Microbiological screening of corneas stored in organ culture medium at Lions Eye Bank of Western Australia from 2011 to 2022.

PURPOSE: The purpose of this study was to analyse the contamination rate of corneal samples stored in OCM at Lions Eye Bank of Western Australia over a 12-year period. METHODS: All OCM samples used to preserve corneas from 2011 to 2022 (inclusive) underwent microbiological testing. Samples were collected into aerobic and anaerobic culture bottles on day 3-5 of corneal preservation and 24 h after transfer to thinning medium. Samples were tested for 7 days using the BACTEC FX system. Corneas remained in quarantine until clearance was obtained. RESULTS: From 2011 to 2022, 3009 corneas were retrieved and 2756 corneas were stored in OCM. Thirty one (1.1%) positive samples were reported, with 20 growths of bacterial origin and 11 fungal. Microbial contamination was mostly identified on day 1 of culture (77.5%). Donors of contaminated samples had a mean age of 55 years, with 17 male and 14 female donors. The highest incidence of contamination came from donors whose cause of death was cancer. Death to enucleation times of contaminated samples ranged from 3.5 to 25.5 h (mean = 13.5 ± 7.3) and death to preservation time ranged from 4.1 to 27.5 h (mean = 14.8 ± 7.2). These did not significantly differ from the average time from death to enucleation (mean = 13.9 ± 3) and death to preservation (mean = 16.3 ± 4.2) of non-contaminated samples. CONCLUSION: Microbiological screening of corneas stored in OCM at LEBWA showed a very low rate of positive cultures with no predictive donor characteristics.

Keeping an 'eye' on ocular GVHD.

Ocular graft versus host disease (GVHD) is a common manifestation in patients undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT). Ocular GVHD affects approximately 10% of patients with acute GVHD and more than 50% of patients with chronic GVHD. Symptoms of dry eye disease are one of the clinical hallmarks of ocular GVHD, and inflammatory changes to the ocular surface, cornea, conjunctiva, eyelids and lacrimal glands have been observed. Less commonly, the posterior segment of the eye is involved in the form of microvascular retinopathy, scleritis or intraretinal and vitreous haemorrhage. Although ocular GVHD does not usually result in permanent visual loss, it often impairs the patient's quality of life and activities of daily living. Regular and more consistent ocular assessment of allo-HSCT patients, including screening prior to transplantation will allow for the earlier detection and treatment of ocular complications associated with GVHD and potentially prevent more severe outcomes. The implementation of additional screening including corneal endothelial cell density assessment and non-invasive analysis of tear biomarkers may be valuable additions to current clinical testing and assist in better detection and clinical intervention in patients with GVHD. This review describes the clinical features, diagnostic criteria and clinical scoring of ocular GVHD, as well as current treatment strategies and potential ophthalmic screening tools for common ocular complications. Further, we describe the clinical and histopathological features of ocular GVHD in preclinical mouse models.

Effects of age on retinal macrophage responses to acute elevation of intraocular pressure.

There is accumulating evidence that aging shifts the central nervous system milieu towards a proinflammatory state, with increased reactivity of microglia in the aging eye and brain having been implicated in the development of age-related neurodegenerative conditions. Indeed, alterations to microglial morphology and function have been recognized as a part of normal aging. Here, we sought to assess the effects of age on the retinal microglial and macrophage response to acute intraocular pressure (IOP) elevation. Further, we performed experiments whereby bone marrow from young or middle-aged mice was used to reconstitute the bone marrow of whole-body irradiated 12 month old mice. Bone marrow chimeric mice then underwent cannulation and IOP elevation 8 weeks after whole-body irradiation and bone marrow transplantation in order to determine whether the age of bone marrow alters the macrophage response to retinal injury. Our data show retinal macrophage reactivity and microglial morphological changes were enhanced in older mice when compared to younger mice in response to injury. When IOP elevation was performed after whole-body irradiation and bone marrow rescue, we noted subretinal macrophage accumulation and glial reactivity was reduced compared to non-irradiated mice that had also undergone IOP elevation. This effect was evident in both groups of chimeric mice that had received either young or middle-aged bone marrow, suggesting irradiation itself may alter the macrophage and glial response to injury rather than the age of bone marrow.

