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Astrocytes Have the Capacity to Act As Antigen-Presenting Cells in The Rostami et al. Journal of Neuroinflammation (2020) 17:119 https://doi.org/10.1186/s12974-020-01776-7 RESEARCH Open Access Astrocytes have the capacity to act as antigen-presenting cells in the Parkinson’s disease brain Jinar Rostami1, Grammatiki Fotaki2, Julien Sirois3, Ropafadzo Mzezewa1, Joakim Bergström1, Magnus Essand2, Luke Healy3 and Anna Erlandsson1* Abstract Background: Many lines of evidence suggest that accumulation of aggregated alpha-synuclein (αSYN) in the Parkinson’s disease (PD) brain causes infiltration of T cells. However, in which ways the stationary brain cells interact with the T cells remain elusive. Here, we identify astrocytes as potential antigen-presenting cells capable of activating T cells in the PD brain. Astrocytes are a major component of the nervous system, and accumulating data indicate that astrocytes can play a central role during PD progression. Methods: To investigate the role of astrocytes in antigen presentation and T-cell activation in the PD brain, we analyzed post mortem brain tissue from PD patients and controls. Moreover, we studied the capacity of cultured human astrocytes and adult human microglia to act as professional antigen-presenting cells following exposure to preformed αSYN fibrils. Results: Our analysis of post mortem brain tissue demonstrated that PD patients express high levels of MHC-II, which correlated with the load of pathological, phosphorylated αSYN. Interestingly, a very high proportion of the MHC-II co-localized with astrocytic markers. Importantly, we found both perivascular and infiltrated CD4+ T cells to be surrounded by MHC-II expressing astrocytes, confirming an astrocyte T cell cross-talk in the PD brain. Moreover, we showed that αSYN accumulation in cultured human astrocytes triggered surface expression of co-stimulatory molecules critical for T-cell activation, while cultured human microglia displayed very poor antigen presentation capacity. Notably, intercellular transfer of αSYN/MHC-II deposits occurred between astrocytes via tunneling nanotubes, indicating spreading of inflammation in addition to toxic protein aggregates. Conclusions: In conclusion, our data from histology and cell culture studies suggest an important role for astrocytes in antigen presentation and T-cell activation in the PD brain, highlighting astrocytes as a promising therapeutic target in the context of chronic inflammation. Keywords: Astrocytes, T-cell infiltration, MHC-II, Antigen presentation, Alpha-synuclein, Parkinson’s disease, Tunneling nanotubes * Correspondence: [email protected] 1Molecular Geriatrics, Department of Public Health and Caring Sciences/ Molecular Geriatrics, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Rostami et al. Journal of Neuroinflammation (2020) 17:119 Page 2 of 18 Background For decades, there has been a debate whether APCs Parkinson’s disease (PD) is a progressive neurodegenera- are present in the brain. Microglial cells arise from the tive disorder with complex pathophysiology that de- yolk sac and invade the brain during embryonic develop- velops over decades. The main characteristics of PD are ment. They have been considered to be the resident intracellular deposits of aggregated αSYN and chronic CNS macrophages and consequently presumed to be the neuroinflammation [1, 2]. Nonetheless, the interplay be- most likely brain APCs. However, several studies indi- tween these two processes and their respective impact cate that microglia are in fact poor antigen presenters on disease progression remain elusive. A recent study [19–21]. Our previous observations that human astro- showed that CD4+ T helper cells derived from PD cytes accumulate and transfer aggregated αSYN raised patients recognize αSYN fibrils as an antigen [3]. In the question whether infiltrating T cells interact with the accordance, the presence of infiltrating T cells has been αSYN containing astrocytes and if the astrocytes can act confirmed in animal models of PD as well as in post as APCs in the PD brain. Hence, the aim with the mortem brain tissues of PD patients [4–6]. However, present study was to investigate the role of αSYN accu- which cell types that act as antigen-presenting cells mulating astrocytes in the chronic neuroinflammation in (APCs) in the PD brain and activate the infiltrating