Proinflammatory Pathways Are Activated in the Human Q344X Rhodopsin Knock-In Mouse Model of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a hereditary disease of the retina that results in complete blindness. Currently, there are very few treatments for the disease and those that exist work only for the recessively inherited forms. To better understand the pathogenesis of RP, multiple mouse models have been generated bearing mutations found in human patients including the human Q344X rhodopsin knock-in mouse. In recent years, the immune system was shown to play an increasingly important role in RP degeneration. By way of electroretinography, optical coherence tomography, funduscopy, fluorescein angiography, and fluorescent immunohistochemistry, we show degenerative and vascular phenotypes, microglial activation, photoreceptor phagocytosis, and upregulation of proinflammatory pathway proteins in the retinas of the human Q344X rhodopsin knock-in mouse. We also show that an FDA-approved pharmacological agent indicated for the treatment of rheumatoid arthritis is able to halt activation of pro-inflammatory signaling in cultured retinal cells, setting the stage for pre-clinical trials using these mice to inhibit proinflammatory signaling in an attempt to preserve vision. We conclude from this work that pro- and autoinflammatory upregulation likely act to enhance the progression of the degenerative phenotype of rhodopsin Q344X-mediated RP and that inhibition of these pathways may lead to longer-lasting vision in not only the Q344X rhodopsin knock-in mice, but humans as well.

Serum anti-recoverin antibodies is found in elderly patients with retinitis pigmentosa and cancer.

PURPOSE: To screen for anti-recoverin antibodies in elderly patients with retinitis pigmentosa (RP) with or without cancer and cross-sectionally characterize the seropositive patients clinically. METHODS: Serum from 75 RP patients who had been tested for mutations in a panel of 83 RP genes and 73 normal controls, all aged 50-80 years, were screened for anti-recoverin antibodies by Western blot using recombinant recoverin, retinal lysate from a marmoset and commercial anti-recoverin antibodies as a control. RESULTS: Three RP patients with typical pigmentary degeneration of the 75 (4.0%) were seropositive for anti-recoverin antibody. Pathogenic mutations were identified in two seropositive RP patients. All three patients had visual impairment since childhood and were diagnosed as RP by the age of 30. The severity of the retinopathy varied greatly among these three patients, ranging in visual acuity from light perception OU to 20/30 OU. Retinitis pigmentosa (RP) patients with a history of cancer were more likely to have anti-recoverin antibodies (3/14; 21.4%) than those without (0/61; 0%; p = 0.005, Fischer exact test). All 73 healthy controls with no history of cancer were also seronegative. CONCLUSION: Our results show that serum anti-recoverin antibodies can be detected in typical RP patients with identified pathogenic mutations and that a history of cancer may increase the risk of developing anti-recoverin antibodies.

C3- and CR3-dependent microglial clearance protects photoreceptors in retinitis pigmentosa.

Complement activation has been implicated as contributing to neurodegeneration in retinal and brain pathologies, but its role in retinitis pigmentosa (RP), an inherited and largely incurable photoreceptor degenerative disease, is unclear. We found that multiple complement components were markedly up-regulated in retinas with human RP and the rd10 mouse model, coinciding spatiotemporally with photoreceptor degeneration, with increased C3 expression and activation localizing to activated retinal microglia. Genetic ablation of C3 accelerated structural and functional photoreceptor degeneration and altered retinal inflammatory gene expression. These phenotypes were recapitulated by genetic deletion of CR3, a microglia-expressed receptor for the C3 activation product iC3b, implicating C3-CR3 signaling as a regulator of microglia-photoreceptor interactions. Deficiency of C3 or CR3 decreased microglial phagocytosis of apoptotic photoreceptors and increased microglial neurotoxicity to photoreceptors, demonstrating a novel adaptive role for complement-mediated microglial clearance of apoptotic photoreceptors in RP. These homeostatic neuroinflammatory mechanisms are relevant to the design and interpretation of immunomodulatory therapeutic approaches to retinal degenerative disease.

