A Targeted Inhibitor of the Alternative Complement Pathway Accelerates Recovery From Smoke-Induced Ocular Injury

Abstract

Purpose: Morphologic 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.

Methods: Mice 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.

Results: The 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.

Conclusions: Taken 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.

Figure 1

Posttreatment with CR2-fH reverses smoke-induced impairment of contrast sensitivity. Optomotor responses were analyzed in C57BL/6J mice after 6 months of cigarette smoke exposure (CSE) or room air followed by 3 months of treatment with nothing, PBS, or alternative complement pathway inhibitor, CR2-fH. (A) Visual acuity was measured by identifying the spatial frequency threshold at a constant speed (12°/s) and contrast (100%). Spatial frequency thresholds were not affected by CSE or treatment. (B) Contrast sensitivity was measured by taking the reciprocal of the contrast threshold at a fixed spatial frequency (0.131 cyc/deg) and speed (12°/s). We previously determined that this spatial frequency falls within the range of maximal contrast sensitivity for 9-month-old C57BL/6J mice (data not shown). After CSE, mice showed a significant reduction in contrast sensitivity compared to controls, which was still maintained after 3 months of CSE cessation. However, smoke-exposed mice treated with CR2-FH showed a contrast sensitivity that was similar to room air controls and significantly higher than in PBS-treated mice following CSE. Data are expressed as mean ± SEM (n = 4–6 per condition; *P < 0.05).

Figure 2

Gene expression changes in ocular tissues following cessation of smoke and treatment with CR2-fH. Analysis of marker gene expression in smoke-exposed mice following cessation of cigarette smoke exposure (CSE) and treatment with PBS or alternative complement pathway inhibitor, CR2-fH, using quantitative RT-PCR on cDNA generated from retina and RPE/choroid/sclera fraction. Please note that with the exception of Rho, Opn1sw, and Opn1mw, which were examined in the retina fraction, all other genes were examined in the RPE/choroid/sclera fraction. Quantitative values were obtained by cycle number (Ct value), determining the difference between the mean experimental and control (Actb) ΔCt values for PBS- and CR2-fH–treated mice following CSE versus room air–exposed mice (fold difference). Candidates were examined from a number of categories including photoreceptor cell function (Rho, Opn1sw, Opn1mw, Rpe65), complement activation (C3, Hc, Cfb, Cfd, Cfh, Cd55, Cd59a), control of angiogenesis (Vegfa, Serpinf1), oxidative stress (Hif1a, Cp), autophagy (Lyz1, Lamp2, Klc3), mitochondrial function (Mfn1, mt-Co1, Dnm1l, Ndufb8, Pfkfb1, Hmox1), and immune response (IL-17 and RoRγ). Significant changes were identified in several categories for CR2-fH–treated mice, in particular, suggesting decreased complement activation and enhanced energy production. Statistics represent comparisons between PBS- and CR2-fH-treated animals following CSE versus never smokers and differences between CSE mice treated with PBS versus CR2-fH. Data are expressed as mean ± SEM (n = 3 per condition; *P < 0.05 analyzing fold changes between treatment groups versus never smokers; #P < 0.01 analyzing fold change differences between CSE mice treated with PBS versus CR2-fH).

Figure 3

CR2-fH rescues ultrastructural changes in the outer retina in mice following cigarette exposure. Electron micrographs of the outer retina obtained from age-matched mice after 6 months of cigarette smoke exposure (CSE) or room air followed by 3 months of treatment with PBS (B) (smoke + PBS), CR2-fH (C) (smoke + alternative complement pathway inhibitor, CR2-fH), or 3 additional months of room air (A) (control, never smokers) were compared. While all animals had the same number of photoreceptors per row (∼9), the nuclei were more densely packed in the smoke + PBS– when compared to the control and CR2-fH–treated animals. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments.

Figure 4

CR2-fH rescues ultrastructural changes in RPE/BrM/CC complex in mice following cigarette exposure. Electron micrographs of the retinal pigment epithelium/Bruch's membrane/choriocapillaris complex (RPE/BrM/CC) obtained from age-matched mice after 6 months of cigarette smoke exposure (CSE) or room air followed by 3 months of treatment with PBS (smoke + PBS), CR2-fH (smoke + alternative complement pathway inhibitor, CR2-fH), or 3 additional months of room air (control, never smokers) were compared, providing an RPE/BrM/CC overview (A–C), or focusing on mitochondria (D–F) and BrM (G–I). In an animal raised in room air (control, never smoker), BrM exhibits an organized pentalaminar structure consisting of RPE basement membrane, inner collagenous layer, middle elastic layer, outer collagenous layer, and choriocapillaris basement membrane, and the choriocapillaris endothelium has fenestrations (arrowheads) along the entire membrane (A, G). (B, H) The RPE/BrM/CC in a mouse following CSE (smoke + PBS) exhibits pathologic changes. Bruch's membrane is disorganized, no longer showing a pentalaminar structure, and large deposits (asterisks) are present in the outer collagen layer (OCL). Notably, there is significant fenestration loss and/or endothelial cell thickening adjacent to OCL deposits. (C, I) The RPE/BrM/CC appears normal in mice treated with CR2-fH following CSE (smoke + CR2-fH). The morphologic features of mitochondria shown in mice exposed to smoke mitochondria have degraded outer membranes and disorganized cristae (E), but normal appearance in never smokers (D) and mice treated with CR2-fH following CSE (F).

Figure 5

Mitochondrial localization is normal 3 months after cessation of smoke exposure. Mitochondrial position was determined from electron micrographs (depicted in Fig. 3) by determining their centroid coordinates as a percentage of the corresponding retinal pigment epithelium (RPE) length and thickness, respectively. Each centroid was subsequently assigned to one of four bins (basolateral, basal, central, or apical) and expressed as percent of total mitochondria (MITO). Mitochondria are localized predominantly to the basolateral and basal compartments of the RPE cells with fewer localized in the central and apical portions in never smokers. While we previously reported that cigarette smoke exposure (CSE) affects the mitochondrial distribution in C57BL/6J mice after 6 months, with mitochondria exhibiting an apical shift from the basal to central compartment, mitochondrial distribution is back to normal 3 months after the cessation of CSE (smoke + PBS), and is not further affected by complement inhibition (smoke + CR2-fH).