Systemic exposure to CpG-ODN elicits low-grade inflammation in the retina.

Previous studies have reported that topical exposure to the toll-like receptor (TLR) 9 ligand CpG-ODN causes widespread ocular inflammation, including retinal microglial activation and posterior segment inflammation. Here we sought to determine the effects of systemic exposure to CpG-ODN in the retina and whether this inflammatory response was altered with Cx3cr1 deficiency or hyperglycemia. Male non-diabetic Cx3cr1+/gfp and Cx3cr1gfp/gfp littermates (normoglycemic controls) and Cx3cr1+/gfpIns2Akitaand Cx3cr1gfp/gfpIns2Akita diabetic mice were injected intraperitoneally with 40 µg CpG-ODN. Immunofluorescence staining was performed 1 week later to assess the expression of MHC Class II and glial fibrillary acidic protein (GFAP), as well as to identify morphological changes to microglia and changes in retinal macrophage cell density. Systemic exposure to CpG-ODN induced the upregulated expression of both GFAP on retinal Müller cells and MHC Class II on the retinal vasculature. Additionally, there was an increased accumulation of macrophages in the subretinal space 1 week after exposure to systemic CpG-ODN as well as characteristic morphological changes to microglia indicative of an activated phenotype. These preliminary studies demonstrate that low-grade inflammatory changes were not enhanced in Cx3cr1-deficient or diabetic mice, indicating that the inflammatory response to systemic CpG-ODN in the retina is unaltered in the context of Cx3cr1 deficiency or prolonged hyperglycemia.

Ocular antigen does not cause disease unless presented in the context of inflammation.

Ocular antigens are sequestered behind the blood-retina barrier and the ocular environment protects ocular tissues from autoimmune attack. The signals required to activate autoreactive T cells and allow them to cause disease in the eye remain in part unclear. In particular, the consequences of peripheral presentation of ocular antigens are not fully understood. We examined peripheral expression and presentation of ocular neo-self-antigen in transgenic mice expressing hen egg lysozyme (HEL) under a retina-specific promoter. High levels of HEL were expressed in the eye compared to low expression throughout the lymphoid system. Adoptively transferred naïve HEL-specific CD4+ T cells proliferated in the eye draining lymph nodes, but did not induce uveitis. By contrast, systemic infection with a murine cytomegalovirus (MCMV) engineered to express HEL induced extensive proliferation of transferred naïve CD4+ T cells, and significant uveoretinitis. In this model, wild-type MCMV, lacking HEL, did not induce overt uveitis, suggesting that disease is mediated by antigen-specific peripherally activated CD4+ T cells that infiltrate the retina. Our results demonstrate that retinal antigen is presented to T cells in the periphery under physiological conditions. However, when the same antigen is presented during viral infection, antigen-specific T cells access the retina and autoimmune uveitis ensues.

Chronic ocular hypertension induced by circumlimbal suture in rats.

PURPOSE: To induce chronic intraocular pressure (IOP) elevation in rat eyes by circumlimbal suture. METHODS: Anesthetized (isoflurane) Long-Evans rats underwent unilateral circumlimbal suture implantation while the fellow eyes served as untreated controls (n = 15). A sham group (n = 8) received the same procedure except that the suture was loosely tied. Intraocular pressure, electroretinography (ERG), and optical coherence tomography (OCT) were monitored for 15 weeks, after which retinal histology and immunofluorescence staining for glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule-1 (Iba-1) were undertaken. RESULTS: Both IOP and ERG remained unaltered in the sham and all control eyes over 15 weeks. In the ocular hypertensive eye, IOP spiked from 17 ± 1 to 58 ± 3 mm Hg immediately after suture application, recovering to 32 ± 2 mm Hg by 24 hours, and remained elevated by 7 to 10 mm Hg above baseline for 15 weeks. At week 2, there was a small reduction of ERG components involving the photoreceptor a-wave, bipolar cell b-wave, and ganglion cell-mediated scotopic threshold response (pSTR). The reduction in a- and b-wave remained stable, while the pSTR became more affected from week 8 onward (P < 0.05). By week 12, there was progressive retinal nerve fiber layer (RNFL) thinning. At week 15, GFAP expression was upregulated in inner retina and on Müller cells. The ganglion cell dysfunction was associated with RNFL thinning and cell loss in the ganglion cell layer. CONCLUSIONS: Circumlimbal suture provides a simple and cost-effective way to induce mild chronic ocular hypertension in rat eyes. This model produces preferential ganglion cell dysfunction and RNFL reduction.