Soluble CX3CL1 gene therapy improves cone survival and function in mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a disease that initially presents as night blindness due to genetic deficits in the rod photoreceptors of the retina. Rods then die, causing dysfunction and death of cone photoreceptors, the cell type that mediates high acuity and color vision, ultimately leading to blindness. We investigated immune responses in mouse models of RP and found evidence of microglia activation throughout the period of cone degeneration. Using adeno-associated vectors (AAVs), delivery of genes encoding microglial regulatory signals led to the identification of AAV serotype 8 (AAV8) soluble CX3CL1 (sCX3CL1) as a promising therapy for degenerating cones. Subretinal injection of AAV8-sCX3CL1 significantly prolonged cone survival in three strains of RP mice. Rescue of cones was accompanied by improvements in visual function. AAV8-sCX3CL1 did not affect rod survival, microglia localization, or inflammatory cytokine levels in the retina. Furthermore, although RNA sequencing of microglia demonstrated marked transcriptional changes with AAV8-sCX3CL1, pharmacological depletion of up to ∼99% of microglia failed to abrogate the effect of AAV8-sCX3CL1 on cone survival. These findings indicate that AAV8-sCX3CL1 can rescue cones in multiple mouse models of RP via a pathway that does not require normal numbers of microglia. Gene therapy with sCX3CL1 is a promising mutation-independent approach to preserve vision in RP and potentially other forms of retinal degeneration.

Neuroinflammation in Retinitis Pigmentosa, Diabetic Retinopathy, and Age-Related Macular Degeneration: A Minireview.

The eye is an immuno-privileged organ. However, certain diseases such as uveitis are intrinsically linked to inflammation. In several retinal degenerative diseases, there is a unique damage at the onset of the disease, but evidence suggests that chronic and low-grade inflammatory processes play an important role in their progression. Studies have identified similar signaling pathways and changes in resident immune cells within the retina among these diseases. Herein, we will discuss some of these studies and propose how understanding this inflammatory response could aid in the development of therapies.

A review of the immunologic findings observed in retinitis pigmentosa.

Most patients suffering from retinitis pigmentosa (RP) inherit the disorder; however, the immune-pathologic features associated with this disease have yet to be extensively studied. Six reports correlate antiretinal immune activity with vision deterioration in RP patients. Some of these patients have sporadic RP that occurs in excess of expected gene segregation during inheritance. The hypothesis that a primary immune-mediated disease process occurs in this sporadic group is supported by significant associations of RP with autoimmune endocrinopathies and other immune-related conditions or factors; however, no immunologic difference regarding RP family history is reported in the peripheral blood studies of RP patients. Twenty-one percent to 51% of RP patients display antiretinal antibodies, whereas 19-58% have antiretinal lymphocyte reactivity to retinal extract, and 60-85% have activated T cells. Mutations in animal models of RP have been shown to cause endoplasmic reticulum stress that may initiate immunopathology for genetic RP, but oxidative stress also encourages immune cytotoxicity. In addition, necrotic cell death is evident, which promotes inflammatory conditions. We review mechanisms and evidence for an occult inflammation in genetic RP and examine reports of efficacy in retarding RP progression with anti-inflammatory agents in clinical trials.

Involvement of Innate Immune System in Late Stages of Inherited Photoreceptor Degeneration.

Retinitis pigmentosa (RP) is a group of inherited retinal degenerations that lead to progressive vision loss. Over 200 mutations in 60 different genes have been shown to cause RP. Given the diversity of genes and mutations that cause RP, corrective gene therapy approaches currently in development may prove both time-consuming and cost-prohibitive for treatment of all forms of RP. An alternative approach is to find common biological pathways that cause retinal degeneration in various forms of RP, and identify new molecular targets. With this goal, we analyzed the retinal transcriptome of two non-allelic forms of RP in dogs, rcd1 and xlpra2, at clinically relevant advanced stages of the two diseases. Both diseases showed very similar trends in changes in gene expression compared to control normal dogs. Pathway analysis revealed upregulation of various components of the innate immune system in both diseases, including inflammasome and complement pathways. Our results show that the retinal transcriptome at advanced stages of RP is very similar to that of other retinal degenerative diseases such as age-related macular degeneration and diabetic retinopathy. Thus, drugs and therapeutics already in development for targeting these retinopathies may also prove useful for the treatment of many forms of RP.

