The online version of this article (https://doi.org/10.1186/s12974-018-1062-3) contains supplementary material, which is available to authorized users.
Age-related macular degeneration (AMD) is a devastating eye disease causing irreversible vision loss in the elderly. Retinal pigment epithelium (RPE), the primary cell type that is afflicted in AMD, undergoes programmed cell death in the late stages of the disease. However, the exact mechanisms for RPE degeneration in AMD are still unresolved. The prevailing theories consider that each cell death pathway works independently and without regulation of each other. Building upon our previous work in which we induced a short burst of inflammasome activity in vivo, we now investigate the effects of prolonged inflammasome activity on RPE cell death mechanisms in rats.
Long-Evans rats received three intravitreal injections of amyloid beta (Aβ), once every 4 days, and were sacrificed at day 14. The vitreous samples were collected to assess the levels of secreted cytokines. The inflammasome activity was evaluated by both immunohistochemistry and western blot. The types of RPE cell death mechanisms were determined using specific cell death markers and morphological characterizations.
We found robust inflammasome activation evident by enhanced caspase-1 immunoreactivity, augmented NF-κB nuclear translocalization, increased IL-1β vitreal secretion, and IL-18 protein levels. Moreover, we observed elevated proteolytic cleavage of caspase-3 and gasdermin D, markers for apoptosis and pyroptosis, respectively, in RPE-choroid tissues. There was also a significant reduction in the anti-apoptotic factor, X-linked inhibitor of apoptosis protein, consistent with the overall changes of RPE cells. Morphological analysis showed phenotypic characteristics of pyroptosis including RPE cell swelling.
Our data suggest that two cell death pathways, pyroptosis and apoptosis, were activated in RPE cells after exposure to prolonged inflammasome activation, induced by a drusen component, Aβ. The involvement of two distinct cell death pathways in RPE sheds light on the potential interplay between these pathways and provides insights on the future development of therapeutic strategies for AMD.
Additional file 1: Figure S1. Preparation of oligomeric Aβ. (A) Various incubation conditions were tested for the optimal oligomeric Aβ generation. Small oligomeric (dimeric and trimeric) and monomeric Aβ species were generated after a shorter period of incubation time (24 h, 100 μM). Note that the higher molecular Aβ species began to increase after 48 h (MW > 170 kDa). (B) Western blot of the 48 h-incubated oligomeric Aβ solution, prepared for intraocular injections (7 μg/5 μL). The control peptide was prepared the same way as Aβ, but was non-reactive to the 6E10 antibody detection. (EPS 5439 kb)12974_2018_1062_MOESM1_ESM.eps
Additional file 2: Figure S2. Additional vitreal cytokine levels following sequential Aβ injections. A list of rat cytokines in addition to Fig. 1 was examined in vitreous samples from both the Aβ-stimulated and the control animals’ eyes using the ELISA-based premade assay. Unlike Fig. 1’s data, all these cytokines showed less than 50% change of concentration levels between the two animal groups, many of which had even lower measurements in the Aβ-stimulated group, for instance, IL-18. N = 7, Student’s t test, * p < 0.05. RANTES, regulated on activation normal T cell expressed and secreted; GM-CSF, granulocyte macrophage colony stimulating factor; EPO, erythropoietin; G-CSF, granulocyte colony stimulating factor; M-CSF, macrophage colony stimulating factor. (EPS 170 kb)
