Monocyte Function in Parkinson's Disease and the Impact of Autologous Serum on Phagocytosis.

Background: Increasing evidence implicates involvement of the innate immune system in the initiation and progression of Parkinson's disease (PD). Monocytes and monocyte-derived cells perform a number of functions, such as phagocytosis, chemotaxis, and cytokine secretion, which may be particularly relevant to PD pathology. The behavior of these cells in early-moderate disease, in conditions more similar to the in-vivo environment has not been fully evaluated. Research Question: Does monocyte function, including phagocytosis, chemotaxis and cytokine secretion, differ in early-moderate PD compared to age and gender-matched controls? Methods: Participants included PD patients (n = 41) with early-moderate stage disease (Hoehn and Yahr ≤2) and age and gender matched controls (n = 41). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and monocytes were further separated using CD14 magnetic beads. Functional assays, including bead phagocytosis (in standard medium and autologous serum), Boyden chamber trans-well chemotaxis, and cytokine secretion on lipopolysaccharide stimulation were performed. Monocyte surface markers relating to chemotaxis were measured using immunohistochemistry and flow cytometry. Between-group analysis was performed using paired t-tests. Results: An autologous serum environment significantly increased bead phagocytosis compared to standard medium as expected, in both patients and controls. When in autologous serum, PD monocytes demonstrated enhanced phagocytosis compared to control monocytes (p = 0.029). The level of serum-based phagocytosis was influenced by complement inactivation and the origin of the serum. There were no significant differences between PD and controls in terms of standard medium based monocyte migration or cytokine secretion in this cohort. Conclusions: Autologous serum has a significant influence on monocyte phagocytosis and reveals increased phagocytic capacity in early-moderate PD compared to controls. These conditions may better reflect the function of monocytes in-vivo in PD patients than standard medium based phagocytosis assays. Further studies will be required to replicate these results in larger cohorts, including earlier and later stages of disease, and to understand which serum factors are responsible for this observation and the potential mechanistic relevance to PD pathogenesis.Funding for this work was provided by Addenbrooke’s Charitable Trust, the Rosetrees Trust and the NIHR Cambridge Biomedical Research Centre. RSW was supported by a Fellowship from Addenbrooke’s Charitable Trust. DKV is supported by a Junior Research Fellowship from Homerton College, Cambridge. KMS is supported by a Fellowship from the Wellcome Trust. CHWG is supported by a Clinician Scientist Fellowship from the Medical Research Council. RAB is an NIHR Senior Investigator and is supported by the Wellcome Trust-MRC Cambridge Stem Cell Institute

Oligodendrocyte progenitor cells become regionally diverse and heterogeneous with age

Oligodendrocyte progenitor cells (OPCs), which differentiate into myelinating oligodendrocytes during central nervous system (CNS) development, are the main proliferative cells in the adult brain. OPCs are conventionally considered a homogeneous population, particularly with respect to their electrophysiological properties, but this has been debated. We show, by using single-cell electrophysiological recordings, that OPCs start out as a homogeneous population, but become functionally heterogeneous, varying both within and between brain regions and with age. These electrophysiological changes in OPCs correlate with the differentiation potential of OPCs; thus, they may underlie the differentiational differences in OPCs between regions and likewise differentiation failure with age.We acknowledge the support of the Wellcome - MRC Cambridge Stem Cell Institute core facility managers, in particular for this work Dr Maike Paramor and Miss Victoria Murray with RNA sequencing, and all staff members of the University Biomedical Services (UBS). This project has received funding from: the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 771411; R.T.K, K.A.E); the Wellcome Trust, a Research Career Development Fellowship (R.T.K. and K.A.E. 091543/Z/10/Z) and a Studentship (102160/Z/13/Z; Y.K); The Paul G Allen Frontiers Group, Allen Distinguished Investigator Award (12076, R.T.K., D.K.V.); The Medical Research Council, a studentship (S.O.S.); The Gates Foundation, a Gates Scholarship (S.S.), The Biotechnology and Biological Sciences Research Council, a studentship (S.A.); Homerton College Cambridge, a Junior Research Fellowship (D.K.V); The UK MS Society, a Cambridge Myelin Repair Centre grant (50; R.T.K, O.D.F.); The Fonds de recherche du Québec-Santé, a scholarship (Y.K.); The Cambridge Commonwealth European & International Trust, a scholarship (Y.K.); and the Lister Institute, a Research Prize (R.T.K., K.A.E, SOS)