PD, in regards to the adaptive immunity. CD4+ T cells remain unclear. Astrocytes, the most numerous glial cell type in the Materials and methods central nervous system, are crucial for maintaining brain Immunohistochemistry homeostasis, but are also highly involved in neuroin- Deparaffinization flammation [7]. In the PD brain, astrocytes are converted Brain sections from mesencephalon and striatum of 10 to a reactive, inflammatory state in which they phagocyt- PD patients and 10 controls (Table 1)wereused(kindly ose aggregated proteins and cell debris as well as secrete provided from Uppsala Biobank by Professor Irina Alafuz- various cytokines and chemokines [8, 9]. Moreover, off, ethical number 2012/494). The thickness of the ana- αSYN deposits appear frequently in astrocytes at all dis- lyzed sections was 5 μm. To insure that exactly the same ease stages [10–14]. We have previously demonstrated region was analyzed from all patients and controls, a cut- that αSYN accumulation in cultured human astrocytes ting mold was used during the sectioning procedure. Prior result in severe cellular stress, indicated by lysosomal, to immunostaining, the sections were deparaffinized using mitochondrial, and endoplasmic reticulum deficiencies. xylene 2× for 10 min, rehydrated using 99.5% ethanol 2× The stressed astrocytes respond by sending out tunnel- for 10 min, 95% ethanol for 10 min, and 70% ethanol for ing nanotubes, enabling intercellular transfer of αSYN 10 min and washed in PBS 3 × 5 min. inclusions to nearby cells, indicating astrocytes as crucial players in the progression of PD pathology [15, 16]. Antigen retrieval and permeabilization All nucleated cells in the body express major histocom- Antigen retrieval was performed by boiling the samples in patibility class I (MHC-I) for presentation of antigenic citric acid at pH 6 for 2 min in 550 W. The samples were peptides to CD8+ T cells, while so called antigen- left to cool down in room temperature (RT) for 20–30 presenting cells (APCs) also express MHC class II (MHC- + II), enabling them to present antigenic peptides to CD4 Table 1 Sex, age, and post mortem fixation time of the 10 PD helper T cells. Dendritic cells (DCs), B cells, and macro- patients and the 10 controls analyzed in this study phages are considered classical or professional APCs as PD Control they also express the co-stimulatory molecules, including Sex Age Post mortem Sex Age Post mortem CD80, CD86, and CD40, that are required for activation fixation time fixation time of CD8+ TcellsandCD4+ Tcells[17]. The cell surface (weeks) (weeks) expression of the co-stimulatory factors by the profes- Male 83 6 Male 72 4 sional APCs can for example be induced by the cytokine Male 84 4 Male 67 4 interferon-gamma (IFNγ). APCs degrade ingested anti- Male 74 4 Male 72 5 gens via the endo-lysosomal pathway and digest the anti- Male 77 3 Male 71 3 gen to smaller fragments that are presented on MHC-II. It is believed that DCs are required for activation of naive T Male 89 3 Male 68 5 cells and a cytolytic T-cell mediated immune response as Male 74 3 Male 61 5 they can also cross-present exogenously ingested peptides Male 81 4 Male 75 5 + on their MHC-I for activation of CD8 T cells. Another Male 79 5 Female 77 5 feature of professional APCs is their ability to cross-talk Female 80 5 Female 61 6 and transfer MHC-II molecules between each other via Female 78 4 Female 75 6 cell-to-cell transfer [18]. Rostami et al. Journal of Neuroinflammation (2020) 17:119 Page 3 of 18 min before permeabilization with 0.4% triton for 20 min. performed to ensure pellet formation of insoluble, large Endogenous peroxidase was blocked with 1% H2O2 fibrils. ThT solution (100 μM) was added to recombinant (Thermo Fischer) for 30 min and blocked with TNB monomers (αSYN-M) (diluted 1:100 from 2 mg/ml in blocking buffer (0.1 M Tris-HCl and 0.15 M NaCl and PBS), αSYN-F (diluted 1:100 from 2 mg/ml in PBS), and 0.5% blocking reagent FP1020, Perkin Elmer) for 30 min. read instantly at a wave length of 420 nm. For electron microscopy, αSYN-M, αSYN-F, and sonicated αSYN-F Primary antibody incubation (all diluted 1:5 in PBS) were dropped onto carbon coated Primary antibodies were prepared in PBS and incubated 300-mesh copper grids, negatively stained with 1% ur- with the sections at 4 °C overnight. The primary anti- anyl acetate for 5 min and air dried.
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