LncRNA MRF drives the regulatory function on monocyte recruitment and polarization through HNRNPD-MCP1 axis in mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) exhibit two bidirectional immunomodulatory abilities: proinflammatory and anti-inflammatory regulatory effects. Long noncoding RNAs (lncRNAs) have important functions in the immune system. Previously, we performed high-throughput sequencing comparing lncRNA expression profiles between MSCs cocultured with or without CD14+ monocytes and screened out a new lncRNA termed lncRNA MCP1 regulatory factor (MRF). However, the mechanism of MRF in MSCs is still unknown. METHODS: MRF expression was quantified via qRT-PCR. RNA interference and lentiviruses were used to regulate MRF expression. The immunomodulatory effects of MSCs on monocytes were evaluated via monocyte migration and macrophage polarization assays. RNA pull-down and mass spectrometry were utilized to identify downstream factors of MRF. A dual-luciferase reporter assay was applied to analyze the transcription factors regulating MRF. qRT-PCR, western blotting and ELISAs were used to assess MCP1 expression. A human monocyte adoptive transfer mouse model was applied to verify the function of MRF in vivo. RESULTS: MRF was upregulated in MSCs during coculture with CD14+ monocytes. MRF increased monocyte recruitment by upregulating the expression of monocyte chemotactic protein (MCP1). Knockdown of MRF enhanced the regulatory effect of MSCs on restraining M1 polarization and facilitating M2 polarization. Mechanistically, MRF bound to the downstream protein heterogeneous nuclear ribonucleoprotein D (HNRNPD) to upregulate MCP1 expression, and the transcription factor interferon regulatory factor 1 (IRF1) activated MRF transcription early during coculture. The human monocyte adoptive transfer model showed that MRF downregulation in MSCs inhibited monocyte chemotaxis and enhanced the effects of MSCs to inhibit M1 macrophage polarization and promote M2 polarization in vivo. CONCLUSION: We identified the new lncRNA MRF, which exhibits proinflammatory characteristics. MRF regulates the ability of MSCs to accelerate monocyte recruitment and modulate macrophage polarization through the HNRNPD-MCP1 axis and initiates the proinflammatory regulatory process in MSCs, suggesting that MRF is a potential target to improve the clinical effect of MSC-based therapy or correct MSC-related immunomodulatory dysfunction under pathological conditions.

AUF1-RGG peptides up-regulate the VEGF antagonist, soluble VEGF receptor-1 (sFlt-1).

The macrophage is essential to the innate immune response, but also contributes to human disease by aggravating inflammation. Under severe inflammation, macrophages and other immune cells over-produce immune mediators, including vascular endothelial growth factor (VEGF). The VEGF protein stimulates macrophage activation and induces macrophage migration. A natural inhibitor of VEGF, the soluble VEGF receptor (sFlt-1) is also produced by macrophages and sFlt-1 has been used clinically to block VEGF. In macrophages, we have shown that the mRNA regulatory protein AUF1/hnRNP D represses VEGF gene expression by inhibiting translation of AURE-regulated VEGF mRNA. Peptides (AUF1-RGG peptides) that are modeled on the arginine-glycine-glycine (RGG) motif in AUF1 also block VEGF expression. This report shows that the AUF1-RGG peptides reduce two other AURE-regulated genes, TNF and GLUT1. Three alternative splice variants of sFlt-1 contain AURE in their 3'UTR, and in an apparent paradox, AUF1-RGG peptides stimulate expression of these three sFlt-1 Variants. The AUF1-RGG peptides likely act by distinct mechanisms with complimentary effects to repress VEGF gene expression and over-express the endogenous VEGF blocking agent, sFlt-1. The AUF1-RGG peptides are novel reagents that reduce VEGF and other inflammatory mediators, and may be useful tools to suppress severe inflammation.