The Role of Retinal Pigment Epithelial Cells in Regulation of Macrophages/Microglial Cells in Retinal Immunobiology.

The ocular tissue microenvironment is immune privileged and uses several mechanisms of immunosuppression to prevent the induction of inflammation. Besides being a blood-barrier and source of photoreceptor nutrients, the retinal pigment epithelial cells (RPE) regulate the activity of immune cells within the retina. These mechanisms involve the expression of immunomodulating molecules that make macrophages and microglial cells suppress inflammation and promote immune tolerance. The RPE have an important role in ocular immune privilege to regulate the behavior of immune cells within the retina. Reviewed is the current understanding of how RPE mediate this regulation and the changes seen under pathological conditions.

A2E-induced inflammation and angiogenesis in RPE cells in vitro are modulated by PPAR-α, -ß/δ, -γ, and RXR antagonists and by norbixin.

N-retinylidene-N-retinylethanolamine (A2E) plays a central role in age-related macular degeneration (AMD) by inducing angiogenesis and inflammation. A2E effects are mediated at least partly via the retinoic acid receptor (RAR)-α. Here we show that A2E binds and transactivates also peroxisome proliferator-activated receptors (PPAR) and retinoid X receptors (RXR). 9'-cis-norbixin, a di-apocarotenoid is also a ligand of these nuclear receptors (NR). Norbixin inhibits PPAR and RXR transactivation induced by A2E. Moreover, norbixin reduces protein kinase B (AKT) phosphorylation, NF-κB and AP-1 transactivation and mRNA expression of the inflammatory interleukins (IL) -6 and -8 and of vascular endothelial growth factor (VEGF) enhanced by A2E. By contrast, norbixin increases matrix metalloproteinase 9 (MMP9) and C-C motif chemokine ligand 2 (CCL2) mRNA expression in response to A2E. Selective PPAR-α, -ß/δ and -γ antagonists inhibit the expression of IL-6 and IL-8 while only the antagonist of PPAR-γ inhibits the transactivation of NF-κB following A2E exposure. In addition, a cocktail of all three PPARs antagonists and also HX531, an antagonist of RXR reproduce norbixin effects on inflammation. Altogether, A2E's deleterious biological effects could be inhibited through PPAR and RXR regulation. Moreover, the modulation of these NR by norbixin may open new avenues for the treatment of AMD.

CFH Loss in Human RPE Cells Leads to Inflammation and Complement System Dysregulation via the NF-κB Pathway.

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is a degenerative disease of the macula, where retinal pigment epithelium (RPE) cells are damaged in the early stages of the disease, and chronic inflammatory processes may be involved. Besides aging and lifestyle factors as drivers of AMD, a strong genetic association to AMD is found in genes of the complement system, with a single polymorphism in the complement factor H gene (CFH), accounting for the majority of AMD risk. However, the exact mechanism of CFH dysregulation confers such a great risk for AMD and its role in RPE cell homeostasis is unclear. To explore the role of endogenous CFH locally in RPE cells, we silenced CFH in human hTERT-RPE1 cells. We demonstrate that endogenously expressed CFH in RPE cells modulates inflammatory cytokine production and complement regulation, independent of external complement sources, or stressors. We show that loss of the factor H protein (FH) results in increased levels of inflammatory mediators (e.g., IL-6, IL-8, GM-CSF) and altered levels of complement proteins (e.g., C3, CFB upregulation, and C5 downregulation) that are known to play a role in AMD. Moreover, our results identify the NF-κB pathway as the major pathway involved in regulating these inflammatory and complement factors. Our findings suggest that in RPE cells, FH and the NF-κB pathway work in synergy to maintain inflammatory and complement balance, and in case either one of them is dysregulated, the RPE microenvironment changes towards a proinflammatory AMD-like phenotype.

Mechanism of Nucleic Acid Sensing in Retinal Pigment Epithelium (RPE): RIG-I Mediates Type I Interferon Response in Human RPE.