Oligodendrocyte Progenitor Cells Become Regionally Diverse and Heterogeneous with Age

We thank J. Trotter (Johannes Gutenberg University, Mainz, Germany) for the NG2-EYFP mice, Dr. Moritz Matthey for help with minipump transplantation, Miss Mariann Kovacs with embryonic dissection, and Dr. Katrin Volbracht for critical comments on the work. We acknowledge the support of the Wellcome – MRC Cambridge Stem Cell Institute core facility managers, in particular for this work Dr. Maike Paramor and Miss Victoria Murray with RNA-seq, and all staff members of the University Biomedical Services (UBS). This project has received the following funding: funding from the European Research Council (ERC) under the European Union Horizon 2020 Research and Innovation Program (grant agreement 771411 to R.T.K. and K.A.E.), a Wellcome Trust research career development fellowship (091543/Z/10/Z to R.T.K. and K.A.E.) and studentship (102160/Z/13/Z to Y.K.), Paul G. Allen Frontiers Group Allen Distinguished Investigator Award 12076 (to R.T.K., D.K.-V., and K.A.E.), a Medical Research Council studentship (to S.O.S.), a Gates Cambridge Trust Gates scholarship (to S.S.), a Biotechnology and Biological Sciences Research Council studentship (to S.A.), a Homerton College Cambridge junior research fellowship (to D.K.-V.), UK MS Society Cambridge Myelin Repair Centre grant 50 (to R.T.K. and O.d.F.), a Fonds de Recherche du Québec - Santé scholarship (to Y.K.), a Cambridge Commonwealth, European and International Trust scholarship (to Y.K.), and a Lister Institute research prize (to R.T.K., K.A.E., and S.O.S.). Publisher Copyright: © 2018 The Author(s)Spitzer et al. show that oligodendrocyte progenitor cells (OPCs) acquire ion channels and sensitivity to neuronal activity that differ between region and age. The onset and decline of ion channels follow developmental milestones. This heterogeneity indicates different functional states of OPCs.Peer reviewe

Prominent microglial inclusions in transgenic mouse models of α-synucleinopathy that are distinct from neuronal lesions.

Alpha-synucleinopathies are a group of progressive neurodegenerative disorders, characterized by intracellular deposits of aggregated α-synuclein (αS). The clinical heterogeneity of these diseases is thought to be attributed to conformers (or strains) of αS but the contribution of inclusions in various cell types is unclear. The aim of the present work was to study αS conformers among different transgenic (TG) mouse models of α-synucleinopathies. To this end, four different TG mouse models were studied (Prnp-h[A53T]αS; Thy1-h[A53T]αS; Thy1-h[A30P]αS; Thy1-mαS) that overexpress human or murine αS and differed in their age-of-symptom onset and subsequent disease progression. Postmortem analysis of end-stage brains revealed robust neuronal αS pathology as evidenced by accumulation of αS serine 129 (p-αS) phosphorylation in the brainstem of all four TG mouse lines. Overall appearance of the pathology was similar and only modest differences were observed among additionally affected brain regions. To study αS conformers in these mice, we used pentameric formyl thiophene acetic acid (pFTAA), a fluorescent dye with amyloid conformation-dependent spectral properties. Unexpectedly, besides the neuronal αS pathology, we also found abundant pFTAA-positive inclusions in microglia of all four TG mouse lines. These microglial inclusions were also positive for Thioflavin S and showed immunoreactivity with antibodies recognizing the N-terminus of αS, but were largely p-αS-negative. In all four lines, spectral pFTAA analysis revealed conformational differences between microglia and neuronal inclusions but not among the different mouse models. Concomitant with neuronal lesions, microglial inclusions were already present at presymptomatic stages and could also be induced by seeded αS aggregation. Although nature and significance of microglial inclusions for human α-synucleinopathies remain to be clarified, the previously overlooked abundance of microglial inclusions in TG mouse models of α-synucleinopathy bears importance for mechanistic and preclinical-translational studies

Peripheral innate immune and bacterial signals relate to clinical heterogeneity in Parkinson's disease.

