Preparation of oligomeric Aβ

Animals

Adult female Long-Evans rats at the age of 4.5 month (Charles River, Wilmington, MA) were randomly divided into two groups. Group 1 (N = 16) comprised rats receiving intravitreal injections of Aβ (5 μL at 1.4 μg/μL as previously published [13]) once every 4 days for a total of three injections. Group 2 (N = 16) rats received intravitreal injections of the control solution (reverse Aβ40–1 peptide) the same way as described for group 1. All rats were sacrificed on the 14th day after initial injection (day 14). Eyes were immediately enucleated and frozen for WB, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) or fixed in 4% paraformaldehyde diluted in Dulbecco’s PBS (Invitrogen, Carlsbad, CA) for 48–72 h prior to paraffin embedding.

Immunohistochemistry (caspase-1, IL-18, NF-κB, active caspase-3)

Paraffin-embedded rat eye tissues were processed following established protocols [13]. Sections from both the Aβ and the control groups were processed simultaneously in an effort to minimize variability in immunoreactivity conditions (N = 3 per group). Primary antibodies recognizing total caspase-1, IL-18, NF-κB, and active caspase-3 are described in Table 1. Non-specific isotype IgGs (Sigma Aldrich) matching the species of primary antibodies are used on negative control tissue sections. For visualization, the slides were developed using the Vector® AEC substrate kit (Vector Laboratories, Burlington ON, Canada) and were counterstained with Mayer’s Hematoxylin (Sigma Aldrich) for the nuclei. Sections processed simultaneously were analyzed and scored so as to avoid difference in immunostaining due to conditions such as temperature and stock of antibodies. Caspase-1, IL-18, and active caspase-3 immunoreactivity was scored in a masked fashion and semi-quantitatively based on a 0–3 point scale. A score of 0 indicates no detectable staining above the background level as compared to the negative control sections, whereas a score of 1, 2, or 3 suggests weak, intermediate, and robust intensity of the immunoreactivity, respectively. The immunoreactivity scores of caspase-1, IL-18, and active caspase-3 were averaged and normalized to the control group.

To detect NF-κB translocalization, an antibody recognizing the phosphorylated Ser 276 locus on NF-κB p65 subunit was used (Table 1). Immunoreactivity was measured quantitatively, in a masked fashion, using a × 60 objective lens and × 10 eyepieces. Positive RPE nuclei were identified as containing both the red AEC chromogen and blue hematoxylin counterstain, thus resulting in a purple appearance distinct from the unlabeled RPE nuclei that were blue in color due to the hematoxylin counterstain alone. The number of NF-κB positive nuclei was converted to percentage of all RPE nuclei in the sample area and was normalized to the control group.

Suspension array for vitreal cytokines

An ELISA-based cytokine assay for vitreal cytokines was carried out (Bio-Plex 200 System, Bio-Rad Laboratories, Hercules, CA). The assay targeted the following cytokines: erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), chemokine (C-X-C motif) ligand 1 (GRO/KC), interferon-gamma (IFN-γ), IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p70, IL-13, IL-17, IL-18, macrophage colony stimulating factor (M-CSF), macrophage inflammatory protein 1alpha (MIP-1α), MIP-3α, regulated on activation, normal T cell expressed and secreted (RANTES), TNF-α, and vascular endothelial growth factor (VEGF). Vitreous from rat eyes in the same group (either Aβ or control) were pooled (N = 7). Experiments were carried out following methods in our earlier publication [15].

Western blot

To determine the level of X-linked inhibitor of apoptosis (XIAP), whole retina tissues (including neuroretina, RPE, Bruch’s membrane, and choroid) from each of the two injection groups were used (N = 5). To investigate IL-18 secretion and gasdermin D (GSDMD) cleavage, RPE-choroid tissues from each of the two injection groups were dissected out and pooled to use (N ≥ 3). Tissues were homogenized in ice-cold RIPA buffer (Thermo Fisher Scientific, Waltham, MA) containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Protein lysates were run under reducing conditions and established blotting procedures were followed. Detailed information on the primary antibodies used in western blotting can be found in Table 1 [11]. As an internal protein loading control, GAPDH was detected either on the stripped membrane or using freshly thawed protein lysates (Table 1). The protein band intensity of XIAP (57 kDa), IL-18 (18 kDa), pro-GSDMD (53 kDa), N-GSDMD (30 kDa), and GAPDH (36 kDa) was individually measured using ImageJ (NIH, Bethesda, MD) and was converted into ratios relative to GAPDH. The final relative intensity of XIAP, IL-18, pro-GSDMD, or N-GSDMD was normalized to the control group.

