As in Real Estate, Location Matters: Cellular Expression of Complement Varies Between Macular and Peripheral Regions of the Retina and Supporting Tissues

Abstract

The cellular events that dictate the initiation of the complement pathway in ocular degeneration, such as age-related macular degeneration (AMD), is poorly understood. Using gene expression analysis (single cell and bulk), mass spectrometry, and immunohistochemistry, we dissected the role of multiple retinal and choroidal cell types in determining the complement homeostasis. Our scRNA-seq data show that the cellular response to early AMD is more robust in the choroid, particularly in fibroblasts, pericytes and endothelial cells. In late AMD, complement changes were more prominent in the retina especially with the expression of the classical pathway initiators. Notably, we found a spatial preference for these differences. Overall, this study provides insights into the heterogeneity of cellular responses for complement expression and the cooperation of neighboring cells to complete the pathway in healthy and AMD eyes. Further, our findings provide new cellular targets for therapies directed at complement.

Keywords: RPE/choroid; age-related macular degeneration; complement; retina; single cell.

Figure 1

Cell type specific complement expression in the combined macula and peripheral human retina, choroid and RPE. (A) Dot plot showing expression pattern of genes of the complement pathways across cell types identified in the combined macula and peripheral retina. The dot plot was generated using Dotplot in the R Seurat package. The size of the dots represents the percentage of cells that expressed gene markers while color shows average expression levels of gene markers. (B) Reprocessed combined macula and peripheral RPE data from Voigt et al. (51). (C) Dot plot showing expression pattern of known gene markers across cell types identified in the combined macula and peripheral choroid.

Figure 2

Validation of scRNA-seq for retinal complement component expression by in situ hybridization. (A) Factors driving complement activation were chosen for detection by in situ hybridization. CFD expression from microglia was detected in the inner nuclear layer (INL), outer plexiform layer (OPL) and outer nuclear layer (ONL), but was mostly associated with microglia expressing AIF1 (alias IBA1) in the OPL. C3 expression was mostly limited to microglia located in OPL. C7 transcripts were detected in the OPL with overlap of signal with the horizontal marker Onecut1 in the OPL. (B) Soluble (CFH, CFI) and membrane-bound (CD55) complement regulators primarily colocalized with vascular cells and microglia. Signals of CFH transcript were strongest from INL and OPL and overlapped with the endothelial marker CD34. CFI expression associated with the endothelial marker CLDN5 was strongest in the INL and OPL. CD55 was robustly expressed in microglia expressing AIF. (C) Complement receptor C3AR1 transcripts were detected at low levels in the INL and OPL and co-localized with the microglia marker AIF1. ITGAX was robustly expressed in the OPL and co-localized with the microglia marker AIF1 in the OPL. ITGB2 transcripts were less abundantand co-localized with AIF1 in the OPL. Also VSIG4 was strongly expressed in OPL and ONL co-localizing with the microglia marker AIF1. (A–C) Arrows indicate co-localization of the gene of interest with respective cell marker. Scale bars, 20 µm.

Figure 3

Complement proteome of macroglia (Müller cell, astrocytes), retinal neurons and RPE/choroid in the macular and peripheral human eye. Müller cells and retinal neurons were purified from peripheral and macular retinal tissue punches (6 mm in diameter) from 5 donor retinae ( Table S1 ) by magnetic-activated cell sorting (MACS) and were submitted to quantitative LC MS/MS mass spectrometry. Contamination with astrocytes is likely, because no surface marker yet separates them clearly from Müller cells. Müller cells definitely outnumber astrocytes. Macroglia and neurons were depleted from ITGAM (alias CD11B)-positive microglia/macrophages and CD31-positive vascular cells. RPE/choroid was collected after the removal of retinal tissue and comprised a mixture of RPE and choroidal cell types including pericytes, endothelial cells, fibroblast and immune cells. Given the intense perfusion of choroidal tissues, these samples do not allow unequivocal discrimination of the source especially of soluble complement components as they could be expressed by local cell types or by liver cells and enter the choroid via the circulation.

Figure 4

C1s protein expression in the human retina. (A) Full C1s (~75 kDa) and the C1s heavy chain (~47 kDa) were reliably detected by Western blot using purified C1s protein and human serum (HS). C1s-depleted HS still contained remaining C1s heavy chain, while full length C1s was absent. (B) Western blot analysis of C1s on purified retinal cell types from peripheral human retina and RPE/choroid. The total protein extracted per cell population was loaded. PDHB (pyruvate dehydrogenase beta subunit) served as housekeeper that has been shown to be expressed at equal levels in all investigated cell types. In contrast to HS, primarily the heavy chain of C1s was detected in retinal samples. C1s levels were highest in Müller cells (MC) and vascular cells (VC). MG, microglia; NR, whole neuroretina. RPE, retinal pigment epithelium including choroid. (C) Representative micrographs of C1s-stainings from macular (left) and peripheral (right) retinae. Photoreceptors and cells of the ganglion cell layer (GCL) as well as punctate structures in the inner plexiform layer (IPL) displayed highest labeling intensities. Sections were co-stained for the Müller cell marker glutamine synthetase (GLUL). (D) Higher magnification of the outer nuclear layer (ONL) and Henle fiber layer (HL) in the macular retina showed a partial overlap of C1s and GLUL. (E) Co-staining of C1s with calretinin, a marker of inner retinal neurons such as ganglion and amacrine cells, yielded no considerable overlap. (C-E) INL, inner nuclear layer; OPL, outer plexiform layer; PRS, photoreceptor inner and outer segments. Scale bars, 20 µm.

