A, Faber-Rod JC, Bauer J, Lucchinetti CF, Sorensen PS, Laursen H, Stadelmann C, Brck W, Rauschka H, Schmidbauer M (2007) Widespread demyelination in the cerebellar cortex in multiple sclerosis. Cookies policy. Front Cell Neurosci 9:111. Clin Immunol 189:6374. Subsequently, tissue was washed in collection medium and chopped using a sterile razor blade. J Neuroinflammation 9:111. https://doi.org/10.1007/s00401-016-1653-y, Van Der Valk P, Amor S (2009) Preactive lesions in multiple sclerosis. 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. Privacy (2017) [9]. Subsequently, sections were deparaffinized in xylene replacement (100%) and graded ethanol series (100, 96, 80 and 70%) to demi-water. From the obtained cortical and subcortical tissue paraffin blocks, 10m sections were cut on a microtome and mounted on positively charged glass slides (Permafrost) and incubated on a heated plate for 1h at 43C. Neuropathology 36:3949. Correspondence to In particular, whereas immunoreactivity for TMEM119 and P2RY12 is decreased in the center of WMLs, immunoreactivity for both markers is not altered in GMLs. Provided by the Springer Nature SharedIt content-sharing initiative. https://doi.org/10.1016/S1474-4422(14)70231-5, Article Google Scholar, Peferoen LAN, Vogel DYS, Ummenthum K, Breur M, Heijnen PDAM, Gerritsen WH, Peferoen-Baert RMB, Van Der Valk P, Dijkstra CD, Amor S (2015) Activation status of human microglia is dependent on lesion formation stage and remyelination in multiple sclerosis. In contrast to WMLs, characterization of lesions in the GM is based on location rather than MHC-II+ activity [28]. T.A. Post-mortem brain material of MS patients was obtained from the Netherlands Brain Bank (NBB, Amsterdam, The Netherlands) and from the Biobank of the Amsterdam MS center (Amsterdam, The Netherlands). MHC-II immunoreactivity was significant between NAWM and NAGM (p<0.05) and NAWM and active WML (p<0.05, Fig. Scalebar=50m. 2h, i). 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. Rod-shaped microglia have been proposed to play a role in synaptic stripping, representing neurodegeneration which is not necessarily mediated by inflammation [36, 37], but is present in various neurodegenerative diseases [38]. Google Scholar, McFarland HF, Martin R (2007) Multiple sclerosis: a complicated picture of autoimmunity. Ann Neurol 68:477493. https://doi.org/10.1002/glia.23097, Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, Eliceiri KW (2017) ImageJ2: ImageJ for the next generation of scientific image data. 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. Anne-Marie van Dam. 7, Table4). Even in subpial GMLs close to meninges containing infiltrated CD3+ and CD20+ cells, we did not observe a difference in the level of immunoreactivity for TMEM119 and P2RY12. To this end, we used post-mortem human MS brain material containing subpial GMLs and various WML types, and leukocortical lesions to perform immunohistochemical analysis of TMEM119 and P2RY12. PubMed Glia 62:18161830. Furthermore, immunoreactivity for TMEM119 and P2RY12 is observed in preactive lesions in the NAWM as well as along the edge of active WMLs and GML. However, it must be noted that variation in P2RY12 and TMEM119 mRNA levels was high in all conditions studied (see Fig. 3l). Compared to NAWM, where MHC-II+ microglia showed a ramified appearance (Fig. Boxplot of semi-automatic quantification of the of the DAB stained area as percentage of the ROI. to block endogenous peroxidase activity. Similar to MHC-II immunoreactivity, active subpial GML showed Iba-1 immunoreactivity in microglia with a more reactive phenotype (Fig. Medium was changed every three days by replacing half of the medium with culture medium. PubMedGoogle Scholar. 2b). Data analysis was performed on the normalized N0 values. Google Scholar, Lassmann H (2014) Mechanisms of white matter damage in multiple sclerosis. 2o) and P2RY12 (Fig. BMC Bioinformatics 18:529. https://doi.org/10.1186/s12859-017-1934-z, Ruifrok AC, Johnston DA (2001) Quantification of histochemical staining by color deconvolution. Per treatment condition, 35 wells containing microglial cells were lysed in a total of 1ml TRIzol (Invitrogen). While studying WM-derived microglia, others have shown similar results upon IFN+LPS treatment, but also increased expression upon IL-4 treatment which we observed to be significantly altered in GM-derived microglia only [15, 18]. This indicates that, although recent evidence points to a role for meningeal infiltration in neuronal loss and glial activation status in MS cortex [5], microglial homeostatic status as indicated by expression of TMEM119 and P2RY12 in demyelinated subpial GM is not altered by the presence of meningeal lymphocytes and still ongoing meningeal inflammation. 