Reverse transcription PCR (RT-PCR)

RPE morphological assessment

To evaluate the morphological changes of RPE cells due to induced inflammasome activity, we developed a custom Photoshop algorithm to measure the area of a set length of RPE monolayer based on RPE pigmentation. For each animal group, a total of nine sections from each animal were used for the analysis. All RPE micrographs were taken under × 60 magnification and subsequently cropped into 12 cm × 2 cm rectangular areas for further processing. Next, choroidal pigments were manually removed and the RPE-only areas were selected by applying “fuzziness”. Then, the pixel count for the cropped area was obtained. The RPE area measurement was expressed as number of pixels. This area measure was equivalent to, but more accurate than measuring the thickness of RPE monolayer.

RPE cell nuclei count and retinal thickness measurement

Following our established protocol, retinal cross sections within 200 μm distance from the optic disc were chosen for the analysis because of their uniform retinal thickness regardless of embedding orientation [13, 18]. Briefly, RPE nuclei were counted by scanning the whole retinal section under × 20 magnification in 10³ μm increments. Retinal thickness was measured from the inner limiting membrane to the photoreceptor outer segments/RPE junction. The mean values of RPE nuclei count per increment, retinal thickness as well as ONL thickness were averaged over a minimum of 4–6 retinal sections per animal at each time point.

Statistical analyses

Data are presented as mean ± SD. Non-parametric tests were used throughout the study except the vitreal cytokine levels and RT-PCR were analyzed by one-tailed Student’s t test. For the non-parametric comparisons between the two groups (Aβ vs control), one-tailed Mann-Whitney U tests were used. All analyses were conducted with GraphPad Prism version 6 (GraphPad Software, La Jolla, CA). Statistical significance was set at p ≤ 0.05.

Pro-inflammatory cytokine secretion in vitreous

To understand the overall inflammatory status in the rat eyes, we collected and examined the vitreous samples using an ELISA-based cytokine profile assay. We found monocytes chemoattractant protein 1 (MCP-1), chemokine (C-X-C motif) ligand 1 (GRO/KC), vascular endothelial growth factor (VEGF), and macrophage inflammatory protein 3 alpha (MIP-3α) were increased more than 50% in the Aβ injected eyes compared to those in the reverse Aβ injected (hereafter, referred to as “control”) eyes (Fig. 1). A smaller, but significant, increase was associated with vitreal IL-1β, a mature secreted product following NLRP3 inflammasome activation. However, IL-18, another NLRP3 inflammasome product, was downregulated in the vitreous sample from the Aβ injection group compared to the control (Aβ 1142.67 ± 64.15 pg/mL; control 1258.13 ± 56.49 pg/mL), which led us to further look at the inflammasome activity in Aβ-injected rat eyes. Measurements of the additional cytokines studied here are given in Additional file 2: Figure S2.

NLRP3 inflammasome activation by multiple Aβ injections

NLRP3 inflammasome activation is achieved by a sequential stimulation of a priming signal and an activation signal. To examine inflammasome activation in RPE, we first looked at the nuclear translocalization of NF-κB, a signature event of NF-κB activation. Using an antibody specifically targeting the phosphorylated p65 subunit of NF-κB, we found strong immunoreactivity in the nuclei of RPE cells in Aβ-injected eyes compared to control eyes (Fig. 2a–c). Next, we immunolabeled caspase-1 in the RPE layer of both Aβ-injected and control eyes, revealing that caspase-1 immunoreactivity was enhanced by 77% compared to the control eyes (Fig. 3a). Consistent with the elevation of IL-1β in the vitreous, we found the other inflammasome activation product, IL-18, was also upregulated, showing more than 6-fold higher immunoreactivity in the RPE layer of Aβ-injected eyes (Fig. 3b–c) and a 58% increase of IL-18 band intensity in protein lysates from the Aβ injected eyes, compared to the control eyes (Fig. 3d–e).

Cell death pathways triggered by Aβ injections

To study the forms of RPE cell death often seen in late AMD, especially GA, we next analyzed morphological changes in RPE after Aβ injections. Using a custom Photoshop algorithm, we first applied a pigment threshold to identify the area occupied by RPE within a standard 12 cm × 2 cm boxed area centered on the RPE monolayer. Next, the number of pixels within the pigment threshold of that area was obtained as an index of the area measurement occupied by the RPE. Higher pixel values indicated thicker RPE monolayers. By comparing the Aβ-injected to the control groups, we observed a 2-fold increase in the number of pixels, suggesting a significant area increase in the RPE, presumably due to swelling of the RPE cells (Fig. 4).

