Specific myeloid signatures in peripheral blood differentiate active and rare clinical phenotypes of multiple sclerosis

Current understanding of Multiple Sclerosis (MS) pathophysiology implicates perturbations in adaptive cellular immune responses, predominantly T cells, in Relapsing-Remitting forms (RRMS). Nevertheless, from a clinical perspective MS is a heterogeneous disease reflecting the heterogeneity of involved biological systems. This complexity requires advanced analysis tools at the single-cell level to discover biomarkers for better patient-group stratification. We designed a novel 44-parameter mass cytometry panel to interrogate predominantly the role of effector and regulatory subpopulations of peripheral blood myeloid subsets along with B and T-cells (excluding granulocytes) in MS, assessing three different patient cohorts: RRMS, PPMS (Primary Progressive) and Tumefactive MS patients (TMS) (n=10, 8, 14 respectively). We further subgrouped our cohort into inactive or active disease stages to capture the early underlying events in disease pathophysiology. Peripheral blood analysis showed that TMS cases belonged to the spectrum of RRMS, whereas PPMS cases displayed different features. In particular, TMS patients during a relapse stage were characterized by a specific subset of CD11c+CD14+ CD33+, CD192+, CD172+-myeloid cells with an alternative phenotype of monocyte-derived macrophages (high arginase-1, CD38, HLA-DR-low and endogenous TNF-a production). Moreover, TMS patients in relapse displayed a selective CD4 T-cell lymphopenia of cells with a Th2-like polarised phenotype. PPMS patients did not display substantial differences from healthy controls, apart from a trend toward higher expansion of NK cell subsets. Importantly, we found that myeloid cell populations are reshaped under effective disease-modifying therapy predominantly with glatiramer acetate and to a lesser extent with anti-CD20, suggesting that the identified cell signature represents a specific therapeutic target in TMS. The expanded myeloid signature in TMS patients was also confirmed by flow cytometry. Serum neurofilament light-chain levels confirmed the correlation of this myeloid cell signature with indices of axonal injury. More in-depth analysis of myeloid subsets revealed an increase of a subset of highly cytolytic and terminally differentiated NK cells in PPMS patients with leptomeningeal enhancement (active-PPMS), compared to those without (inactive-PPMS). We have identified previously uncharacterized subsets of circulating myeloid cells and shown them to correlate with distinct disease forms of MS as well as with specific disease states (relapse/remission)

Novel CSF Biomarkers Tracking Autoimmune Inflammatory and Neurodegenerative Aspects of CNS Diseases

The accurate diagnosis of neuroinflammatory (NIDs) and neurodegenerative (NDDs) diseases and the stratification of patients into disease subgroups with distinct disease-related characteristics that reflect the underlying pathology represents an unmet clinical need that is of particular interest in the era of emerging disease-modifying therapies (DMT). Proper patient selection for clinical trials and identifying those in the prodromal stages of the diseases or those at high risk will pave the way for precision medicine approaches and halt neuroinflammation and/or neurodegeneration in early stages where this is possible. Towards this direction, novel cerebrospinal fluid (CSF) biomarker candidates were developed to reflect the diseased organ’s pathology better. Μisfolded protein accumulation, microglial activation, synaptic dysfunction, and finally, neuronal death are some of the pathophysiological aspects captured by these biomarkers to support proper diagnosis and screening. We also describe advances in the field of molecular biomarkers, including miRNAs and extracellular nucleic acids known as cell-free DNA and mitochondrial DNA molecules. Here we review the most important of these novel CSF biomarkers of NIDs and NDDs, focusing on their involvement in disease development and emphasizing their ability to define homogeneous disease phenotypes and track potential treatment outcomes that can be mirrored in the CSF compartment

Perturbation of transcriptome in non-neoplastic salivary gland epithelial cell lines derived from patients with primary Sjögren's syndrome

The data presented here are related to the research article titled “Impaired anti-inflammatory activity of PPARγ in the salivary epithelia of Sjögren's syndrome patients imposed by intrinsic NF-κB activation” (Vakrakou et al., Journal of Autoimmunity, in press, 2017). In the cited manuscript, using comparative analyses of salivary gland biopsy specimens and ductal salivary gland epithelial cell (SGEC) lines from SS patients and disease controls, we have demonstrated that the ductal epithelia of SS patients display constitutively reduced PPARγ expression, transcriptional activity and anti-inflammatory function that were associated with cell-autonomously activated NF-κB and IL-1β pathways in these cells. Herein, the comparative transcriptome analysis of SGEC lines is presented. We show that the ductal epithelia of SS patients with severe lymphoepithelial lesions manifest constitutive perturbation in various inflammation- and metabolism related signaling pathways

Impaired clearance of early apoptotic cells mediated by inhibitory IgG antibodies in patients with primary Sjögren's syndrome.

