Background
The complement cascade comprises three pathways – classical, mannose-binding lectin, and alternative pathway – all of which converge at the proteolysis of complement component 3 (C3), promoting complement activation and downstream assembly of membrane attack complex (MAC) [
1]. Activation of the classical pathway is triggered by complement component 1q (C1q) binding to immune complexes on pathogenic cell surfaces, or atypical activators such as modified lipids, apoptotic cells [
2], advanced glycation end products [
3] and C-reactive protein [
4]. C1q binding promotes the proteolysis of C4, C2, C3 and the downstream activation of the complement cascade, propagating the effector functions of complement, including the lysis of target cells [
1].
In neurodegenerative diseases such as Age-Related Macular Degeneration (AMD), complement dysregulation as a consequence of environmental pressures and/or gene mutations, is known to contribute to disease progression [
5‐
7]. AMD is the leading cause of blindness in the Western World, posing a significant economic burden with an annual global cost of 350 billion dollars [
8]. Atrophic or “dry” late-stage AMD is characterised by the emergence of geographic atrophy, in which a central retinal lesion in the photoreceptors and retinal pigment epithelium (RPE) develops and progressively expands over time [
8]. Genome-wide association studies (GWAS) indicate a link between dysregulation of multiple complement pathways (e.g. C3, C4, C1s, CFI, SERPING1) and the progressive expansion of the macular lesion in geographic atrophy [
9]. In addition, histopathological investigations show a range of complement components and factors from all pathways present in drusen of AMD donor eyes (reviewed in [
7]). These drusen components are associated with the accrual of activated microglia and infiltrating blood-borne macrophages in the outer retina, which is a well-established feature of AMD pathogenesis [
10].
Our previous studies have shown upregulation of a range of complement pathway genes, including
Cfb,
C3 and
Cfh after photo-oxidative retinal degeneration, and demonstrate a critical role for C3-expressing retinal macrophages [
11,
12]. However, the contribution of each complement pathway to C3 activation is still largely unknown, impeding the development of effective anti-complement drugs for retinal degenerations [
13,
14]. Other investigators have utilised
Cfb−/−,
C1qa −/− and
Mbl−/− mice to demonstrate the involvement of all complement pathways to experimental choroidal neovascularization [
15]. Despite strong evidence supporting the involvement of the alternative pathway in retinal degenerations [
16], it has been suggested that the alternative pathway is not the only contributor towards retinal degenerations, and that other complement pathways may also facilitate disease progression [
15].
Emerging evidence has implicated the classical pathway in AMD, including genetic polymorphisms [
17] and an abundance of autoantibodies associated with AMD [
18‐
20]. C1q, the activator molecule of the classical complement pathway, has also been implicated in a number of complement-independent processes including activation of the inflammasome, another key component of the innate immune system [
21], and by acting as a bridging molecule between the innate and adaptive immune system [
22,
23]. Recently, C1q was found to promote microglial activation and degeneration in retinal ischemia/reperfusion (I/R) injury [
24]. However, the role of C1q in the progression of retinal degenerations remains unclear. In this study, we aimed to elucidate the contribution of the classical complement pathway to disease progression, using a model of photo-oxidative damage (PD), in which complement activation by microglia and macrophages is a key feature leading to progressive photoreceptor loss [
11]. Our findings illustrate the role of the classical complement pathway in focal retinal degeneration, showing subretinal macrophages as key players in delivering C1q, and that blocking C1q activity during degeneration may be a useful strategy to slow the progression of retinal atrophy.
Methods
Animal experimentation
All procedures were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for Use of Animals in Ophthalmic and Vision Research. The study was approved by the Australian National University Animal Experimentation Ethics Committee (Ethics ID: A2014/56 and A2017/41). Complement component 1, q subcomponent, alpha polypeptide knock out (C1qa−/−) mice were used for this study (C1qa < tm1Mjw>). C57BL/6 J mice were used as wild-type (WT) controls. Animals were born and raised in dim (5 lx) cyclic light conditions in 12:12 h cycle, and house in individual vented cages (IVCs). Food and water was provided in constant supply and cages changed on a weekly basis. Adult mice aged between postnatal day (P) 60-90 were used for all experiments. Equal numbers of male and female mice were used throughout the study to avoid any gender biases. Animals with no exposure to photo-oxidative damage were used as dim-reared controls.