In vivo multi-modal imaging of experimental autoimmune uveoretinitis in transgenic reporter mice reveals the dynamic nature of inflammatory changes during disease progression.

BACKGROUND: Experimental autoimmune uveoretinitis (EAU) is a widely used experimental animal model of human endogenous posterior uveoretinitis. In the present study, we performed in vivo imaging of the retina in transgenic reporter mice to investigate dynamic changes in exogenous inflammatory cells and endogenous immune cells during the disease process. METHODS: Transgenic mice (C57Bl/6 J Cx 3 cr1 (GFP/+) , C57Bl/6 N CD11c-eYFP, and C57Bl/6 J LysM-eGFP) were used to visualize the dynamic changes of myeloid-derived cells, putative dendritic cells and neutrophils during EAU. Transgenic mice were monitored with multi-modal fundus imaging camera over five time points following disease induction with the retinal auto-antigen, interphotoreceptor retinoid binding protein (IRBP1-20). Disease severity was quantified with both clinical and histopathological grading. RESULTS: In the normal C57Bl/6 J Cx 3 cr1 (GFP/+) mouse Cx3cr1-expressing microglia were evenly distributed in the retina. In C57Bl/6 N CD11c-eYFP mice clusters of CD11c-expressing cells were noted in the retina and in C57Bl/6 J LysM-eGFP mice very low numbers of LysM-expressing neutrophils were observed in the fundus. Following immunization with IRBP1-20, fundus examination revealed accumulations of Cx3cr1-GFP(+) myeloid cells, CD11c-eYFP(+) cells and LysM-eGFP(+) myelomonocytic cells around the optic nerve head and along retinal vessels as early as day 14 post-immunization. CD11c-eYFP(+) cells appear to resolve marginally earlier (day 21 post-immunization) than Cx3cr1-GFP(+) and LysM-eGFP(+) cells. The clinical grading of EAU in transgenic mice correlated closely with histopathological grading. CONCLUSIONS: These results illustrate that in vivo fundus imaging of transgenic reporter mice allows direct visualization of various exogenously and endogenously derived leukocyte types during EAU progression. This approach acts as a valuable adjunct to other methods of studying the clinical course of EAU.

Forced exercise protects the aged optic nerve against intraocular pressure injury.

We have previously shown that the optic nerve of mice becomes increasingly vulnerable to injury with advancing age. Here, we investigated whether regular exercise can modify this age-related vulnerability and improve optic nerve recovery after injury. Aged (12-month-old) C57BL/6J mice were exercised by swimming for 60 min/d, 5 d/wk for 6 weeks. After 5 weeks, injury to the optic nerve was induced by short-term elevation of intraocular pressure. Retinal function was recorded using the electroretinogram and the cellular and biochemical changes induced by injury were assessed using immunohistochemistry and quantitative polymerase chain reaction. We found that exercise almost completely reversed age-related vulnerability of the optic nerve to injury such that exercised aged mice had a similar functional response to injury as non-exercised young (3-month-old) mice. Exercise also abrogated injury-induced astrocytic gliosis and macrophage activation in the aged retina. These data suggest that the known benefits of exercise also extend to the visual system and support further investigation of physical activity as a means of protecting against injury, dysfunction, and degeneration in the aging eye.

Rd8 mutation in the Crb1 gene of CD11c-eYFP transgenic reporter mice results in abnormal numbers of CD11c-positive cells in the retina.