OBJECTIVES: Deficient efferocytosis (i.e. phagocytic clearance of apoptotic cells) has been frequently reported in systemic lupus erythematosus (SLE). Todate, patients with primary Sjögren's syndrome (SS) have not been assessed for phagocytosis of apoptotic cells (ApoCell-phagocytosis) and of particulate targets (microbeads, MB-phagocytosis). DESIGN: ApoCell-phagocytosis and MB-phagocytosis were comparatively assessed by flow cytometry in peripheral blood specimens and monocyte-derived macrophage (MDM) preparations from healthy blood donors (HBD) and consecutive SS, SLE and rheumatoid arthritis (RA) patients. Cross-admixture ApoCell-phagocytosis experiments were also performed using phagocytes from HBD or patients, and apoptotic cells pretreated with whole sera or purified serum IgG derived from patients or HBD. RESULTS: Compared to HBD, approximately half of SS and SLE patients studied (but not RA) manifested significantly reduced ApoCell-phagocytosis (p<0.001) and MB-phagocytosis (p<0.003) by blood-borne phagocytes that correlated inversely with disease activity (p≤0.004). In cross-admixture assays, healthy monocytes showed significantly reduced ApoCell-phagocytosis when fed with apoptotic cells that were pretreated with sera or purified serum IgG preparations from SS and SLE patients (p<0.0001, compared to those from HBD or RA). Such aberrant effect of the SS and SLE sera and IgG preparations correlated linearly with their content of IgG antibodies against apoptotic cells (p≤0.0001). Phagocytic dysfunction maybe also present in certain SS and SLE patients, as supported by deficient capacity of MDM for ApoCell-phagocytosis and MB-phagocytosis under patients' serum-free conditions. CONCLUSION: Similarly to SLE, efferocytosis is frequently impaired in SS and is primarily due to the presence of inhibitory IgG anti-ApoCell antibodies and secondarily to phagocytes' dysfunction

Impaired MB-phagocytosis by peripheral blood (PB) phagocytes (A; monocytes B; granulocytes) and by monocyte-derived macrophages (MDM, C) obtained from SS and SLE patients, but not from RA patients.

<p>The horizontal lines indicate the median levels in each group, whereas the numbers in boxes indicate the percentages of individuals with decreased MB-phagocytosis, as defined by the presence of MB-PhI values that were two standard deviations below the corresponding mean of HBD. Statistically significant comparisons of patient groups to HBD are shown.</p

An Updated Evaluation of Intrathecal IgG Synthesis Markers in Relation to Oligoclonal Bands

The aim was to evaluate the performance of the latest quantitative marker for intrathecal IgG synthesis and to compare it with other established markers used for the same purpose. We retrospectively applied Auer’s and Reiber’s intrathecal IgG synthesis formulae in a cohort of 372 patients under investigation for central nervous system demyelination who had undergone lumbar puncture and oligoclonal bands (OCBs) detection for demonstrating intrathecal IgG synthesis. A ROC analysis revealed Auer’s formula had lower sensitivity (68%) compared to Reiber’s formula (83%) and IgG index (89%), in our cohort of patients that exhibited normal to mildly elevated albumin quotients (4.48 ± 3.93). By excluding possible sources of errors, we assume that Auer’s formula is less sensitive than other established tools for the “prediction” of the detection of OCBs in routine cerebrospinal fluid (CSF) analyses due to the mathematical model used. Given the ability of Reiber’s hyperbolic formula to describe the blood–CSF IgG distribution across a wide range of blood–brain barrier functionality, its use and the use of similar formulae are recommended for the discrimination between CNS-derived and blood-derived molecules in clinical laboratories

Inhibitory effect of IgG on ApoCell-phagocytosis.

<p>Purified serum IgG preparations from SS and SLE patients display inhibitory activity on ApoCell-phagocytosis by healthy MDM that correlates with its binding activity to early apoptotic cells. <b>A.</b> Purified serum IgG preparations from SS and SLE patients (but not RA) display significantly increased binding to early apoptotic cells. Binding index was normalized and expressed as fold increase over the binding of a purified IgG preparation from a HBD used in all experiments. <b>B–D</b>. Cross-admixture ApoCell-phagocytosis experiments demonstrating that the pretreatment of early apoptotic cells with purified serum IgG preparations derived from SS and SLE (but not from RA) results in decreased ApoCell-phagocytosis by healthy MDM, as compared to treatment with IgG from HBD. In <b>B</b>, data are expressed as percent of baseline ApoCell-phagocytosis values (e.g. treatment of apoptotic cells with PBS only, considered as 100%). Similar results were obtained by slightly different experimental setups assaying the ingestion of CFSE-labelled IgG-pretreated apoptotic cells by electronically gated CD14-stained MDM in dual-color flow cytometry (<b>C</b>; representative results from 3 independent experiments) or the uptake of pHrodo-SE-labelled apoptotic cells in single-color flow cytometry (<b>D</b>). <b>E</b>. Highly significant inverse correlation between the rates of ApoCell-phagocytosis obtained by the various purified serum IgG preparations used (shown in A) and the levels of anti-ApoCell antibodies in those preparations (shown in A).</p