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Susan Hannes, Baoying Liu, Zhiyu Li, Diamond Ling, H Nida Sen, Robert B Nussenblatt; Peripheral Monocyte Subset-Derived Macrophages Show Distinct Expression Kinetics of Immune Biomarkers Associated with Ocular Autoimmunity. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):884.
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Human peripheral blood monocytes are precursors of some tissue-resident macrophages and have been implicated in the pathogenesis of non-infectious uveitis. They can be categorized into three subgroups: CD14++CD16- (classical), CD14++CD16+ (intermediate), and CD14+CD16++ (non-classical). Previously, we observed that monocytes isolated from the circulating blood of non-infectious uveitis patients exhibit a skewed phenotype toward an elevated level of the CD14++CD16+ subset. However, the destiny of these monocyte subsets after differentiation has not been fully investigated. Here, we sought to evaluate the expression kinetics of immune biomarkers associated with non-infectious uveitis in both M1 (pro-inflammatory) and M2 (alternative) macrophages in vitro.
Human peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors and uveitis patients using a Ficoll gradient centrifugation protocol. Subsets of monocytes were purified by flow cytometry (BDFACSAria II) based on CD14 and CD16 staining. The subsets were differentiated over a time course using GM-CSF and M-CSF to produce M1 and M2 macrophages, respectively. An Annexin V Apoptosis Assay was used to monitor viability. The following cell surface markers were assayed with flow cytometry: CD1a, CD11b, CD11c, CD69, CD80, CD86, and CD163.
We observed a time dependent effect in biomarker changes during polarization procedures using either GM-CSF or M-CSF by monitoring cell-surface marker changes. During M1 and M2 differentiation conditions, marker expression levels varied, with perceptible increases in CD80, CD86, and CD163 for GM-CSF differentiated<br /> CD14++CD16- and CD14++CD16+ subpopulations. Regarding GM-CSF versus M-CSF conditions, CD80 exhibited increased expression with M-CSF polarization in the CD14+CD16++ subpopulation. CD86 levels are higher with M-CSF polarization in CD14++CD16+ and CD14+CD16++ subpopulations. During either polarization condition, the CD14+CD16++ population was found to die more quickly than the other two.
Our study showed that under both M1 and M2 polarization conditions, peripheral monocyte subset-derived macrophages exhibit distinctive expression kinetics of immune biomarkers associated with ocular autoimmunity. Our results contribute to knowledge of the innate immune mechanisms involved in non-infectious uveitis.
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