There has been considerable debate about whether dendritic cells (DCs), which are potent antigen-presenting cells pivotal to adaptive immune responses, are present in CNS parenchyma. In studies aimed at answering this issue, we discovered that while the neural retina of young naive transgenic C57BL/6 CD11c-eYFP reporter mice contained more than 800 CD11c-positive cells/retina, these cells were virtually absent in C57BL/6 CD11c-DTR/GFP mice. Clinical fundus examination, confocal imaging of retinal whole mounts, and sections revealed colocalization of CD11c-positive cells with classic mild to severe retinal dystrophic lesions. Immunophenotypic analysis revealed that CD11c-positive cells in the neural retina of these mice had the characteristic profile of activated microglia and not DCs. Genotypic analysis confirmed that the cause of the retinal dystrophic lesions in CD11c-eYFP transgenic mice was the occurrence of the Crb1(rd8) mutation, which affects all mice of the C57BL/6N strain but not the C57BL/6J strain. Comparison of 2 different types of CD11c reporter transgenic mice revealed that a mutation in the Crb1 gene leads to retinal degeneration resulting in the activation of large numbers of local microglia that could be readily mistaken for CD11c-positive putative DCs.

The effects of CX3CR1 deficiency and irradiation on the homing of monocyte-derived cell populations in the mouse eye.

This study examined whether CX3CR1 deficiency altered monocytic cell replenishment dynamics in ocular tissues in the context of radiation chimeras. Long-term effects of irradiation and effects of sublethal irradiation on ocular macrophages were also assessed. Bone marrow from BALB/c Cx 3 cr1 (+/gfp) or Cx 3 cr1 (gfp/gfp) mice was used to reconstitute full body irradiated WT mice and donor cell densities in the uveal tract were compared at 4 and 8 weeks post-transplantation. BALB/c and C57BL/6J chimeric mice were examined at 6 months of age to determine strain-related differences in microglial replenishment and radiation sensitivity. A separate cohort of mice were sublethally irradiated (5.5 Gy) and retinal tissue assessed 8 and 12 weeks later. CX3CR1 deficiency altered the early replenishment of monocytes in the posterior iris but not in the iris stroma, choroid or retina. In six month old chimeric mice, there were significantly higher GFP(+) cell densities in the uveal tract when compared to non-irradiated 8-12 week old Cx 3 cr1 (+/gfp) mice. Additionally, MHC Class II expression was upregulated on hyalocytes and GFP(+) cells in the peripheral retina and the repopulation of microglia appeared to be more rapid in C57BL/6J mice compared to BALB/c mice. Transient expression of MHC Class II was observed on retinal vasculature in sublethally irradiated mice. These data indicate CX3CR1-deficiency only slightly alters monocyte-derived cell replenishment in the murine uveal tract. Lethal irradiation leads to long-term increase in monocytic cell density in the uveal tract and retinal microglial activation, possibly as a sequelae to local irradiation induced injury. Microglial replenishment in this model appears to be strain dependent.

Effect of anterior chamber cannulation and acute IOP elevation on retinal macrophages in the adult mouse.

PURPOSE: To assess the retinal macrophage response to cannulation of the anterior chamber (AC) and acute elevation of intraocular pressure (IOP) in adult mice. METHODS: Eyes from 12-month-old C57BL/6J WT mice were subject to IOP increase (50 mm Hg for 30 minutes) by direct cannulation of the AC. Fellow eyes were either cannulated without pressure increase or left untreated. Electroretinography was carried out prior to IOP elevation and 1 week later. Immunofluorescence staining was performed on frozen sections and retinal wholemounts 1 week after sham and IOP elevation. Eyes were assessed by epifluorescence and confocal microscopy and the density of vitreal hyalocytes and subretinal macrophages was calculated. RESULTS: The density of hyalocytes and subretinal macrophages was significantly increased 1 week after IOP elevation and AC cannulation compared with naïve eyes. CD68 and MHC Class II expression was upregulated in both cannulated eyes and eyes with elevated IOP. Electroretinographic signals derived from retinal ganglion cells were significantly reduced in response to acute IOP elevation, but not in response to cannulation alone. CONCLUSIONS: Cannulation of the AC causes an increase in hyalocyte density, microglial activation, and accumulation of macrophages in the subretinal space. These macrophage changes are similar to those observed in eyes subject to IOP elevation. Additional IOP elevation led to significant Müller cell activation, which was not evident after cannulation alone. These data highlight the importance of using appropriate controls in models of acute retinal injury.

