Jared E. Knickelbein

Modulation of Immune Responses by Extracellular Vesicles From Retinal Pigment Epithelium.

Purpose Extracellular vesicles (EV), such as exosomes, are important mediators of intercellular communication and have been implicated in modulation of the immune system. We investigated if EV released from retinal pigment epithelium (RPE) modulate immune responses in vitro. Methods Extracellular vesicles were isolated from ARPE-19 cultures stimulated or not with the inflammatory cytokines IL-1β, IFN-γ, and TNF-α. Isolated EV were characterized by nanoparticle flow and Western blot analyses. Retinal pigment epithelium–derived EV were cultured with human peripheral blood mononuclear cells, which were assayed for T-cell proliferation by 3H-thymidine incorporation. Retinal pigment epithelium–derived EV were also independently cultured with enriched lymphocytes or monocytes. Cell phenotype and cell death were evaluated by flow cytometric analysis. Cytokine levels were assayed in culture supernatants by multiplex bead analysis. Results The concentration of ARPE-derived EV from cytokine-stimulated cultures was slightly higher than from nonstimulated cultures. The size of EV was approximately 100 nm in both groups. Extracellular vesicles from both nonstimulated and cytokine-stimulated ARPE-19 significantly inhibited T-cell proliferation without affecting T-cell viability. Culture of EV from nonstimulated ARPE-19 with undifferentiated human monocytes induced an immunoregulatory phenotype with a significantly higher percentage of CD14++CD16+ monocytes and upregulation of TGF-β1. Culture of EV from cytokine-stimulated ARPE-19 cells with human monocytes induced upregulation of several proinflammatory cytokines and monocyte death. Conclusions Retinal pigment epithelium cells constitutively secrete EV in the size range of exosomes, with increased release from RPE cells stimulated with inflammatory cytokines. Extracellular vesicles from both nonstimulated and cytokine-stimulated RPE inhibited T-cell stimulation. Extracellular vesicles from nonstimulated ARPE-19 cells promoted an immunoregulatory CD14++CD16+ phenotype in human monocytes, while EV from cytokine-stimulated ARPE-19 cells caused human monocyte death. These findings suggest that RPE cells use EV to induce a downregulatory immune environment under homeostatic conditions. In an inflammatory milieu, RPE-derived EV may mitigate a potentially harmful inflammatory response through killing of monocytes.

Inflammatory Mechanisms of Age-related Macular Degeneration.

Late age-related macular degeneration (AMD), specifically central geographic atrophy (GA) and choroidal neovascularization (CVN),1 is the leading cause of irreversible vision loss in the elderly in the developed world.2 AMD is categorized as either i) non-neovascular, or dry, when CNV is not present, or ii) neovascular, or wet, when CNV is present. Increasing age is the most significant risk factor for AMD development.3 In the United States, AMD is more common in Caucasians, and smoking is strongly associated. The hallmark of AMD clinically is the presence of drusen situated under the retinal pigment epithelium (RPE). Drusen size is categorized as small (≤62μm), medium (63–124μm), or large (≥125μm), with presence of large drusen being a significant predictor of progression to late AMD and associated vision loss.4 The Age-Related Eye Disease Studies (AREDS and AREDS2) have shown that high-dose antioxidant and zinc supplementation delay the progression of AMD in moderate- and high-risk patients.5, 6 Anti-vascular endothelial growth factor (VEGF) treatment is the mainstay of therapy for wet AMD.7 Currently, there is no treatment for GA. While AMD pathogenesis is undoubtedly multifactorial, including the effects of aging and oxidative stress as well as genetic and environmental factors, significant evidence has emerged implicating inflammation and the immune system. The major role of the immune system is to identify and respond to physiologic insults, such as infection, malignancy, and tissue damage. Often this takes the form of robust inflammatory responses, such as those seen in various forms of uveitis, despite the potent down-regulatory immune environment within the eye.8 In AMD, immune dysregulation in the form of overt intraocular inflammation is not clinically apparent. It has been proposed that the role of inflammation is not always negative. The concept of parainflammation has been suggested to describe inflammatory responses to tissue stress that are intermediate between basal and robust inflammatory states and function in a reparative manner.9, 10 As AMD is not accompanied by an intense inflammatory reaction, it is possible that dysregulation of reparative parainflammatory mechanisms, in the context of the aging eye, lead to a low grade chronic inflammatory response and subsequent AMD pathology. This review will focus on the potential inflammatory mechanisms of AMD pathogenesis based on evidence from human studies.

