Antigen-Specific Intraocular Cytokine Responses Distinguish Ocular Tuberculosis From Undifferentiated Uveitis in Tuberculosis-Immunoreactive Patients.

PURPOSE: To compare antigen-specific intraocular immune responses between different clinical phenotypes of tuberculin skin test (TST)-positive and TST-negative uveitis. DESIGN: Single center, retrospective cross-sectional study. METHODS: Patients requiring diagnostic or therapeutic vitrectomy for the management of intraocular inflammation were divided into 3 groups based on Standardization of Uveitis Nomenclature (SUN) classification criteria for tubercular uveitis. Group 1 included patients with ocular tuberculosis (OTB; n = 23) who were TST-positive patients, met the SUN criteria, and/or had a polymerase chain reaction (PCR)-positive test for TB. Group 2 included patients with uveitis of unknown origin (UNK; n = 24) who were undifferentiated TST-positive patients who had not met SUN criteria. Group 3 included non-TB uveitis patients (n = 24) who were TST-negative either with or without a well-defined non-TB diagnosis. Total vitreous cells were activated with Mycobacterium tuberculosis-specific Early Secreted Antigenic Target-6 (ESAT-6) or the retinal autoantigen, interphotoreceptor retinoid-binding protein peptide (pIRBP 1-20), stained for intracellular interferon gamma (IFNγ), tumor necrosis factor-alfa (TNFα), and interleukin 17 (IL-17), and analyzed by flow cytometry. Antigen-specific single and dual (polyfunctional) cytokine responses to ESAT-6 and IRBP were compared between the 3 groups. RESULTS: All cytokine responses to ESAT-6 were higher in the UNK group compared with the non-TB control subjects, while all except IL-17 were comparable between the OTB and non-TB groups. Polyfunctional responses-IFNγ/IL-17 (P = .002), TNFα/IL-17 (P = .02), and TNFα/IFNγ (P = .01)-were significantly greater for UNK than the OTB group. Polyfunctional cells also produced more cytokine per cell than respective monofunctional cells. IRBP cytokine responses were comparable between different groups and were not affected by the clinical phenotype or duration of disease. CONCLUSION: The intraocular polyfunctional cytokine response is stronger in undifferentiated TST-positive uveitis than in OTB patients, likely representing an exaggerated anti-TB immune response rather than active infection.

The Role of Inflammation in Diabetic Retinopathy.

Inflammation is central to pathogenic processes in diabetes mellitus and the metabolic syndrome and particularly implicates innate immunity in the development of complications. Inflammation is a primary event in Type 1 diabetes where infectious (viral) and/or autoimmune processes initiate disease; in contrast, chronic inflammation is typical in Type 2 diabetes and is considered a sequel to increasing insulin resistance and disturbed glucose metabolism. Diabetic retinopathy (DR) is perceived as a vascular and neurodegenerative disease which occurs after some years of poorly controlled diabetes. However, many of the clinical features of DR are late events and reflect the nature of the retinal architecture and its cellular composition. Retinal microvascular disease is, in fact, an early event pathogenetically, induced by low grade, persistent leukocyte activation which causes repeated episodes of capillary occlusion and, progressive, attritional retinal ischemia. The later, overt clinical signs of DR are a consequence of the retinal ischemia. Metabolic dysregulation involving both lipid and glucose metabolism may lead to leukocyte activation. On a molecular level, we have shown that macrophage-restricted protein tyrosine phosphatase 1B (PTP1B) is a key regulator of inflammation in the metabolic syndrome involving insulin resistance and it is possible that PTP1B dysregulation may underlie retinal microvascular disease. We have also shown that adherent CCR5+CD11b+ monocyte macrophages appear to be selectively involved in retinal microvascular occlusion. In this review, we discuss the relationship between early leukocyte activation and the later features of DR, common pathogenetic processes between diabetic microvascular disease and other vascular retinopathies, the mechanisms whereby leukocyte activation is induced in hyperglycemia and dyslipidemia, the signaling mechanisms involved in diabetic microvascular disease, and possible interventions which may prevent these retinopathies. We also address a possible role for adaptive immunity in DR. Although significant improvements in treatment of DR have been made with intravitreal anti-VEGF therapy, a sizeable proportion of patients, particularly with sight-threatening macular edema, fail to respond. Alternative therapies targeting inflammatory processes may offer an advantage.

