Introduction
Uveitis is the fourth leading cause of blindness in the working age population in developed countries [
1,
2]. Endogenous posterior uveoretinitis makes up 22% of uveitis cases [
3]. The aetiology of non-infectious uveoretinitis is unknown in most cases and has been considered to have an autoimmune basis [
4,
5]. Well-established animal models of experimental autoimmune uveoretinitis have provided a valuable experimental platform for improving our understanding of the disease pathogenesis and mechanisms of autoimmune uveoretinitis [
4-
8].
Experimental autoimmune uveoretinitis (EAU) is an organ-specific, T-cell mediated disease that can be induced in susceptible mouse strains by direct immunization with retinal antigens, including interphotoreceptor retinoid binding protein (IRBP) or arrestin (retinal soluble antigen, S-antigen), in complete Freund’s adjuvant and a simultaneous intraperitoneal injection of pertussis toxin. Alternatively, EAU can be induced indirectly by adoptive transfer of retinal antigen-specific effector T cells [
4]. Disease usually develops around 10 to 14 days after immunization and is clinically evident within the retina as inflammatory cell infiltration which will include macrophages [
9], dendritic cells (DCs) [
10], neutrophils [
11] and T cells [
9,
11]. In addition to the influx of blood-derived leukocytes, the resident myeloid-derived macrophages, retinal microglia, are also activated during EAU [
7,
8].
The Cx
3cr1
gfp/+ transgenic knock-in mouse [
12] has enabled exquisite
in vivo and
ex vivo visualization of the dynamic changes in microglia in steady and diseased states in the eye [
13-
17], as well as in the brain [
18,
19], and in particular their rapid responsiveness to injury and presence of noxious stimuli [
14,
15]. The study of
Cx
3
cr1-bearing brain microglia
in vivo in these reporter mice requires the surgical creation of a defect or window in the calvaria and two-photon microscopic examination of the superficial cortex [
20]. By contrast, the eye offers unique advantages for direct
in vivo visualization of infiltrating and resident immune cells in reporter mice with minimal experimental manipulation [
16,
21-
23]. However, to date there has been limited use of reporter mice to investigate the dynamics of infiltrating leukocytes and resident myeloid cells in an ocular model of autoimmunity.
In the present study, we demonstrate that
in vivo fundus examination of transgenic reporter mice facilitates monitoring of the dynamic changes in various exogenously and endogenously derived cells of myeloid lineage during EAU progression. In particular we chose to examine EAU in transgenic mice (
C57Bl/6 J Cx
3
cr1
GFP/+
,
C57Bl/6 CD11c-eYFP, and
C57Bl/6 J LysM-eGFP) in which promoter elements of the myeloid-specific
Cx
3
cr1,
CD11c and
lysM genes are expressed alongside a specific fluorescent reporter in an attempt to characterize the relative temporal pattern of resident and infiltrating myeloid cells, DCs, and neutrophils, respectively. Whilst we appreciate that none of these transgenic reporter mice provide definitive identification of any of the above myeloid cell subsets and thus have their limitations [
24], our results do provide novel insights into the cell mediated immune events in this model of human endogenous posterior uveoretinitis and allow accurate clinical grading of disease severity that correlates with histopathological changes.
Discussion
Intravital imaging using genetically modified reporter mice in which leukocyte subtypes are endogenously labelled with a fluorescent reporter gene transcript has greatly enhanced our understanding of cellular and immunological mechanisms during inflammation of several tissues [
38-
40]. Many of these experimental approaches are partly hindered or complicated by the potential effects of surgical intervention needed to exteriorise or surgically alter the tissue under investigation such as mesentery [
41,
42], cremaster muscle [
42-
44], liver [
45], lung [
37], kidney [
40,
43], and skin [
46]. In the case of intravital imaging of the brain a craniotomy window is required [
47,
48]. The eye has several advantages over most organs because by its very nature it provides a clear transparent window on both neural tissue (retina) and connective tissues (cornea, iris) [
33,
49], thus avoiding surgical intervention. We sought to exploit the recent development of multi-modal imaging techniques which allow high-quality examination of the mouse fundus [
16,
22] to investigate the behaviour of cells of myeloid origin during the course of EAU, a widely used model of ocular autoimmune disease.