TNFa knockdown in the retina promotes cone survival in a mouse model of autosomal dominant retinitis pigmentosa.

Expression of T17M rhodopsin (T17M) in rods activates the Unfolded Protein Response (UPR) and leads to the development of autosomal dominant retinitis pigmentosa (adRP). The rod death occurs in adRP retinas prior to cone photoreceptor death, so the mechanism by which cone photoreceptors die remains unclear. Therefore, the goal of the study was to verify whether UPR in rods induces TNFa-mediated signaling to the cones and to determine whether the TNFa deficit could prevent adRP cone cell death. Primary rod photoreceptors and cone-derived 661W cells transfected with siRNA against TNFa were treated with tunicamycin to mimic activation of UPR in T17M retinas expressing normal and reduced TNFa levels. The 661W cells were then exposed to recombinant TNFa to evaluate cell viability. In vivo, the role of TNFa was assessed in T17M TNFa+/- mice by electroretinography, optical coherence tomography, histology, immunohistochemistry, and a cytokine enzyme-linked immunosorbent assay. Rods overexpressed and secreted TNFa in response to UPR activation. The recombinant TNFa treatment lowered the number of viable cones, inducing cell death through elevation of pro-inflammatory cytokines and caspase-3/7 activity. The TNFa deficiency significantly protected adRP retinas. The photopic ERG amplitudes and the number of surviving cones dramatically increased in T17M TNFa+/- mice. This neuroprotection was associated with a reduced level of pro-inflammatory cytokines. Our results indicate that rod photoreceptors, following UPR activation during adRP progression, secrete TNFa and signal a self-destructive program to the cones, resulting in their cell death. TNFa therefore holds promise as a therapeutic target for treatment of adRP.

A profile of transcriptomic changes in the rd10 mouse model of retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP) is a photoreceptor disease that affects approximately 100,000 people in the United States. Treatment options are limited, and the prognosis for most patients is progressive vision loss. Unfortunately, understanding of the molecular underpinnings of RP initiation and progression is still limited. However, the development of animal models of RP, coupled with high-throughput sequencing, has provided an opportunity to study the underlying cellular and molecular changes in this disease. METHODS: Using RNA-Seq, we present the first retinal transcriptome analysis of the rd10 murine model of retinal degeneration. RESULTS: Our data confirm the loss of rod-specific transcripts and the increased relative expression of Müller-specific transcripts, emphasizing the important role of reactive gliosis and innate immune activation in RP. Moreover, we report substantial changes in relative isoform usage among neuronal differentiation and morphogenesis genes, including a marked shift to shorter transcripts. CONCLUSIONS: Our analyses implicate remodeling of the inner retina and possible Müller cell dedifferentiation.

Infliximab reduces Zaprinast-induced retinal degeneration in cultures of porcine retina.