Treatment With FoxP3+ Antigen-Experienced T Regulatory Cells Arrests Progressive Retinal Damage in a Spontaneous Model of Uveitis.

We specify the clinical features of a spontaneous experimental autoimmune uveitis (EAU) model, in which foreign hen-egg lysozyme (HEL) is expressed in the retina, controlled by the promoter for interphotoreceptor retinol binding protein (IRBP). We previously reported 100% P21 (post-partum day) IRBP:HEL single transgenic (sTg) mice, when crossed to transgenic T cell receptor mice (3A9) generating the double transgenic (dTg) genotype, develop EAU despite profound lymphopenia (thymic HEL-specific T cell deletion). In this work, we characterized the immune component of this model and found conventional dTg CD4+ T cells were less anergic than those from 3A9 controls. Furthermore, prior in vitro HEL-activation of 3A9 anergic T cells (Tan) rendered them uveitogenic upon adoptive transfer (Tx) to sTg mice, while antigen-experienced (AgX, dTg), but not naïve (3A9) T cells halted disease in P21 dTg mice. Flow cytometric analysis of the AgX cells elucidated the underlying pathology: FoxP3+CD25hiCD4+ T regulatory cells (Treg) comprised ∼18%, while FR4+CD73+FoxP3-CD25lo/-CD4+ Tan comprised ∼1.2% of total cells. Further Treg-enrichment (∼80%) of the AgX population indicated FoxP3+CD25hiCD4+ Treg played a key role in EAU-suppression while FoxP3-CD25lo/-CD4+ T cells did not. Here we present the novel concept of dual immunological tolerance where spontaneous EAU is due to escape from anergy with consequent failure of Treg induction and subsequent imbalance in the [Treg:Teffector] cell ratio. The reduced numbers of Tan, normally sustaining Treg to prevent autoimmunity, are the trigger for disease, while immune homeostasis can be restored by supplementation with AgX, but not naïve, antigen-specific Treg.

Activation of dendritic cells by crosslinked collagen hydrogels (artificial corneas) varies with their composition.

Activated T cells are known to promote fibrosis, a major complication limiting the range of polymeric hydrogels as artificial corneal implants. As T cells are activated by dendritic cells (DC), minimally activating hydrogels would be optimal. In this study, we evaluated the ability of a series of engineered (manufactured/fabricated) and natural collagen matrices to either activate DC or conversely induce DC apoptosis in vitro. Bone marrow DC were cultured on a series of singly and doubly crosslinked hydrogels (made from recombinant human collagen III [RHCIII] or collagen mimetic peptide [CMP]) or on natural collagen-containing matrices, MatrigelTM and de-cellularised mouse corneal stroma. DC surface expression of major histocompatibility complex Class II and CD86 as well as apoptosis markers were examined. Natural matrices induced low levels of DC activation and maintained a "tolerogenic" phenotype. The same applied to singly crosslinked CMP-PEG gels. RHCIII gels singly crosslinked using either N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide with the coinitiator N-hydroxy succinimide (EDC-NHS) or N-cyclohexyl-N-(2-morpholinoethyl)carbodiimide metho-p-toulenesulfonate with NHS (CMC-NHS) induced varying levels of DC activation. In contrast, however, RHCIII hydrogels incorporating an additional polymeric network of 2-methacryloyloxyethyl phosphorylcholine did not activate DC but instead induced DC apoptosis, a phenomenon observed in natural matrices. This correlated with increased DC expression of leukocyte-associated immunoglobulin-like receptor-1. Despite low immunogenic potential, viable tolerogenic DC migrated into and through both natural and manufactured RHCIII gels. These data show that the immunogenic potential of RHCIII gels varies with the nature and composition of the gel. Preclinical evaluation of hydrogel immunogenic/fibrogenic potential is recommended.

Polarized retinal pigment epithelium generates electrical signals that diminish with age and regulate retinal pathology.