The effects of age and Cx3cr1 deficiency on retinal microglia in the Ins2(Akita) diabetic mouse.

PURPOSE: Diabetic retinopathy (DR) is a major cause of visual impairment in developed countries. While DR has been described classically as a microvascular disease, recent evidence suggests that changes to retinal microglia are an early feature of retinopathy. In our study, we assessed changes in microglial distribution and morphology in vivo and ex vivo in a mouse model of non-proliferative DR, and further examined effects of age and the absence of the functional chemokine receptor Cx(3)cr1 on the progression of these changes. METHODS: To isolate the effects of the three variables: diabetic status, age, and role of Cx(3)cr1, the Ins2(Akita) mouse was crossed with Cx(3)cr1-eGFP reporter mice. Eyes were assessed clinically in vivo at 10, 20, 30, and 46 weeks of age, and the retinal structure and arrangement of GFP(+) microglia was examined ex vivo using whole mount immunofluorescence staining and confocal microscopy. RESULTS: clinical examination of the fundus, vasculature, or GFP(+) microglial distribution did not reveal any macroscopic changes related to diabetic status: however, ex vivo microscopic analysis revealed alterations in microglial network organization, and evidence of cell shape changes regarded classically as signs of activation, in Ins2(Akita) mice from 10 weeks of age. These changes were exacerbated in older diabetic mice whose microglia lacked Cx(3)cr1 (Ins2(Akita) Cx(3)cr1(gfp/gfp) mice). Diabetic status and Cx(3)cr1 deficiency led to accumulations of Iba-1(+) hyalocytes (vitreal macrophages) and subretinal macrophages. CONCLUSIONS: These data showed that changes to murine retinal microglia occur in response to systemic diabetic status in the absence of overt retinopathy and inflammation. These changes are exaggerated in mice lacking Cx(3)cr1, suggesting fractalkine- Cx(3)cr1 interactions may have a role in early neuronal changes in preproliferative DR.

Changes in murine hyalocytes are valuable early indicators of ocular disease.

PURPOSE: The distribution, density, and phenotype of hyalocytes or vitreous macrophages in mouse eyes was examined during normal aging and in models of background diabetic retinopathy, retinal vascular proliferation, and exposure to TLR4 and TLR9 ligands. METHODS: The phenotype and density of hyalocytes were investigated in retinal and ciliary body wholemounts of normal wild-type (WT; C57BL/6) mice at 7, 17, and 120 weeks of age, Ins2(Akita) mice, transgenic Kimba mice (VEGF-induced retinal neovascularization), and WT mice 24 hours after single intraperitoneal injection with lipopolysaccharide (LPS) or 1 week after three identical doses administered 2 weeks apart. Another group of mice each received a single topical drop of 20 µg CpG-oligodeoxynucleotides (CpG-ODN) to the abraded corneal surface and were euthanized 1 week later. RESULTS: The data revealed an approximately fivefold increase in the density of preretinal hyalocytes in 120-week-old mice. Some hyalocytes in older eyes contained phagocytosed melanin. Hyalocyte density was doubled in Ins2(Akita) mice after only 3 to 4 weeks of hyperglycemia. Kimba mice had an eightfold increase in the density of hyalocytes, and many displayed signs of activation. WT mice exposed to single or multiple systemic doses of LPS showed a twofold to threefold increase in hyalocytes. Topical CpG-ODN treatment led to a very marked (sevenfold) increase in preretinal hyalocyte density. CONCLUSIONS: The present study demonstrated that murine hyalocytes were responsive to aging, hyperglycemia, locally produced VEGF, and both systemic and ocular-derived TLR ligands. Thus hyalocytes or vitreous macrophages may be a valuable and previously unrecognized sensitive indicator of pathologic changes in the eye.

Neutralization of IL-17 ameliorates uveitis but damages photoreceptors in a murine model of spondyloarthritis.