Age-related macular degeneration (AMD), a degenerative disease of the outer retina, is the leading cause of blindness among the elderly. A hallmark of geographic atrophy (GA), an advanced type of nonneovascular AMD (dry AMD), is photoreceptor and retinal pigment epithelium (RPE) cell death. Currently, there are no FDA-approved therapies for GA due to a lack of understanding of the disease-causing mechanisms. Increasing evidence suggests that chronic inflammation plays a predominant role in the pathogenesis of dry AMD. Dead or stressed cells release danger signals and inflammatory factors, which causes further damage to neighboring cells. It has been reported that type I interferon (IFN) response is activated in RPE cells in patients with AMD. However, how RPE cells sense stress to initiate IFN response and cause further damage to the retina are still unknown. Although it has been reported that RPE can respond to extracellularly added dsRNA, it is unknown whether and how RPE detects and senses internally generated or internalized nucleic acids. Here, we elucidated the molecular mechanism by which RPE cells sense intracellular nucleic acids. Our data demonstrate that RPE cells can respond to intracellular RNA and induce type I IFN responses via the RIG-I (DExD/H-box helicase 58, DDX58) RNA helicase. In contrast, we showed that RPE cells were unable to directly sense and respond to DNA through the cGAS-STING pathway. We demonstrated that this was due to the absence of the cyclic GMP-AMP synthase (cGAS) DNA sensor in these cells. The activation of IFN response via RIG-I induced expression of cell death effectors and caused barrier function loss in RPE cells. These data suggested that RPE-intrinsic pathways of nucleic acid sensing are biased toward RNA sensing.

Extracellular Soluble Membranes from Retinal Pigment Epithelial Cells Mediate Apoptosis in Macrophages.

A central characterization of retinal immunobiology is the prevention of proinflammatory activity by macrophages. The retinal pigment epithelial cells (RPEs) are a major source of soluble anti-inflammatory factors. This includes a soluble factor that induces macrophage apoptosis when the activity of the immunomodulating neuropeptide alpha-melanocyte-stimulating hormone (α-MSH) is neutralized. In this manuscript, isolated extracellular soluble membranes (ESMs) from primary RPE were assayed to see if they could be the soluble mediator of apoptosis. Our results demonstrated that RPE ESMs mediated the induction of macrophage apoptosis that was suppressed by α-MSH. In contrast, the RPE line ARPE-19, cultured under conditions that induce similar anti-inflammatory activity to primary RPEs, did not activate apoptosis in the macrophages. Moreover, only the ESMs from primary RPE cultures, and not those from the ARPE-19 cell cultures, expressed mFasL. The results demonstrate that RPE ESMs are a soluble mediator of apoptosis and that this may be a mechanism by which the RPEs select for the survival of α-MSH-induced suppressor cells.

Complement C5 is not critical for the formation of sub-RPE deposits in Efemp1 mutant mice.

The complement system plays a role in the formation of sub-retinal pigment epithelial (RPE) deposits in early stages of age-related macular degeneration (AMD). But the specific mechanisms that connect complement activation and deposit formation in AMD patients are unknown, which limits the development of efficient therapies to reduce or stop disease progression. We have previously demonstrated that C3 blockage prevents the formation of sub-RPE deposits in a mouse model of EFEMP1-associated macular degeneration. In this study, we have used double mutant Efemp1R345W/R345W:C5-/- mice to investigate the role of C5 in the formation of sub-RPE deposits in vivo and in vitro. The data revealed that the genetic ablation of C5 does not eliminate the formation of sub-RPE deposits. Contrarily, the absence of C5 in RPE cultures promotes complement dysregulation that results in increased activation of C3, which likely contributes to deposit formation even in the absence of EFEMP1-R345W mutant protein. The results also suggest that genetic ablation of C5 alters the extracellular matrix turnover through an effect on matrix metalloproteinases in RPE cell cultures. These results confirm that C3 rather than C5 could be an effective therapeutic target to treat early AMD.

Mitochondrial C3a Receptor Activation in Oxidatively Stressed Epithelial Cells Reduces Mitochondrial Respiration and Metabolism.

