In diabetic macular oedema (DMO), blood components passing through the disrupted blood–retinal barrier cause neuroinflammation, but the mechanism by which autoantibodies induce neuroglial dysfunction is unknown. The aim of this study was to identify a novel autoantibody and to evaluate its pathological effects on clinically relevant photoreceptor injuries.
Biochemical purification and subsequent peptide fingerprinting were applied to identify autoantigens. The titres of autoantibodies in DMO sera were quantified and their associations with clinical variables were evaluated. Two animal models (i.e. passive transfer of autoantibodies and active immunisation) were characterised with respect to autoimmune mechanisms underlying photoreceptor injuries.
After screening serum IgG from individuals with DMO, fumarase, a Krebs cycle enzyme expressed in inner segments, was identified as an autoantigen. Serum levels of anti-fumarase IgG in participants with DMO were higher than those in diabetic participants without DMO (p < 0.001) and were related to photoreceptor damage and visual dysfunction. Passively transferred fumarase IgG from DMO sera in concert with complement impaired the function and structure of rodent photoreceptors. This was consistent with complement activation in the damaged photoreceptors of mice immunised with fumarase. Fumarase was recruited to the cell surface by complement and reacted to this autoantibody. Subsequently, combined administration of anti-fumarase antibody and complement elicited mitochondrial disruption and caspase-3 activation.
This study has identified anti-fumarase antibody as a serum biomarker and demonstrates that the generation of this autoantibody might be a pathological mechanism of autoimmune photoreceptor injuries in DMO.
Diabetic retinopathy often leads to severe vision loss [1, 2]. An increasing number of studies have determined that disturbances in both vascular cells and neuroglial components reciprocally promote the pathogenesis of diabetic retinopathy [3,4,5]. In particular, diabetes mellitus exacerbates blood–retinal barrier (BRB) breakdown and concomitant diabetic macular oedema (DMO), which often leads to severe vision disturbances. However, it remains largely unknown how the extravasated blood components damage neuroglial cells.
Advances in optical coherence tomography (OCT) have enabled clinicians to evaluate retinal status . Retinal thickening in the macula is one of the major indicators of DMO , and high-resolution OCT enables assessment of the status of the ellipsoid zone of the photoreceptors as a representative of photoreceptor integrity. Histologically, this region contains a high number of mitochondria that support the increased level of metabolism of photoreceptor cells . In eyes with DMO the ellipsoid zone line is often disrupted, which significantly correlates with vision impairment [9, 10]. However, the molecular or cellular mechanisms behind the photoreceptor damage must be elucidated in order to establish novel therapeutic strategies for DMO [11, 12].
Subretinal spaces are limited by the barrier properties of the external limiting membrane and the retinal pigment epithelium and, in healthy retinas, are known to be isolated from immunity (‘immunoprivileged’) . In chorioretinal diseases, impairment of neurovascular units leads to microvascular pathogenicity and neuroinflammation, as seen in neurological diseases . In cancer-associated retinopathy (CAR), autoantibodies against proteins expressed in photoreceptor cells, such as recoverin and arrestin, contribute to progressive photoreceptor degeneration . This evidence encouraged us to speculate that BRB breakdown in diabetic eyes might lead to neuroglial damage through various neurotoxic blood components, including immunological agents, which is to some extent supported by publications regarding the recruitment and activation of inflammatory cells in chorioretinal diseases [16, 17].
In the current study, we aimed to identify autoantibodies against photoreceptor cells in a subgroup of individuals with DMO and to investigate their contribution to photoreceptor damage.
Participants and biosampling
Participants with type 2 diabetes were consecutively recruited at the Department of Ophthalmology of Kyoto University Hospital and divided into the following three groups: (1) DM group, comprising participants with type 2 diabetes but not diabetic retinopathy; (2) DR group, comprising participants with diabetic retinopathy but not centre-involved DMO; and (3) DMO group, comprising participants with centre-involved DMO.
In the discovery cohort recruited from July 2014 to February 2016, we first included participants as shown in the flow chart (see electronic supplementary material [ESM] Fig. 1). Samples from these participants were used for screening by western blot or immunostaining, immunoprecipitation and subsequent MS, ELISA to measure the autoantibody titre, and animal or cell culture experiments (ESM Fig. 1). In the validation cohort for ELISA, participants were included from March 2016 to December 2017. Participants of the same sex and similar age and HbA1c level to the individual groups were included in the ELISA experiments. After excluding 33 eyes with media opacity affecting visual acuity according to participants’ medical records, we included 106 eyes in the DMO group of both cohorts and analysed the association of anti-fumarase IgG with other clinical variables (ESM Fig. 1). We collected serum samples in 21 participants before and after anti-vascular endothelial growth factor (anti-VEGF) treatment (with three initial consecutive monthly injections followed by an ‘as-needed’ phase) and evaluated the changes in autoantibody titres. The characteristics of participants in the individual studies are detailed in ESM Tables 1–10.