3i). 2h), TMEM119 + cells were visible at the edge of active WMLs and active subpial GML, but not in the rim of chronic-active WMLs (Fig. https://doi.org/10.1177/1352458517739139, Article https://doi.org/10.3389/fncel.2015.00084, Grabert K, Michoel T, Karavolos MH, Clohisey S, Baillie JK, Stevens MP, Freeman TC, Summers KM, Mccoll BW (2016) Microglial brain region-dependent diversity and selective regional sensitivities to ageing. MHC-II + cells and Iba1+ cells were present in the rim of active WMLs, chronic-active WMLs and active subpial GMLs (Fig. 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. Based on these in vitro observations, we next explored the possibility that the presence of IL-4 and IFN immunoreactivity varies between GMLs and WMLs, which would affect microglial expression of TMEM119 and P2RY12 in both lesion types. By using this website, you agree to our Lesion location was determined by the relative absence of PLP immunoreactivity indicating demyelinating/demyelinated areas. Exp Mol Med 49:e329e329. In total 7 housekeeping genes were tested, of which SDHA and POLR2F expression were selected for gene expression normalization using NormFinder [34]. MicroElute RNA Clean Up kit (Omega Bio-Tek, Norcross, USA) and analyzed for quality and quantity using a NanoDrop spectrophotometer (Thermo Scientific). California Privacy Statement, 6). Indeed, P2RY12 mRNA is reduced by IFN+LPS in microglia from WM and GM. Microglia are proposed to play a role in MS lesion formation, however previous literature has not been able to distinguish infiltrated macrophages from microglia. Compared to NAGM, where MHC-II immunoreactivity was present in a small amount of ramified microglia, MHC-II+ microglia showed a more reactive phenotype in the active subpial GML (Fig. In subpial lesions with infiltration of CD3+ cells (a) and CD20+ cells (b) show immunoreactivity for TMEM119 (c) and P2RY12 (d). When comparing immunoreactivity present in GMLs versus NAGM, no differences were found. 4b, X2(6)=21.202, p<0.01). https://doi.org/10.1038/emm.2017.41. For immunohistochemical purposes, a total of 27 tissue blocks from 18 clinically diagnosed and pathologically verified MS patients were used. Scalebar inserts=50m. The PCR reaction was performed using the StepOnePlus Real-Time PCR system (Applied Biosystems). Graphs of TMEM119 and P2RY12 mRNA levels in cultured primary human microglia derived from WM enriched areas or GM enriched areas treated with IFN+LPSor with IL-4 compared to untreated WM microglia. All images analyzed had a region of interest (ROI) of 622466m. Brain 140:19001913. 2a), active WMLs showed numerous amoeboid, MHC-II+ cells (Fig. Therefore, in this study we utilize the microglia-specific, homeostatic markers TMEM119 and P2RY12 tocharacterize their immunoreactivity in MS grey matter lesionsin comparison to white matter lesions. Kooij: Nothing to discloseC.A.M. Chronic-active WMLs showed MHC-II+ cells with a more reactive phenotype (Fig. In addition, preactive lesions in the white matter show immunoreactivity for TMEM119 and P2RY12. It is a chronic inflammatory, degenerative disease of the central nervous system (CNS), pathologically characterized by leukocyte infiltration of the CNS, demyelination of the white and grey matter, and subsequent axonal loss. 6). Subsequent post-hoc analysis revealed that active WMLs had more CD3+ (p<0.01) and IFN + (p<0.05) cells compared to NAWM while chronic-active WMLs presented with more CD3+ (p<0.01) and IL-4+cells (p<0.05) (Fig. eNeuro 6:ENEURO.0448-18.2019. However, recent studies utilizing single-cell RNA-seq have shown that microglia in normal appearing white matter (NAWM) and normal-appearing grey matter (NAGM) of MS patients differ in their gene expression pattern [19]. https://doi.org/10.1093/brain/awv398, Article Therefore, in order to expand the existing literature we identified and compared the expression of the homeostatic markers TMEM119 and P2RY12 in MS GMLs to WMLs. Nat Immunol 8:913919. It is therefore plausible that blocking the entrance of lymphocytes into the CNS of MS patients may not interfere with all possible effects of microglia in both WMLs