Neovascular age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). An overactive complement system is associated with AMD pathogenesis, and serum pro-inflammatory cytokines, including IL-17, are elevated in AMD patients. IL-17 is produced by complement C5a-receptor-expressing T-cells. In murine CNV, infiltrating γδT- rather than Th17-cells produce the IL-17 measurable in lesioned eyes. Here we asked whether C5a generated locally in response to CNV recruits IL-17-producing T-cells to the eye. CNV lesions were generated using laser photocoagulation and quantified by imaging; T-lymphocytes were characterized by QRT-PCR. CNV resulted in an increase in splenic IL-17-producing γδT- and Th17-cells; yet in the CNV eye, only elevated levels of γδT-cells were observed. Systemic administration of anti-C5- or anti-C5a-blocking antibodies blunted the CNV-induced production of splenic Th17- and γδT-cells, reduced CNV size and eliminated ocular γδT-cell infiltration. In ARPE-19 cell monolayers, IL-17 triggered a pro-inflammatory state; and splenocyte proliferation was elevated in response to ocular proteins. Thus, we demonstrated that CNV lesions trigger a systemic immune response, augmenting local ocular inflammation via the infiltration of IL-17-producing γδT-cells, which are presumably recruited to the eye in a C5a-dependent manner. Understanding the complexity of complement-mediated pathological mechanisms will aid in the development of an AMD treatment.

PurposeMorphologic and genetic evidence exists that an overactive complement system driven by the complement alternative pathway (AP) is involved in pathogenesis of age-related macular degeneration (AMD). Smoking is the only modifiable risk factor for AMD. As we have shown that smoke-related ocular pathology can be prevented in mice that lack an essential activator of AP, we ask here whether this pathology can be reversed by increasing inhibition in AP.MethodsMice were exposed to either cigarette smoke (CS) or filtered air (6 hours/day, 5 days/week, 6 months). Smoke-exposed animals were then treated with the AP inhibitor (CR2-fH) or vehicle control (PBS) for the following 3 months. Spatial frequency and contrast sensitivity were assessed by optokinetic response paradigms at 6 and 9 months; additional readouts included assessment of retinal morphology by electron microscopy (EM) and gene expression analysis by quantitative RT-PCR.ResultsThe CS mice treated with CR2-fH showed significant improvement in contrast threshold compared to PBS-treated mice, whereas spatial frequency was unaffected by CS or pharmacologic intervention. Treatment with CR2-fH in CS animals reversed thinning of the retina observed in PBS-treated mice as analyzed by spectral-domain optical coherence tomography, and reversed most morphologic changes in RPE and Bruch's membrane seen in CS animals by EM.ConclusionsTaken together, these findings suggest that AP inhibitors not only prevent, but have the potential to accelerate the clearance of complement-mediated ocular injury. Improving our understanding of the regulation of the AP is paramount to developing novel treatment approaches for AMD.

Complement activation plays a significant role in age-related macular degeneration (AMD) pathogenesis, and polymorphisms interfering with factor H (fH) function, a complement alternative pathway (AP) inhibitor, are associated with increased AMD risk. We have previously validated an AP inhibitor, a fusion protein consisting of a complement receptor 2 fragment linked to the inhibitory domain of fH (CR2-fH) as an efficacious treatment for choroidal neovascularization (CNV) when delivered intravenously. Here we tested an alternative approach of AAV-mediated delivery (AAV5-VMD2-CR2-fH or AAV5-VMD2-mCherry) using subretinal delivery in C57BL/6J mice. Secretion of CR2-fH was confirmed in polarized retinal pigment epithelium (RPE) cells. A safe concentration of AAV5-VMD2-CR2-fH was identified using electroretinography, optical coherence tomography (OCT), RPE morphology, and antibody profiling. One month after gene delivery, CNV was induced using argon laser photocoagulation. OCT assessment demonstrated reduced CNV with AAV5-VMD2-CR2-fH administration. Bioavailability studies revealed that gene-therapy delivered similar levels of CR2-fH to the RPE/choroid as treatment by intravenous injections, and C3a ELISA verified reduced CNV-associated ocular C3a production. These results contribute to existing data illustrating the importance of the AP of complement in CNV development and its potential role in AMD treatment. Demonstration of AAV-vector efficacy opens new avenues for the development of treatment strategies.

Recently, a novel Slc22 gene family member expressed in murine olfactory mucosa was identified and based on sequence homology proposed to be an organic anion transporter [Oat6 (Slc22a20); J. C. Monte, M. A. Nagle, S. A. Eraly, and S. K. Nigam. Biochem Biophys Res Commun 323: 429-436, 2004]. However, no functional data for Oat6 was reported. In the present study, we demonstrate that murine Oat6 mediates the inhibitable transport of estrone sulfate using both Xenopus oocyte expression assay and Chinese hamster ovary (CHO) cells stably transfected with mOat6 (CHO-mOat6). Uptake was virtually eliminated by probenecid and the anionic herbicide 2,4-dichlorophenoxyacetate. The organic anions ochratoxin A, salicylate, penicillin G, p-aminohippurate, and urate inhibited mOat6-mediated accumulation to varying degrees. Transport of estrone sulfate by mOat6 was demonstrated to be saturable, and K(m) estimates of 109.8 +/- 22.6 microM in oocytes and 44.8 +/- 7.3 microM in CHO-mOat6 cells were obtained. Inhibitory constants for 2,4-dichlorophenoxyacetate (15.7 +/- 2.0 microM), salicylate (49.0 +/- 4.4 microM), probenecid (8.3 +/- 2.5 microM), and penicillin G (1,450 +/- 480 microM) were also determined. Accumulation of estrone sulfate mediated by mOat6 was significantly trans-stimulated by glutarate, indicating that mOat6 functions as an organic anion/dicarboxylate exchanger. These data demonstrate for the first time that the novel murine gene Oat6 (Slc22a20) encodes a functional organic anion transporter and mOat6 is indeed the newest member of the OAT gene family.

Mouse RPE cells express and secrete CFB sufficient to promote RPE damage and CNV. This further supports that local complement production may regulate disease processes; however, the reconstitution experiments suggest that additional components may be sequestered from the bloodstream. Understanding the process of ocular complement production and regulation will further our understanding of the AMD disease process and the requirements of a complement-based therapeutic.