The transepithelial potential difference (TEP) across the retinal pigment epithelial (RPE) is dependent on ionic pumps and tight junction "seals" between epithelial cells. RPE cells release neurotrophic growth factors such as pigment epithelial derived factor (PEDF), which is reduced in age-related macular degeneration (AMD). The mechanisms that control the secretion of PEDF from RPE cells are not well understood. Using the CCL2/CX3CR1 double knockout mouse model (DKO), which demonstrates RPE damage and retinal degeneration, we uncovered an interaction between PEDF and the TEP which is likely to play an important role in retinal ageing and in the pathogenesis of AMD. We found that: (a) the expression of ATP1B1 (the Na+ /K+ -ATPase ß1 subunit) was reduced significantly in RPE from aged mice, in patients with CNV (Choroidal Neovascularization) and in DKO mice; (b) the expression of PEDF also was decreased in aged persons and in DKO mice; (c) the TEP across RPE was reduced markedly in RPE cells from DKO mice and (d) an applied electric field (EF) of 50-100 mV/mm, used to mimic the natural TEP, increased the expression and secretion of PEDF in primary RPE cells. In conclusion, the TEP across the RPE depends on the expression of ATP1B1 and this regulates the secretion of PEDF by RPE cells and so may regulate the onset of retinal disease. Increasing the expression of PEDF using an applied EF to replenish a disease or age-reduced TEP may offer a new way of preventing or reversing retinal dysfunction.

Endogenous bioelectric currents promote differentiation of the mammalian lens.

The functional roles of bioelectrical signals (ES) created by the flow of specific ions at the mammalian lens equator are poorly understood. We detected that mature, denucleated lens fibers expressed high levels of the α1 and ß1 subunits of Na+ /K+ -ATPase (ATP1A1 and ATP1B1 of the sodium pump) and had a hyperpolarized membrane potential difference (Vmem ). In contrast, differentiating, nucleated lens fiber cells had little ATP1A1 and ATP1B1 and a depolarized Vmem . Mimicking the natural equatorial ES with an applied electrical field (EF) induced a striking reorientation of lens epithelial cells to lie perpendicular to the direction of the EF. An EF also promoted the expression of ß-crystallin, aquaporin-0 (AQP0) and the Beaded Filament Structural Protein 2 (BFSP2) in lens epithelial cells (LECs), all of which are hallmarks of differentiation. In addition, applied EF activated the AKT and CDC2 and inhibition of AKT reduced the activation of CDC2. Our results indicate that the endogenous bioelectrical signal at the lens equator promotes differentiation of LECs into denucleated lens fiber cells via depolarization of Vmem. Development of methods and devices of EF application or amplification in vivo may supply a novel treatment for lens diseases and even promote regeneration of a complete new lens following cataract surgery.

Mesenchymal stem cell therapy for retro-corneal membrane - A clinical challenge in full-thickness transplantation of biosynthetic corneal equivalents.

Artificial corneas (keratoprostheses) and biosynthetic collagen-based corneal equivalents are surgical implants designed to ease the global burden of corneal blindness. However, keratoprostheses in many cases fail due to development of fibrous retro-corneal membranes (RCM). Fibrous membranes which develop in the anterior chamber after prosthesis implantation do so on a matrix of fibrin. This study investigated fibrin deposition and RCM formation after full-thickness collagen-based hydrogel implants and compared them with syngeneic and allogeneic corneal grafts in mice. Fibrin cleared from the anterior chamber within 14 days in both allo- and syn-grafts but, persisted in hydrogel implants and developed into dense retro-corneal membrane (RCM) which were heavily infiltrated by activated myofibroblasts. In contrast, the number of CD11b+ macrophages infiltrating the initial deposition of fibrin in the anterior chamber (AC) after hydrogel implantation was markedly reduced compared to syn- and allo-grafts. Inoculation of mesenchymal stem cells prior to collagen gel implant promoted clearance of gel-associated fibrin from the anterior chamber. We propose that a failure of macrophage-mediated clearance of fibrin may be the cause of RCM formation after collagen-based hydrogel implants and that mesenchymal stem cell therapy promotes clearance of fibrin and prevents RCM formation. STATEMENT OF SIGNIFICANCE: The manuscript addresses the potential value of bone marrow-derived mesenchymal stem cell therapy for retro-corneal membrane (RCM) formation in full-thickness transplantation of biosynthetic corneal equivalents. This work reports the pathophysiological changes in the anterior chamber of the mouse eye following full-thickness recombinant human cross-linked collagen-based hydrogel implants in which persistent fibrin promotes the development of dense RCM. Furthermore, pre-treatment with mesenchymal stem cells reduces RCM formation and enhances corneal transparency.

Lipopolysaccharide-primed heterotolerant dendritic cells suppress experimental autoimmune uveoretinitis by multiple mechanisms.

