Abstract
Purpose :
The avascular cornea is endowed with resident plasmacytoid dendritic cells (pDCs). We have recently shown that corneal pDCs are involved in maintaining cornea avascularity through expression of angiostatic proteins, including endostatin (ES), platelet factor (PF)-4, tissue inhibitor of metalloprotease (TIMP)-3 and thrombospondin (TSP)-1. The purpose of this study was to examine if corneal pDCs express neuropeptide receptors and if neuropeptides may regulate the angiostatic activity of pDCs
Methods :
Primary splenic pDCs from DPE-GFP×RAG1-/- transgenic mice (pDC-GFP) were isolated by fluorescence activated cell sorting (FACS) and cultured with primary C57BL/6 trigeminal ganglion (TG) neurons, isolated by Percoll differential gradient, in Ham’s F-12 with 10% heat inactivated FBS. Corneal pDC gene and protein expression of ES, PF-4, TSP-1, TIMP-3 were quantified by single cell qRT-PCR as well as by flow cytometry. Expression of melanocortin (MC-4) receptor and angiostatic molecules of splenic and corneal pDCs was quantified by flow cytometry ± MC-4 agonist (THIQ). Translocation of nuclear factor kappa B (NF-κB) to the nucleus was examined by confocal imaging of pDCs following treatment with THIQ. Significance was determined by ANOVA and t-test
Results :
At 24 hours of pDC and TG neuron co-culture, expression of ES, TSP-1, PF-4, and TIMP-3 by pDCs at the gene and protein levels was significantly increased compared to baseline pDCs and TG neurons alone (p<0.05). The expression of MC receptors 1-5 was assessed on pDCs, and revealed that pDCs express the gene and protein of the MC-4 receptor at greater levels compared to conventional dendritic cells (cDCs) and macrophages (p<0.05). pDCs incubated for 24 hours with a highly selective agonist for MC-4, THIQ, demonstrated significantly greater mRNA and protein levels of ES, PF-4, TSP-1, and TIMP-3 compared to baseline non MC-4 activated pDCs (p<0.05). pDCs treated with THIQ showed significantly increased NF-κB nuclear translocation as compared to untreated pDCs (p<0.05)
Conclusions :
Our findings demonstrate that co-culture of pDCs with TG neurons increases pDC expression of proteins that suppress neovascularization. pDCs expression of angiostatic molecules is, at least in part, mediated by MC-4 through NF-κB signaling
This is a 2020 ARVO Annual Meeting abstract.