Ellen J Lee

Epifluorescence intravital microscopy of murine corneal dendritic cells.

Purpose. Dendritic cells (DCs) are antigen-presenting cells vital for initiating immune responses. In this study the authors examined the in vivo migratory capability of resident corneal DCs to various stimuli. Methods. The authors used mice expressing enhanced yellow fluorescent protein (eYFP) under control of the CD11c promoter to visualize corneal DCs. To assess the distribution and mobility of DCs, normal corneas were imaged in vivo and ex vivo with fluorescence microscopy. Intravital microscopy was used to examine the responses of resident central and peripheral corneal DCs to silver nitrate injury, lipopolysaccharide, microspheres, and tumor necrosis factor (TNF-alpha). In some experiments, TNF-alpha injection was used to first induce centripetal migration of DCs to the central cornea, which was subsequently reinjected with microspheres. Results. In normal corneas, DCs were sparsely distributed centrally and were denser in the periphery, with epithelial-level DCs extending into the epithelium. Videomicroscopy showed that though cell processes were in continuous movement, cells generally did not migrate. Within the first 6 hours after stimulation, neither central nor peripheral corneal DCs exhibited significant lateral migration, but central corneal DCs assumed extreme morphologic changes. An increased number of DCs in the TNF-alpha-stimulated central cornea were responsive to subsequent microsphere injection by adopting a migratory behavior, but not with increased speed. Conclusions. In vivo imaging reveals minimal lateral migration of corneal DCs after various stimuli. In contrast, DCs within the central cornea after initial TNF-alpha injection are more likely to respond to a secondary insult with lateral migration.

In vivo imaging of the immune response in the eye

The immune system is governed by dynamic events involving in part direct intercellular interactions between an immune cell and other cells or the cell’s environment. Owing to its unique optical characteristics, the eye offers remarkable opportunities for the analysis of the immune system by intravital microscopy. In this review, we present a brief overview of the current state of knowledge of leukocyte trafficking in each of three anatomically distinct and medically important regions of the eye (cornea, iris, retina) as determined by the application of intravital microscopy to animal models of disease. Additionally, we discuss the use of ocular imaging in patients and volunteers. Finally, we examine the future prospects for this field in terms of its potential for impacting our understanding of fundamental immunological phenomena.

Feasibility Study of Lamellar Keratoplasty in a Murine Model

Purpose: In contrast to penetrating keratoplasty (PK), the donor cornea in lamellar keratoplasty (LK) remains separated from the host aqueous humor. There is debate about relative merits of each approach, but experimental comparisons have never been performed in animal models. Therefore, the authors developed a murine LK model. Methods: For allogeneic PK and LK surgeries, corneas of C57BL/6 mice were transplanted to BALB/c mice, assessed by slit lamp, and scored for opacity, edema, and neovascularization up to 46 d post-transplantation. Additional PK or LK surgeries were performed for histological assessment. Results: Graft rejection rate was less in LK vs. PK (69.2 vs. 100%), as was neovascularization (84.6 vs. 100%). In LK, inflammatory cells infiltrated primarily the button; in PK, heavier infiltration was observed throughout the cornea. Conclusions: This study demonstrates the feasibility of LK in mice and presents data suggesting that the inflammatory response in LK differs from that in PK.

Investigation of the relationship between the onset of arthritis and uveitis in genetically predisposed SKG mice

IntroductionSystemic rheumatic conditions are often accompanied by intraocular inflammatory disease (termed uveitis). Despite the frequent manifestation of uveitis with arthritis, very little is understood of the underlying mechanisms that mediate the eye’s susceptibility to disease. The genetically susceptible SKG mouse strain develops arthritis that arises from an inherent mutation that disrupts T-cell antigen receptor signal transduction and thymic selection. The ensuing T-cell–mediated disease is further modulated through exposure to microbial triggers. The purpose of this study was to elucidate how a genetically determined shift in the T-cell repertoire toward self-reactive T cells that drive arthritis influences uveitis in SKG mice.MethodsSKG mice (BALB/c mice that harbor the W163C point mutation in zeta-chain-associated protein kinase 70 [i.e., ZAP-70]) were housed under arthritis-resistant, specific pathogen–free conditions. Arthritis was induced by intraperitoneal injection with fungal glucans (zymosan or curdlan). Arthritis onset and severity were evaluated by clinical scoring, histopathology and infrared imaging within the joints. Periocular traits involving blepharoconjunctivitis were evaluated by clinical scoring and histology. Eyes were evaluated for signs of anterior uveitis using intravital videomicroscopy to document cell-trafficking responses within the iris vasculature and stroma and by histology to detect inflammatory infiltrate and tissue damage within the anterior and posterior eye segments.ResultsExposure to zymosan resulted in the predicted arthritic, sexually dimorphic phenotype in SKG mice. The eyes of SKG mice exhibited episodic intravascular cellular responses to zymosan or curdlan as indicated by significant increases in leukocyte–endothelium interactions akin to ocular vasculitis. However, despite the significant increase in early cell-trafficking responses, cellular infiltration into the iris stroma was not observed and histopathological signs indicative of a sustained uveitis were absent. Instead, eyes of SKG mice developed blepharoconjunctivitis that coincided with arthritis and exhibited sexual dimorphism.ConclusionsThis study underscores the complexity surrounding the pathogenesis of uveitis and its relationship with arthritis. The findings suggest that distinct mechanisms exist by which pathogenic autoimmune T cells target the eyes versus joints, which likely involves the environmental context but nonetheless should be taken into account in the identification and development of effective therapies for each organ.