The innate immune system is implicated in Parkinson's disease (PD), but peripheral in-vivo clinical evidence of the components and driving mechanisms involved and their relationship with clinical heterogeneity and progression to dementia remain poorly explored. We examined changes in peripheral innate immune-related markers in PD cases (n = 41) stratified according to risk of developing early dementia. 'Higher Risk'(HR) (n = 23) and 'Lower Risk' (LR) (n = 18) groups were defined according to neuropsychological predictors and MAPT H1/H2 genotype, and compared to age, gender and genotype-matched controls. Monocyte subsets and expression of key surface markers were measured using flow cytometry. Serum markers including alpha-synuclein, inflammasome-related caspase-1 and bacterial translocation-related endotoxin were measured using quantitative immuno-based assays. Specific markers were further investigated using monocyte assays and validated in plasma samples from a larger incident PD cohort (n = 95). We found that classical monocyte frequency was elevated in PD cases compared to controls, driven predominantly by the HR group, in whom Toll-Like Receptor (TLR)4+ monocytes and monocyte Triggering Receptor Expressed on Myeloid cells-2 (TREM2) expression were also increased. Monocyte Human Leukocyte Antigen (HLA)-DR expression correlated with clinical variables, with lower levels associated with worse cognitive/motor performance. Notably, monocyte changes were accompanied by elevated serum bacterial endotoxin, again predominantly in the HR group. Serum alpha-synuclein and inflammasome-related caspase-1 were decreased in PD cases compared to controls regardless of group, with decreased monocyte alpha-synuclein secretion in HR cases. Further, alpha-synuclein and caspase-1 correlated positively in serum and monocyte lysates, and in plasma from the larger cohort, though no associations were seen with baseline or 36-month longitudinal clinical data. Principal Components Analysis of all monocyte and significant serum markers indicated 3 major components. Component 1 (alpha-synuclein, caspase-1, TLR2+ monocytes) differentiated PD cases and controls in both groups, while Component 2 (endotoxin, monocyte TREM2, alpha-synuclein) did so predominantly in the HR group. Component 3 (classical monocytes, alpha-synuclein) also differentiated cases and controls overall in both groups. These findings demonstrate that systemic innate immune changes are present in PD and are greatest in those at higher risk of rapid progression to dementia. Markers associated with PD per-se (alpha-synuclein, caspase-1), differ from those related to cognitive progression and clinical heterogeneity (endotoxin, TREM2, TLR4, classical monocytes, HLA-DR), with mechanistic and therapeutic implications. Alpha-synuclein and caspase-1 are associated, suggesting inflammasome involvement common to all PD, while bacterial translocation associated changes may contribute towards progression to Parkinson's dementia. Additionally, HLA-DR-associated variations in antigen presentation/clearance may modulate existing clinical disease

Microglial inclusions and neurofilament light chain release follow neuronal α-synuclein lesions in long-term brain slice cultures.

BACKGROUND: Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression. METHODS: Alpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed. RESULTS: To study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2-3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture's genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions. CONCLUSION: The successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments

Microglial expression of CD83 governs cellular activation and restrains neuroinflammation in experimental autoimmune encephalomyelitis

AbstractMicroglial activation during neuroinflammation is crucial for coordinating the immune response against neuronal tissue, and the initial response of microglia determines the severity of neuro-inflammatory diseases. The CD83 molecule has been recently shown to modulate the activation status of dendritic cells and macrophages. Although the expression of CD83 is associated with early microglia activation in various disease settings, its functional relevance for microglial biology has been elusive. Here, we describe a thorough assessment of CD83 regulation in microglia and show that CD83 expression in murine microglia is not only associated with cellular activation but also with pro-resolving functions. Using single-cell RNA-sequencing, we reveal that conditional deletion of CD83 results in an over-activated state during neuroinflammation in the experimental autoimmune encephalomyelitis model. Subsequently, CD83-deficient microglia recruit more pathogenic immune cells to the central nervous system, deteriorating resolving mechanisms and exacerbating the disease. Thus, CD83 in murine microglia orchestrates cellular activation and, consequently, also the resolution of neuroinflammation.</jats:p

Monocyte Function in Parkinson's Disease and the Impact of Autologous Serum on Phagocytosis