and GMLs. https://doi.org/10.1186/s40478-019-0850-z, DOI: https://doi.org/10.1186/s40478-019-0850-z. van Wageningen, T.A., Vlaar, E., Kooij, G. et al. Clinicopathological information of patients from which brain material was used in this study, is provided in Table1. J Neuroinflammation 14:116. Brain Pathol 17:3844, Kutzelnigg A, Lucchinetti CF, Stadelmann C, Brck W, Rauschka H, Bergmann M, Schmidbauer M, Parisi JE, Lassmann H (2005) Cortical demyelination and diffuse white matter injury in multiple sclerosis. The present study is the first to identify that in post-mortem material for MS patients, immunoreactivity for TMEM119 and P2RY12 in MS GMLs is different to that in WMLs. Tissue was trypsinized for 30min at 37C using 0.25% trypsin (Difco) dissolved in a trypsinization buffer (8g/l NaCl (Sigma), 0.4g/l KCl (Sigma, Darmstadt, Germany), 0.84g/l NaHCO3 (Merck, Darmstadt, Germany), 0.2g/l EDTA (Promega, Madison, USA), 4.8g/l HEPES (Sigma), and 1g/l glucose dissolved in MilliQ water, pH set at 7.6). https://doi.org/10.1093/brain/awx113, Moore CS, Ase AR, Kinsara A, Rao VTS, Robinson MM, Leong SY, Butovsky O, Ludwin SK, Seguela P, Bar-Or A, Antel JP (2015) P2Y12 expression and function in alternatively activated human microglia. 4a, X2(6)=49.459, p<0.01). 2c). Boxplots represent the mean, the 1st and 4th quartile and the minimum and maximum value. https://doi.org/10.1111/nyas.12841, Geurts JJ, Barkhof F (2008) Grey matter pathology in multiple sclerosis. Isolation of primary microglia was conducted either directly after tissue collection or within 12h thereafter. 3e) whereas Iba-1 immunoreactivity in less inflammatory subpial GMLs often was visible in rod-like microglia (Fig. https://doi.org/10.1038/nn1805, Lou N, Takano T, Pei Y, Xavier AL, Goldman SA, Nedergaard M (2016) Purinergic receptor P2RY12-dependent microglial closure of the injured blood-brain barrier. Cancer Res 64:52455250. Scalebar (a-h)=200m. Upon use for immunohistochemistry, tissue sections were heated to 58C for 30min. 1a, b). Representative images of lesion types used in this study. Subsequently, after washes with TBS, the sections were incubated for 30min. Anal Quant Cytol Histol 23:291299, CAS https://doi.org/10.1038/nn.3554, Beaino W, Janssen B, Kooij G, van der Pol SMA, van Het Hof B, van Horssen J, Windhorst AD, de Vries HE (2017) Purinergic receptors P2Y12R and P2X7R: potential targets for PET imaging of microglia phenotypes in multiple sclerosis. Nat Neurosci 17:131143. Pictures of double labeled sections were taken at wavelengths ranging from 480nm to 680nm at 60x magnification using the Nuance multispectral imaging system (PerkinElmer). 1e, f) [23]. The levels of TMEM119 and P2RY12 mRNA did not differ between untreated microglia derived from WM or GM. If one tissue block featured several lesions of the same type, these values were averaged. Even though immunoreactivity for TMEM119 was absent in the center of active WMLs (Fig. Dysfunction of the blood-brain-barrier leads to infiltration of leukocytes into the CNS, possibly attracted by antigens presented by microglia and/or by infiltrated macrophages [6, 8]. Representative images of IFN 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). After incubation, culture medium consisting of equal amounts of DMEM and Hams F12 supplemented with 10% fetal calf serum (Gibco, Life Technologies), 1% Penicillin/Streptomycin (Invitrogen) and 1% L-glutamine (Invitrogen) was added to de-activate the trypsin and the tissue homogenate was further dissociated using titration with a 10ml pipette into a homogenous suspension which was filtered using a 100m mesh (Greiner-bio-one, Alphen aan de Rijn, The Netherlands). The presence of rod-shaped microglia in GMLs suggests that these cells are responsive irrespective of the relative absence of lymphocytes, and low MHC-II immunoreactivity. Whereas we observed that in (active) WMLs, TMEM119 and P2RY12 immunoreactivity is largely absent compared to NAWM, which is in line with previous findings [16, 35], we now show that the level of TMEM119 and P2RY12 immunoreactivity is not affected in GMLs compared to NAGM. Analysis of MHC-II immunoreactivity revealed a significant difference between all lesion types (Fig. P2RY12+ cells were present in ramified cells similarly to what was observed for MHC-II+ and Iba-1+ cells in the NAWM (Fig. Representative images of MHC-II (a, b, c), Iba-1 (d, e, f), TMEM119 (g, h, i) and P2RY12 (j, k, l) immunoreactivityin normal