Exposure of bone-marrow-derived dendritic cells (BMDC) to high-dose ultrapure lipopolysaccharide for 24 hr (LPS-primed BMDC) enhances their potency in preventing inter-photoreceptor retinoid binding protein: complete Freund's adjuvant-induced experimental autoimmune uveoretinitis (EAU). LPS-primed BMDC are refractory to further exposure to LPS (= endotoxin tolerance), evidenced here by decreased phosphorylation of TANK-binding kinase 1, interferon regulatory factor 3 (IRF3), c-Jun N-terminal kinase and p38 mitogen-activated protein kinase as well as impaired nuclear translocation of nuclear factor κB (NF-κB) and IRF3, resulting in reduced tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-12 and interferon-ß secretion. LPS-primed BMDC also show reduced surface expression of Toll-like receptor-4 and up-regulation of CD14, followed by increased apoptosis, mediated via nuclear factor of activated T cells (NFATc)-2 signalling. LPS-primed BMDC are not only homotolerant to LPS but are heterotolerant to alternative pathogen-associated molecular pattern ligands, such as mycobacterial protein extract (Mycobacterium tuberculosis). Specifically, while M. tuberculosis protein extract induces secretion of IL-1ß, TNF-α and IL-6 in unprimed BMDC, LPS-primed BMDC fail to secrete these cytokines in response to M. tuberculosis. We propose that LPS priming of BMDC, by exposure to high doses of LPS for 24 hr, stabilizes their tolerogenicity rather than promoting immunogenicity, and does so by multiple mechanisms, namely (i) generation of tolerogenic apoptotic BMDC through CD14:NFATc signalling; (ii) reduction of NF-κB and IRF3 signalling and downstream pro-inflammatory cytokine production; and (iii) blockade of inflammasome activation.

The Microbiota Determines Susceptibility to Experimental Autoimmune Uveoretinitis.

The microbiota is a crucial modulator of the immune system. Here, we evaluated how its absence or reduction modifies the inflammatory response in the murine model of experimental autoimmune uveoretinitis (EAU). We induced EAU in germ-free (GF) or conventionally housed (CV) mice and in CV mice treated with a combination of broad-spectrum antibiotics either from the day of EAU induction or from one week prior to induction of disease. The severity of the inflammation was assessed by fundus biomicroscopy or by histology, including immunohistology. The immunophenotyping of T cells in local and distant lymph nodes was performed by flow cytometry. We found that GF mice and mice where the microbiota was reduced one week before EAU induction were protected from severe autoimmune inflammation. GF mice had lower numbers of infiltrating macrophages and significantly less T cell infiltration in the retina than CV mice with EAU. GF mice also had reduced numbers of IFN-γ and IL-17-producing T cells and increased numbers of regulatory T cells in the eye-draining lymph nodes. These data suggest that the presence of microbiota during autoantigen recognition regulates the inflammatory response by influencing the adaptive immune response.

High-Risk Corneal Graft Rejection in the Setting of Previous Corneal Herpes Simplex Virus (HSV)-1 Infection.

PURPOSE: The "high-risk phenotype" of corneal graft recipients is considered to be related to preexisting vascularization such as that associated with herpes simplex virus-1 (HSV-1) keratitis (HSK). The purpose of this study was to investigate the immunologic mechanisms underlying accelerated corneal graft rejection using a mouse model of HSK. METHODS: Herpes simplex virus type 1 keratitis was induced in BALB/c mice. Syngeneic and allogeneic (C57BL/6 mice) corneal grafts were performed in mice with HSK at different times after infection. Some grafts were performed on HSV-infected CD4 T cell-deficient BALB/c mice. Clinical, histologic, immunologic, and virus detection studies were performed on samples of cornea, draining lymph node (LN), and trigeminal ganglion (TG) cells. RESULTS: Corneal grafts in mice with HSK rejected with higher frequency and more rapid tempo compared with grafts in uninfected mice. In corneas with HSK and vascularization at the time of grafting, both syngeneic and allogeneic corneal grafts failed with similar frequency and tempo. However, in the absence of preexisting inflammation and vascularization, syngeneic grafts were accepted when the grafts were performed at a late time point after HSV infection (42 days), whereas allografts were rejected at this time. In contrast, syngeneic grafts in nonvascularized HSV-infected recipients failed if they were performed within 10 days of HSV infection, an effect that was dependent on CD4 T cells, as demonstrated using CD4 deficient mice. Importantly, a variably sustained but strongly positive anti-HSV T-cell response was detected in allografted HSK recipients with a similar but lesser response in syngeneic hosts. CONCLUSIONS: A previous HSV-1 corneal infection predisposes donor grafts to a high risk of failure by both innate and adaptive immune mechanisms in which an anti-HSV CD4 T-cell response plays a prominent role.