INTRODUCTION: Uveitis, or intraocular inflammatory disease, is a frequent extra-articular manifestation of several forms of arthritis. Despite the frequent co-occurrence of uveitis and arthritis, little is understood of the eye's predisposition to this disease. We recently described a previously unreported uveitis in a murine model of spondyloarthropathy triggered by autoimmunity to aggrecan, a prominent proteoglycan (PG) macromolecule in cartilage. In contrast to the joint and spine, wherein interferon-gamma (IFNγ) deficiency reduced disease, IFNγ deficiency worsened uveitis. Given the regulatory role of IFNγ on the Th17 response and the current focus of anti-interleukin-17 therapeutics in patients with uveitis and spondyloarthritis, we sought to determine the extent to which interleukin (IL)-17 mediates uveitis in the absence of IFNγ. METHODS: Antigen specific T cell cytokine production was measured in splenocyte cultures using multiplex-ELISA. Transgenic (Tg) mice expressing the T cell receptor (TCR) recognizing the dominant arthritogenic epitope in the G1 domain of PG (TCR-Tg), also lacking IFNγ, were immunized with PG. Mice were then systemically administered an anti-IL-17 neutralizing antibody. The onset and severity of peripheral arthritis was evaluated by clinical scoring criteria and histology. Uveitis was assessed using intravital videomicroscopy, which visualizes leukocyte trafficking within the vasculature and tissue of the iris, and by histology. RESULTS: TCR-Tg splenocytes stimulated in vitro with recombinant G1 peptide demonstrated exacerbated production of cytokines, such as macrophage inflammatory protein (MIP)-1α, MIP-1ß, IL-1ß, and most notably IL-17A as a consequence of IFNγ deficiency. In vivo, IL-17 inhibition prevented the component of PG-induced arthritis that occurs independently of IFNγ. Blockade of IL-17 ameliorated the ongoing leukocyte trafficking responses within the iris vasculature and tissue, which coincided with reduced infiltration of leukocytes within the anterior and posterior eye segments. However, the anti-IL-17 treatment resulted in unanticipated photoreceptor toxicity. CONCLUSIONS: These data support a protective, regulatory role for IFNγ in suppression of IL-17-mediated intraocular disease and to a lesser extent, joint disease. The unanticipated photoreceptor toxicity raises some caution regarding the use of anti-IL-17 therapeutics until the mechanism of this potential effect is determined.

Accumulation of murine subretinal macrophages: effects of age, pigmentation and CX3CR1.

Macrophages or activated microglia in the subretinal space are considered a hallmark of some retinal pathologies. We investigated the effects of age, pigmentation and CX(3)CR1 deficiency on the accumulation of macrophages/activated microglia in the outer retina of young and old Cx(3)cr1(gfp/gfp) (CX(3)CR1-deficient) or Cx(3)cr1(gfp/+) mice on either a pigmented (C57BL/6) or albino (BALB/c) background. Quantitative analysis of immunostained retinal-choroidal whole mounts revealed an increase in subretinal macrophage (SRMΦ) numbers in young Cx(3)cr1(gfp/gfp) mice compared with Cx(3)cr1(gfp/+) mice, however the increase was more marked in albino Cx(3)cr1(gfp/gfp) mice. In aged mice, large numbers of SRMΦ/activated microglia replete with autofluorescent debris were noted in both old pigmented Cx(3)cr1(gfp/gfp) and Cx(3)cr1(gfp/+) mice proving this accumulation was not CX(3)CR1-dependent. While CX(3)CR1 deficiency leads to an early onset of SRMΦ accumulation, our data reveal that this change occurs in both aged Cx(3)cr1(gfp/+) and Cx(3)cr1(gfp/gfp) pigmented mice in the absence of marked retinal degeneration and is likely a normal response to aging.

Interferon-γ regulates discordant mechanisms of uveitis versus joint and axial disease in a murine model resembling spondylarthritis.