Additional file 3: Figure S3. Retinal distribution of injected Aβ. To ensure the effectiveness of sequential Aβ injections, retinal cross-sections from day 14 (indicated as “3X Aβ day 14”) were stained for the injected Aβ using the 6E10 antibody (Table 1). As visualized by the red AEC, Aβ exhibited good penetration of all retinal layers from retinal ganglion cell layer to RPE. Compared to its counterpart from our previous acute Aβ model (indicated as “1X Aβ day 14”), the current 3X Aβ day 14 retinal cross-sections showed much stronger Aβ immunoreactivity, suggesting a higher retinal Aβ concentration maintained by the sequential Aβ injections. Such robust Aβ immunoreactivity in the 3X Aβ day 14 tissue (neuroretina + RPE) was comparable to those of 1X Aβ retinal tissues at days 1 and 4, with even more enhanced labeling in the outer nuclei layer. RGC, retinal ganglion cell; IPL, inner plexiform layer; INL, inner nuclei layer; OPL, outer plexiform layer; ONL, outer nuclei layer; IS/OS, inner/outer segments. Scale bar 20 μm. (EPS 13727 kb)
Age-Related Eye Disease Study Research G. A simplified severity scale for age-related macular degeneration: AREDS report no. 18. Arch Ophthalmol. 2005;123:1570–4. CrossRef
Amoaku WM, Chakravarthy U, Gale R, Gavin M, Ghanchi F, Gibson J, Harding S, Johnston RL, Kelly SP, Lotery A, et al. Defining response to anti-VEGF therapies in neovascular AMD. Eye (Lond). 2015;29:721–31. CrossRef
Sarks JP, Sarks SH, Killingsworth MC. Evolution of geographic atrophy of the retinal pigment epithelium. Eye (Lond). 1988;2(Pt 5):552–77. CrossRef
Hageman GS, Luthert PJ, Victor Chong NH, Johnson LV, Anderson DH, Mullins RF. An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch’s membrane interface in aging and age-related macular degeneration. Prog Retin Eye Res. 2001;20:705–32. CrossRefPubMed
Gao J, Liu RT, Cao S, Cui JZ, Wang A, To E, Matsubara JA. NLRP3 inflammasome: activation and regulation in age-related macular degeneration. Mediat Inflamm. 2015;2015:690243. CrossRef
Hoh Kam J, Lenassi E, Jeffery G. Viewing ageing eyes: diverse sites of amyloid Beta accumulation in the ageing mouse retina and the up-regulation of macrophages. PLoS One. 2010;5(10):e13127.
Wang K, Yao Y, Zhu X, Zhang K, Zhou F, Zhu L. Amyloid β induces NLRP3 inflammasome activation in retinal pigment epithelial cells via NADPH oxidase- and mitochondria-dependent ROS production. J Biochem Mol Toxicol. 2016;31:e21887.
Kerur N, Hirano Y, Tarallo V, Fowler BJ, Bastos-Carvalho A, Yasuma T, Yasuma R, Kim Y, Hinton DR, Kirschning CJ, et al. TLR-independent and P2X7-dependent signaling mediate Alu RNA-induced NLRP3 inflammasome activation in geographic atrophy. Invest Ophthalmol Vis Sci. 2013;54:7395–401. CrossRefPubMedPubMedCentral
Doyle SL, Ozaki E, Brennan K, Humphries MM, Mulfaul K, Keaney J, Kenna PF, Maminishkis A, Kiang AS, Saunders SP, et al. IL-18 attenuates experimental choroidal neovascularization as a potential therapy for wet age-related macular degeneration. Sci Transl Med. 2014;6:230ra244. CrossRef
Newman AM, Gallo NB, Hancox LS, Miller NJ, Radeke CM, Maloney MA, Cooper JB, Hageman GS, Anderson DH, Johnson LV, Radeke MJ. Systems-level analysis of age-related macular degeneration reveals global biomarkers and phenotype-specific functional networks. Genome Med. 2012;4:16. CrossRefPubMedPubMedCentral
Bressler SB, Bressler, N.M.: Age-related macular degeneration: non-neovascular early AMD, intermediate AMD, and geographic atrophy . In Retina (Ryan SJ ed., vol. 2, 5th edition. London: Elsevier; 2014. p. 1150–82.
- Evidence for the activation of pyroptotic and apoptotic pathways in RPE cells associated with NLRP3 inflammasome in the rodent eye
Jing Z. Cui
Joanne A. Matsubara
- BioMed Central