Sera were aliquoted within 1 h after sampling and immediately stored at −80°C. We conducted this study in accordance with the Declaration of Helsinki. The study was approved by the Kyoto University Graduate School and Faculty of Medicine Ethics Committee and registered at the UMIN Japan Clinical Trial Registry (UMIN000014015). All participants provided written informed consent before inclusion in the study. More details of the patients and biosampling are provided in ESM Methods.
Enrichment of photoreceptor cells
Eyeballs from dead pigs were obtained from an abattoir and stored on ice soon after slaughter. After the isolation of the retinas, photoreceptor cells from porcine retinas were prepared by centrifugation in a stepwise Percoll gradient (see ESM Methods).
Cell culture and gene knockout
Human embryonic kidney (HEK) 293 cells and 661W cells were cultured. The gene encoding fumarate hydratase (FH) was knocked out in HEK 293 cells using the CRISPR/Cas9 system (see ESM Methods).
After approval by the institutional review board of the Kyoto University Graduate School of Medicine, wild-type or Ins2Akita C57BL/6 mice and C57BL/10.RIII-H2r H2-T18b/(71NS)SnJ (B10.RIII) mice were housed in accordance with the Institutional Animal Care and Use Committee guidelines as well as with the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research. Eyes were harvested under a lethal dose of anaesthesia. The exclusion criterion was based on the media opacity, which affects the in vivo assessment. We performed subretinal injections of 290 eyes and excluded 14 eyes with media opacities (see ESM Methods).
The mRNA expression of murine Fh (also known as Fh1; accession number: NM_010209.2) in retinas from C57BL/6 mice (wild-type or Ins2Akita diabetic) was determined using PCR (see ESM Methods).
Porcine retinal lysates were applied to SDS-PAGE. Sera from participants were incubated as primary antibodies to detect retinal autoantigens.
We evaluated protein levels of fumarase in HEK 293 cells and levels of cytochrome C, apoptosis-inducing factor and β-actin in the cytosolic fraction of 661W cells using commercially available antibodies (ESM Table 11). The antibodies were used according to the manufacturers’ instructions. See ESM Methods for more information.
Cell lysates of porcine photoreceptor cells were incubated with serum IgG conjugated to protein G-coated beads to immunoprecipitate antigens against autoantibodies (see ESM Methods).
After in-gel digestion of immunoprecipitated proteins from porcine photoreceptor cells, tryptic digests were introduced into a mass spectrometer (see ESM Methods).
Preparation of cytosol fractions
Cytosol fractions were prepared from 661W cells using a subcellular fractionation kit (Mitochondria Isolation Kit for Cultured Cells; Thermo Scientific, Waltham, MA, USA) (see ESM Methods).
Measurements of the intracellular Ca2+ concentration
The intracellular Ca2+ concentration was measured using a Fluo 4-AM kit (Calcium kit-Fluo 4; Dojindo Laboratories, Tokyo, Japan) in 661W cells (see ESM Methods).
Measurements of mitochondrial membrane potential
After incubation with JC-1 dye (Thermo Scientific), fluorescence levels were measured in HEK 293 cells according to the manufacturer’s protocol (see ESM Methods).
Caspase-3 activity in 661W cells was assessed by the colorimetric assay (APOPCYTO Caspase-3 Colorimetric Assay Kit; MBL, Aichi, Japan) (see ESM Methods).
After HEK 293 or 661W cells had been incubated with fluorescence-labelled anti-fumarase antibody, the fluorescence intensity was analysed using FACSCalibur (BD Biosciences, San Jose, CA, USA) (see ESM Methods).
Wells in which the bottom was coated with recombinant human fumarase were incubated in diluted serum as the primary antibodies (see ESM Methods).
Antibody depletion and subretinal injection of serum
Agarose beads conjugated to recombinant human fumarase were incubated with DMO sera to prepare sera depleted of anti-fumarase antibodies. These specimens were injected into the subretinal spaces in C57BL/6 mice (see ESM Methods).
Purification of anti-fumarase antibodies from DMO sera and preparation of complement components
Anti-fumarase antibodies from DMO sera were captured using NHS-Activated Sepharose 4 Fast Flow (GE Healthcare, Piscataway, NJ, USA) coupled to human recombinant fumarase. The purified autoantibodies were administered into the subretinal spaces in C57BL/6 mice (passive transfer model; see ESM Methods).
Immunisation with fumarase
B10.RIII mice were immunised using fumarase in combination with complete Freund’s adjuvant and pertussis toxin (active immunisation model; see ESM Methods).
Retinal sections from mice (C57BL/6 wild-type, C57BL/6 Ins2Akita or B10.RIII) retinas were incubated with DMO sera or antibodies against cytochrome C oxidase (COX) IV, fumarase, recoverin, CD59a and C5b-9, followed by fluorescence-labelled secondary antibodies (ESM Table 11). Human retinas from deceased donors were obtained from Eversight (https://www.eversightvision.org/researchers/researcher/#request-tissue; see ESM Table 4 for donor details) and were stained with anti-fumarase antibody. HEK 293 or 661W cells were also incubated with primary antibodies against fumarase and C5b-9 (ESM Table 11).