Fenofibrate and Diabetic Retinopathy

Diabetic retinopathy, a common and sight-threatening microvascular complication of diabetes mellitus, is a leading cause of blindness among working-aged adults. Medical therapies including intensive control of hyperglycemia and hypertension have been shown to reduce the incidence and progression of diabetic retinopathy. The association of dyslipidemia and treatment with statins with diabetic retinopathy is inconsistent in epidemiologic studies. However, two recent randomized clinical trials have demonstrated beneficial effects of systemic fenofibrate therapy in reducing the progression of diabetic retinopathy independently of serum lipid levels. These findings suggest that fenofibrate may be an effective strategy for reducing the progression of diabetic retinopathy, thus reducing the large and growing public health burden of treating the sight-threatening complications of diabetic retinopathy.

Clinical trials in noninfectious uveitis.

The treatment of noninfectious uveitis continues to remain a challenge for many ophthalmologists. Historically, clinical trials in uveitis have been sparse, and thus, most treatment decisions have largely been based on clinical experience and consensus guidelines. The current treatment paradigm favors initiation then tapering of corticosteroids with addition of steroid-sparing immunosuppressive agents for persistence or recurrence of disease. Unfortunately, in spite of a multitude of highly unfavorable systemic effects, corticosteroids are still regarded as the mainstay of treatment for many patients with chronic and refractory noninfectious uveitis. However, with the success of other conventional and biologic immunomodulatory agents in treating systemic inflammatory and autoimmune conditions, interest in targeted treatment strategies for uveitis has been renewed. Multiple clinical trials on steroid-sparing immunosuppressive agents, biologic agents, intraocular corticosteroid implants, and topical ophthalmic solutions have already been completed, and many more are ongoing. This review discusses the results and implications of these clinical trials investigating both alternative and novel treatment options for noninfectious uveitis.

Vogt-Koyanagi-Harada Syndrome

Vogt-Koyanagi-Harada (VKH) syndrome is a rare multisystem autoimmune disease affecting the eyes, brain, inner ear, and skin.

Multimodal Imaging of the White Dot Syndromes and Related Diseases.

The white dot syndromes encompass a group of rare posterior uveitis conditions that are characterized by outer retinal and/or choroidal hypopigmented lesions that are thought to be inflammatory in nature. The size, shape, and location of lesions in the fundus aid in differentiating these conditions. Multimodal imaging, including modalities such as fundus autofluorescence, optical coherence tomography, fluorescein angiography, and indocyanine green angiography, among others, has become integral in diagnosing and monitoring many of the white dot syndromes. Furthermore, multimodal imaging modalities have provided insights into the pathogenesis and exact sites within the retina and choroid affected by white dot syndromes.

Therapeutic options for the treatment of non-infectious uveitis

Non-infectious uveitis encompasses a group of site-threatening intraocular inflammatory diseases. Several classes of immunosuppressive medications, including corticosteroids, T cell inhibitors, antimetabolites and, more recently, biologic agents have been shown to be effective in reducing intraocular inflammation in non-infectious uveitis. However, these agents also pose significant risks if not administered and monitored properly. Further research into the pathogenic mechanisms of non-infectious uveitis will hopefully aid in the development of more targeted and safer therapies.

Systemic Sunitinib Malate Treatment for Advanced Juxtapapillary Retinal Hemangioblastomas Associated with von Hippel-Lindau Disease