Disease severity of EAU is routinely determined using histopathological grading methods [
28,
29,
50]. To circumvent the issue of single time point post-mortem grading, several research groups have developed non-invasive clinical grading methods including topical endoscopic fundus imaging [
51], otoscope imaging [
6], scanning laser ophthalmoscopy [
52] and optical coherence tomography [
53-
55]. In this present study, multi-modal fundus ophthalmoscopy was chosen to grade the disease severity as it has the advantages of being relatively inexpensive and has the capability of capturing both video and still frame images in brightfield, together with green and red fluorescence wavelengths.
Many previous phenotypic analyses of the inflammatory cell infiltrate during EAU have used multi-parameter flow cytometry to show that the majority of infiltrating cells are myeloid-derived with a peak in T cell infiltration around d14 p.i. [
11,
56,
57]. Alternative approaches to visualize leukocyte trafficking
in vivo in the eye during EAU have included use of acridine orange, a non-specific nuclear dye which can be visualized by fluorography. This method revealed leukocytes rolling along the retinal veins as early as d14 p.i. [
58,
59]; however, their specific phenotype was obviously not determined. More recently,
in vivo imaging of the leukocyte subtypes with more specificity has become easier with the availability of genetically modified mouse models in which genes regulating leukocyte subtypes are used as promoters to express fluorescent reporter proteins. Although these transgenic mouse models are useful for providing insights to myeloid lineage cell types in normal and diseased state, some authors have warned of cautious interpretation of these mice as the sole means of identifying and distinguishing macrophages and DCs [
24]. Such limitations are also true of the three transgenic mouse lines chosen for the present study.
There are several subpopulations of Cx
3cr1-GFP
+ myeloid-derived cells in the normal retina including the hyalocytes on the retinal surface [
60], subretinal macrophages on the other aspect of the neural retinal [
61-
63], and the extensive network of microglial populations in the retinal parenchyma [
13,
64].
C57Bl/6 J Cx
3
cr1
GFP/+
mice [
12] have been widely used to investigate the role of microglia in numerous ocular conditions that may have an inflammatory element in their pathogenesis including potential models of retinal degeneration [
65], retinopathy of prematurity [
66,
67] and diabetic retinopathy [
16]. In the present study we demonstrate highly distinctive perivenular infiltrates of Cx
3cr1-GFP
+ cells at d14 p.i. to d35 p.i. which is in agreement with previous studies [
8,
68]. The perivenular infiltrate could theoretically represent haematogenous Cx
3cr1-GPF
+ (GFP
low) myeloid cells recently extravasated into the retina or the chemotactic migration of resident Cx
3cr1-GPF
+ (GFP
high) microglia towards the vasculature. We believe the former is the case as the Cx
3cr1-GPF
+ (GFP
high) microglia network seemed largely undisturbed, something that was subsequently confirmed by retinal whole mount analysis (data not shown).