Ultrathin slices of HEK 293 cells were observed by transmission electron microscopy (H-7650; Hitachi, Tokyo, Japan) (see ESM Methods).
Cell viability assay
Treated HEK 293 or 661W cells were applied to a cell viability/cytotoxicity assay kit (LIVE/DEAD assay; Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s instructions (see ESM Methods).
Amplitudes of the mixed cone and rod responses were evaluated in C57BL/6 mice (see ESM Methods).
After retinal sectional images were obtained using spectral domain OCT in participants with diabetic retinopathy, we evaluated the central subfield thickness, ellipsoid zone status and disorganisation of the retinal inner layers. Spectral domain OCT images of C57BL/6 mice were also acquired (see ESM Methods).
No experiments were randomised. All experiments were blind to group assignment and outcome assessment. After confirming normality with the Kolmogorov–Smirnov test, the Kruskal–Wallis test of ANOVA with Dunnett’s multiple comparison, Mann–Whitney U test or Wilcoxon signed-rank test was performed to evaluate non-parametric data. Spearman’s rank correlation coefficient was used to show statistical correlations. Paired t tests, Student’s t tests or one-way ANOVA with Bonferroni correction were used to assess statistical differences in parametric parameters. Sampling distributions in participants’ characteristics were evaluated using Fisher’s exact test or the χ2 test. We further applied univariate or multivariate logistic and linear regression analyses. These statistical analyses were performed using commercial software (PASW Statistics, version 18; SPSS, Chicago, IL, USA), and p < 0.05 was considered significant. Values are shown as means ± SD.
Identification of fumarase as an autoantibody target
During autoantibody screening, western blot analyses showed that serum IgG from DMO participants had high levels of immunoreactivity to proteins expressed in porcine retinas and photoreceptor cells, suggesting the presence of autoantibodies specific to individuals with DMO (Fig. 1). We therefore planned the translational research to compare OCT and immunoreactivity to retinal autoantigens (Fig. 2). Among several bands, an approximately 50 kDa band was detected in sera from 19 of 27 DMO participants (70.4%). In addition, immunostaining using serum IgG revealed specific signals in photoreceptor cells, including inner segments, of C57BL/6 mice in 23 of 27 DMO participants (85.2%) (Fig. 2).
To identify the targets of the autoantibodies in DMO serum, we performed a combination of immunoprecipitation and MS, as described previously . Porcine photoreceptor lysates were immunoprecipitated with serum IgG according to protein G sepharose bead-based procedures. SDS-PAGE followed by silver staining revealed that a unique, approximately 50 kDa band was precipitated with DMO serum (Fig. 3a, b). After in-gel digestion, peptide mass fingerprinting identified fumarase (predicted molecular weight 55 kDa) as an autoantigen, which was confirmed by immunoblotting using mouse monoclonal anti-fumarase antibody and recombinant human fumarase (Fig. 3c, d).
Fumarase, one of the main metabolic enzymes of the tricarboxylic acid cycle, is transported mainly into the mitochondrial matrix, although some forms are cytosolic . In the retinas of both control and diabetic mice (male heterozygous Ins2Akita mice), fumarase mRNA was amplified by PCR (ESM Fig. 2). Immunostaining showed that fumarase was highly expressed in photoreceptor inner segments containing accumulated mitochondria in both human and rodent retinas (Fig. 3e and ESM Fig. 2b, respectively), although there were no definite differences in its expression between control and diabetic donors.
Quantitative analyses of anti-fumarase antibody
ELISA analysis revealed that the titres of total serum IgG against fumarase from DMO participants were significantly higher than those from participants in the DM and DR groups (Fig. 4a) . According to the post hoc comparisons, the titres of anti-fumarase IgG were not different among participants with moderate non-proliferative diabetic retinopathy, severe non-proliferative diabetic retinopathy and proliferative diabetic retinopathy (data not shown) . Furthermore, an independent cohort confirmed that the DMO group had higher titres than the DR group (p < 0.001; ESM Table 6). Multivariate logistic regression analyses indicated that the titre of anti-fumarase IgG may be a serum biomarker for a subgroup of DMO among individuals with type 2 diabetes or diabetic retinopathy (ESM Tables 12 and 13).
In 106 eyes with centre-involved DMO, the serum titre of anti-fumarase IgG was modestly correlated to logarithm of the minimum angle of resolution (logMAR) visual acuity and ellipsoid zone disruption on spectral domain OCT images, rather than to disorganisation of retinal inner layers or central subfield thickness (Fig. 4b, c, ESM Table 14). Interestingly, the titre of this autoantibody was decreased after anti-VEGF treatment (Fig. 4d).