Complement component 3 fragment C3a is an anaphylatoxin involved in promoting cellular responses important in immune response and host defense. Its receptor (C3a receptor, C3aR) is distributed on the plasma membrane; however, lysosomal localization in immune cells has been reported. Oxidative stress increases intracellular reactive oxygen species (ROS), and ROS activate complement signaling in immune cells and metabolic reprogramming. Here we tested oxidative stress and intracellular complement in mitochondrial dysfunction in RPE cells using high resolution live-cell imaging, and metabolism analysis in isolated mitochondria using Seahorse technology. While C3aR levels were unaffected by oxidative stress, its cell membrane levels decreased and mitochondrial (mt) localization increased. Trafficking was dependent on endocytosis, utilizing endosomal-to-mitochondrial cargo transfer. H2O2-treatment also increased C3a-mtC3aR co-localization dose-dependently. In isolated mitochondria from H2O2-treated cells C3a increased mitochondrial Ca2+ uptake, that could be inhibited by C3aR antagonism (SB290157), mitochondrial Ca2+ uniporter blocker (Ru360), and Gαi-protein inhibition (pertussis toxin, PTX); and inhibited mitochondrial repiration in an SB290157- and PTX-dependent manner. Specifically, mtC3aR activation inhibited state III ADP-driven respiration and maximal respiratory capacity. Mitochondria from control cells did not respond to C3a. Furthermore, transmitochondrial cybrid ARPE-19 cells harboring J haplogroup mitochondria that confer risk for age-related macular degeneration, showed high levels of mtC3aR and reduced ATP production upon C3a stimulation. Our findings suggest that oxidative stress increases mtC3aR, leading to altered mitochondrial calcium uptake and ATP production. These studies will have important implication in our understanding on the balance of extra- and intracellular complement signaling in controlling cellular health and dysfunction.

Inhibiting TLR7 Expression in the Retinal Pigment Epithelium Suppresses Experimental Autoimmune Uveitis.

Experimental autoimmune uveitis (EAU), a model of human uveitis, is an organ-specific, T cell-mediated autoimmune disease. Autoreactive T cells can penetrate the blood-retinal barrier, which is a physical defense composed of tight junction-linked retinal pigment epithelial (RPE) cells. RPE cells serve as antigen-presenting cells (APCs) in the eye since they express MHC class I and II and Toll-like receptors (TLRs). Although previous studies have shown that supplementation with TLR agonists exacerbates uveitis, little is known about how TLR signaling in the RPE contributes to the development of uveitis. In this study, we isolated the RPE from EAU mice, which were induced by active immunization (aEAU) or adoptive transfer of antigen-specific T cells (tEAU). The expression of TLRs on RPE was determined, and both aEAU and tEAU mice exhibited induced tlr7 expression. The TLR7 agonist R848 was shown to induce aggressive disease progression, along with significantly elevated levels of the uveopathogenic cytokine IL-17. Furthermore, not only IL-17 but also R848 appeared to enhance the inflammatory response and to impair the barrier function of the RPE, indicating that TLR7 signaling is involved in the pathogenesis of EAU by affecting the behaviors of the RPE and consequently allowing the infiltration of autoreactive T cells intraocularly. Finally, local application of shRNA against TLR7 delivered by recombinant AAV effectively inhibited disease severity and reduced IFN-γ and IL-17. Our findings highlight an immunomodulatory role of RPE TLR7 in EAU development and provide a potential therapeutic strategy for autoimmune uveitis.

The Protective Effects of VVN001 on LPS-Induced Inflammatory Responses in Human RPE Cells and in a Mouse Model of EIU.

To investigate protective effects of VVN001 on lipopolysaccharide (LPS)-induced inflammatory response in human retinal pigment epithelial (RPE) cells and in a mouse model of endotoxin-induced uveitis (EIU), and to explore the underlying mechanisms. Human primary RPE (hRPE) and ARPE-19 cells were pretreated with or without VVN001 for 1 h followed by 10 µg/mL LPS stimulation for 24 h. mRNA, and protein levels of inflammatory cytokines were analyzed with real-time PCR, western blotting, and ELISA. EIU was induced by intravitreal injection of 125 ng LPS in female BALB/c mice. VVN001 eye drops (1%) were locally administrated every 4 h for 24 h after LPS injection. Clinical scores were assessed with a slit lamp. mRNA and protein levels of inflammatory cytokines were investigated simultaneously. Compared with the LPS group, VVN001 pretreatment significantly reduced mRNA expressions of intercellular adhesion molecule-1 (ICAM-1), IL-6, IL-8, TNF-α, IL-1ß, IL-18, caspase-1 in hRPE, and ARPE-19 cells. Protein overproduction of ICAM-1, TNF-α, IL-1ß, NLRP3, caspase-1 P20, and p-IκBα/IκBα stimulated by LPS was suppressed by VVN001 pretreatment. In vivo, VVN001 significantly reduced the average clinical score from 5.0 to 1.3 in EIU mice. Furthermore, overproduction of ICAM-1, IL-1ß, NLRP3, caspase-1 P20, and p-IκBα/IκBα at mRNA and protein levels were remarkably suppressed by VVN001. VVN001 alleviated the inflammatory response induced by LPS both in vitro and in vivo. The effect of anti-inflammation is associated with inhibiting the overproduction of ICAM-1 and blocking the activation of NLRP3 inflammasome and the NF-κB signaling pathway.

