Turnover of Resident Plasmacytoid Dendritic Cells in the Cornea

Abstract

Purpose : Plasmacytoid dendritic cells (pDCs), which orchestrate innate and adaptive immune responses, have been shown to reside in the naïve cornea by our group. This study aimed to evaluate the turnover of corneal pDCs.

Methods : Parabiosis and bone marrow (BM) chimera models were adopted to explore pDC turnover. To generate BM chimeras, CD45.1 mice were irradiated while protecting their skull with lead shields. 5×10⁵ BM cells of DPE-GFP×RAG1^-/- (pDC-GFP) mice (with GFP⁺ pDCs) were adoptively transferred to replenish the BM in the irradiated mice. Parabiosis was performed by joining CD45.1 and pDC-GFP mice. Corneal suture placement or cautery was performed in wildtype C57BL/6 or chimeric mice. Corneas were stained with combinations of CD45 (pan-leukocyte marker), CD45.1 (recipient leukocytes marker), CD45.2 (donor leukocyte marker), PDCA-1, and CD45R/B220 (pDC markers), and Ki-67 (proliferation marker), followed by confocal microscopy or flow cytometry. T test was applied to compare the groups. p<0.05 was considered significant.

Results : Resident pDCs were observed in both peripheral (82.9±5.1 cells/mm²) and central (51.6±4.9) corneas of naïve WT mice. We did not observe recruitment of any pDCs to the naïve parabiont corneas for up to 8 months in the periphery and 12 months in the center. However, as early as day 1 after corneal suture placement in BM chimeras, GFP⁺ donor pDCs were recruited to the peripheral (52.0±9.3) and central (35.5±5.9) corneas. On day 7, the density of GFP⁺ pDCs was further increased in both corneal periphery (196.3±52.8; p=0.004) and center (110.9±22.6, p=0.002). We also observed an increase in the density of GFP^neg resident recipient pDCs in the peripheral cornea on day 7 (55.4±15.5) compared with day 1 (16.8±6.2, p=0.003), where they had initially declined after suturing. In contrast to lack of turnover in naïve corneas of parabionts, corneal inflammation resulted in rapid turnover of pDCs, albeit without reaching equilibrium. This finding, in line with the increase in density of GFP^neg pDCs in BM chimeras, suggests possible proliferation of resident pDCs. In fact, confocal micrographs showed co-staining of Ki-67 with pDCs in both naïve (20.8%) and cauterized (64.8%) corneas.

Conclusions : Following inflammation, rapid recruitment of pDCs into the cornea is evident. Additionally, resident pDCs may proliferate in situ during both steady state and inflammation.

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