BACKGROUND: cGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4 to 5% of retinitis pigmentosa (RP), a rare form of retinal dystrophy. Growing evidence suggests that inflammation is involved in the progression of RP. The aims of this study were to corroborate the presence of high TNFα concentration in the eyes of RP patients and to evaluate whether the blockade of TNFα with Infliximab, a monoclonal anti-TNFα antibody, prevented retinal degeneration induced by PDE6 inhibition in cultures of porcine retina. METHODS: Aqueous humor from 30 patients with RP and 13 healthy controls were used to quantify the inflammatory mediators IL-6, TNFα, IL-1ß, IL-10 by a multiplex enzyme-linked immunosorbent assay (ELISA) system. Retinal explants from pig were exposed to Zaprinast, a PDE6 inhibitor, for 24 hours in the absence or the presence of Infliximab. Cell death was evaluated by TUNEL assay. The number and distribution of caspase-3 positive cells, indirect poly(ADP)ribose polymerase (PARP) activation and glial fibrillary acidic protein (GFAP) content were visualized by immunolabeling. Antioxidant total capacity, nitrites and thiobarbituric acid reactive substances (TBARS) formation were determined to evaluate antioxidant-oxidant status. RESULTS: IL-6 and TNFα concentrations were higher in the aqueous humor of RP patients than in controls. Infliximab prevented retinal degeneration, as judging by the reduced presence of TUNEL-positive cells, the reduction of caspase-3 activation and also reduction of glial activation, in an ex vivo model of porcine retina. Additionally, Infliximab partially reduced oxidative stress in retinal explants exposed to Zaprinast. CONCLUSIONS: Inflammatory mediators IL-6 and TNFα were elevated in the aqueous humor of RP patients corroborating previous studies suggesting sustained chronic inflammation. Our study suggests that TNFα is playing an important role in cell death in an ex vivo model of retinal degeneration by activating different cell pathways at different cell layers of the retina that should be further studied.

Functional endothelial progenitor cells selectively recruit neurovascular protective monocyte-derived F4/80(+) /Ly6c(+) macrophages in a mouse model of retinal degeneration.

Retinitis pigmentosa is a group of inherited eye disorders that result in profound vision loss with characteristic retinal neuronal degeneration and vasculature attenuation. In a mouse model of retinitis pigmentosa, endothelial progenitor cells (EPC) from bone marrow rescued the vasculature and photoreceptors. However, the mechanisms and cell types underlying these protective effects were uncertain. We divided EPC, which contribute to angiogenesis, into two subpopulations based on their aldehyde dehydrogenase (ALDH) activity and observed that EPC with low ALDH activity (Alde-Low) had greater neuroprotection and vasoprotection capabilities after injection into the eyes of an rd1 mouse model of retinitis pigmentosa compared with EPC with high ALDH activity (Alde-High). Of note, Alde-Low EPC selectively recruited F4/80(+) /Ly6c(+) monocyte-derived macrophages from bone marrow into retina through CCL2 secretion. In addition, the mRNA levels of CCR2, the neurotrophic factors TGF-ß1 and IGF-1, and the anti-inflammatory mediator interleukin-10 were higher in migrated F4/80(+) /Ly6c(+) monocyte-derived macrophages as compared with F4/80(+) /Ly6c(-) resident retinal microglial cells. These results suggest a novel therapeutic approach using EPC to recruit neuroprotective macrophages that delay the progression of neural degenerative disease.

Phosphodiesterase inhibition induces retinal degeneration, oxidative stress and inflammation in cone-enriched cultures of porcine retina.

Inherited retinal degenerations affecting both rod and cone photoreceptors constitute one of the causes of incurable blindness in the developed world. Cyclic guanosine monophosphate (cGMP) is crucial in the phototransduction and, mutations in genes related to its metabolism are responsible for different retinal dystrophies. cGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4-5% of the retinitis pigmentosa, a rare form of retinal degeneration. The aim of this study was to evaluate whether pharmacological PDE6 inhibition induced retinal degeneration in cone-enriched cultures of porcine retina similar to that found in murine models. PDE6 inhibition was induced in cone-enriched retinal explants from pigs by Zaprinast. PDE6 inhibition induced cGMP accumulation and triggered retinal degeneration, as determined by TUNEL assay. Western blot analysis and immunostaining indicated that degeneration was accompanied by caspase-3, calpain-2 activation and poly (ADP-ribose) accumulation. Oxidative stress markers, total antioxidant capacity, thiobarbituric acid reactive substances (TBARS) and nitric oxide measurements revealed the presence of oxidative damage. Elevated TNF-alpha and IL-6, as determined by enzyme immunoassay, were also found in cone-enriched retinal explants treated with Zaprinast. Our study suggests that this ex vivo model of retinal degeneration in porcine retina could be an alternative model for therapeutic research into the mechanisms of photoreceptor death in cone-related diseases, thus replacing or reducing animal experiments.