Figure 5

C3 protein expression in the human retina. (A) The C3 α-chain (~120 kDa) and its cleavage products were robustly detected by Western blot if purified C3 or human serum (HS) was loaded. No characteristic bands were detected in C3-depleted HS. (B) Western blot analysis of C3 on purified retinal cell types from peripheral human retina and RPE/choroid. The total protein extracted per cell population was loaded. PDHB served as housekeeper since not the exact same amount of protein could be loaded given the low protein yield from the microglial (MG) and vascular cell (VC) populations. The C3 α-chain and its cleavage product C3d were detected in every retinal cell population at comparable levels. MC, Müller cells; N, retinal neurons; NR, whole neuroretina; RPE, RPE/choroid mixed samples. (C) Representative micrographs of C3-stainings from macular (left) and peripheral (right) retinae. Photoreceptors, photoreceptor terminals and Müller cell processes were positively labeled. Co-staining for the Müller cell marker glutamine synthetase (GLUL) demonstrated an overlap primarily in cells from the peripheral retina. (D) Higher magnification of the inner nuclear layer (INL) highlighting the co-localization of C3 and GLUL (arrows). (E) Co-staining of C3 and GLUL did not result in an overlap in the outer retina. C3 stained cone photoreceptors (asterisk) which form a single row excluding rod photoreceptors from that layer in the central retina. (C–E) GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRS, photoreceptor inner and outer segments. Scale bars, 20 µm.

Figure 6

CFI, CFH and C7 protein expression in the human retina. (A) Loading purified C7 or human serum (HS), full length C7 was detected as a single band (~90 kDa) and cleavage products <90 kDa. (B) Full-length C7 was detected at low levels in purified cell types, but robustly in RPE/choroid. Cleavage products were detected in all cell populations. C7 immunoreactivity was similar in macular and peripheral retinal sections. (D) Higher magnification of the C7 staining in RPE. (E) Loading purified CFI or HS, only the CFI heavy chain (~50 kDa) was unequivocally detected by Western blot. (F) CFI was present in Müller cells (MC), microglia (MG), vascular cells (VC) and RPE/choroid, but not in neurons. (G) Comparable CFI-labeling of structures of the macular and peripheral retina. CFI-immunoreactivity partially overlapped with that of the Müller cell marker glutamine synthetase (GLUL). (H) Higher magnification of the inner plexiform layer (IPL) demonstrating co-localization of CFI with IBA1-positive microglia. (I) Detection of CFH (~150 kDa) by Western blot using purified CFH and HS. (J) CFH was only detected in RPE/choroid. A contamination with CFH from the system circulation cannot be excluded. (K) CFH immunoreactivity was confined to vessel lumens. (L) Minor CFH immunoreactivity at the RPE – Bruch’s membrane interface at higher magnification. (B, F, J) PDHB or GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) served as housekeepers. N, neurons; NR, whole neuroretina. (C, D, G, H, K, L) GCL, ganglion cell layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRS, photoreceptor segments. Scale bars, 20 µm.

Figure 7

Comparison of cell type-specific complement expression between peripheral human (H) and mouse (M) retina. Expression of complement components was determined by scRNA-seq. For cross-species comparison data from the present study on human retina was compared to recently published data from mouse retina (48). Expression is measured in units of FPKM (Fragments Per Kilobase of transcript per Million mapped reads) and color-coded on a logarithmic scale.

Figure 8

Log fold change (FC) of complement gene transcription within retina and choroidal cells from donors diagnosed with early AMD. (A) LogFC of Differentially expressed (DE) retinal genes between early AMD macula and normal macula were defined as a gene showing expression in at least 10% of cells within a cell type and a P ≤ 0.05. (B) LogFC of DE choroidal genes between early AMD macula and normal macula were defined as in A.

Figure 9

Heat map of complement gene expression determined using bulk RNA-seq for macular retina (MR), peripheral retina (PR), macular RPE/choroid/sclera (MRCS) and peripheral RPE/choroid/sclera (PRCS) from normal, early AMD and advanced AMD donors. Expression is measured in units of FPKM and color-coded on a logarithmic scale.