Functional analysis of anti-fumarase antibody
These results encouraged us to investigate the in vivo effects of anti-fumarase antibody on photoreceptor damage. DMO sera with anti-fumarase antibodies were randomly selected and injected into the subretinal spaces of C57BL/6 mice (Fig. 5). Both the IgG in DMO serum and C5b-9, which is a component of the membrane attack complex (MAC) of the complement system, showed diffuse immunostaining with a punctate appearance mainly in both inner and outer segments and little cellular infiltration 24 h after the injection. In contrast, these signals were completely diminished in retinas injected with serum depleted of anti-fumarase antibodies (Fig. 5d, e).
Histological analyses revealed that outer segments and parts of inner segments, but not the outer nuclear layer, disappeared in some areas of the retinas 7 days after the administration of DMO serum containing anti-fumarase IgG. In contrast, both inner and outer segments were preserved in retinas that received sera depleted of anti-fumarase antibody. This was consistent with spectral domain OCT findings that the ellipsoid zone line was not complete and outer retinal thickness was decreased in retinas administrated DMO serum, compared with retinas injected with serum depleted of fumarase antibodies (Fig. 5f–i).
The deposition of MAC and minimal cellular infiltration in photoreceptor outer segments were consistent with the higher titres of IgG1 and/or IgG3 and the absence of CD59a, a membrane-bound inhibitor of the MAC (Figs 4e–h, 5c–e). These results encouraged us to investigate the function of anti-fumarase antibody in concert with the complement cascade among several mechanisms of autoantibody-mediated autoimmunity. We first evaluated photoreceptor damage in C57BL/6 mice in which autoantibodies and complement were passively transferred. Histological and electroretinogram experiments confirmed that the photoreceptors were morphologically and functionally ablated 7 days after subretinal injection with anti-fumarase IgG + complement, as observed in eyes injected with DMO sera, whereas these changes were not observed after injection of fumarase IgG alone or control IgG + complement (Fig. 6a–c, ESM Fig. 3). In six of 14 (42.9%) B10.RIII mice (H2r) immunised with fumarase, both haematoxylin and eosin staining and immunofluorescence delineated structural damage in the photoreceptor inner and outer segments, although cells rarely migrated into the subretinal spaces (Fig. 6d). Mouse IgG was partly co-localised with C5b-9 in the inner and outer segments (Fig. 6e).
We further planned cell culture experiments to investigate the mechanisms by which this autoantibody and complement induced cellular damage. In vitro experiments revealed that the autoantibody was partially co-localised with fumarase and C5b-9 on the cell surface after incubation with anti-fumarase IgG purified from DMO sera and complement, whereas co-staining was not observed in FH−/− cells (Fig. 7a, b, ESM Fig. 4a–c).
The intracellular Ca2+ concentration was transiently increased under control IgG and complement, as in the case of sublytic MAC-induced Ca2+ influx in nucleated cells . High levels of Ca2+ influx persisted after treatment with both anti-fumarase antibody and complement, compared with no alteration of the Ca2+ concentration by anti-fumarase antibody alone (Fig. 7c). Ca2+ chelators reversed the immunoreactivity of fumarase on the cell surface (ESM Fig. 4d, e). Consistent with Ca2+ burst-induced mitochondrial disruption , the mitochondrial membrane potential was decreased and proapoptotic signal transducing molecules, cytochrome C and apoptosis-inducing factor, leaked into the cytosol under anti-fumarase antibody in combination with complement (Fig. 7d, e). Caspase-3, an apoptosis executioner, was also activated in a Ca2+-dependent manner (Fig. 7f).
Transmission electron microscopy revealed multiple processes during cell death, including vacuolation in the cytosol and mitochondria as well as nuclear deformation under anti-fumarase antibody and complement (Fig. 7g) . Actually, the number of dead cells increased after administration of the combination, which was reversed by FH gene ablation. Furthermore, this cell death partly depended on caspase-3 activation (Fig. 7h, i). Pruning of axons and dendrites is mediated by mitochondrial damage and a caspase pathway in developmental or pathological neurons [25, 26], which might allow us to speculate that these in vitro experiments support subcellular damage of photoreceptor inner and outer segments.
Many publications have shown that, in addition to retinal thickening, foveal photoreceptor damage is related to visual dysfunction in eyes with DMO [7, 9, 10], although the cellular and molecular mechanisms are unknown. A disrupted BRB in diabetic retinas allows blood components, including autoantibodies, to pour into the extravascular space and exacerbate neuroinflammation [3, 4, 27]. In the current study, anti-fumarase autoantibody, which was newly identified in sera from a subgroup of DMO participants, was clinically associated with photoreceptor damage and vision impairment. This fumarase autoantibody, in concert with complement activation, induced the dropout of photoreceptor inner and outer segments. Our results suggest a novel target of autoantibodies in DMO sera as well as a novel pathological mechanism for photoreceptor damage, which is clinically evident on spectral domain OCT images in DMO [9, 10].
After screening using immunoblotting, in which protein denaturation provides higher sensitivity for epitopes, we used immunoprecipitation with a non-denaturing buffer (high salt concentration and neutral pH) to mimic the three-dimensional antigen–antibody reaction and to concentrate autoantigens for subsequent MS . The second screening excluded many autoantigen candidates, allowing the identification of several putative antigens. Of these, fumarase was both a target for autoantibodies in DMO sera and was highly expressed in the inner segment of photoreceptors, which prompted us to focus on qualitative and quantitative analyses of anti-fumarase antibody.