Cellular targets of anti-alpha-enolase autoantibodies of patients with autoimmune retinopathy.

Autoantibodies against alpha-enolase are often associated with visual loss in patients with autoimmune retinopathy. Anti-recoverin autoantibodies have been the most extensively studied for their pathologic association with cancer-associated retinopathy (CAR). It has been shown that anti-recoverin antibodies penetrate retinal layers corresponding to the cellular location of recoverin and cause the death of photoreceptors and bipolar cells. However, the pathogenic effects of anti-alpha-enolase antibodies have not been studied. In this study, we tested the labeling and apoptotic effects of such autoantibodies on retinal cells. Serum antibodies against alpha-enolase from patients with autoimmune retinopathy were tested ex vivo and in vivo in Sprague-Dawley rats. Autoantibodies to alpha-enolase specifically labeled the retinal ganglion cells and inner nuclear layer cells. Using ex vivo experiments and intravitreal injections, we observed that antibodies were capable of penetrating retinal tissue to target ganglion cell and inner nuclear layers and, consequently, were able to induce cell death through an apoptotic process. The apoptotic nuclei detected by a DNA fragmentation assay and caspase 3-positive cells were co-localized in the ganglion cell layer and inner nuclear layer. The results showed that antibodies against alpha-enolase target antigens in these layers and induce the apoptotic death of sensitive cells. Rat retinal explants and the intravitreal injection of antibodies provide us with a good model to identify antibody pathogenic targets in the retina. Such identification may help explain the complex of clinical symptoms for autoimmune retinopathy mediated by autoantibody and may help guide treatment strategies.

Clinical and immunologic aspects of cancer-associated retinopathy.

PURPOSE: To report clinical and immunologic aspects of cancer-associated retinopathy (CAR). DESIGN: Observational consecutive case series. METHODS: A retrospective review was made of 18 consecutive patients with cancer-associated retinopathy who had antiretinal antibody determination by Western blot testing. RESULTS: Clinically, a variety of ophthalmic observations including electroretinography impairment, retinal vessel narrowing, deterioration of visual acuity, visual field changes, and uveitis were frequently observed. As retinal autoantigens in the 18 cases, recoverin was found in all 18 cases (100%), heat shock cognate protein 70 (HSC70) was found in six cases (33%), and other proteins were found in four cases (20%). These antibodies were detected in only 60% of the patients at the initial examination, however, and then became increasingly apparent on the subsequent testing that was performed three times on serum samples obtained sequentially during the following months. CONCLUSION: For diagnosis of cancer-associated retinopathy, the presence of serum autoantibody toward recoverin is essentially required in addition to the characteristic clinical aspects noted above.

An atypical phenotype of macular and peripapillary retinal atrophy caused by a mutation in the RP2 gene.

AIMS: To determine the molecular basis and describe the phenotype of an atypical retinal dystrophy in a family presenting with bilateral, progressive central visual loss. METHODS: Family members were examined. Investigations included Goldman perimetry, electrophysiology, and autofluorescence imaging. Candidate gene screening was performed using SSCP and sequence analysis. The proband's lymphoblastoid cells were examined for protein expression. RESULTS: Fundal examination of the proband, his mother, and brother revealed peripapillary and macular atrophy. Autosomal dominant retinal dystrophy was suspected, but less severe disease in the mother led to screening for mutations in X linked genes. A 4 bp microdeletion in exon 3 of the RP2 gene, segregating with disease, was identified. No RP2 protein expression was detected. CONCLUSION: The distinct phenotype in this family, caused by this frameshifting mutation in RP2, broadens the phenotypic spectrum of X linked retinitis pigmentosa. The absence of RP2 protein suggests that loss of protein function and not novel gain of function could account for the atypical phenotype. A definitive diagnosis of X linked retinitis pigmentosa permits appropriate genetic counselling with important implications for other family members. Clinicians should have a low threshold for screening RP2 in families with retinal dystrophy, including posterior retinal disease, not immediately suggestive of X linked inheritance.