Distinct effects of complement and of NLRP3- and non-NLRP3 inflammasomes for choroidal neovascularization.

NLRP3 inflammasome activation and complement-mediated inflammation have been implicated in promoting choroidal neovascularization (CNV) in age-related macular degeneration (AMD), but central questions regarding their contributions to AMD pathogenesis remain unanswered. Key open questions are (1) whether NLRP3 inflammasome activation mainly in retinal pigment epithelium (RPE) or rather in non-RPE cells promotes CNV, (2) whether inflammasome activation in CNV occurs via NLRP3 or also through NLRP3-independent mechanisms, and (3) whether complement activation induces inflammasome activation in CNV. Here we show in a neovascular AMD mouse model that NLRP3 inflammasome activation in non-RPE cells but not in RPE cells promotes CNV. We demonstrate that both NLRP3-dependent and NLRP3-independent inflammasome activation mechanisms induce CNV. Finally, we find that complement and inflammasomes promote CNV through independent mechanisms. Our findings uncover an unexpected role of non-NLRP3 inflammasomes for CNV and suggest that combination therapies targeting inflammasomes and complement may offer synergistic benefits to inhibit CNV.

Phagocytosis by the Retinal Pigment Epithelium: Recognition, Resolution, Recycling.

Tissue-resident phagocytes are responsible for the routine binding, engulfment, and resolution of their meals. Such populations of cells express appropriate surface receptors that are tailored to recognize the phagocytic targets of their niche and initiate the actin polymerization that drives internalization. Tissue-resident phagocytes also harbor enzymes and transporters along the endocytic pathway that orchestrate the resolution of ingested macromolecules from the phagolysosome. Solutes fluxed from the endocytic pathway and into the cytosol can then be reutilized by the phagocyte or exported for their use by neighboring cells. Such a fundamental metabolic coupling between resident phagocytes and the tissue in which they reside is well-emphasized in the case of retinal pigment epithelial (RPE) cells; specialized phagocytes that are responsible for the turnover of photoreceptor outer segments (POS). Photoreceptors are prone to photo-oxidative damage and their long-term health depends enormously on the disposal of aged portions of the outer segment. The phagocytosis of the POS by the RPE is the sole means of this turnover and clearance. RPE are themselves mitotically quiescent and therefore must resolve the ingested material to prevent their toxic accumulation in the lysosome that otherwise leads to retinal disorders. Here we describe the sequence of events underlying the healthy turnover of photoreceptors by the RPE with an emphasis on the signaling that ensures the phagocytosis of the distal POS and on the transport of solutes from the phagosome that supersedes its resolution. While other systems may utilize different receptors and transporters, the biophysical and metabolic manifestations of such events are expected to apply to all tissue-resident phagocytes that perform regular phagocytic programs.

Retinal Pigment Epithelial Cells Derived from Induced Pluripotent Stem (iPS) Cells Suppress or Activate T Cells via Costimulatory Signals.

Human retinal pigment epithelial (RPE) cells derived from induced pluripotent stem (iPS) cells have immunosuppressive properties. However, RPE cells are also known as immunogenic cells, and they have major histocompatibility complex expression and produce inflammatory proteins, and thus experience immune rejection after transplantation. In this study, to confirm the immunological properties of IPS-RPE cells, we examined whether human RPE cells derived from iPS cells could suppress or stimulate inflammatory T cells from uveitis patients via costimulatory signals. We established T cells from patients with active uveitis as target cells and used iPS-RPE cells as effector cells. As a result, cultured iPS-RPE cells inhibited cell proliferation and the production of IFN-γ by activated uveitis CD4+ T cells, especially Th1-type T cells. In contrast, iPS-RPE cells stimulated T cells of uveitis patients. The iPS-RPE cells constitutively expressed B7-H1/CD274 and B7-DC/CD273, and suppressed the activation of T cells via the PD-1 receptor. iPS-RPE expressed these negative costimulatory molecules, especially when RPE cells were pretreated with recombinant IFN-γ. In addition, iPS-RPE cells also expressed B7-H3/CD276 costimulatory molecules and activated uveitis T cells through the B7-H3-TLT-2 receptor. Thus, cultured iPS-derived retinal cells can suppress or activate inflammatory T cells in vitro through costimulatory interactions.