OBJECTIVE: The spondylarthritides (such as ankylosing spondylitis) are multisystem inflammatory diseases that frequently result in uveitis. Despite the common co-occurrence of uveitis with arthritis, there has been no explanation for the susceptibility of the eye to inflammation. Using an innovative intravital videomicroscopic approach, we discovered the coexistence of uveitis with axial and peripheral joint inflammation in mice immunized with cartilage proteoglycan (PG). The aim of this study was to elucidate the characteristics of uveitis and test the impact of interferon-γ (IFNγ) deficiency on the eye versus the joint and spine. METHODS: Female T cell receptor (TCR)-transgenic mice or IFNγ-knockout mice crossed to TCR-transgenic mice were immunized with PG. Uveitis was assessed by intravital videomicroscopy and histology. The clinical and histopathologic severity of arthritis and spondylitis were evaluated. The bone remodeling process within the spine was assessed by whole-body near-infrared imaging. Immunoblotting and immunofluorescence staining were used to examine the expression of PG and ADAMTS-5 and to examine the cellular composition of eyes with uveitis. RESULTS: PG neoepitopes along with the aggrecanase ADAMTS-5 were present in the eye, as they were the joint. Anterior uveitis developed in response to PG immunization. The cellular infiltrate consisted mainly of neutrophils and eosinophils. Unexpectedly, IFNγ deficiency markedly exacerbated uveitis while ameliorating joint and spine disease, indicating divergent mechanisms that drive diseases in the eye versus the joints and spine. CONCLUSION: This study provides the first detailed description of a murine disease model in which uveitis coincides with arthritis and spondylitis. Our observations provide a great opportunity for understanding the pathogenesis of a relatively common but poorly understood disease.

Contrasting ocular effects of local versus systemic endotoxin.

PURPOSE. A marked cellular infiltrate has been observed when endotoxin (lipopolysaccharide [LPS]) is injected into the mouse eye, but systemically injected LPS does not produce a comparable effect. Several hypotheses were tested to reconcile this discordance. METHODS. BALB/c mice were injected intravitreally (ivt) or intraperitoneally (ip) with Escherichia coli LPS. Uveitis was assessed by traditional and intravital microscopy. Cytokine levels in the eye, plasma, or spleen were measured by single or multiplex ELISA assays. RESULTS. The eye's higher sensitivity was confirmed to local LPS exposure, as 250 ng ivt LPS produced a brisk leukocytic infiltrate whereas ip injection of 100 µg LPS did not. The hypothesis was tested that the lack of a cellular infiltrate after ip LPS is explained by less induction of cytokines in the eye, but surprisingly, ip LPS resulted in comparable cytokine levels to ivt LPS. The hypothesis was disproved that the eye's sensitivity to local LPS is due to lack of expression of intracellular inhibitors of LPS such as A20, IRAK-M, or SARM. Finally, the hypothesis that systemic LPS inhibits diapedesis was tested by injection of LPS ip and ivt simultaneously, a strategy that did not significantly reduce leukocyte rolling or sticking in iris vessels but blocked the cellular infiltrate normally seen with ivt LPS. CONCLUSIONS. Systemic and local LPS exposures produce discordant effects within the murine eye. The hypothesis that systemic LPS desensitizes leukocytes to the stimuli responsible for transmigration offers a plausible explanation for this discordance.

The monocyte chemokine receptor CX3CR1 does not play a significant role in the pathogenesis of experimental autoimmune uveoretinitis.

PURPOSE: To examine the role of the monocyte chemokine receptor CX(3)CR1 in experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in naive WT, Cx(3)cr1(gfp/+), and Cx(3)cr1(gfp/gfp) C57Bl/6 mice or chimeric mice. Ocular disease severity was graded by histologic analysis of resin sections. In addition, immunohistochemistry and confocal microscopy were performed on retinal whole mounts to characterize the monocytic infiltrate and changes in retinal microglia. To determine the relative roles of resident and blood-borne monocyte-derived cells in the active phase of uveoretinitis, EAU was induced 4 weeks after transplantation in chimeric mice (Cx(3)cr1(gfp/gfp)→WT and Cx(3)cr1(gfp/+)→WT), and analysis was performed at days 14, 16, 21, and 28 after immunization. RESULTS: After EAU induction, disease scores were not significantly different in WT, Cx(3)cr1(gfp/+), and Cx(3)cr1(gfp/gfp) mice. Chimeric studies revealed both donor- and host-derived monocyte-derived cells in the inner retinal layers during early EAU; however, it was donor monocytic cells that infiltrated the photoreceptors, the site of the target antigen. The absence of CX(3)CR1 did not impede the ability of monocyte-derived cells from Cx(3)cr1(gfp/gfp) donor mice to infiltrate during the peak of EAU. CONCLUSIONS: The lack of CX(3)CR1 on monocyte-derived cells does not significantly influence the onset or severity of EAU. In addition, chimeric studies revealed that it is primarily blood-derived monocytes that mediate photoreceptor damage in the effector phase of EAU, and this process is not CX(3)CR1 dependent.

