Elsevier

The Ocular Surface

Volume 18, Issue 2, April 2020, Pages 277-285
The Ocular Surface

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

https://doi.org/10.1016/j.jtos.2020.02.005Get rights and content

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+ CD11clow 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.

Introduction

Since the initial description of plasmacytoid dendritic cells (pDCs) by the pathologists Lennert and Remmele in 1958, several aspects of their vital properties in immune response have emerged [1]. They were initially categorized as lymphoblasts, purely based on their localization in interfollicular areas of human reactive lymph nodes and their morphology [1]. In 1980s, availability of lineage markers enabled detection of some T-cell markers including CD4 (OKT4) on these cells, which along with their well-developed rough endoplasmic reticulum, resembling plasma cells, led to re-naming them to plasmacytoid T cells [[2], [3], [4], [5], [6]] or T-associated plasma cells [7]. By further immunophenotyping, Facchetti et al. demonstrated expression of several myelomonocytic markers and lack of granulocytes, B or T cell associated antigens including CD20, CD22, or T cell receptor component CD3 on these cells, designating them as plasmacytoid monocytes [8]. Later, extensive studies showed that plasmacytoid T cells/monocytes can differentiate into mature conventional dendritic cells (cDCs) in vitro, coining the term plasmacytoid pre-dendritic cells and later pDC for them [9]. Further studies revealed the phenotypic similarities between pDCs and natural interferon-producing cells (IPCs) [[10], [11], [12], [13]] and eventually, Siegal et al. found that pDCs can secret large amounts of interferon-α (IFN-α) [14]. This observation, confirmed by others [15,16], provided the necessary evidence to conclude pDCs and IPCs represent the same cell entity.

Although pDCs share many features with cDCs, such as sensing pathogens and the capacity to capture and present antigens [17], they show distinct properties, which contrast cDCs. For instance, in marked contrast to cDCs, pDCs exhibit no dendrites in blood smears and contain abundant rough endoplasmic reticulum, numerous mitochondria, and a small Golgi apparatus in scanning electron microscopy [18]. Further, contrary to cDCs, pDCs express recombination-activating gene products, exhibit D-J rearrangements of immunoglobulin heavy chains [19], and express high levels of endosomal receptors, toll-like receptor (TLR)-7 and −9, which enables them to recognize single stranded RNA and double stranded DNA of pathogens, respectively [20,21].

Multifaceted properties of pDCs encompass a broad spectrum of immune responses, ranging from pathogen challenge to tumor immunology, and atherosclerosis [[22], [23], [24], [25], [26]]. During antiviral immunity, pDCs serve as the first line of innate immune cell defense, through their early production of IFN-α [14,16,27], as well as indirectly by bridging innate and adaptive immune responses through activating, attracting, and differentiating natural killer (NK) cells [28,29], T cells [30], as well as B cells, and plasma cells [31,32]. pDCs also modulate adverse immune reactions, by inducing tolerance through several strategies. They express 2,3-indoleamine dioxygenase [33], and promote CD4+ and CD8+ T regulatory cell (Treg) differentiation [[34], [35], [36], [37]]. They also enhance secretion of interleukin (IL)-10 by inducible T cell co-stimulator (ICOS)-expressing Tregs [38], and additionally reduce the proliferative capacity of alloreactive T cells [39,40]. Recently, it has been shown that pDCs in gut-associated lymphoid tissues are key players in establishing oral tolerance by inhibiting antigen-specific T cells, followed by generation of regulatory T cells [41,42]. pDCs are constantly generated in the bone marrow and subsequently enter the blood stream, where they constitute 0.2–0.8% of mononuclear cells [18,43,44]. pDCs then home to the thymus, spleen, liver, lymph nodes, mucosal-associated lymphoid tissues, and Peyer's patches [9,[45], [46], [47], [48], [49], [50], [51]]. Although pDCs can be recruited to the sites of inflammation in peripheral tissues during disease, current evidence suggests that they are typically confined to lymphoid tissues during steady state.

The conjunctiva is the mucosal barrier protecting the ocular surface and is divided into the bulbar conjunctiva, which covers the majority of the ocular surface, and the tarsal conjunctiva, which lines the inner surface of the eyelids. In addition to contributing mucin to the tear film, as a common feature of mucosal barriers, the conjunctiva plays an active role in mediating immune responses following exposure to antigens from the external environment [52,53]. In addition, similar to the gut, the conjunctiva can induce tolerance to foreign antigens under physiological conditions. However, our knowledge is currently limited on mechanisms through which tolerance is maintained in the conjunctiva. Considering recent findings on the presence and critical role of pDCs in inducing tolerance in mucosal tissue of gut [41,42], we aimed to assess if the conjunctiva, another mucosal tissue, and the limbus host resident pDCs during steady state.

In the current study, we report the presence of resident pDCs in the bulbar conjunctiva and limbus of wild-type mouse during steady state. We observe a higher density of pDCs in the limbus, where they line limbal vessels and patrol the intravascular spaces. We also demonstrate that pDCs residing in the naïve bulbar conjunctiva are mature and express T cell co-inhibitory and lower to negligible co-stimulatory markers. We further show that pDCs are attracted to vascular endothelial cells and interact with them in vitro during endothelial cell tube formation.

Section snippets

Mice

Wild-type 6-8-week-old C57BL/6N mice were obtained through Charles River Laboratories (Wilmington, MA); DPE-GFP×RAG1−/− transgenic mice were kindly provided by Dr. Ulrich H. von Andrian (Harvard Medical School, Boston, MA) and were bred in house at the animal facilities of Schepens Eye Research Institute and Tufts Comparative Medicine Services, Tufts Medical Center. Experiments were performed in concordance with the Statement for the Use of Animals in Ophthalmic and Visual Research (Association

Plasmacytoid dendritic cells reside in the naïve bulbar conjunctiva

In order to assess if pDCs are present in the naïve bulbar conjunctiva, we initially performed IF staining with CD45 (pan-leukocyte marker), CD11c (cDC and pDC marker), and PDCA-1 (pDC marker) on the excised bulbar conjunctival whole-mounts of naïve wild-type C57BL/6N mice. As demonstrated in Fig. 1a, we observed a population of CD45+ CD11clow PDCA-1+ cells in the naïve bulbar conjunctiva. Fig. 1b demonstrates a magnified confocal micrograph of CD45+ CD11clow PDCA-1+ cells in this tissue. As

Discussion

In homeostatic conditions, distribution of pDCs is largely limited to primary and secondary lymphoid organs. Our study demonstrates that pDCs reside in the naïve bulbar conjunctiva as well as the limbus. We show that pDCs are less frequently located in the bulbar conjunctiva in comparison with the limbus where they accompany limbal vessels. To verify the presence of pDCs in these peripheral tissues, we applied three different laboratory methodologies. Initially, we performed fluorescence

Financial support

NIH R01-EY022695 (PH), NIH R01-EY026963 (PH), Research to Prevent Blindness Career Development Award (PH), Massachusetts Lions Research Fund, Inc. (PH), Research to Prevent Blindness Challenge Grant, and Tufts Institutional Support.

Acknowledgement

We would like to express our gratitude to Mr. Allen Parmelee and Stephen Kwok for their technical assistance in sorting splenic cells. We would also like to acknowledge Dr. Ulrich von Andrian (Harvard Medical School, Boston, MA) for generously sharing DPE-GFP×RAG1−/− transgenic mice and FLT3L-secreting B16 melanoma cell line.

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