To induce photo-oxidative damage (PD), mice were housed in Perspex boxes coated with a reflective interior, and exposed to 100Klux of natural white LED for 1, 3, 5 and 7 days, with free access to food and water, as described in our previous methodology [
25]. Some animals were recovered in dim light conditions and were collected at 8, 10 and 14 days following the onset of PD. Each animal was administered with pupil dilator eye-drops twice daily during PD (Atropine Sulphate 1%
w/
v eye-drops; Bausch and Lomb, NSW, Australia). Electroretinography (ERG) was used to measure mouse retinal function in response to full-field flash stimuli under scotopic conditions as described previously [
25]. Animals were euthanized with CO
2 prior to tissue collection. Eyes were collected and processed for cryosections or RNA extraction, as previously described in our publications [
12,
25].
C1q neutralisation using ANX-M1
C1q neutralisation strategies included local pre-treatment, local post-treatment and systemic treatment following the paradigms listed below. Briefly, a neutralising antibody to C1q, ANX-M1 and a non-specific isotype control IgG monoclonal antibody (mAb) were provided by Annexon Biosciences (CA, USA). Monoclonal antibody M1 binds and neutralises C1q thereby preventing the activation of the classical complement pathway. ANX-M1 anti-C1q antibody or an IgG isotype control antibody were formulated in endotoxin-free 0.1 M phosphate-buffered saline (Thermo Fisher Scientific, MA, USA). All experiments were performed on adult C57BL/6 J mice under double-blind conditions. Intravitreal injections were performed as described in our previous publication, with a volume of 1 μl injected into each mouse eye [
26].
Local inhibition
Pre-treatment
A 1 μl solution containing either the ANX-M1 anti-C1q antibody or an IgG isotype control antibody (7.5 μg/μl) was injected into each mouse eye, prior to the commencement of photo-oxidative damage (day 0). One animal from each cohort (ANX-M1 anti-C1q antibody and IgG control) were placed in the same light box for 7 days of photo-oxidative damage. Retinas were assessed at 7 days after photo-oxidative damage (day 14) and analysed (N = 10/group).
Local inhibition
Post-treatment
A 1 μl solution of ANX-M1 anti-C1q antibody or IgG isotype control antibody (7.5 μg/μl) was administered intravitreally immediately after completion of the 7-day photo-oxidative damage procedure (day 7) and placed in dim-cyclic light until day 14, for retinal assessment (N = 10/group).
Systemic inhibition
100 mg/kg of ANX-M1 anti-C1q antibody or an IgG isotype control antibody was administered intraperitoneally on day 0 (prior to the PD procedure), day 4 (during the PD procedure) and on day 8 (the day after completion of the PD procedure) in order to sustain complement inhibition. Hemolytic assay and retinal assessment were performed at day 12 (N = 10/group) as per our previous publications [
11,
27].
TUNEL and immunohistochemistry
A terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) assay (Roche Diagnostics, Basel, Switzerland) was used to detect photoreceptor cell death. Retinal cryosections were used for the TUNEL assay according to our previously described protocols, with minor modifications [
28,
29]. Sections were counterstained with the DNA-specific dye bisbenzimide (1:10000; Sigma Aldrich, MO, USA) for visualisation of cell nuclei.
To detect and localise specific proteins, immunohistochemistry was performed on retinal cryosections according to previously published methodology, with minor modifications [
12,
30]. A list of primary antibodies is provided in Table
1.