PURPOSE To describe the clinical course of advanced juxtapapillary retinal capillary hemangioblastomas (RCH) associated with von Hippel-Lindau (VHL) disease treated with systemic sunitinib malate, an agent that inhibits both anti-vascular endothelial growth factor and anti-platelet-derived growth factor signaling. DESIGN Observational case review. PARTICIPANTS Three patients with advanced VHL-related juxtapapillary RCH treated with systemic sunitinib malate. METHODS Patient 1 was followed routinely every 4 months while on systemic sunitinib prescribed by her oncologist for metastatic pancreatic neuroendocrine and kidney tumors. Patients 2 and 3 were part of a prospective clinical trial evaluating the use of systemic sunitinib for ocular VHL lesions during a period of 9 months. Visual acuity, size of RCH, and degree of exudation were recorded at each visit. Optical coherence tomography (OCT) and fluorescein angiography were also obtained at some visits. MAIN OUTCOME MEASURES Visual acuity, size of RCH, and degree of exudation. RESULTS Three patients with advanced VHL-associated juxtapapillary RCH were treated with systemic sunitinib malate. While none of the patients lost vision during therapy, treatment with sunitinib malate did not improve visual acuity or reduce the size of RCH. Improvements in RCH-associated retinal edema were observed in two patients. All patients experienced multiple adverse effects, including thyroid toxicity, thrombocytopenia, nausea, fatigue, jaundice, and muscle aches. Two of the three patients had to discontinue treatment prematurely and the third required dose reduction. CONCLUSIONS Systemic sunitinib malate may be useful in slowing progression of ocular disease from VHL-associated RCH. However, significant systemic adverse effects limited its use in this small series, and systemic sunitinib malate may not be safe for treatment of RCH when used at the doses described in this report. Further studies are required to determine if this medication used at lower doses with different treatment strategies, other medications in the same class or drugs directed at multiple targets in the tumor, may be safer and more effective for the treatment of advanced VHL-associated RCH.

Elevated CD1c+ Myeloid Dendritic Cell Proportions Associate With Clinical Activity and Predict Disease Reactivation in Noninfectious Uveitis.

Purpose To test the association between elevated proportions of CD1c+ myeloid dendritic cells (mDCs) and disease activation/reactivation in noninfectious uveitis. Methods Noninfectious uveitis patients (n = 89) and healthy controls (n = 111) were recruited. The proportion of CD1c+ mDCs in the total dendritic cell (DC) population of peripheral blood was measured by flow cytometry (CD1c+ mDCs gated on Lineage 1+HLADR+ DCs). Disease activity was assessed per Standardization of Uveitis Nomenclature criteria. Uveitis reactivation was ascribed to clinically quiescent patients who developed reactivation of intraocular inflammation within 6 months. Results The proportions of CD1c+ mDCs were increased in noninfectious uveitis patients, especially in active disease, compared to healthy controls. This CD1c+ mDC elevation was not associated with underlying systemic diseases, anatomic locations of uveitis, medications, or demographic factors. Longitudinal data showed that the dynamics of CD1c+ mDC levels were correlated with disease activity. The average proportion of CD1c+ mDCs in active uveitis patients was 60% so we set this as the cutoff between high and low CD1c+ mDC levels. Although 74% of quiescent patients had low proportions of CD1c+ mDCs, 26% still had high proportions. Quiescent patients with high CD1c+ mDC proportions showed increased risk of disease reactivation, compared to quiescent patients with low CD1c+ mDC proportions. Conclusions Increased proportions of CD1c+ mDCs were associated with clinical activity, and quiescent patients with elevated CD1c+ mDCs were more likely to undergo reactivation. This suggests that CD1c+ mDC proportion may be a potential biomarker for assessing clinical activation and reactivation in noninfectious uveitis.

Non-invasive method of monitoring retinal vasculitis in patients with birdshot chorioretinopathy using optical coherence tomography

Background/aims To investigate the utility of using montaged optical coherence tomography (OCT) thickness maps to monitor perivascular thickness as a marker of vasculitic activity in patients with large-vessel retinal vasculitis. Methods This is a retrospective cohort study of 22 eyes of 11 patients with a history of retinal vasculitis associated with birdshot chorioretinopathy (BCR). Patients had serial spectral domain 6×6 mm cube OCT scans centred on the fovea, optic nerve and proximal branches of the superior and inferior retinal vessels. OCT thickness change maps for each respective region were analysed. Changes in perivascular thickness were confirmed by assessing vasculitic activity on fluorescein angiography (FA), when clinically indicated. Results In three patients, montaged OCT scans were acquired at diagnosis and serially through initial treatment. In all three patients, montaged OCT demonstrated reduced perivascular thickening with oral prednisone treatment, which was confirmed by FA showing reduced vascular leakage in both eyes. Eight patients had serial montaged OCT scans after diagnosis and initial treatment of BCR. Four of these patients showed fluctuations in perivascular thickness during flares and treatment that were confirmed by either increased or decreased vascular leakage on FA. The other four patients remained quiet on their immunosuppressive treatment regimens, and no changes in perivascular thickness were detected. Conclusions Evaluating large-vessel perivascular thickness on OCT scans may be a useful method for non-invasively monitoring posterior pole large-vessel retinal vasculitis.