MiR‐106b‐25 Dysregulation in Complex Regional Pain Syndrome Contributes to T Cell Dysfunction

Abstract

BackgroundComplex regional pain syndrome (CRPS) is a debilitating chronic pain disorder with no effective treatments. Growing evidence implicates aberrant immune regulation in the skin and T cell dysfunction in CRPS pathology. MicroRNA (miRNA) show promise in identifying biomarkers and disease mechanisms. miRNA cluster miR‐106b‐25 is dysregulated in CRPS patient whole blood and serum derived small extracellular vesicles (sEVs) compared to healthy controls. miR‐106b‐25 members are predicted to target several immune genes related to T cell function including CD69. We hypothesize that miR‐106b‐25 cluster plays a role in T‐cell dysfunction by regulating members of CD69 signaling pathway. Here we examine miR‐106b‐25 mediated signaling in sEVs, whole blood, and skin from the tibia fracture model (TFM) of CRPS.MethodsMouse TFM was generated, and pain hypersensitivity was confirmed by von Frey and dynamic weight bearing. Serum, whole blood, and hind limb skin were collected from TFM and control mice. sEVs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. miRNA from sEVs were assessed through HTG EdgeSeq. T cells were isolated from single cell suspensions of hind limb skin. Whole blood samples were obtained from CRPS patients or healthy controls. miRNA and gene expression changes in blood and T cells were assessed by qPCR. Luciferase reporter assay was used to confirm miR‐25 binding to CD69. Jurkat cells were transfected with miR‐25 mimic or inhibitor to assess regulation of gene expression by qPCR and flow cytometry. T cell phenotypes and expression of CD69 and other residency markers were determined by flow cytometry.ResultsCharacterization of sEVs showed no difference between TFM and control mice. miRNA analysis of TFM sEVs confirmed dysregulation of 30 miRNA previously associated with CRPS patients including miR‐106b‐25. There was an inverse correlation of miR‐25 and CD69 in blood samples from CRPS patients compared to controls. Reporter assay confirmed miR‐25 binding to CD69. miR‐25 nucleofection in Jurkat cells downregulated CD69 as measured by flow cytometry. Analysis of miR‐106b‐25 and target gene expression in TFM whole blood and skin T cells and T cell flow cytometry experiments are ongoing.ConclusionsmiRNA signatures in CRPS patients and TFM mice show common alterations including miR‐106b‐25. miR‐25 can regulate CD69 expression in vitro. miR‐25 may contribute to localized T cell dysfunction in the skin in CRPS.