Table 1
Primary antibodies used for immunohistochemistry and Western blot
Rabbit α-IBA1 | Ionised calcium binding protein 1 | 1:500 | Wako Chemicals | 019-19,741 |
Goat α-IL-1β | IL-1 beta/IL1F2 | 1:1000 | R&D Systems | AF-501 |
Goat α-C3d | Complement component C3d | 1:2000 | R&D Systems | AF-2655 |
Rabbit α-C1q | C1q, clone 4.8 | 1:400 | Abcam | AB182451 |
Rabbit α-C1q | C1q complement isolated from human plasma | 1:1000 | Dako | F0254 |
Rabbit α-NALP3/NLRP3 | 1-50 amino acids of human NLRP3 | 1:500 | Novus Biologicals | NPB2-12446SS |
Rat α-F4/80 | F4/80 clone A3-1 | 1:100 | Abcam | AB6640 |
Rabbit α-GAPDH | GAPDH | 1:4000 | Sigma Aldrich | G9545 |
Fluorescence analysis and imaging
TUNEL+ and IBA1+ cells were quantified along the full length of retinal cryosection in duplicate sections per animal. For photoreceptor cell death, only TUNEL+ cells in the ONL were counted. IBA1+ cells in the outer retina (including subretinal space, ONL-RPE) were taken across the cryosection. For each section, number of TUNEL+ or IBA1+ cells was recorded (superior to inferior) in increments of 0.5 mm across the full length of retinal cryosection. Total cell counts were averaged from at least 2 sections per animal with 6 animals for each experimental group.
To quantify photoreceptor loss in retinal cryosections, the number of rows of photoreceptor nuclei was counted within the retinal lesion site (superior retina, 1 mm away from the optic nerve head). Five measurements were taken per retina in duplicate per animal.
Fluorescence in retinal cryosections was visualised and imaged using a laser-scanning A1+ confocal microscope (Nikon, Tokyo, Japan). Images were acquired using NIS-Elements AR software (Nikon). Negative control slides (no primary antibody) were imaged with each immunolabelled slide to determine specificity of staining and set up the threshold for fluorescence intensity for comparison to positively-stained slides. Images were processed and assembled into panels using Photoshop CS6 software (Adobe Systems, CA, USA).
Fluorescence-activated cell sorting (FACS)
Mouse retinal microglia and macrophages (CD11b
+) were isolated using a fluorescence-activated cell sorter (BD FACSAria II; BD Biosciences, NJ, USA), using previously described protocols with minor modifications [
25,
30,
31]. Mouse retinas were pooled from 2 animals (4 retinas) for each sample. Cells were stained using an anti-mouse CD11b-Phycoerythrin (PE) conjugated antibody (#101207, 1:500; Biolegend, CA, USA). FACS-isolated cells were then used immediately for RNA extraction.
Quantitative real-time polymerase chain reaction (qPCR)
RNA extraction and purification was performed on retinas and isolated CD11b
+ cells using a combination of TRIzol reagent (Thermo Fisher Scientific) and an RNAqueous Total RNA Isolation Kit (Thermo Fisher Scientific) as described in our previous publication [
32]. cDNA was prepared from 500 ng of each RNA sample using a Tetro cDNA Synthesis Kit (Bioline Reagents, London, UK) according to the manufacturer’s protocol.
Gene expression changes were measured via qPCR using Taqman hydrolysis probes (listed in Table
2) and Taqman Gene Expression Master Mix (Thermo Fisher Scientific). Each qPCR was run using a QuantStudio 12 K Flex instrument (Thermo Fisher Scientific) at the Biomolecular Resource Facility (JCSMR, ANU). Analysis was performed using the comparative cycle threshold method (ΔΔC
t) which was normalised to the expression of
Gapdh and
Actb reference genes, as established previously [
33,
34].