Lactobacillus paracasei KW3110 Suppresses Inflammatory Stress-Induced Premature Cellular Senescence of Human Retinal Pigment Epithelium Cells and Reduces Ocular Disorders in Healthy Humans.

Lactobacillus paracasei KW3110 (KW3110) has anti-inflammatory effects and mitigates retinal pigment epithelium (RPE) cell damage caused by blue-light exposure. We investigated whether KW3110 suppresses chronic inflammatory stress-induced RPE cell damage by modulating immune cell activity and whether it improves ocular disorders in healthy humans. First, we showed that KW3110 treatment of mouse macrophages (J774A.1) produced significantly higher levels of interleukin-10 as compared with other lactic acid bacterium strains (all p < 0.01). Transferring supernatant from KW3110- and E. coli 0111:B4 strain and adenosine 5'-triphosphate (LPS/ATP)-stimulated J774A.1 cells to human retinal pigment epithelium (ARPE-19) cells suppressed senescence-associated phenotypes, including proliferation arrest, abnormal appearance, cell cycle arrest, and upregulation of cytokines, and also suppressed expression of tight junction molecule claudin-1. A randomized, double-blind, placebo-controlled parallel-group study of healthy subjects (n = 88; 35 to below 50 years) ingesting placebo or KW3110-containing supplements for 8 weeks showed that changes in critical flicker frequency, an indicator of eye fatigue, from the week-0 value were significantly larger in the KW3110 group at weeks 4 (p = 0.040) and 8 (p = 0.036). These results suggest that KW3110 protects ARPE-19 cells against premature senescence and aberrant expression of tight junction molecules caused by chronic inflammatory stress, and may improve chronic eye disorders including eye fatigue.

Only IL-1ß release is inflammasome-dependent upon ultraviolet B irradiation although IL-18 is also secreted.

DNA damage accumulates in aged postmitotic retinal pigment epithelium (RPE) cells, a phenomenon associated with the development of age-related macular degeneration. In this study, we have experimentally induced DNA damage by ultraviolet B (UVB) irradiation in interleukin-1α (IL-1α)-primed ARPE-19 cells and examined inflammasome-mediated signaling. To reveal the mechanisms of inflammasome activation, cells were additionally exposed to high levels of extracellular potassium chloride, n-acetyl-cysteine, or mitochondria-targeted antioxidant MitoTEMPO, prior to UVB irradiation. Levels of interleukin-18 (IL-18) and IL-1ß mRNAs were detected with qRT-PCR and secreted amounts of IL-1ß, IL-18, and caspase-1 were measured with ELISA. The role of nucleotide-binding domain and leucine-rich repeat pyrin containing protein 3 (NLRP3) in UVB-induced inflammasome activation was verified by using the NLRP3-specific siRNA. Reactive oxygen species (ROS) levels were measured immediately after UVB exposure using the cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2 DCFDA) indicator, the levels of cyclobutane pyrimidine dimers were assayed by cell-based ELISA, and the extracellular levels of adenosine triphosphate (ATP) determined using a commercial bioluminescence assay. We found that pro-IL-18 was constitutively expressed by ARPE-19 cells, whereas the expression of pro-IL-1ß was inducible by IL-1α priming. UVB induced the release of mature IL-18 and IL-1ß but NLRP3 contributed only to the secretion of IL-1ß. At the mechanistic level, the release of IL-1ß was regulated by K+ efflux, whereas the secretion of IL-18 was dependent on ROS production. As well as K+ efflux, the cells released ATP following UVB exposure. Collectively, our data suggest that UVB clearly stimulates the secretion of mature IL-18 as a result of ROS induction, and this response is associated with DNA damage. Moreover, in human RPE cells, K+ efflux mediates the UVB-activated NLRP3 inflammasome signaling, leading to the processing of IL-1ß.

mTORC1 signaling pathway regulates macrophages in choroidal neovascularization.