Retinal Pigment Epithelial Cell Apoptosis is Influenced by a Combination of Macrophages and Soluble Mediators Present in Age-Related Macular Degeneration.

PURPOSE: Age-related macular degeneration (AMD) is one of the leading causes of blindness in the elderly population aged ≥60 years. Previous studies have shown that retinal pigment epithelial cell (RPE) degeneration is one of the early and crucial stages in AMD. It has been suggested that microglia and macrophages may be involved in the impairment of RPE, but how they and RPE are influenced by other factors present as AMD develops is unclear. Therefore the purpose of this study was to determine the role of macrophages in RPE degeneration in the presence of cytokines and oxidative stress likely to be present as AMD develops. METHODS: A co-culture model system was set up using bone marrow-derived macrophages and brain or retinal microglia cultured with RPE. Cytokines (IL-1ß, TNF-α, IFN-γ, and IL-6) and oxidized low-density lipoprotein were included in the culture at concentrations estimated to be likely during AMD, and apoptosis of RPE cells determined using flow cytometry to detect annexin V. RESULTS: Macrophages were shown capable of enhancing the apoptosis of RPE cells in a contact-dependent manner. IL-1ß, IFN-γ, IL-6, TNF-α, and oxLDL increased apoptosis; they increased RPE cell apoptosis directly, increased the susceptibility of RPE to subsequent apoptosis in the presence of microglia/macrophages, and increased the ability of microglia/macrophages to cause apoptosis. CONCLUSIONS: These findings indicate that microglia and macrophages are capable of enhancing the degeneration of RPE, which are crucial in AMD development. However this is dependent on the microenvironment present as AMD develops.

In-vivo evidence that high mobility group box 1 exerts deleterious effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model and Parkinson's disease which can be attenuated by glycyrrhizin.

High-mobility group box 1 (HMGB1) is a nuclear and cytosolic protein that is released during tissue damage from immune and non-immune cells - including microglia and neurons. HMGB1 can contribute to progression of numerous chronic inflammatory and autoimmune diseases which is mediated in part by interaction with the receptor for advanced glycation endproducts (RAGE). There is increasing evidence from in vitro studies that HMGB1 may link the two main pathophysiological components of Parkinson's disease (PD), i.e. progressive dopaminergic degeneration and chronic neuroinflammation which underlie the mechanistic basis of PD progression. Analysis of tissue and biofluid samples from PD patients, showed increased HMGB1 levels in human postmortem substantia nigra specimens as well as in the cerebrospinal fluid and serum of PD patients. In a mouse model of PD induced by sub-acute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), systemic administration of neutralizing antibodies to HMGB1 partly inhibited the dopaminergic cell death, and reduced the increase of RAGE and tumour necrosis factor-alpha. The small natural molecule glycyrrhizin, a component from liquorice root which can directly bind to HMGB1, both suppressed MPTP-induced HMGB1 and RAGE upregulation while reducing MPTP-induced dopaminergic cell death in a dose dependent manner. These results provide first in vivo evidence that HMGB1 serves as a powerful bridge between progressive dopaminergic neurodegeneration and chronic neuroinflammation in a model of PD, suggesting that HMGB1 is a suitable target for neuroprotective trials in PD.

Evidence for a role of adaptive immune response in the disease pathogenesis of the MPTP mouse model of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease and results from the loss of dopaminergic neurons of the nigrostriatal pathway. The pathogenesis of PD is poorly understood, but inflammatory processes have been implicated. Indeed increases in the number of major histocompatibility complex II (MHC II) reactive cells have long been recognised in the brains of PD patients at post-mortem. However whether cells expressing MHC II play an active role in PD pathogenesis has not been delineated. This was addressed utilising a transgenic mouse null for MHC II and the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In wild-type mice MHC II levels in the ventral midbrain were upregulated 1-2 days after MPTP treatment and MHC II was localized in both astrocytes and microglia. MHC II null mice showed significant reductions in MPTP-induced dopaminergic neuron loss and a significantly reduced invasion of astrocytes and microglia in MHC II null mice receiving MPTP compared with controls. In addition, MHC II null mice failed to show increases in interferon-γ or tumour necrosis factor-α in the brain after MPTP treatment, as was found in wild-type mice. However, interleukin-1ß was significantly increased in both wild-type and MHC II null mice. These data indicate that in addition to microglial cell/myeloid cell activation MHC Class II-mediated T cell activation is required for the full expression of pathology in this model of PD.