Background: Increasing evidence implicates involvement of the innate immune system in the initiation and progression of Parkinson's disease (PD). Monocytes and monocyte-derived cells perform a number of functions, such as phagocytosis, chemotaxis, and cytokine secretion, which may be particularly relevant to PD pathology. The behavior of these cells in early-moderate disease, in conditions more similar to the in-vivo environment has not been fully evaluated.Research Question: Does monocyte function, including phagocytosis, chemotaxis and cytokine secretion, differ in early-moderate PD compared to age and gender-matched controls?Methods: Participants included PD patients (n = 41) with early-moderate stage disease (Hoehn and Yahr ≤2) and age and gender matched controls (n = 41). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and monocytes were further separated using CD14 magnetic beads. Functional assays, including bead phagocytosis (in standard medium and autologous serum), Boyden chamber trans-well chemotaxis, and cytokine secretion on lipopolysaccharide stimulation were performed. Monocyte surface markers relating to chemotaxis were measured using immunohistochemistry and flow cytometry. Between-group analysis was performed using paired t-tests.Results: An autologous serum environment significantly increased bead phagocytosis compared to standard medium as expected, in both patients and controls. When in autologous serum, PD monocytes demonstrated enhanced phagocytosis compared to control monocytes (p = 0.029). The level of serum-based phagocytosis was influenced by complement inactivation and the origin of the serum. There were no significant differences between PD and controls in terms of standard medium based monocyte migration or cytokine secretion in this cohort.Conclusions: Autologous serum has a significant influence on monocyte phagocytosis and reveals increased phagocytic capacity in early-moderate PD compared to controls. These conditions may better reflect the function of monocytes in-vivo in PD patients than standard medium based phagocytosis assays. Further studies will be required to replicate these results in larger cohorts, including earlier and later stages of disease, and to understand which serum factors are responsible for this observation and the potential mechanistic relevance to PD pathogenesis

Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes

This is an author-created, uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association (ADA), publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version is available in Diabetes in print and online at http://diabetes.diabetesjournals.orgThe erratum to this article is available in ORE at http://hdl.handle.net/10871/40335Studies in type 1 diabetes indicate potential disease heterogeneity, notably in the rate of β-cell loss, responsiveness to immunotherapies, and, in limited studies, islet pathology. We sought evidence for different immunological phenotypes using two approaches. First, we defined blood autoimmune response phenotypes by combinatorial, multiparameter analysis of autoantibodies and autoreactive T-cell responses in 33 children/adolescents with newly diagnosed diabetes. Multidimensional cluster analysis showed two equal-sized patient agglomerations characterized by proinflammatory (interferon-γ-positive, multiautoantibody-positive) and partially regulated (interleukin-10-positive, pauci-autoantibody-positive) responses. Multiautoantibody-positive nondiabetic siblings at high risk of disease progression showed similar clustering. Additionally, pancreas samples obtained post mortem from a separate cohort of 21 children/adolescents with recently diagnosed type 1 diabetes were examined immunohistologically. This revealed two distinct types of insulitic lesions distinguishable by the degree of cellular infiltrate and presence of B cells that we termed "hyper-immune CD20Hi" and "pauci-immune CD20Lo." Of note, subjects had only one infiltration phenotype and were partitioned by this into two equal-sized groups that differed significantly by age at diagnosis, with hyper-immune CD20Hi subjects being 5 years younger. These data indicate potentially related islet and blood autoimmune response phenotypes that coincide with and precede disease. We conclude that different immunopathological processes (endotypes) may underlie type 1 diabetes, carrying important implications for treatment and prevention strategies.JDRFNational Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College LondonEuropean Union (EU FP7) award - Persistent Virus Infection in Diabetes Network Study Group (PEVNET)EU FP7 Large-Scale Focused Collaborative Research Project on Natural Immunomodulators as Novel Immunotherapies for Type 1 Diabetes (NAIMIT)EU FP7 Large-Scale Focused Collaborative Research Project on β-cell preservation through antigen-specific immunotherapy in Type 1 Diabetes: Enhanced Epidermal Antigen Delivery Systems (EE-ASI)National Institutes of Health (NIH)National Institute of Diabetes and Digestive and Kidney DiseasesNational Institute of Allergy and Infectious DiseasesEunice Kennedy Shriver National Institute of Child Health and Human DevelopmentNational Center for Research ResourcesGeneral Clinical Research CenterAmerican Diabetes Association (ADA