Novel Characterization of Resident Plasmacytoid Dendritic Cells in the Retina and Choroid

Abstract

Purpose : Plasmacytoid dendritic cells (pDCs), as a major orchestrator of innate and adaptive immune responses, are typically not present in peripheral tissues during steady state. This study aimed to evaluate if pDCs are present in naïve retina and choroid.

Methods : Retina and choroid of naïve 6-8 week-old male wild-type C57BL/6 mice were digested via collagenase and DNase. Single cell suspensions of retina/choroid underwent immunofluorescence (IF) staining with CD45 (pan-leukocyte marker), PDCA-1 (specific pDC marker), B220/CD45R, Gr-1 (both expressed by pDCs), CD11c (conventional and pDC marker), CD3 (T cell marker), CD19 (B cell marker), or their respective isotype controls, followed by flow cytometry. Retina/choroid of 6-8 week-old DPE-GFP×RAG1^-/- (pDC-GFP) transgenic mice (with GFP-tagged pDCs) underwent fluorescent microcopy (Nikon Eclipse) to assess distribution of pDCs in these tissues. Further, to assess if retinal/choroidal pDCs express Toll-like Receptor (TLR)-7 and TLR-9, GFP⁺ cells were isolated from the choroid and retina, and splenic macrophages were sorted as controls via IF staining with F4/80 (macrophage marker) using flow cytometry. Isolated cells underwent single cell qRT-PCR using TLR7, TLR9, and GAPDH (house-keeping gene control) primers. ANOVA with Bonferroni correction was applied to compare the groups. p<0.05 was considered statistically significant.

Results : Flow cytometric analysis showed presence of resident CD45⁺PDCA-1⁺B220/CD45RA⁺ pDCs in naïve retina (3.6% of CD45⁺ leukocytes) and choroid (5.1% of CD45⁺ leukocytes). Identity of pDCs in these tissues was confirmed by expression of CD11c^low and Gr-1 as well as lack of expression of B and T cell markers of CD19 and CD3, respectively. Presence of resident pDCs was confirmed by observing GFP⁺ cells in the retina/choroid of pDC-GFP mice via fluorescent microscopy (n=4). Retinal and choroidal GFP-tagged pDCs expressed 34.1±16.1 and 153.3±73.6-folds higher TLR-7 mRNA transcripts compared to splenic macrophages (p=0.04 and p<0.001, respectively). Similarly, mRNA levels of TLR9 were 295.1±9.0 and 276.3±80.3 times higher in retinal and choroidal GFP-expressing pDCs compared to splenic macrophages (p=0.002 and p=0.006, respectively).

Conclusions : The naïve retina and choroid host resident pDCs, which express high levels of TLR7 and TLR9. Unraveling the roles of these cells require further studies.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.