Investigation of the peptidoglycan sensing molecule, PGLYRP-2, in murine inflammatory uveitis

Background/aim Peptidoglycan (PGN) recognition proteins (PGLYRPs) are innate immune molecules that recognise bacterial cell wall PGN, and participate in several inflammatory diseases such as arthritis. We sought to elucidate the contribution of PGLYRPs in murine uveitis (intraocular inflammatory disease) elicited by PGN, and the extent to which systemically administered PGN alters uveitis compared with arthritis versus locally triggered ocular responses. Methods Mice deficient for PGLYRP-2, PGLYRP-3 or PGLYRP-4 were administered PGN by an intraperitoneal or intraocular injection. Arthritis was assessed by near-infrared imaging and histopathology, while uveitis was measured by intravital videomicroscopy and histopathology. Results Systemic PGN exposure predisposed to arthritis through a PGLYRP-2 dependent mechanism. By contrast, systemic PGN exposure did not predispose to uveitis, and PGLYRP-2 deficiency had no impact on the development the uveitis. When PGN was administered locally, a robust uveitis ensued, which occurred independently of PGLYRP-2. Regardless of whether PGN was administered systemically or locally, neither PGLYRP-3 nor PGLYRP-4 deficiency significantly altered ocular inflammation compared with wild-type control animals. Conclusions Our findings highlight the complexity of PGLYRPs and how PGLYRP-2 may use different molecular pathways in the joints versus eyes. Collectively, our results support a non-essential or redundant role for PGLYRPs-2, -3, -4 in uveitis.

The effects of corneal endothelium on graft survival in a murine model of lamellar keratoplasty.

Purpose: Here we investigate the role of donor endothelium on allograft rejection in a lamellar keratoplasty (LK) model using grafts with or without donor endothelium. Methods: Corneal buttons of donor C57BL/6 mice (2.0 mm) were transplanted to lamellar recipient beds (1.5 mm) in BALB/c mice. Two variations of the LK procedure were performed: (1) standard LK (SLK) (n = 13) without donor endothelium and (2) modified LK (MLK) (n = 14) with retained donor endothelium. The graft status was assessed by slit lamp biomicroscopy and scored for stromal opacity, corneal edema, neovascularization, and anterior chamber reaction up to 46 days post-transplantation. Corneas were also observed histologically. Results: The presence of a grafted corneal endothelium promoted graft rejection; 92.9% (13/14) of grafts were rejected in MLK after an average of 8.3 days, while 69.2% (9/13) of grafts were rejected in SLK on average 10.8 days after transplantation. The former’s stromal opacity was significantly greater at all time points after day 14 except for day 21 (p = 0.77) and day 32 (p = 0.25). Corneal edema was significantly greater in the former at all time points after day 10 except for day 21 (p = 0.16). Neovascularization was significantly greater in the former at all time points after day 10 except for day 25 (p = 0.22). Conclusion: Variations of this model of LK may be useful for studies of immunological mechanisms in corneal transplantation. The donor corneal endothelium may serve as a target of the immune response which promotes inflammation, neovascularization, and graft rejection.

Aberrant interleukin-1 signalling does not increase susceptibility of mice to NOD2-dependent uveitis

NOD2 is the genetic cause of Blau syndrome, an autoinflammatory disease that manifests as coincident uveitis and arthritis. Since dysregulation of IL‐1 signalling is considered a pathogenic mechanism in a number of related autoinflammatory conditions, we examined the extent to which unimpeded interleukin (IL)‐1 signalling influences NOD2‐dependent inflammation of the eye versus the joint.

Nod2 Deficiency Augments Th17 Responses and Exacerbates Autoimmune Arthritis

Arthritis in a genetically susceptible SKG strain of mice models a theoretical paradigm wherein autoimmune arthritis arises because of interplay between preexisting autoreactive T cells and environmental stimuli. SKG mice have a point mutation in ZAP-70 that results in attenuated TCR signaling, altered thymic selection, and spontaneous production of autoreactive T cells that cause arthritis following exposure to microbial β-glucans. In this study, we identify Nod2, an innate immune receptor, as a critical suppressor of arthritis in SKG mice. SKG mice deficient in Nod2 (Nod2−/−SKG) developed a dramatically exacerbated form of arthritis, which was independent of sex and microbiota, but required the skg mutation in T cells. Worsened arthritis in Nod2−/−SKG mice was accompanied by expansion of Th17 cells, which to some measure coproduced TNF, GM-CSF, and IL-22, along with elevated IL-17A levels within joint synovial fluid. Importantly, neutralization of IL-17A mitigated arthritis in Nod2−/−SKG mice, indicating that Nod2-mediated protection occurs through suppression of the Th17 response. Nod2 deficiency did not alter regulatory T cell development or function. Instead, Nod2 deficiency resulted in an enhanced fundamental ability of SKG CD4+ T cells (from naive mice) to produce increased levels of IL-17 and to passively transfer arthritis to lymphopenic recipients on a single-cell level. These data reveal a previously unconsidered role for T cell–intrinsic Nod2 as an endogenous negative regulator of Th17 responses and arthritogenic T cells. Based on our findings, future studies aimed at understanding a negative regulatory function of Nod2 within autoreactive T cells could provide novel therapeutic strategies for treatment of patients with arthritis.

Molecular Signaling and Ocular Inflammation: An Update on the NOD-Like Receptors (NLRs)

Ellen J Lee1,2 and Holly L Rosenzweig1,2,3* 1Department of Ophthalmology, Oregon Health & Science University, Portland, OR, USA 2VA Medical Center, Portland, OR, USA 3Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA *Corresponding author: Holly L. Rosenzweig, PhD, VA Medical Center, 3710 SW US, Veterans Hospital Rd. Bldg 103, Room E-221A, Mail stop: VA RD E-mail: rosenzwh@ohsu.edu