To understand what caused the RPE to enlarge or swell, we prepared RPE-choroid tissue homogenates from both injection groups and tested them for gasdermin D (GSDMD), a protein executing pyroptosis and whose proteolytic cleavage is triggered by both canonical and non-canonical inflammasome activation in immune cells [19–21]. The cleaved GSDMD N-terminal fragment (N-GSDMD) at 30 kDa was increased, while the uncleaved pro-GSDMD full-length protein at 53 kDa was decreased, in the Aβ group compared to the control group (Fig. 5).

Next, we also tested for apoptosis involvement using caspase-3 activation as a surrogate marker. By immunohistochemistry, we found 2.5- to 4.5-fold higher immunoreactivity levels of active caspase-3 in photoreceptor inner segments and RPE of the Aβ group, respectively, compared to the control group (Fig. 6a–b). Two examples of active caspase-3 immunoreactivity from both Aβ-injected and control eyes were provided to demonstrate the enhanced red AEC labeling in photoreceptor inner segments and RPE from Aβ-injected eyes (Fig. 6c). To further support this finding, we measured the mRNA and protein levels of X-chromosome-linked inhibitor of apoptosis (XIAP), a classic anti-apoptosis factor. At the transcriptional level, there was a 10-fold reduction in the Aβ group compared to the controls (Fig. 7a). Concomitant with the mRNA data, there was a 33% reduction in XIAP protein compared to the control group (Fig. 7b–c).

Finally, to determine whether the activation of pyroptotic and apoptotic pathways caused overt anatomical changes to the retina, we counted the RPE nuclei and assessed retinal thickness. Our results showed that there was no significant RPE nuclei loss or retinal thickness changes, including the ONL thickness, in the retinal cross sections of Aβ-injected eyes compared to the control eyes (Fig. 8). These measurements indicated no overt anatomical/histological changes were evident at the time point studied here.

RPE inflammasome activation is a feature of the model

NLRP3 inflammasome activation, one of the fundamental innate immune defense mechanisms, has recently been studied for its role in the development of AMD [22]. Our earlier study indicated that a single Aβ injection resulted in a peak in pro-inflammation at 4 days post injection, but then dramatically subsided [13]. In the current study, we extended the duration of pro-inflammation from 4 to 14 days by making sequential injections of Aβ every 4 days to achieve better retinal penetration and mimic a chronic inflammatory microenvironment in the outer retina (Additional file 3: Figure S3). Our results demonstrated that longer exposure to pro-inflammation triggered robust NF-κB p65 subunit translocalization in RPE nuclei, elevated levels of total caspase-1 immunoreactivity, and enhanced secretion of IL-1β in vitreous and increased IL-18 presence in retina. Collectively, these results support inflammasome activity in the RPE (Fig. 3, Additional file 3: Figure S3). When compared with other inflammasome studies on RPE cells including ours, the current study demonstrated significant increase of IL-18 in neuroretina and RPE-choroid, but not in the vitreous where IL-1β was found upregulated by more than 50% (Figs. 1 and 3, Additional file 2: Figure S2). This secretion pattern is unique in that it diversifies the downstream biological events of these inflammasome cytokines: in our previous acute model of single Aβ injection, vitreal IL-18 increased by more than 3-fold at day 14 whereas vitreal IL-1β elevated by less than 50% compared to the controls [13]. Such discrepancy in IL-1β/18 vitreal levels can be partially explained by the “distinct licensing requirements” for processing these two cytokines by NLRP3 inflammasomes, which might involve further regulation of caspase-11 and/or reactive oxygen species (ROS), as evident in immune cells [23]. This mechanism is also very likely to be true in RPE cells since Aβ has recently been shown to induce NLRP3 inflammasome activation via NADPH oxidase- and mitochondria-dependent ROS pathway in vitro [24]. Other studies suggest that a high level of retinal IL-18 (including RPE-choroid) is more related to the cell death events in GA [6, 25], which support our findings that higher IL-18 immunoreactivity levels in Aβ-injected retina are correlated with the activation of pyroptotic pathway.

