Search

Background: There is significant interest in understanding the nature of the inflammatory response and its role in Alzheimers disease (AD) pathophysiology. Immune cell phenotypes and their key pathway activation by AD stage is unclear. We therefore evaluated immune cell phenotypes in the cerebrospinal fluid (CSF) and their transcriptional profile comparing AD‐dementia, Mild Cognitive Impairment (MCI)‐AD and normal cognition controls using transcriptomics. Method: We characterized the gene expression profiles of immune cells in the CSF using both bulk RNA sequencing (RNAseq) and complementary single cell transcriptomics (scRNAseq). First, in a cross‐sectional cohort study, including 6 individuals with MCI‐AD meeting NIA/AA 2011 criteria and 9 amyloid negative controls with normal cognition, (mean age 68.75 years (SD 5.2), 58.8% Female), we identified significant pathways using gene set enrichment analysis (GSEA). Next, using scRNAseq analysis on2 AD‐dementia, 2 MCI‐AD and 2 normal controls we further elucidated the gene expression level changes in distinct immune cells. After clustering into immune cell types, we assessed their activation by GSEA and Gene set variation analysis (GSVA). Result: RNAseq analysis identified 47 genes that were differentially expressed between MCI‐AD and normal control samples (adj‐p‐val<0.05). GSEA identified downregulation of antigen processing, interferon signaling pathways, and neutrophil degranulation (adj‐p‐val<0.05). Next, scRNAseq, analysis defined immune cells within each sample and identified altered pathways that were in each cell type. We observed that CD4+CD8+ T cells were highly represented in AD‐dementia (17%) and MCI‐AD(14%) but not in normal controls(11%) (p<0.00001). The immune cell types that differed by disease stage between AD‐dementia and MCI‐AD were monocytes; increasing disease severity was associated with a transition from more classical monocytes to intermediate and nonclassical monocytes. The key pathways associated with monocytes were IL6 JAK STAT3 signaling, particularly STAT3, which is involved in monocyte‐to‐macrophage differentiation and the transition of classic monocytes to other subsets. Additionally, the pathways include interferon gamma (IFNg) response and TNF signaling via NF‐kB, which play important roles in activating macrophages and monocytes and promoting differentiation. Conclusion: Our data show enrichment in the proportion of CD4+CD8+ T cells and pathways related to monocyte differentiation that differ across AD stage. [ABSTRACT FROM AUTHOR]

The availability of robust protocols to differentiate induced pluripotent stem cells (iPSCs) into many human cell lineages has transformed research into the origins of human disease. The efficacy of differentiating iPSCs into specific cellular models is influenced by many factors including both intrinsic and extrinsic features. Among the most challenging models is the generation of human bronchial epithelium at air‐liquid interface (HBE‐ALI), which is the gold standard for many studies of respiratory diseases including cystic fibrosis. Here, we perform open chromatin mapping by ATAC‐seq and transcriptomics by RNA‐seq in parallel, to define the functional genomics of key stages of the iPSC to HBE‐ALI differentiation. Within open chromatin peaks, the overrepresented motifs include the architectural protein CTCF at all stages, while motifs for the FOXA pioneer and GATA factor families are seen more often at early stages, and those regulating key airway epithelial functions, such as EHF, are limited to later stages. The RNA‐seq data illustrate dynamic pathways during the iPSC to HBE‐ALI differentiation, and also the marked functional divergence of different iPSC lines at the ALI stages of differentiation. Moreover, a comparison of iPSC‐derived and lung donor‐derived HBE‐ALI cultures reveals substantial differences between these models. [ABSTRACT FROM AUTHOR]

Objective: To determine the inflammatory analytes that predict clinical progression and evaluate their performance against biomarkers of neurodegeneration. Methods: A longitudinal study of MCI‐AD patients in a Discovery cohort over 15 months, with replication in the Alzheimer's Disease Neuroimaging Initiative (ADNI) MCI cohort over 36 months. Fifty‐three inflammatory analytes were measured in the CSF and plasma with a RBM multiplex analyte platform. Inflammatory analytes that predict clinical progression on Clinical Dementia Rating Scale‐Sum of Boxes (CDR‐SB) and Mini Mental State Exam scores were assessed in multivariate regression models. To provide context, key analyte results in ADNI were compared against biomarkers of neurodegeneration, hippocampal volume, and CSF neurofilament light (NfL), in receiver operating characteristic (ROC) analyses evaluating highest quartile of CDR‐SB change over two years (≥3 points). Results: Cerebrospinal fluid inflammatory analytes in relation to cognitive decline were best described by gene ontology terms, natural killer cell chemotaxis, and endothelial cell apoptotic process and in plasma, extracellular matrix organization, blood coagulation, and fibrin clot formation described the analytes. CSF CCL2 was most robust in predicting rate of cognitive change and analytes that correlated to CCL2 suggest IL‐10 pathway dysregulation. The ROC curves for ≥3 points change in CDR‐SB over 2 years when comparing baseline hippocampal volume, CSF NfL, and CCL2 were not significantly different. Interpretation: Baseline levels of immune cell chemotactic cytokine CCL2 in the CSF and IL‐10 pathway dysregulation impact longitudinal cognitive and functional decline in MCI‐AD. CCL2's utility appears comparable to biomarkers of neurodegeneration in predicting rapid decline. [ABSTRACT FROM AUTHOR]