Table 2
Taqman hydrolysis probes used for qPCR (Thermo Fisher Scientific)
Actb | Actin-beta | AK078935 | Mm01205647_g1 |
C1qa | Component 1, q subcomponent, alpha polypeptide | NM_007572.2 | Mm00432142_m1 |
C2 | Component 2 | NM_013484 | Mm00442726_m1 |
C4 | Component 4 | NM_011413 | Mm01132415_g1 |
Casp-1 | Caspase-1 | NM_009807 | Mm00438023_m1 |
Casp-8 | Caspase-8 | NM_001080126 | Mm01255716_m1 |
Cfb | Factor B | NM_001142706.1 | Mm00433918_g1 |
Cfh | Factor H | NM_009888 | Mm01299248_m1 |
Cntf | Ciliary neurotrophic factor | NM_170786.2 | Mm00446373_m1 |
Fgf2 | Fibroblast growth factor 2 | NM_008006.2 | Mm01285715_m1 |
Gapdh | Glyceraldehyde-3-phosphate dehydrogenase | NM_001289726 | Mm99999915_g1 |
Gfap | Glial fibrillary acid protein | NM_010277.3 | Mm01253033_m1 |
Il-18 | Interleukin 18 | NM_008360.1 | Mm00434226_m1 |
Il-1b | Interleukin 1-beta | NM_008361 | Mm00434228_m1 |
Nlrp3 | NLR Pyrin Domain Family 3 | NM_145827 | Mm00840904_m1 |
Serping1 | Serine peptidase inhibitor member 1 | NM_009776 | Mm00437834_m1 |
Western blot
Retinas were collected into Cellytic M buffer (Sigma-Aldrich) containing a Protease Inhibitor Cocktail (Sigma-Aldrich). Western blotting was performed on retinal protein lysates according to previously described methods with minor modifications [
35,
36]. 20 μg of denatured protein was loaded onto a 4-20% Mini-Protean TGX Precast Protein gel (Bio-Rad, CA, USA) followed by semi-dry transfer to a nitrocellulose membrane. A list of primary antibodies used for Western blot are provided in Table
1. A secondary antibody-peroxidase conjugate was used for visualisation (Bio-Rad). The protein was visualised with chemiluminescence using a Clarity Western ECL kit (Bio-Rad) and images were captured and analysed using a Chemidoc MP with Image Lab software (Bio-Rad). The expression of the protein of interest was normalised to GAPDH.
Statistical analysis
All statistical analysis was performed using Prism 6 (GraphPad Software, CA, USA). Unpaired student t-test, one-way analysis of variance (ANOVA) or two-way ANOVA were run as appropriate for each dataset. Multiple comparisons were run with appropriate post-hoc tests to determine statistical significance for the interactions between each experimental group (P < 0.05). Graphs are displayed as mean ± SEM.
Discussion
This study investigated the contribution of C1q and the classical complement pathway in the timing of disease progression induced in retinal degeneration. Our data illustrate the role of locally-derived retinal C1q in driving the progression of retinal atrophy and the possibility of therapeutic interventions using C1q neutralisation. The findings indicate that: First, genetic ablation of C1qa indicated variable and inconclusive effects on retinal function during the 7 days of photo-oxidative damage. At 1 week following photo-oxidative damage (day 14), the absence of C1q was found to protect retinal structure, reduce photoreceptor cell death and inflammation, and improve retinal function. Second, we demonstrate a possible role for the classical complement pathway in the activation of the NLRP3 inflammasome and secretion of IL-1β in the degenerating retina, which may promote retinal damage during this post-exposure period. Third, by comparing neutralization of classical complement activator C1q systemically and locally in the retina, we demonstrate that local C1q expressed by subretinal microglia/macrophages, and not serum complement, plays a vital role in the progression of retinal degeneration. Together, the results indicate that the classical complement pathway, mediated by C1q, contributes to progression of retinal degeneration. These results further indicate that a therapeutic approach targeting the classical complement initiator C1q may be beneficial in slowing the progression of focal retinal degenerations, where dysregulated complement is a major feature of disease pathogenesis, such as AMD.