Macrophages are involved in choroidal neovascularization (CNV). The mechanistic target of rapamycin complex 1 (mTORC1) is a central cell regulator, but mTORC1 function in macrophages in CNV is not fully understood. We explored the effect of mTORC1 pathway regulation on macrophages in CNV. A laser-induced murine CNV model was performed. Expression of phospho-S6 and F4/80 in CNV lesions was analyzed by immunofluorescence. Macrophages in CNV lesions were found at 1 day after laser treatment, reached a peak at 5 days, and decreased at 7 and 14 days. mTORC1 activity of cells in CNV lesions was increased from 3 to 7 days, and deceased at 14 days. Most infiltrating macrophages in CNV lesions had strong mTORC1 activity at 3 and 5 days that subsequently decreased. In vitro, THP-1 macrophages were polarized to M1 or M2 with rapamycin or siRNA treatment. The human retinal pigment epithelium (RPE) cell line ARPE-19 was co-cultured with macrophages. Cytokine expression of macrophages and ARPE-19 cells was detected by quantitative PCR. Inhibiting mTORC1 activity of macrophages reduced M1 and strengthened M2, which was reversed by mTORC1 hyperactivation. Both M1 and M2 macrophages induced RPE cells to express less PEDF and more MMP9, IL-1ß and MCP-1. Inhibiting or enhancing mTORC1 activity of macrophages changed cytokine expression of RPE cells. Together, we demonstrated that macrophage functions in CNV were regulated partly by the mTORC1 pathway, and mTORC1 activity of macrophages influenced the expression of cytokines that are associated with CNV development in RPE cells. This study provides more understanding about the regulatory mechanism of macrophages in CNV.

Caspase-1-dependent inflammasomes mediate photoreceptor cell death in photo-oxidative damage-induced retinal degeneration.

Activation of the inflammasome is involved in the progression of retinal degenerative diseases, in particular, in the pathogenesis of Age-Related Macular Degeneration (AMD), with NLRP3 activation the focus of many investigations. In this study, we used genetic and pharmacological approaches to explore the role of the inflammasome in a mouse model of retinal degeneration. We identify that Casp1/11-/- mice have better-preserved retinal function, reduced inflammation and increased photoreceptor survivability. While Nlrp3-/- mice display some level of preservation of retinal function compared to controls, pharmacological inhibition of NLRP3 did not protect against photoreceptor cell death. Further, Aim2-/-, Nlrc4-/-, Asc-/-, and Casp11-/- mice show no substantial retinal protection. We propose that CASP-1-associated photoreceptor cell death occurs largely independently of NLRP3 and other established inflammasome sensor proteins, or that inhibition of a single sensor is not sufficient to repress the inflammatory cascade. Therapeutic targeting of CASP-1 may offer a more promising avenue to delay the progression of retinal degenerations.

A20 Inhibits Intraocular Inflammation in Mice by Regulating the Function of CD4+T Cells and RPE Cells.

A20 is a negative regulator of inflammation and immunity and plays a role in several autoimmune and inflammatory diseases. Here, we demonstrate that A20 overexpression significantly ameliorates severity of EAU by inhibiting the infiltration of Th1 and Th17 cells, and by protecting integrity of the blood retinal barrier. In vitro studies showed that A20 silencing could promote CD4+T cells toward a Th1 and Th17 phenotype. A decreased expression of A20 in CD4+T cells was noticed in active BD patients but not in VKH patients. Furthermore, silencing of A20 in hRPE cells induced the production of IL-6, IL-8, and MCP-1 and downregulated ZO-1 and occludin expression which is mediated by inhibition of MAPK and NF-κB pathways. This study reveals a mechanism by which A20 prevents autoimmune uveitis.

Complement Receptor 1 (CR1/CD35)-expressing retinal pigment epithelial cells as a potential therapy for age-related macular degeneration.