A key role for PTP1B in dendritic cell maturation, migration, and T cell activation.

Dendritic cells (DC) are the major antigen-presenting cells bridging innate and adaptive immunity, a function they perform by converting quiescent DC to active, mature DC with the capacity to activate naïve T cells. They do this by migrating from the tissues to the T cell area of the secondary lymphoid tissues. Here, we demonstrate that myeloid cell-specific genetic deletion of PTP1B (LysM PTP1B) leads to defects in lipopolysaccharide-driven bone marrow-derived DC (BMDC) activation associated with increased levels of phosphorylated Stat3. We show that myeloid cell-specific PTP1B deletion also causes decreased migratory capacity of epidermal DC, as well as reduced CCR7 expression and chemotaxis to CCL19 by BMDC. PTP1B deficiency in BMDC also impairs their migration in vivo. Further, immature LysM PTP1B BMDC display fewer podosomes, increased levels of phosphorylated Src at tyrosine 527, and loss of Src localization to podosome puncta. In co-culture with T cells, LysM PTP1B BMDC establish fewer and shorter contacts than control BMDC. Finally, LysM PTP1B BMDC fail to present antigen to T cells as efficiently as control BMDC. These data provide first evidence for a key regulatory role for PTP1B in mediating a central DC function of initiating adaptive immune responses in response to innate immune cell activation.

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.

High Chlamydia Burden Promotes Tumor Necrosis Factor-Dependent Reactive Arthritis in SKG Mice.

OBJECTIVE: Chlamydia trachomatis is a sexually transmitted obligate intracellular pathogen that causes inflammatory reactive arthritis, spondylitis, psoriasiform dermatitis, and conjunctivitis in some individuals after genital infection. The immunologic basis for this inflammatory response in susceptible hosts is poorly understood. As ZAP-70(W163C) -mutant BALB/c (SKG) mice are susceptible to spondylo-arthritis after systemic exposure to microbial ß-glucan, we undertook the present study to compare responses to infection with Chlamydia muridarum in SKG mice and BALB/c mice. METHODS: After genital or respiratory infection with C muridarum, conjunctivitis and arthritis were assessed clinically, and eye, skin, and joint specimens were analyzed histologically. Chlamydial major outer membrane protein antigen-specific responses were assessed in splenocytes. Treg cells were depleted from FoxP3-DTR BALB/c or SKG mice, and chlamydial DNA was quantified by polymerase chain reaction. RESULTS: Five weeks after vaginal infection with live C muridarum, arthritis, spondylitis, and psoriasiform dermatitis developed in female SKG mice, but not in BALB/c mice. Inflammatory bowel disease did not occur in mice of either strain. The severity of inflammatory disease was correlated with C muridarum inoculum size and vaginal burden postinoculation. Treatment with combination antibiotics starting 1 day postinoculation prevented disease. Chlamydial antigen was present in macrophages and spread from the infection site to lymphoid organs and peripheral tissue. In response to chlamydial antigen, production of interferon-γ and interleukin-17 was impaired in T cells from SKG mice but tumor necrosis factor (TNF) responses were exaggerated, compared to findings in T cells from BALB/c mice. Unlike previous observations in arthritis triggered by ß-glucan, no autoantibodies developed. Accelerated disease triggered by depletion of Treg cells was TNF dependent. CONCLUSION: In the susceptible SKG strain, Chlamydia-induced reactive arthritis develops as a result of deficient intracellular pathogen control, with antigen-specific TNF production upon dissemination of antigen, and TNF-dependent inflammatory disease.

TLR9 and TLR7/8 activation induces formation of keratic precipitates and giant macrophages in the mouse cornea.