The classical pathway in retinal atrophy
C1q is associated with pathological features of age-related neurodegenerative diseases such as amyloid-β accumulation in Alzheimer’s Disease (AD), glaucomatous damage in a model of glaucoma and drusen deposition in AMD [
37‐
39]. Evidence from other studies indicates that non-canonical functions of C1q influence local inflammation in the central nervous system (CNS), such as by promoting the dynamic shift of astrocytes to a more pro-inflammatory state which is associated with AD and Huntington’s Disease [
40]. Moreover, C1q is involved in the elimination of synapses in a mouse model of AD, and blockade of C1q significantly preserved synaptic function [
41,
42]. The ablation of
C1qa further prevented the progressive loss of retinal ganglion cells in a mouse model of glaucoma [
43]. However, a recent study demonstrated that numerous complement components, including C1q, were important in maintaining inner nuclear layer (INL) retinal integrity, and that the absence of C1q in the developing retina reduced retinal function in the aging retina [
44]. Others have reported a neuroprotective influence of C1q on the survival of cone photoreceptors in
Rho−/− mice and elucidated that C1q was involved in cone cell preservation in hereditary retinopathies [
45], indicating a role of C1q in normal retinal development.
Our present findings suggest that C1q facilitates the progression of late-stage retinal atrophy, following the onset of retinal damage. Studies in our laboratory have previously shown upregulation of classical complement components
C1s, C4a and
C2 in retinas following photo-oxidative damage [
11], but their specific roles have not been described. Other studies on the pathological effects of
C1qa in neurodegeneration have been implicated in mouse models of retinal ischemic injury [
24], and glaucoma [
43]. The findings presented here show that initially (days 1-7 of photo-oxidative damage) the absence of
C1qa has no definitive impact on retinal degeneration induced by photo-oxidative damage; however, over an extended timeframe, sustained expression of
C1qa is a driver of progressive loss of photoreceptors. Rohrer et al. (2010) suggest an involvement of the activation of the classical complement in the development of choroidal neovascularisation, in the late stage of AMD in a laser-induced rodent model of choroidal neovascularisation, since the alternative complement is not sufficient to produce the pathology [
15]. Consistent with this concept, the data from the current study demonstrates that localised post-treatment with ANX-M1 C1q inhibition protects against retinal degeneration during the progression phase of damage (7-14 days), but not earlier during the onset of retinal degeneration. The findings suggest a potential role of C1q in mediating the progression of retinal degeneration.
C1q and inflammasome activation by subretinal macrophages in photo-oxidative damage
C1q stimulation activates the NLRP3 inflammasome and the secretion of IL-1β in CEP-primed BMDMs [
21]. NLRP3 inflammasome activation in AMD has been proposed to occur in response to several stimuli including drusen components, RPE and complement proteins, oxidative stress, oxidative by-products and DNA [
21,
46,
47]. In this study, in the absence of classical pathway activation, there was a reduced NLRP3 inflammasome expression in a specific population of cells - the subretinal microglia/macrophages.
This finding is consistent with previous studies showing that C1q can activate NLRP3 in BMDMs of humans, and in mice after immunization with CEP-adducted mouse serum albumin, to model a dry AMD-like pathology [
21]. Other evidence has demonstrated the capability of RPE cells to induce NLRP3 inflammasome expression and propagate photoreceptor toxicity in the presence of blue-light photo-oxidative damage [
48]. We have previously shown that in photo-oxidative damage, inflammasome expression and IL-1β production were observed in retinal degeneration, where IL-1β was found to be expressed by microglia and macrophages at the site of photoreceptor degeneration [
49]. Our findings from the current study show significant down-regulation of IL-1β in isolated retinal macrophages from
C1qa−/− retinas compared to wild-type controls, which correlated to a more preserved photoreceptor population at 14 days. The findings indicate that the reduced activity in the classical pathway dampens NLRP3 inflammasome expression along with pro-inflammatory IL-1β secretion, resulting in protection of the photoreceptor population.
It is of interest that we detected no significant difference in C3 expression by Western blot between WT and
C1qa−/− mice at day 14. This may indicate that the effector functions enabled by the classical complement pathway may be independent of hydrolysis of C3 [
38,
50,
51], which is predominantly amplified by the alternative complement pathway [
11] in photo-oxidative damage models of retinal degeneration. Our findings indicate that one of the non-canonical mechanisms of complement activation is associated with inflammasome activation, as indicated by decreased NLRP3 labelling in
C1qa−/− microglia. Further, C1q can trigger a rapid enhancement of phagocytosis upon recognition of apoptotic cells, independent of the classical component pathway activation [
52]. For these reasons, we hypothesise that both inflammasome activation and enhanced phagocytosis are mediated by C1q, explaining why we see protection of retinal function in the
C1qa−/− mice, even though no changes in C3 were detected at 14 days. These findings highlight the intricate association between the classical pathway and inflammasome activation, although the precise role of C1q in inflammasome activation and other non-canonical functions, such as phagocytosis, warrants further investigation.