The purpose of this study was to identify a membrane-bound complement inhibitor that could be overexpressed on retinal pigment epithelial cells (RPE) providing a potential therapy for age-related macular degeneration (AMD). This type of therapy may allow replacement of damaged RPE with cells that are able to limit complement activation in the retina. Complement Receptor 1 (CR1) is a membrane-bound complement inhibitor commonly found on erythrocytes and immune cells. In this study, QPCR and flow cytometry data demonstrated that CR1 is not well-expressed by RPE, indicating that its overexpression may provide extra protection from complement activation. To screen CR1 for this ability, a stable CR1-expressing ARPE19 line was created using a combination of antibiotic selection and FACS. Cell-based assays were used to demonstrate that addition of CR1 inhibited deposition of complement proteins C3b and C6 on the transfected line. In the end, this study identifies CR1 as a complement inhibitor that may be overexpressed on stem cell-derived RPE to create a potential "enhanced" cell therapy for AMD. A combination cell/complement therapy may create transplantable RPE better suited to avoid complement-mediated lysis and limit chronic inflammation in the retina.

Autoimmune-Mediated Retinopathy in CXCR5-Deficient Mice as the Result of Age-Related Macular Degeneration Associated Proteins Accumulation.

Previous research has shown that CXCR5-/- mice develop retinal degeneration (RD) with age, a characteristic related to age macular degeneration (AMD). RD in these mice is not well-understood, and in this study, we sought to characterize further the RD phenotype and to gain mechanistic insights into the function of CXCR5 in the retina. CXCR5-/- and WT control mice were used. Fundus images demonstrated a significant (p < 0.001) increase of hypo-pigmented spots in the retina of aged CXCR5-/- mice compared with WT control mice. PAS staining indicated localization of deposits in the sub-retinal pigment epithelia (RPE) layer. AMD-associated proteins Cryab, amyloid beta, and C3d were detected within the RPE/sub-RPE tissues by immunofluorescence (IF). In addition, western blot analysis of COX-2, Arg1, and VEGF-a revealed an increase in the signaling of these molecules within the RPE/choroid complex. Transmission electron microscopy (TEM) indicated a drusen-like structure of sub-RPE deposits with an accumulation of vacuolated cellular debris. Loss of photoreceptors was detected by peanut lectin staining and was corroborated by a reduction in MAP2 signaling. Loss of blood-retinal barrier integrity was demonstrated by a reduction of ZO-1 expression. Inflammatory cells were detected in the sub-RPE space, with an increase in IBA-1 positive microglia cells on the surface of the RPE. Mass spectrometry analysis of CXCR5-/- mouse RPE/choroid proteins extracts, separated by SDS-page and incubated with autologous serum, identified autoantibodies against AMD-associated proteins: Cryaa, Cryab, and Anxa2. In vitro evaluations in BV-2 cell culture indicated a significant increase in production of Arg-1 (p < 0.001) and COX-2 (p < 0.01) in the presence of anti-CXCR5 antibody when compared with Igg-treated control BV-2 cells stimulated with IL-4 and TNFα/IFNγ, respectively. Anti-CXCR5 antibody treatment without stimulating agents did not affect Arg-1 and COX-2 expression; this suggests that CXCR5 may have a regulatory role in microglia cells activation. These results indicate that with age, CXCR5-/- mice develop RD characterized by microglia dysfunction, increased production of CXCL13 in the RPE progressive photoreceptor, neuronal loss, and sub-RPE deposition of cellular debris, resulting in the production of immunogenic proteins and autoimmune-mediated RD.

Tissue-specific production of MicroRNA-155 inhibits melanocortin 5 receptor-dependent suppressor macrophages to promote experimental autoimmune uveitis.

Tissue-specific immune regulation is an important component of the immune response relevant to many areas of immunology. The focus of this study is on tissue-specific mechanisms that contribute to autoimmune uveitis. Precise gene regulation is necessary for the proper expression of an inflammatory or regulatory response. This precision gene regulation can be accomplished by microRNA at the level of the mRNA transcript. miR-155, in particular, has a complicated role in the immune response with positive and negative inflammatory effects. In this work, we identify a decrease in miR-155 in suppressor macrophages and further examine how tissue-specific production of miR-155 impacts experimental autoimmune uveitis. Importantly, we show that eliminating miR-155 expression by the target tissue before initiation reduces disease severity, but elimination of miR-155 after the onset of inflammation does not alter the course of disease. Additionally, expression of miR-155 by the target tissue before initiation is necessary for the induction of regulatory immunity that protects from further autoimmune disease, but not after the onset of inflammation. In summary, we find a MC5r-dependent decrease in miR-155 in postexperimental autoimmune uveitis APC, miR-155 production by the target tissue is necessary for the initiation of autoimmune uveitis, and may have a role in establishing protective regulatory immunity.