According to the quantitative ELISA data, 32 of 65 participants (49.2%) with DMO had a moderate titre of anti-fumarase antibody (above the mean + 2SD in the DM group), whereas western blot detected an approximately 50 kDa band in 19 of 27 (70.4%) DMO serum. The discrepancy might depend on the different biochemical methods used to detect the different epitopes. Another possibility might be the presence of additional autoantigens with almost the same molecular weight, such as arrestin and enolase, both of which have been reported as targets for autoantibodies in sera from individuals with CAR or age-related macular degeneration [28, 29]. Furthermore, immunoblotting revealed that IgG in DMO sera had immunoreactivity to several antigens with other molecular weights in photoreceptor cells, and immunostaining using serum IgG also showed immunoreactivity to photoreceptor cells in 23 of 27 DMO participants (85.2%). Some participants with the lower titre of anti-fumarase IgG had photoreceptor damage and poor visual function. These results suggest additional autoantigens in photoreceptor cells. Using immunofluorescence, we often observed definite fluorescent signals in the ganglion cell layer and inner nuclear layer. Taken together, systematic analyses of anti-retinal antibodies would promote our understanding of the immunological aspects of neuroinflammation in DMO.
Statistical analyses of the clinical data demonstrated that anti-fumarase IgG was increased in a subgroup of DMO participants with photoreceptor damage and vision impairment, although whether this autoantibody is the cause or result of photoreceptor damage remains to be determined. In vitro and in vivo experiments in the current study suggest that this autoantibody might promote photoreceptor damage in DMO. In contrast, whether autoantibody production is induced by the intracellular antigens derived from photoreceptor cells remains ill defined. We speculate that the disrupted BRB could allow retinal autoantigens to enter the blood or to recruit or activate immune cells to migrate to and from the retina , and a future study should elucidate the molecular mechanisms by which vascular permeability contributes to the elicitation of autoimmunity. As another possible explanation, anti-fumarase IgG might represent mitochondria-related systemic complications due to diabetes, which are clinically associated with DMO . Antigen-specific regulatory T cells, which lead to immune tolerance, might be modulated by diabetic states . Diabetes induces mitochondrial damage that is at least partly mediated via oxidative stress, which might contribute to the exposure of intracellular autoantigens and immunological tolerance loss by the post-translational modification of autoantigens [31,32,33,34].
Complement MAC was formed in photoreceptor inner and outer segments in the passive transfer model. This encouraged us to speculate that photoreceptor damage is mediated via complement activation at least partly among several autoantibody-related mechanisms [35, 36]. Some autoantibodies can, by themselves, inhibit the function of antigens and concomitantly promote pathological mechanisms, as in the case of myasthenia gravis. In contrast, anti-fumarase IgG did not promote cellular injury in the absence of complement. Cell death was not increased in FH−/− cells, and a few publications have documented oncological findings induced by genetic mutations [37, 38]. These results suggest that functional inhibition by this autoantibody alone does not induce photoreceptor damage. Immunofluorescence in cultured cells might suggest that the antibody reacted with intracellular autoantigens released from damaged cells and concomitantly contributed to secondary inflammatory responses .
We cannot conclude how the autoantibody for fumarase, a ubiquitously expressed protein, specifically injures photoreceptor cells in DMO; similarly, the mechanism of anti-mitochondrial antibodies in primary biliary cirrhosis has not been determined . Photoreceptor cells highly express fumarase and are the main source of reactive oxygen species in diabetic states, which might promote an antigen–antibody reaction in these cells as discussed above [33, 34]. The absence of CD59 might allow complement-mediated autoimmune mechanisms in the photoreceptor inner and outer segments. In addition, the barrier properties of the retinal pigment epithelium and external limiting membrane might inhibit the diffusion of autoantibodies or complement components and cellular infiltration from or into the subretinal spaces and confine cellular damage to these layers .
Generally, intracellular autoantigens are released during non-apoptotic cell death, and the deposition of antigen–antibody complex subsequently exacerbates cellular damage via several inflammatory mechanisms. Alternatively, anti-recoverin and anti-enolase antibodies from individuals with CAR are internalised into cells and induce cytotoxicity in the absence of complement [41, 42]. Histological findings in animal models led us to investigate complement-mediated mechanisms rather than inflammatory responses. Surprisingly, fumarase was recruited to the cell surface after complement treatment, and reacted to anti-fumarase antibody derived from DMO sera with subsequent MAC formation, as in the case of autoantigens at the cell surface. Such changes depend on calcium signalling, although the downstream executioners remain to be elucidated. This result is consistent with the enigmatic finding that anti-fumarase antibody reacted with antigens in both photoreceptor inner and outer segments in mice administered with DMO sera, although immunostaining with the same sera was limited to the photoreceptor inner segments in untreated retinas. This is a novel molecular mechanism by which this autoantibody reacts to intracellular antigens on the cell surface. Future studies should elucidate whether sublytic MAC formation promotes the elicitation of autoimmune responses to intracellular autoantigens. Such biological changes are to some extent analogous to autoimmune responses to mitochondrial molecules in a few diseases .