appearing matter and in the demyelinated center of active WMLs and chronic-active WMLs and in pre-active lesions (m, n, o, p). in a conventional steam cooker. WMLs showing a rim of MHC-II+cells around the demyelinated lesion with less, but still apparent amoeboid MHC-II+ cells in the center of the demyelinated lesion were deemed chronic-active WMLs (or mixed active/inactive according to Kuhlmann et al., (2017) (Fig. 8). P-values <0.05 were considered statistically significant. P2RY12+ cells were absent along the rim of active WMLs but present in the rim of chronic-active WMLs (Fig. Glia 55:360368. Moreover, there is a clear tendency that IFN+LPS reduces TMEM119 expression in microglia from both origins. Input of RNA for cDNA synthesis for all samples was normalized based on the sample with the lowest concentration of RNA. mRNA levels from GM derived cells are represented in the grey-coloured box. Brain 139:807815. We subsequently questioned whether this different expression of TMEM119 and P2RY12 of microglia in the center of GMLs versus WMLs could be explained by intrinsic differences in responsivity of WM and GM derived microglia. Iba-1 immunoreactivity was also significantly different between all lesion types (Fig. Of interest is that in a subpial GML with a clear rim of MHC-II+ microglial cells, we observed that these microglia are TMEM119+ but not P2RY12+. All authors contributed to and approved the final manuscript. 3a, b). Thus, in conclusion, these data suggest that the continued presence of TMEM119 and P2RY12 immunoreactivity in subpial GMLs could reflect the absence of IL-4 and IFN and low presence of infiltrating lymphocytes in the lesion parenchyma (and not meninges) compared to WMLs. PubMed Central Dashed lines indicate the edge of the lesion. Lesion activity in GMLs is defined by the presence or absence of a rim of activated microglia surrounding the lesion area as defined by Peterson et al., (2001) [30]. The type 1 lesions were added to the analysis to exclude that the differences in immunoreactivity found between GMLs and WMLs were either due to location, or due to time of lesion development. One case showed a subpial GML with a clear rim of activated MHC-II+ cells, this lesion was deemed an active subpial GML (Fig. https://doi.org/10.1186/s12974-017-1034-z, CAS Subpial demyelinated (g) GMLs hardly show MHC-II+ cells (h). 5a-f). Neurology 79:13691376. Article In addition, we studied whether the absence or presence of CD3+ and CD20+ cells in the meninges close to the subpial GMLs is of relevance for TMEM119 and P2RY12 immunoreactivity in subpial GMLs. The mRNA levels observed of various genes expressed in microglia of these patients did not differ from that of the five patients with other diagnoses (data not shown). Cite this article. The PCR protocol was adapted from the manufacturers description and featured 40cycles with an annealing temperature of 60C, followed by a melt curve analysis. From the subsequently acquired DAB images without heamatoxylin signal, an auto-threshold method was applied. In compliance with all local ethical and legal guidelines, informed consent for brain autopsy and the use of brain tissue and clinical information for scientific research was given by either the donor or the next of kin. https://doi.org/10.1158/0008-5472.CAN-04-0496, Lassmann H, van Horssen J (2016) Oxidative stress and its impact on neurons and glia in multiple sclerosis lesions. Google Scholar, Ichi SJ, Kino Y, Asahina N, Takitani M, Miyoshi J, Ishida T, Saito Y (2016) TMEM119 marks a subset of microglia in the human brain. Whereas in the center of active WMLs TMEM119 and P2RY12 immunoreactivity is absent, TMEM119+ microglia are visible surrounding the lesion, and both TMEM119+ and P2RY12+ microglia are visible in the rim of chronic-active WMLs. Microglia derived from both WM and GM showed downregulation of the anti-inflammatory marker mannose receptor (MRC) after treatment with IFN+LPS and upregulation of the pro-inflammatory marker interleukin (IL)-1 whereas treatment with IL-4 did not affect these markers (Additional file 1: Figure S3) [11, 15, 18]. Lancet Neurol 7:841851. Glia 65:375387. We conclude that the observed difference in immunoreactivity for TMEM119 and P2RY12 in GMLs and WMLs could be due to the absence or presence of lymphocytes and inflammatory mediators in the parenchyma. Data from the semi-quantitative RT-PCR also did not show a normal distribution. To determine whether the