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01.12.2018 | Research | Ausgabe 1/2018 Open Access

Journal of Neuroinflammation 1/2018

Evidence for the activation of pyroptotic and apoptotic pathways in RPE cells associated with NLRP3 inflammasome in the rodent eye

Zeitschrift:
Journal of Neuroinflammation > Ausgabe 1/2018
Autoren:
Jiangyuan Gao, Jing Z. Cui, Eleanor To, Sijia Cao, Joanne A. Matsubara
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12974-018-1062-3) contains supplementary material, which is available to authorized users.

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.
Zusatzmaterial
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)
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