Anti-fumarase antibody and complement impaired the structure and function of mitochondria. Generally, overload of calcium signalling damages mitochondria [23, 44], which allowed us to speculate that the persistent Ca2+ influx would contribute to mitochondrial injuries at least in part in cultured cells incubated with this autoantibody and complement. Mitochondrial disruption is one of the initial changes in neurodegenerative diseases . In several pathological states, mitochondrial damage is one of the main regulators of synaptic plasticity . Several mechanisms of mitochondrial damage are generally accepted: dysregulated Ca2+ levels, generation of reactive oxygen species, loss of mitochondrial membrane potential (Δψm) and efflux of apoptotic or necrotic factors, some of which have been shown in this study [26, 31, 45, 46]. The other mechanism is caspase activation. Several publications have reported that the pruning of axons and dendrites is mediated via a caspase cascade . We thus speculated that caspase-3 activation partly contributed to the subcellular damage in the inner and outer segments, which corresponded to the dendrites specialised for photoreception.
Improvement in visual acuity is the primary endpoint for the efficacy of anti-VEGF drugs for DMO, which is supported by the general surrogate marker of decreased macular thickness . Recent publications have documented the restoration of the ellipsoid zone line on spectral domain OCT images after anti-VEGF therapy . Re-establishment of the BRB by this standard treatment might decrease intraocular anti-fumarase IgG or complement components derived from the blood and concomitant photoreceptor damage. The decrease in this autoantibody might be consistent with photoreceptor restoration after treatment, although further studies should elucidate the underlying molecular mechanisms. A translational research study has demonstrated the involvement of complement under anti-VEGF treatment in age-related macular degeneration . Future systematic and comparative studies may reveal the cellular and histological injuries induced by individual autoantibodies and/or complement in individual diseases [9, 10, 49, 50].
In conclusion, we have identified fumarase as a target of autoantibodies in a subgroup of DMO sera and a potential contributor to photoreceptor damage in DMO, although whether this autoantibody is the result of this pathogenesis remains to be elucidated. Further characterisation of autoantibodies against photoreceptor cells may facilitate the future development of antigen-specific immunomodulatory therapies for photoreceptor damage in DMO.
All data generated or analysed during this study are included in this published article and its supplementary information files.
Cytochrome C oxidase
Diabetic macular oedema
Human embryonic kidney
Logarithm of the minimum angle of resolution
Membrane attack complex
Optical coherence tomography
Vascular endothelial growth factor
Frank RN (2004) Diabetic retinopathy. N Engl J Med 350(1):48–58. https://doi.org/10.1056/NEJMra021678
Yau JW, Rogers SL, Kawasaki R et al (2012) Global prevalence and major risk factors of diabetic retinopathy. Diabetes Care 35(3):556–564. https://doi.org/10.2337/dc11-1909
Gardner TW, Antonetti DA, Barber AJ, LaNoue KF, Levison SW (2002) Diabetic retinopathy: more than meets the eye. Surv Ophthalmol 47(Suppl 2):S253–S262. https://doi.org/10.1016/S0039-6257(02)00387-9
Antonetti DA, Klein R, Gardner TW (2012) Diabetic retinopathy. N Engl J Med 366(13):1227–1239. https://doi.org/10.1056/NEJMra1005073
Murakami T, Frey T, Lin C, Antonetti DA (2012) Protein kinase cβ phosphorylates occludin regulating tight junction trafficking in vascular endothelial growth factor-induced permeability in vivo. Diabetes 61(6):1573–1583. https://doi.org/10.2337/db11-1367
Huang D, Swanson EA, Lin CP et al (1991) Optical coherence tomography. Science 254(5035):1178–1181. https://doi.org/10.1126/science.1957169
Browning DJ, Glassman AR, Aiello LP et al (2007) Relationship between optical coherence tomography-measured central retinal thickness and visual acuity in diabetic macular edema. Ophthalmology 114(3):525–536. https://doi.org/10.1016/j.ophtha.2006.06.052
Spaide RF, Curcio CA (2011) Anatomical correlates to the bands seen in the outer retina by optical coherence tomography: literature review and model. Retina 31(8):1609–1619. https://doi.org/10.1097/IAE.0b013e3182247535
Sakamoto A, Nishijima K, Kita M et al (2009) Association between foveal photoreceptor status and visual acuity after resolution of diabetic macular edema by pars plana vitrectomy. Graefes Arch Clin Exp Ophthalmol 247(10):1325–1330. https://doi.org/10.1007/s00417-009-1107-5