differences in microglial TMEM119 and P2RY12 immunoreactivity between WMLs and GMLs were due to differences in microglial responsiveness, we isolated primary human microglia from WM (corpus callosum) and GM (cortex) tissue obtained at autopsy and treated those with IFN+LPS or IL-4 as representatives of a pro- or anti-inflammatory stimulus, respectively. P2RY12 mRNA level was attenuated after treatment with IFN+LPS in both WM- and GM-derived microglia (WM: p<0.05, GM: p<0.05), while after IL-4 treatment, P2RY12 expression was enhanced in the GM only (p<0.05) (Fig. Similarly to TMEM119, P2RY12 immunoreactivity showed significant differences between lesion types (X2(6)=31.705, p<0.01) primarily driven by differences between NAWM and active WMLs (p<0.01), and chronic active WMLs (p<0.05). 2k, l). 2022 BioMed Central Ltd unless otherwise stated. In order to compare differences on a group level within WM- or GM-derived conditions, a Friedmans test was used with post-hoc testing done manually by comparing individual data sets within WM- and GM-derived microglia with the Wilcoxon Signed Ranks test. Afterwards, slides were dried overnight in an incubator at 37C before being stored at room temperature (RT). In contrast to TMEM119, P2RY12 immunoreactivity reappeared in the center of chronic-active WMLs, showing a reactive phenotype similar to Iba-1 and MHC-II+ cells in those lesions (Fig. After one day of culturing, cells were cultured in culture medium +25ng/ml human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF; Peprotech, London, the UK). To exclude the possibility that this difference is due to distant locations of the lesions (cortical GM compared to more inflammatory WM) or due to time of development of the lesions (e.g. P values <0.05 were considered statistically significant. 2j), but they were virtually absent in active WMLs. From a clinical point of view, MS is very heterogeneous and is associated with an array of symptoms, including sensory and motor deficits, fatigue, cognitive and psychiatric disturbances [1, 2]. Nat Neurosci 19:504516. Multiple Sclerosis (MS) is the most common cause of acquired neurological disability in young adults. When studying the expression profile of microglia, at least two genes have been related to a homeostatic signature of microglia in the human and rodent brain, i.e. GML develop earlier on in the disease and are therefore less inflammatory), we verified and confirmed that in leukocortical (type 1) lesions, encompassing both WML and GML, this difference in TMEM119 and P2RY12 immunoreactivity is also present. 3j),in active subpial GMLs (Fig. Lancet Neurol 14:194207. Sections were pretreated as described for single labeling above. As we are not aware of any other observations on TMEM119 regulation in human microglia in vitro, we are the first to find that IL-4 treatment significantly reduced its mRNA level in WM-derived microglia. To verify that TMEM119 and P2RY12 were markers for GM microglia in addition to WM microglia, we observed that both markers completely overlap with Iba-1+ microglia in white- and grey normal appearing matter (Additional file 1: Figure S2). Within these ROIs, signals from DAB and hematoxylin were separated using the color deconvolution plug-in [32]. volume7, Articlenumber:206 (2019) 3c). Inserts show magnifications of the area of interest. Statistical analysis was conducted using SPSS Statistics 22 (IBM, Armonk, USA). However, immunologically active GMLs are rarely found in post-mortem MS brain material and are mostly represented by leukocortical lesions [23]. 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. After centrifugation, a myelin layer was formed at the interphase and microglial cells are pelleted. Post-hoc testing was done between WM groups and between GM groups. Google Scholar, Butovsky O, Jedrychowski MP, Moore CS, Cialic R, Lanser AJ, Gabriely G, Koeglsperger T, Dake B, Wu PM, Doykan CE, Fanek Z, Liu L, Chen Z, Rothstein JD, Ransohoff RM, Gygi SP, Antel JP, Weiner HL (2014) Identification of a unique TGF--dependent molecular and functional signature in microglia. The recent development of microglia specific TMEM119 knock-in and CreERT2 mice [28] will be a useful tool to gain more knowledge on the functional role of TMEM119. Proc Natl Acad Sci U S A 113:E1738E1746. Sections were subsequently dehydrated in graded series of ethanol, cleared in xylene and mounted with Entellan.

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