Differential turnover rates of monocyte-derived cells in varied ocular tissue microenvironments.

Monocytes of bone marrow (BM) origin are circulating precursors that replenish dendritic cells and macrophage populations in peripheral tissues during homeostasis. The eye provides a unique range of varying tissue microenvironments in which to compare the different turnover rates of monocyte-derived cells. This was investigated in the present study using radiation chimeras, whereby BM from Cx3cr1(+/gfp) mice was used to rescue myeloablated wild-type (WT) BALB/c mice (conventional chimeras). The use of Cx3cr1(+/gfp) mice as BM donors allowed the clear visualization of newly recruited monocyte-derived cells. Following BM reconstitution, mice were killed at 2, 4, 6, and 8 weeks, and wholemount ocular tissues were processed for immunohistochemistry and confocal microscopy. "Reverse" chimeras (WT into Cx3cr1(+/gfp)) were also created to act as a further method of cross-referencing cell turnover rates. In conventional chimeras, Cx3cr1(+/gfp) cells began repopulating the uveal tract (iris, ciliary body, choroid) 2 weeks post-transplantation with close to complete replenishment by 8 weeks. By contrast, the earliest recruitment of Cx3cr1(+/gfp) cells into the host retina occurred at 4 weeks. In reverse chimeras, a steady accumulation of host Cx3cr1(+/gfp) macrophages in the subretinal space of Cx3cr1(+/gfp) adult mice suggests that these cells arise from long-term resident microglia and not newly recruited WT donor cells. In summary, chimeric mouse models, in which lineage-specific cells carry a fluorescent reporter, have been used in the present study to visualize the turnover of monocyte-derived cells in different tissue compartments of the eye. These data provide valuable insights into differential monocyte turnover rates within a single complex organ.

Retinal microglia and uveal tract dendritic cells and macrophages are not CX3CR1 dependent in their recruitment and distribution in the young mouse eye.

PURPOSE: The chemokine receptor CX3CR1 is expressed by monocyte-derived dendritic cells (DCs) and macrophages. CX3CR1 mediates leukocyte migration and adhesion in homeostatic and inflammatory conditions. Mice lacking Cx3cr1 have altered distribution and function of DC subpopulations in some tissue microenvironments. The present study compares the distribution of monocyte-derived cells in the normal retina and uveal tract as a prelude to the investigation of the role of CX3CR1 in murine models of ocular disease. METHODS: Transgenic mice in which either one (Cx3cr1 gfp/+, heterozygous) or both (Cx3cr1 gfp/gfp, homozygous) copies of the Cx3cr1 gene have been replaced by the enhanced green fluorescent protein (eGFP) reporter gene were used to investigate the role of Cx3cr1 expression on macrophages and DCs in the normal uveal tract and retina. Chimeric mice were used to investigate turnover of these cells in the normal, uninflamed eye. RESULTS: Confocal analysis found no significant differences in the density, phenotype or morphology of eGFP+ cells between Cx3cr1 gfp/+ and Cx3cr1 gfp/+ mice in immunostained iris, ciliary body, or choroidal and retinal wholemounts. Flow cytometry also failed to detect any difference in the density or cell shape of eGFP+ cells between Cx3cr1 gfp/+ and Cx3cr1 gfp/+ mice. Chimeras revealed 73% turnover of monocyte-derived cells in the iris and 63% in the choroid by 6 weeks after transplantation. CONCLUSIONS: These data illustrate that homing or migration of DCs and macrophages to the uveal tract and retina in normal young mice is not Cx3cr1 dependent and provide a solid foundation for future studies of monocyte-derived cells and the role of Cx3cr1 in models of ocular disease.