Murakami T, Nishijima K, Akagi T et al (2012) Optical coherence tomographic reflectivity of photoreceptors beneath cystoid spaces in diabetic macular edema. Invest Ophthalmol Vis Sci 53(3):1506–1511. https://doi.org/10.1167/iovs.11-9231
Aiello LP, Avery RL, Arrigg PG et al (1994) Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 331(22):1480–1487. https://doi.org/10.1056/NEJM199412013312203
Cunningham ET Jr, Adamis AP, Altaweel M et al (2005) A phase II randomized double-masked trial of pegaptanib, an anti-vascular endothelial growth factor aptamer, for diabetic macular edema. Ophthalmology 112(10):1747–1757. https://doi.org/10.1016/j.ophtha.2005.06.007
Bunt-Milam AH, Saari JC, Klock IB, Garwin GG (1985) Zonulae adherentes pore size in the external limiting membrane of the rabbit retina. Invest Ophthalmol Vis Sci 26(10):1377–1380
Lo EH, Dalkara T, Moskowitz MA (2003) Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci 4(5):399–415. https://doi.org/10.1038/nrn1106
Thirkill CE, Roth AM, Keltner JL (1987) Cancer-associated retinopathy. Arch Ophthalmol 105(3):372–375. https://doi.org/10.1001/archopht.1987.01060030092033
Ishida S, Yamashiro K, Usui T et al (2003) Leukocytes mediate retinal vascular remodeling during development and vaso-obliteration in disease. Nat Med 9(6):781–788. https://doi.org/10.1038/nm877
Ambati J, Anand A, Fernandez S et al (2003) An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice. Nat Med 9(11):1390–1397. https://doi.org/10.1038/nm950
Srivastava R, Aslam M, Kalluri SR et al (2012) Potassium channel KIR4.1 as an immune target in multiple sclerosis. N Engl J Med 367(2):115–123. https://doi.org/10.1056/NEJMoa1110740
Tolley E, Craig I (1975) Presence of two forms of fumarase (fumarate hydratase E.C. 22.214.171.124) in mammalian cells: immunological characterization and genetic analysis in somatic cell hybrids. Confirmation of the assignment of a gene necessary for the enzyme expression to human chromosome 1. Biochem Genet 13:867–883
Early Treatment Diabetic Retinopathy Study Research Group (1985) Photocoagulation for diabetic macular edema. Early Treatment Diabetic Retinopathy Study report number 1. Arch Ophthalmol 103:1796–1806
Wilkinson CP, Ferris FL 3rd, Klein RE et al (2003) Proposed international clinical diabetic retinopathy and diabetic macular edema disease severity scales. Ophthalmology 110(9):1677–1682. https://doi.org/10.1016/S0161-6420(03)00475-5
Triantafilou K, Hughes TR, Triantafilou M, Morgan BP (2013) The complement membrane attack complex triggers intracellular Ca2+ fluxes leading to NLRP3 inflammasome activation. J Cell Sci 126(13):2903–2913. https://doi.org/10.1242/jcs.124388
Rizzuto R, De Stefani D, Raffaello A, Mammucari C (2012) Mitochondria as sensors and regulators of calcium signalling. Nat Rev Mol Cell Biol 13(9):566–578. https://doi.org/10.1038/nrm3412
Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516. https://doi.org/10.1080/01926230701320337
Williams DW, Kondo S, Krzyzanowska A, Hiromi Y, Truman JW (2006) Local caspase activity directs engulfment of dendrites during pruning. Nat Neurosci 9(10):1234–1236. https://doi.org/10.1038/nn1774
Mattson MP, Gleichmann M, Cheng A (2008) Mitochondria in neuroplasticity and neurological disorders. Neuron 60(5):748–766. https://doi.org/10.1016/j.neuron.2008.10.010
Funatsu H, Yamashita H, Noma H et al (2002) Increased levels of vascular endothelial growth factor and interleukin-6 in the aqueous humor of diabetics with macular edema. Am J Ophthalmol 133(1):70–77. https://doi.org/10.1016/S0002-9394(01)01269-7
Adamus G, Aptsiauri N, Guy J et al (1996) The occurrence of serum autoantibodies against enolase in cancer-associated retinopathy. Clin Immunol Immunopathol 78(2):120–129. https://doi.org/10.1006/clin.1996.0021
Adamus G, Chew EY, Ferris FL, Klein ML (2014) Prevalence of anti-retinal autoantibodies in different stages of age-related macular degeneration. BMC Ophthalmol 14(1):154. https://doi.org/10.1186/1471-2415-14-154
Shechter R, London A, Schwartz M (2013) Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates. Nat Rev Immunol 13(3):206–218. https://doi.org/10.1038/nri3391
Nishikawa T, Edelstein D, Du XL et al (2000) Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404(6779):787–790. https://doi.org/10.1038/35008121
Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008) Regulatory T cells and immune tolerance. Cell 133(5):775–787. https://doi.org/10.1016/j.cell.2008.05.009