Objective The pathophysiology of the most common joint disease, osteoarthritis (OA), remains poorly understood. Since synovial fluid (SF) bathes joint cartilage and synovium, we reasoned that a comparative analysis of its protein constituents in health and OA could identify pathways involved in joint damage. We undertook this study to perform a proteomic analysis of knee SF from OA patients and control subjects and to compare the results to microarray expression data from cartilage and synovium. Methods Age-matched knee SF samples from 10 control subjects, 10 patients with early-stage OA, and 10 patients with late-stage OA were compared using 2-dimensional difference-in-gel electrophoresis and mass spectrometry (MS). MS with a multiplexed peptide selected reaction monitoring assay was used to confirm differential expression of a subset of proteins in an independent OA patient cohort. Proteomic results were analyzed by Ingenuity Pathways Analysis and compared to published synovial tissue and cartilage messenger RNA profiles. Results Sixty-six proteins were differentially present in healthy and OA SF. Three major pathways were identified among these proteins: the acute-phase response signaling pathway, the complement pathway, and the coagulation pathway. Differential expression of 5 proteins was confirmed by selected reaction monitoring assay. A focused analysis of transcripts corresponding to the differentially present proteins indicated that both synovial and cartilage tissues may contribute to the OA SF proteome. Conclusion Proteins involved in the acute-phase response signaling pathway, the complement pathway, and the coagulation pathway are differentially regulated in SF from OA patients, suggesting that they contribute to joint damage. Validation of these pathways and their utility as biomarkers or therapeutic targets in OA is warranted. [ABSTRACT FROM AUTHOR]

Background/Purpose: We have previously shown that pre-exposure to IL-1β augments the immunosuppressive activity of MSC using the mixed lymphocyte reaction as a readout assay. In this project, we studied the profiles of inflammatory cytokines secreted by MSCs at the protein level to identify biomarkers in in vitroprimed MSCs. The objective of this study was to determine the MSC response to 'danger signals.' Methods: MSCs were isolated from a healthy donor and cultured as per a protocol published earlier by the Skeletal Research Center. After three passages, 200,000 cells per 35 mm culture dish were incubated in 2 ml DMEM with 10% fetal bovine serum at 37°C for 5 days until 90% confluent. The MSCs were then treated with LPS (10 μg/ml), IL-1β (25 ng/ml), TNF-α (50 ng/ml), or poly (I:C) (30 ng/ml) overnight (18 hours). Control and treatment groups were done in triplicate. The culture media were then collected and analyzed for secreted mediators using the RayBiotech® Human Cytokine Array (2000), as per the manufacturer's protocol. Chemiluminescense was detected using Kodak BioMax MR film, and was analyzed using the ImageJ program (). Normalization and fold-difference computations were then performed in MS Excel. Pathway analysis for biomarker detection was performed using the Ingenuity () program by the CWRU proteomics center. Results: There were clear distinctions in the patterns of inflammatory mediators produced by MSC upon exposure to LPS versus poly (I:C). LPS stimulated the production of mediators that affect mostly neutrophil chemotaxis and activation, including IL-8 (1.8 fold), CXCL7 (2 fold), CXCL5 (2.4 fold), CXCL1 (1.6 fold). However, poly (I:C) induced mediators with activities on T cells and NK cells including IL-15 (18 fold), IL-4 (23 fold), TGF-β3 (9.7 fold), CCL-18 (4.6 fold). While poly (I:C) stimulated IL-1β production (2.6 fold), LPS did not (0.7 fold), and neither agent increased TNF-α significantly (1.3 and 2.1 fold). Interestingly, there was marked production of CCL1 (12 fold) upon exposure of MSC to IL-1β but not to LPS (0.6 fold), TNF-α (1.3) or poly (I:C) (2.1 fold). Conclusion: Priming MSC by TLRs ligands triggered different cytokine and chemokine expression profiles. These profile patterns may signify framework of the upcoming immune responses in the tissue. Accordingly, the candidate biomarkers for ongoing MSC immunomodulatory activities should include those for neutrophils versus those for T/NK cells. Furthermore, our results strongly suggest involvement of IL-1β in MSCmediated activation of T-regulatory cells by induction of the CCR-8 ligand, CCL-1. CCR-8 is a known biomarker identifying CD4+ memory T-cells enriched for FoxP3+ T-regulatory cells and is involved in delivery of immunosuppression. [ABSTRACT FROM AUTHOR]