Sequential injections of Aβ also provide a good model to study Aβ’s role in inflammasome activation in the eye. Considered as one of the pathological hallmarks in AD, the deposition of Aβ in the AD brain is associated with elevated NLRP3 inflammasome activity, particularly the elevation of IL-1β production [26–28]. Using microglia culture models, Halle et al. demonstrated the importance of NLRP3 inflammasome activation for the recruitment of microglia to Aβ deposits in the AD brain [29]. Heneka et al. further discovered that in the absence of NLRP3 or caspase-1, mice carrying mutations associated with familial AD were largely protected from spatial memory loss, and demonstrated reduced IL-1β secretion and enhanced Aβ clearance [27]. However, the involvement of these pathways has not been well established in the eye tissue. As a continuation from our previous studies [13], our current Aβ multi-injection model recapitulates seminal features associated with each step of the NLRP3 inflammasome activation cascades. Therefore, by giving the animals sequential Aβ injections, we were able to sustain Aβ-induced pro-inflammatory responses in the neuroretina and RPE at a comparably high level until day 14, when the presence of Aβ was much diminished in the single Aβ injection model [13].

Pyroptosis and apoptosis may contribute to RPE cell death in this model

The involvement of NLRP3 inflammasome activation in RPE has been studied in many different AMD models [30–33]. However, once the inflammasome is activated, little is known of the exact biological events that occur and whether these events lead to cell death. Using rodent and non-human primate models, Doyle and colleagues demonstrate the efficacy of IL-18 treatment as a potential alternative, adjuvant therapy for CNV [34–36]. On the other side, Ambati and collaborators showed evidence that IL-18 drives RPE degeneration after NLRP3 inflammasome activation in murine models [6, 37]. In the current study, we also assessed the changes after activation of the NLRP3 inflammasome by Aβ. Intriguingly, after prolonged pro-inflammation in outer retina, we found enlarged or swollen RPE cells and significant increases in the proteolytic cleavage of full-length GSDMD in the RPE-choroid tissues. As reported by earlier studies, the cytolytic effects of pyroptosis are mediated by the oligomerization of the GSDMD’s N-terminal fragments (N-GSDMD) in cellular membrane, resulting in the cell-burst pore formation [38]. Hence, it confirms the activation of pyroptotic pathway in the Aβ-injected animals [19]. Therefore, we have demonstrated the co-existence of two events that occur after inflammasome activation: the secretion of mature pro-inflammatory cytokines, including the inflammasome products (IL-18 and IL-1β) and morphological and western blot evidence that supports the GSDMD-mediated pyroptotic pathway activation in RPE cells. Such an orchestrated response has also been seen in non-ocular cell types [39].

Unlike pyroptosis, which rapidly lyses the cell, apoptosis is considered as a non-inflammatory and non-cytolytic form of programmed cell death. From a canonical point of view, pyroptosis is biochemically characterized as caspase-1 dependent and caspase-3 independent, whereas apoptosis is often caspase-3 dependent and caspase-1 independent. Interestingly, in the current study, we observed parallel cleavage of both caspase-1 and caspase-3 in the RPE tissue, challenging the dogma of their mutual exclusiveness. Although it is biologically impossible for one single cell to undergo both distinctive cell death pathways, it is still likely for one type of tissue, such as the RPE monolayer in the retina, to accommodate these pathways in a spatially discrete and stimulus dose-dependent manner. One study using murine bone marrow-derived macrophages exploited the potential of crosstalk between pyroptosis and apoptosis. The authors exhibited a DNA-dose dependent integral model for cell death, with apoptosis seen at a lower-dose of DNA stimulation and pyroptosis at higher doses. They further concluded that such an explicit response is regulated through caspase-8 activation [40].

Despite the fact that our data supported the activation of both pyroptotic and apoptotic pathways in the RPE-choroid tissue of multiple Aβ-injected eyes, there were no significant anatomical changes as indicated by RPE nuclei counts and retinal thickness measurements (Fig. 8). All the biochemical markers used in this study (IL-18, IL-1β, NF-κB, caspase-1, caspase-3, GSDMD, XIAP) proved changes leading towards RPE atrophy via both cell death pathways. Combined with the anatomical data, our results suggest a transition between early and middle-to-late stage of dry AMD, where RPE cells begin to show early biochemical signs of cell death and before any observable retinal structural changes.

RPE-specific vs choroidal macrophage-mediated responses in vivo

The ability to dissect RPE-choroid from neuroretina was demonstrated in our earlier work [11, 13] and those of others [41]. However, it is possible that in the RPE-choroid sample, the choroidal component with macrophages, may also contribute to the inflammasome activity in our in vivo work. Testing for activated macrophages by immunohistochemistry in paraffin sections or by microdissection of choroidal tissue alone [42] may help us better understand the migration of immune cells from choroid in our Aβ-injected animal groups. It is possible that the choroidal macrophages, important immune cells associated with AMD [43, 44] in combination with RPE may both work to exacerbate the chronic inflammatory milieu in the AMD eye. Therefore, future in vitro work on cultured RPE cells and choroidal macrophages will allow us to define the role of each cell type without the confounding effects from the other.