Du Y, Veenstra A, Palczewski K, Kern TS (2013) Photoreceptor cells are major contributors to diabetes-induced oxidative stress and local inflammation in the retina. Proc Natl Acad Sci U S A 110(41):16586–16591. https://doi.org/10.1073/pnas.1314575110
Doyle HA, Mamula MJ (2001) Post-translational protein modifications in antigen recognition and autoimmunity. Trends Immunol 22(8):443–449. https://doi.org/10.1016/S1471-4906(01)01976-7
Saadoun S, Waters P, Bell BA et al (2010) Intra-cerebral injection of neuromyelitis optica immunoglobulin G and human complement produces neuromyelitis optica lesions in mice. Brain 133(2):349–361. https://doi.org/10.1093/brain/awp309
Patrick J, Lindstrom J (1973) Autoimmune response to acetylcholine receptor. Science 180(4088):871–872. https://doi.org/10.1126/science.180.4088.871
Toro JR, Nickerson ML, Wei MH et al (2003) Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America. Am J Hum Genet 73(1):95–106. https://doi.org/10.1086/376435
Pollard PJ, Spencer-Dene B, Shukla D et al (2007) Targeted inactivation of fh1 causes proliferative renal cyst development and activation of the hypoxia pathway. Cancer Cell 11(4):311–319. https://doi.org/10.1016/j.ccr.2007.02.005
Hanayama R, Tanaka M, Miyasaka K et al (2004) Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice. Science 304(5674):1147–1150. https://doi.org/10.1126/science.1094359
Kaplan MM, Gershwin ME (2005) Primary biliary cirrhosis. N Engl J Med 353(12):1261–1273. https://doi.org/10.1056/NEJMra043898
Adamus G, Machnicki M, Seigel GM (1997) Apoptotic retinal cell death induced by antirecoverin autoantibodies of cancer-associated retinopathy. Invest Ophthalmol Vis Sci 38(2):283–291
Magrys A, Anekonda T, Ren G, Adamus G (2007) The role of anti-α-enolase autoantibodies in pathogenicity of autoimmune-mediated retinopathy. J Clin Immunol 27(2):181–192. https://doi.org/10.1007/s10875-006-9065-8
Grundtman C, Kreutmayer SB, Almanzar G, Wick MC, Wick G (2011) Heat shock protein 60 and immune inflammatory responses in atherosclerosis. Arterioscler Thromb Vasc Biol 31(5):960–968. https://doi.org/10.1161/ATVBAHA.110.217877
Georgiannakis A, Burgoyne T, Lueck K et al (2015) Retinal pigment epithelial cells mitigate the effects of complement attack by endocytosis of C5b-9. J Immunol 195(7):3382–3389. https://doi.org/10.4049/jimmunol.1500937
Swerdlow RH, Parks JK, Cassarino DS et al (1998) Mitochondria in sporadic amyotrophic lateral sclerosis. Exp Neurol 153(1):135–142. https://doi.org/10.1006/exnr.1998.6866
Galluzzi L, Blomgren K, Kroemer G (2009) Mitochondrial membrane permeabilization in neuronal injury. Nat Rev Neurosci 10(7):481–494. https://doi.org/10.1038/nrn2665
Mori Y, Suzuma K, Uji A et al (2016) Restoration of foveal photoreceptors after intravitreal ranibizumab injections for diabetic macular edema. Sci Rep 6(1):39161. https://doi.org/10.1038/srep39161
Keir LS, Firth R, Aponik L et al (2017) VEGF regulates local inhibitory complement proteins in the eye and kidney. J Clin Invest 127(1):199–214. https://doi.org/10.1172/JCI86418
Sawyer RA, Selhorst JB, Zimmerman LE, Hoyt WF (1976) Blindness caused by photoreceptor degeneration as a remote effect of cancer. Am J Ophthalmol 81(5):606–613. https://doi.org/10.1016/0002-9394(76)90125-2
Ambati J, Atkinson JP, Gelfand BD (2013) Immunology of age-related macular degeneration. Nat Rev Immunol 13(6):438–451. https://doi.org/10.1038/nri3459
We would like to thank R. H. Rosa Jr (Texas A&M Health Science Center, Bryan, TX, USA) for providing discussion, and K. Okamoto-Furuta and H. Kohda (Division of Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University, Kyoto, Japan) for technical assistance in electron microscopy.
The study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (26462637, 17K11423, 18K19610). Immunostaining of human retinas, which used the Core Center for Vision Research funded by P30 EY007003 from the National Eye Institute, was supported by awards from Eversight and the Diabetes Consortium (National Institutes of Health). MS studies were performed at the Medical Research Support Center, Graduate School of Medicine, Kyoto University, which was supported by the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
The authors declare that there is no duality of interest associated with this manuscript.
Electronic supplementary material
About this article
Cite this article
Yoshitake, S., Murakami, T., Suzuma, K. et al. Anti-fumarase antibody promotes the dropout of photoreceptor inner and outer segments in diabetic macular oedema. Diabetologia 62, 504–516 (2019). https://doi.org/10.1007/s00125-018-4773-1
- Anti-fumarase antibody
- Diabetic macular oedema
- Optical coherence tomography
- Photoreceptor damage
- Serum biomarker