Macrophage adherence to the inner corneal surface and formation of MGCs in the stroma are common signs of chronic inflammation following corneal infection. To determine whether macrophage adherence (known clinically as KPs) and giant cell formation were specific to innate immune activation via particular TLR ligands, macrophage activation was examined in a murine model of TLR-mediated corneal inflammation. The corneal epithelium was debrided and highly purified TLR ligands were topically applied once to the cornea of TLR7(-/-), TLR9(-/-), Cx3cr1(gfp/+), CD11c(eYFP), and IL-4(-/-) mice. At 1 week post-treatment macrophage activation and phenotype was evaluated in the cornea. Treatment with TLR2, TLR3, TLR4, and TLR5 ligands caused an increase in the number of activated stromal macrophages in the central cornea at 1 week post-treatment. However, treatment with TLR9 ligand CpG-ODN and the TLR7/8 ligand R848/Resiquimod led to an accumulation of macrophages on the corneal endothelium and formation of multinucleated giant macrophages in the corneal stroma. We suggest that giant cell formation, which is a characteristic feature of granuloma formation in many tissues, may be a unique feature of TLR9- and TLR7/8-mediated macrophage activation.

Local targeting of the CD200-CD200R axis does not promote corneal graft survival.

Corneal graft rejection is primarily a CD4(+) T cell-mediated mechanism in which macrophages may play an important inflammatory role. CD200Fc fusion protein is an artificial agonist of CD200R1, a receptor expressed predominantly on myeloid cells, engagement of which is known to down-regulate macrophage function. We therefore wished to test whether CD200Fc could be used as a therapeutic agent to prolong corneal graft survival. The distribution of CD200R1 and CD200, its natural ligand, was examined by immunohistology in the cornea and conjunctiva of unoperated rats and rats that had received corneal allografts. Mouse CD200Fc was injected subconjunctivally into transplanted rats on six occasions from the day of surgery until day 10 after transplantation. Control groups received injections of mouse IgG or diluent PBS. Allo-transplants were also performed in CD200(-/-) and control mice. The ability of CD200Fc to bind rat macrophages in vitro and to inhibit nitric oxide production was tested. Mean day of rejection in CD200Fc, IgG and PBS-treated rats was 12, 10 and 9 respectively (p=0.24). Mean day of rejection in CD200(-/-) and wild type mice was 17.5 and 16.0 respectively (p=0.07). Mouse CD200Fc bound to rat macrophages in a dose-dependent manner, but was unable to inhibit nitric oxide production. The fact that treatment with CD200Fc did not inhibit graft rejection and the failure of CD200 deficiency to affect graft survival suggests that local targeting of the CD200-CD200R axis to suppress macrophage activation is not a useful therapeutic strategy in corneal graft rejection.

Treatment of diffuse subretinal fibrosis uveitis with rituximab.

Diffuse subretinal fibrosis uveitis (DUS) syndrome is a rare form of granulomatous multifocal choroiditis (MFC) characterised by enlarging areas of subretinal fibrosis (SRF) which coalesce with subsequent macular involvement and visual loss. First described by Palestine, DUS carries a poor visual prognosis despite use of high-dose corticosteroids and systemic immunosuppression. We report two cases of bilateral DUS successfully treated with rituximab. We believe given the B-cell predominance in the underlying pathogenesis of the disease, rituximab should be considered first line in the management of this potentially devastating disease.

IL-33 attenuates the development of experimental autoimmune uveitis.

Interleukin-33 (IL-33) is associated with several important immune-mediated disorders. However, its role in uveitis, an important eye inflammatory disease, is unknown. Here, we investigated the function of IL-33 in the development of experimental autoimmune uveitis (EAU). IL-33 and IL-33 receptor (ST2) were expressed in murine retinal pigment epithelial (RPE) cells in culture, and IL-33 increased the expression of Il33 and Mcp1 mRNA in RPE cells. In situ, IL-33 was highly expressed in the inner nuclear cells of the retina of naïve mice, and its expression was elevated in EAU mice. ST2-deficient mice developed exacerbated EAU compared with WT mice, and administration of IL-33 to WT mice significantly reduced EAU severity. The attenuated EAU in IL-33-treated mice was accompanied by decreased frequency of IFN-γ+ and IL-17(+) CD4+ T cells and reduced IFN-γ and IL-17 production but with increased frequency of IL-5(+) and IL-4(+) CD4 T cells and IL-5 production in the draining lymph node and spleen. Macrophages from the IL-33-treated mice show a significantly higher polarization toward an alternatively activated macrophage phenotype. Our results therefore demonstrate that the endogenous IL-33/ST2 pathway plays an important role in EAU, and suggest that IL-33 represents a potential option for treatment of uveitis.