Jiangyuan Gao

Inflammatory mediators induced by amyloid-beta in the retina and RPE in vivo: implications for inflammasome activation in age-related macular degeneration.

PURPOSE Drusen are hallmarks of age-related macular degeneration (AMD). Amyloid-beta 1-40 (Aβ 1-40), a constituent of drusen, is known to stimulate inflammatory pathways in RPE; however, its effect in vivo is not known. The purpose of this study was to examine the effect of Aβ 1-40 on cytokine expression and inflammasome activation relevant to AMD in an animal model. METHODS Wild-type rats received intravitreal injections of Aβ 1-40, and eyes were taken at days 1, 4, 14, and 49 postinjection. The RPE, neuroretina, and vitreous were analyzed for cytokine expression, inflammasome activation, and microglial response via RT-PCR, immunohistochemistry, and suspension array assay. Retinal cell loss was assessed via apoptotic markers and retinal thickness. RESULTS Aβ 1-40 stimulated upregulation of IL-6, TNF-α, IL-1β, IL-18, caspase-1, NLRP3, and XAF1 genes in the RPE/choroid and the neuroretina. Increased IL-1β and IL-6 immunoreactivity was found in retinal sections, and elevated levels of IL-1β and IL-18 were found in the vitreous of Aβ-injected eyes. Aβ 1-40 induced a moderate increase in CD11b/c-reactive cells on day 1 postinjection only. No evidence of the proapoptotic XAF1 protein, p53, TUNEL immunoreactivity, or retinal thinning was observed. CONCLUSIONS These results confirm earlier in vitro work and support the proinflammatory role of drusen component Aβ 1-40 in the RPE and retina. Inflammasome activation may be responsible for this effect in vivo. This model is useful for understanding cellular triggers of inflammasome activation and proposed early inflammatory events in the outer retina associated with the etiology of AMD.

NLRP3 Inflammasome: Activation and Regulation in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly in industrialized countries. AMD is a multifactorial disease influenced by both genetic and environmental risk factors. Progression of AMD is characterized by an increase in the number and size of drusen, extracellular deposits, which accumulate between the retinal pigment epithelium (RPE) and Bruchs membrane (BM) in outer retina. The major pathways associated with its pathogenesis include oxidative stress and inflammation in the early stages of AMD. Little is known about the interactions among these mechanisms that drive the transition from early to late stages of AMD, such as geographic atrophy (GA) or choroidal neovascularization (CNV). As part of the innate immune system, inflammasome activation has been identified in RPE cells and proposed to be a causal factor for RPE dysfunction and degeneration. Here, we will first review the classic model of inflammasome activation, then discuss the potentials of AMD-related factors to activate the inflammasome in both nonocular immune cells and RPE cells, and finally introduce several novel mechanisms for regulating the inflammasome activity.

CFH Y402H polymorphism and the complement activation product C5a: effects on NF-κB activation and inflammasome gene regulation

Background/aims The Y402H polymorphism in the complement factor H (CFH) gene is an important risk factor for age-related macular degeneration (AMD). Complement activation products and proinflammatory cytokines are associated with this polymorphism at the systemic level, but less is known of the associations in the outer retina of the genotyped eye. Here we investigate complement activation products and their role in nuclear factor (NF)-κB activation and gene expression of the NLRP3 inflammasome pathway. Methods Postmortem donor eyes were genotyped for the CFH Y402H polymorphism and assessed for complement C3a, C5a, interleukin (IL)-18 and tumour necrosis factor (TNF)-α. ARPE19 cells were stimulated basolaterally with C5a or TNF-α in polarised cultures. NF-κB activation was assessed with a reporter cell line. Gene expression of inflammasome-related (NLRP3, caspase-1, IL-1β and IL-18) and classic inflammatory (IL-6 and IL-8) genes was studied. The distribution of inflammasome products, IL-1β and IL-18, was studied in postmortem donor eyes with AMD pathologies. Results Eyes with the homozygous at-risk variant demonstrated higher levels of C5a, IL-18 and TNF-α in Bruchs membrane and choroid. C5a promoted NF-κB activation and upregulation of IL-18 in polarised ARPE19. TNF-α promoted NF-κB activation and gene expression of caspase-1, IL-1β, IL-18, IL-6 and IL-8, but downregulated NLRP3. In eyes with geographic atrophy, strong immunoreactivity was observed for inflammasome products IL-1β and IL-18 compared with age-matched controls. Conclusion The at-risk polymorphism of the CFH Y402H may contribute to AMD disease process through increased complement and NF-κB activation, and the upregulation of IL-18, a product of inflammasome activation.

Drusen and Pro-inflammatory Mediators in the Post-Mortem Human Eye

With age and drusen accumulation, the environment of the eye tends to shift more towards a pro-inflammatory state (Figure 6) through either IL-1β and/or possibly IFN pathways. Little change is induced in the complement inhibitor, CFI, in association with drusen, suggesting a failure to activate protective mechanisms against complement activation. Alternatively, the lack of change in CFI accumulation level could indicate that that the pro-inflammatory actions of drusen may not yet be enough to trigger inhibition of the complement cascade in healthy eye tissue. Here, we identified three inflammatory molecules in the postmortem human eye, p-STAT3, CXCL-10, and CXCL-11, to be strongly correlated with the presence of drusen within a population controlled for age (≤57 years old, Figure 6). This pattern of accumulation suggests that these molecules are closely involved with the presence of drusen rather than with aging, and may represent a predisposition toward AMD pathogenesis. A recent microarray study on mouse RPE cells reported expression changes in over 315 genes associated with advanced age [48]. Many pathways they identified were also found in our prior microarray study (unpublished observation) [16]. Similarly, Chen et al. [49] found that the mouse neuroretina displayed age-related upregulation of the complement cascade and activation of retinal microglia [49]. Curiously, neither study found expression changes in any of the gene products examined in this study. It is important to note that drusen-like deposits have not been documented in wild-type-mouse eyes [50]. Thus, one interpretation for this discrepancy may be that the genes identified in our microarray studies, and specifically the gene products studied here reflect a drusen-specific response rather than a general response to aging. Our studies now set the stage for future experiments testing the function of each of these potential mediators in the pathogenesis of AMD. One approach might involve assessing the response of each mediator following Aβ and/or AGE injection into young wild-type mice to determine degree of mimicry to AMD pathology. Figure 6 Summary diagram depicting the distribution of IL-1β, RSAD2, p-STAT3, and CXCL-10/11 immunoreactivity in post-mortem eye tissue with respect to aging and present of drusen. Icons denote the relative immunostaining of each molecule in a given retinallocation. ... Acknowledgments We thank Jonathan Tang, Jonathan Coleman, and Dr. Ian Clark for discussion and editing of the manuscript. This work was funded by Canadian Institutes of Health Research (CIHR-MOP-97806) to J.A.M. Grant Support Supported by Canadian Institutes of Health Research (CIHR) Grant# MOP-97806 (to JAM).