Resident plasmacytoid dendritic cells patrol vessels in the naïve limbus and conjunctiva

Abstract

Plasmacytoid dendritic cells (pDCs) constitute a unique population of bone marrow-derived cells that play a pivotal role in linking innate and adaptive immune responses. While peripheral tissues are typically devoid of pDCs during steady state, few tissues do host resident pDCs. In the current study, we aim to assess presence and distribution of pDCs in naïve murine limbus and bulbar conjunctiva. Immunofluorescence staining followed by confocal microscopy revealed that the naïve bulbar conjunctiva of wild-type mice hosts CD45⁺ CD11c^low PDCA-1⁺ pDCs. Flow cytometry confirmed the presence of resident pDCs in the bulbar conjunctiva through multiple additional markers, and showed that they express maturation markers, the T cell co-inhibitory molecules PD-L1 and B7-H3, and minor to negligible levels of T cell co-stimulatory molecules CD40, CD86, and ICAM-1. Epi-fluorescent microscopy of DPE-GFP×RAG1^-/- transgenic mice with GFP-tagged pDCs indicated lower density of pDCs in the bulbar conjunctiva compared to the limbus. Further, intravital multiphoton microscopy revealed that resident pDCs accompany the limbal vessels and patrol the intravascular space. In vitro multiphoton microscopy showed that pDCs are attracted to human umbilical vein endothelial cells and interact with them during tube formation. In conclusion, our study shows that the limbus and bulbar conjunctiva are endowed with resident pDCs during steady state, which express maturation and classic T cell co-inhibitory molecules, engulf limbal vessels, and patrol intravascular spaces.

Keywords: Conjunctiva; Intravital microscopy; Limbus; Plasmacytoid dendritic cell; Vascular endothelial cell.

Figure 1.. Identification of resident plasmacytoid dendritic cells in the naïve murine bulbar conjunctiva.

Representative confocal micrograph of whole mount bulbar conjunctiva of naïve C57BL/6N mouse stained with CD45 (pan-leukocyte marker; red), CD11c (conventional and plasmacytoid dendritic cell marker; green), and PDCA-1 (specific pDC marker; white), showing triple-stained CD45⁺ CD11c^low PDCA-1⁺ pDCs with white arrows at lower (a) and higher (b) magnification. White arrow heads indicate double stained CD45⁺ CD11c⁺ PDCA-1^neg conventional dendritic cells. Quantification of density of CD45⁺ bone marrow-derived cells, CD11c⁺ conventional and CD11c^low PDCA-1⁺ plasmacytoid dendritic cells in the bulbar conjunctiva of naïve mice (n=3) (c). Error bars; standard deviation, scale bars; 50 μm

Figure 2.. Characterization of the resident plasmacytoid dendritic cells in the naïve bulbar conjunctiva by flow cytometry.

Excised bulbar conjunctiva of naïve C57BL/6N mice (pooled from 6–8 mice) were digested by collagenase D and DNAse to yield single cells. Afterwards, cells were stained with combinations of fluorochrome-conjugated antibodies against CD45 (pan-leukocyte marker), Ly-6C, PDCA-1, Ly-49Q, CD11c (expressed by pDCs), CD11b, F4/80, Ly-6G, CD3, CD19 or their respective isotype controls. After gating on live CD45⁺ singlets to present bone marrow-derived cells, CD11b^neg Ly-6C⁺ cells were selected (a). Next, among CD45⁺ CD11b^neg Ly-6C⁺ cells, F4/80^neg PDCA-1⁺ cells were selected, representing a population of CD11b^neg F4/80^neg Ly-6C⁺ PDCA-1⁺ cells among CD45⁺ cells in the bulbar conjunctiva (b). Histogram of flow cytometry analysis reveals that CD45⁺ CD11b^neg F4/80^neg Ly-6C⁺ PDCA-1⁺ cells in naïve conjunctiva are in fact pDCs as they express CD11c and Ly-49Q, and are negative for Ly-6G, CD3, and CD19 (c). Fluorescence minus one isotype control staining is presented in gray and staining with designated antibodies are presented in blue open histograms. Plots are representative of 3 independent experiments.

Figure 3.. Immunophenotyping of the conjunctival plasmacytoid dendritic cells by flow cytometry.

Digested single cell suspension of bulbar conjunctiva of naïve C57BL/6N mice (pooled from 6–8 mice) were labeled with combinations of CD45, CD11b, F4/80, Ly-6C, PDCA-1, PD-L1, B7-H3, CD40, CD86, ICAM-1, MHC-II (I-A/I-E) antibodies, or their respective isotype controls. Blue open histograms represent expression of T cell co-inhibitory and co-stimulatory markers of PD-L1, B7-H3, CD40, CD86, ICAM-1 as well as maturity marker MHC-II on resident pDCs (identified as CD45⁺ CD11b^neg F4/80^neg Ly-6C⁺ PDCA-1⁺ singlets) in naïve mouse. Fluorescence minus one isotype control background staining is depicted in dark profile. Plots are representative of 3 independent experiments.

Figure 4.. Distribution of plasmacytoid dendritic cells in the limbus and bulbar conjunctiva.

Representative fluorescent microscope image of the limbus and bulbar conjunctiva of naïve DPE-GFP×RAG1^−/− mice in which pDCs solely express GFP (a). Limbus (indicated by blue star) is defined as the area between two arbitrary solid lines, 100 μm apart, parallel to limbal vessels, and bulbar conjunctiva is marked by the red star, considered as the area between dash and solid lines. Scale bar; 50 μm. Quantification of the density of the pDCs in the limbus and the bulbar conjunctiva (n=8), indicating higher density of pDCs in the limbus (b). Error bars; standard deviaaon, ‡; p=0.001

Figure 5.. Multiphoton micrograph of the limbus and bulbar conjunctiva of GFP⁺ plasmacytoid dendritic cells in DPE-GFP×RAG1^−/− transgenic mouse in homeostatic state.

Intravital MPM of limbus and bulbar conjunctiva of DPE-GFP×RAG1^−/− mouse with specifically GFP-tagged pDCs, reveals presence of resident GFP⁺ pDCs in these tissues (a). Inset is magnified two folds in (b). Note two distinct morphologies of pDCs; pDCs with two thin polar elongated stellates (white arrows) and pDCs with shorter but usually more stellates and thicker cell bodies (white arrow heads). Scale bars; 50 μm

Figure 6.. Plasmacytoid dendritic cells reside in close proximity to limbal vessels and patrol intravascular spaces.

Representative multi-photon micrograph of the limbus of naïve DPE-GFP×RAG1^−/− mice injected with Qdot (a). Quantification of density of pDCs in contact with limbal vessels based on vessel diameter (b). (c-d) Comparison of track displacement length (c) and mean speed (d) between extravascular and intravascular pDCs. (e, f) Representative still images of quantified track of an intravascular pDC, resembling moving in a loop (e) and mixed crawling and loop pattern (f). Error bars; standard deviation, ***; p<0.001, scale bar; 200 μm in (a) and 50 μm (e, f)