Project description:To explore the effects of periostin on tumor-associated macrophagess, we first sorted CD14+ monocytes from peripheral blood mononuclear cells of healthy donors using magnetic-activated cell sorting and then induced their differentiation into macrophages by culture with M-CSF for 5 days. In the absence of IL-4 stimulation, these macrophages expressed CD163 and Arg1 (a marker of M2 macrophage activation) but not CD206, suggesting that they were similar to macrophages in the early stage of mycosis fungoides (low frequency of CD206+ cells and low IL-4 expression). We then stimulated these macrophages with or without 100 ng/ml of periostin for 6 hours and examined gene expression using a cDNA microarray. Periostin stimulated gene expression on monocyte-derived macrophages was measured at 6 hours after exposure to doses of 100 ng/ml periostin.
Project description:Background: Circulating periostin levels are increased in systemic sclerosis (SSc) patients and correlates positively with disease severity. Additionally, monocytes/macrophages are one of the key cells in SSc pathogenesis. However, the effect of periostin on immune cells, particularly monocytes and macrophages, has not been well investigated. Objectives: To examine the effect of periostin on monocytes and monocyte-derived macrophages (MDMs) in SSc. Methods: Monocytes were purified from blood samples of nine diffuse cutaneous SSc patients and five healthy controls (HCs) and were differentiated into macrophages. Cell surface markers were assessed by flow cytometry. RNA sequencing analysis was performed on recombinant periostin-stimulated monocytes from three HCs. Cytokine/chemokine and extracellular matrix (ECM) protein expressions in MDMs differentiated supplemented with recombinant periostin were assessed by quantitative real-time polymerase chain reaction and bead-based immunoassays in six HCs. Moreover, we evaluated whether periostin was involved in monocyte migration by Transwell assay. Results: The modified Rodnan total skin thickness score correlated positively with the proportion of CD80-CD206+ M2 cells. The M2 macrophage proportion was reduced in rPn-stimulated MDMs in HCs, but not in SSc patients. mRNA expression levels of pro-fibrotic cytokine, chemokine, and ECM protein genes were significantly upregulated in recombinant periostin-stimulated monocytes and MDMs as compared to those from control monocytes and MDMs. Similar trends for their protein levels were also observed in MDMs. Moreover, the ratio of migrated cells in recombinant periostin-stimulated monocytes was significantly higher than that in control monocytes.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.