The difficulty in distinguishing subpopulations of macrophages from cells of DC lineage was the motivation for the creation of CD11c transgenic reporter mice, specifically the
CD11c-eYFP [
25] and
CD11c-DTR/GFP mice [
69]. In these transgenic mice the promoter for the
Itgax (CD11c) gene is used to drive eYFP expression (
CD11c-eYFP mice) or GFP and diphtheria toxin receptor (DTR) expression (
CD11c-DTR/GFP mice). CD11c is a leukocyte integrin comprised of an alpha X subunit that along with CD18, a leukocyte beta 2 integrin polypeptide, forms the CD11c/CD18 heterodimer which is important in leukocyte adhesion, migration and cell to cell interaction during immune responses. CD11c is expressed heterogeneously by different populations of DCs [
25] and is important in T cell priming [
69]. However, it is also expressed to at least one log lower than DCs on other immune cells such as natural killer cells, subpopulations of macrophages and activated T cells [
25]. As such immune cells are not normally a feature of the resting central nervous system, we, like other previous investigators [
31,
32,
70], thought is reasonably safe to assume that in the resting and disease state CD11c-eYFP
+ cells may represent predominantly DCs. The view that
CD11c-eYFP mice are valuable for examining the distribution of DCs has recently been strongly challenged by Hume [
24] who points out that CD11c has no function in antigen presentation; not all DCs are CD11c
+ and that not all CD11c
+ cells are antigen-presenting cells. It was thus with caution that we chose to take advantage of the transgenic
C57Bl/6 N CD11c-eYFP mice to examine the dynamic of DCs in EAU. Indeed at the commencement of this study we had the further complication of discovering that these mice had a pre-existing retinal dystrophy due to the presence of the
rd8 mutation in the
crb1 gene [
71] and that the CD11c-eYFP
+ cells in the retina represented activated microglia [
22]. In the present study, CD11c-eYFP
+ cells were recruited into the eye at d14 and d21 p.i. in a similar pattern to that observed in
C57Bl/6 J Cx
3
cr1
GFP/+
mice leading us to conclude that these CD11c-eYFP
+ cells are likely a mixture of myeloid-derived cells.
Interestingly, despite the
C57Bl/6 N CD11c-eYFP mice carrying the
rd8 mutation and the pre-existing disrupted retinal architecture prior to immunization, they did not develop a more severe form of EAU as may have been predicted if one were to assume that this dystrophic condition compromised the immune status of the retina as we have previously concluded [
22], although we have not specifically proven that the blood-ocular barrier was compromised.
In the
C57Bl/6 J LysM-eGFP mouse line generated by Faust and colleagues [
26], homologous recombination was used to insert the eGFP gene into the LysM locus. This was chosen because LysM is expressed specifically in the myelomonocytic cell lineage (macrophages and neutrophil granulocytes). Characterization of LysM-eGFP
+ cells in the blood revealed that the eGFP
high polymorphonuclear granulocytes outnumbered LysM-eGFP
+ monocytes by 50:1. In the present study, we observed exceedingly small numbers (16.6 ± 1.18) of largely static LysM-eGFP
+ myelomonocytic cells around the optic nerve head in the normal
C57Bl/6 J LysM-eGFP fundus. We propose that these are likely of monocyte lineage as it is highly unusual to detect extravasated neutophils in the normal retina. However, video analysis (not shown) revealed many LysM-eGFP
+ travelling at high velocity in retinal vessel lumina, which we conclude are likely to be circulating neutrophils. In these mice we demonstrated increased numbers of LysM-eGFP
+ cells in the peripapillary retinal vessels at d14 to d21 of EAU. Closer examination of the high power images (see inset, Figure
4) suggests that many of these are marginating in vessel lumina, a pattern which differs from the perivascular infiltrates observed in the
C57Bl/6 J Cx
3
cr1
GFP/+
and
C57Bl/6 N CD11c-eYFP mice. Subsequent flow cytometry of retinal tissue during EAU in
C57Bl/6 J LysM-eGFP mice revealed these cells to be largely a neutrophilic infiltrate (Goldberg and colleagues, unpublished data).
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
XC performed the immunization, in vivo analysis, tissue collection and subsequent histology, and drafted the manuscript. JMK was part supervisor of XC during her PhD studies and helped plan the study. JVF and CCB were also part supervisors of XC and advised on the experimental approach and reviewed the manuscript. GLG and IPW provided data from the C57Bl/6 J LysM-eGFP mice. PGM is the principle chief investigator in whose laboratory the studies were performed, and was primarily responsible with XC for the clinical grading, data analysis, data interpretation and writing of the manuscript. All authors read and approved the final manuscript.