Immune response to retinal antigens in patients with gyrate atrophy and other hereditary retinal dystrophies.

PURPOSE: Gyrate atrophy (GA) is a rare hereditary disease that causes retinal destruction. Retinal damage in GA and other heredodegenerative diseases such as retinitis pigmentosa (RP) releases sequestered antigens and may trigger immune response to these molecules. Here, we studied the immune response to retinal antigens in patients with GA and RP and compared it with that of patients with inactive posterior uveitis and normal volunteers. PATIENTS AND METHODS: Peripheral blood was collected from 24 patients with RP, 10 patients with GA, 10 patients with inactive posterior uveitis, and 16 normal volunteers. Cell-mediated immune responses to human S-antigen (HS-Ag), bovine S-antigen (BS-Ag), and interphotoreceptor retinoid-binding protein (IRBP) were investigated by lymphocyte proliferation assay. In addition, serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were studied by ELISA. Immunologic data were correlated with clinical and electrophysiological findings. RESULTS: Patients with GA or RP responded to HS-Ag and BS-Ag more vigorously than patients with uveitis or healthy controls, as shown by higher mean stimulation indices and larger proportions of responders. Unlike S-Ag, IRBP stimulated low lymphocyte responses in only a small proportion of RP patients. The mean sVCAM-1 levels were significantly higher in the sera from patients with GA than in that from normal controls. CONCLUSION: An elevated cellular immune response to S-Ag is common in patients with GA and RP. This elevated cellular immune response to S-Ag may exacerbate retinal destruction in patients with GA and RP.

Autoimmune retinopathy: patients with antirecoverin immunoreactivity and panretinal degeneration.

PURPOSE: To investigate whether antirecoverin antibodies are present in patients with retinitis pigmentosa (RP). Recoverin, a retinal protein, has been implicated as a cause of cancer-associated retinopathy (CAR), which manifests as an RP-like retinal degeneration. The rationale is that the ocular findings in CAR syndrome are similar to those found in many forms of RP, and since 40% of patients with RP have no family history, some patients may have an underlying autoimmune process causing or contributing to their retinopathy. METHODS: Serum samples from 521 patients diagnosed with RP were screened for antiretinal proteins activity by Western blot analysis. Fifty-one patients had antibody reactivity against retinal proteins in the range of 23 to 26 kd and underwent dot-blot analysis for antirecoverin antibody, checking IgG and IgM antibodies. Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the titer of antirecoverin antibodies in patients with positive results on dot-blot analysis. Lymphocyte proliferation assays using recoverin were performed on 26 samples. RESULTS: Ten patients were found to have antirecoverin antibody and/or cellular immunoreactivity. Eight patients had positive dot-blot testing: 6 patients had both IgG and IgM antirecoverin activity, and 1 patient each had IgG or IgM activity. In these 8 patients, numerous other antiretinal protein antibodies were present. Three patients had positive recoverin-mediated lymphocyte proliferation, and all patients were positive for antirecoverin antibodies on ELISA testing. CONCLUSIONS: Antirecoverin immunoreactivity was found in 10 patients without systemic malignancy but with clinical findings consistent with RP. These results suggest that there are other immunogenic mechanisms occurring in the formation of antirecoverin antibodies in addition to the putative tumor-mediated mechanisms. This survey suggests that there may be rare cases of CAR-like syndrome in the category of simplex RP, or that some patients with RP also have antirecoverin antibodies that may be exacerbating their underlying disease. Arch Ophthalmol. 2000;118:1525-1533