Complement-expressing macrophages as potent local mediators of onset and progression of retinal atrophy
Retinal microglia/macrophages express complement components in rodent retinas in response to aging [
31]. Here, we demonstrate that subretinal microglia/macrophages also express
C1q in focal retinal degeneration
. Our data show that subretinal microglia/macrophages in the degenerating retina can express CD68
+ lysosomal protein aggregates, and contain multiple TUNEL
+ apoptotic cells, demonstrating a phagocytic phenotype. It is likely that C1q expression may be found in these phagocytic macrophages of the subretinal space, as we have detected C1q expression co-localising with subretinal microglia/macrophages in the degenerating retina after photo-oxidative damage. It is plausible that C1q is secreted to bind and recognize defective synapses at a stage of disease prior to overt neurodegeneration, as reported in models of neurodegenerative diseases in the brain and retina [
38,
43], however this was not observed directly in this study.
In normal and diseased brain tissues resident microglia express many-fold higher levels of
C1qa,
−b and
–c, the three protein subunits of C1q, compared to macrophages and other myeloid cells [
53,
54]. The capacity of microglia to synthesize
C1qa has been confirmed in rodent brain [
55], however, in the context of AMD the close interdependence and proximity of the retina with Bruch’s membrane/RPE/choroid confounds the argument that microglia are the key synthesizers of C1q. Our findings demonstrate that C1q proteins are localised near drusen and in the subretinal microglia/macrophages of human AMD patients whose RPE atrophy was prominent. The delayed appearance of these C1q-expressing subretinal microglia/macrophages, coincides with RPE and choriocapillaris breakdown [
56] suggesting a population derived from the choriocapillaris. However, a recent study investigating models of RPE breakdown suggests that subretinal populations are predominantly resident microglia, owing to their rapid mobilisation from the inner retina to the site of injury [
57].
Our findings with the local delivery of the C1q neutralisation suggest that locally-derived C1q contributes to the progression of retinal atrophy. Coupled with the association of C1q expressed by myeloid cells with neuroinflammation and age-related neurodegeneration [
58], our findings support the idea that locally administered immunotherapy targeting the classical complement pathway may be effective in slowing the progression of retinal degeneration. We did not detect any ocular inflammation specifically associated with anti-C1q delivery in these experiments. This suggests that administration of an anti-C1q antibody is unlikely to have additional risks to those normally associated with intravitreal injections (reviewed in [
59]). Further investigations are required to explore the mechanisms underlying the protective effect of local C1q neutralisation.
Conclusions
This study illustrates the contribution of the classical complement pathway in facilitating retinal atrophy induced by photo-oxidative damage. The classical pathway, through its initiating molecule C1q, appears to exacerbate photoreceptor damage through interaction with the inflammasome. The efficacy of intravitreal C1q-targeted inhibition in ameliorating the late-stage of photo-oxidative damage-induced retinal atrophy, reinforces the concept that targeting C1q, or the macrophage capability to express complement, may be effective in slowing the progression of retinal degenerations. Further utilisation of strategies that can elucidate the exact origins of complement-expressing macrophages - microglia or monocytes - are warranted. Clarifying the identity and function of these complement-expressing myeloid cells will enable the design of more precise therapeutics targeting the complement system, which may be beneficial for retinal diseases where macrophage recruitment and complement activation are a key pathogenic feature, including AMD.
Acknowledgments
The authors thank the NSW Organ and Tissue Donation Service. The authors also thank Annexon Bioscience (San Francisco, CA, USA) for their C1q neutralizing antibodies.