Background Macular fibrosis causes irreparable vision loss in neovascular age-related macular degeneration (nAMD) even with anti-vascular endothelial growth factor (VEGF) therapy. Inflammation is known to play an important role in macular fibrosis although the underlying mechanism remains poorly defined. The aim of this study was to understand how infiltrating macrophages and complement proteins may contribute to macular fibrosis. Methods Subretinal fibrosis was induced in C57BL/6J mice using the two-stage laser protocol developed by our group. The eyes were collected at 10, 20, 30 and 40 days after the second laser and processed for immunohistochemistry for infiltrating macrophages (F4/80 and Iba-1), complement components (C3a and C3aR) and fibrovascular lesions (collagen-1, Isolectin B4 and α-SMA). Human retinal sections with macular fibrosis were also used in the study. Bone marrow-derived macrophages (BMDMs) from C57BL/6J mice were treated with recombinant C3a, C5a or TGF-β for 48 and 96 h. qPCR, Western blot and immunohistochemistry were used to examine the expression of myofibroblast markers. The involvement of C3a-C3aR pathway in macrophage to myofibroblast transition (MMT) and subretinal fibrosis was further investigated using a C3aR antagonist (C3aRA) and a C3a blocking antibody in vitro and in vivo. Results Approximately 20~30% of F4/80+ (or Iba-1+) infiltrating macrophages co-expressed α-SMA in subretinal fibrotic lesions both in human nAMD eyes and in the mouse model. TGF-β and C3a, but not C5a treatment, significantly upregulated expression of α-SMA, fibronectin and collagen-1 in BMDMs. C3a-induced upregulation of α-SMA, fibronectin and collagen-1 in BMDMs was prevented by C3aRA treatment. In the two-stage laser model of induced subretinal fibrosis, treatment with C3a blocking antibody but not C3aRA significantly reduced vascular leakage and Isolectin B4+ lesions. The treatment did not significantly alter collagen-1+ fibrotic lesions. Conclusions MMT plays a role in macular fibrosis secondary to nAMD. MMT can be induced by TGF-β and C3a but not C5a. Further research is required to fully understand the role of MMT in macular fibrosis. Graphical abstract Macrophage to myofibroblast transition (MMT) contributes to subretinal fibrosis. Subretinal fibrosis lesions contain various cell types, including macrophages and myofibroblasts, and are fibrovascular. Myofibroblasts are key cells driving pathogenic fibrosis, and they do so by producing excessive amount of extracellular matrix proteins. We have found that infiltrating macrophages can transdifferentiate into myofibroblasts, a phenomenon termed macrophage to myofibroblast transition (MMT) in macular fibrosis. In addition to TGF-β1, C3a generated during complement activation in CNV can also induce MMT contributing to macular fibrosis. RPE = retinal pigment epithelium. BM = Bruch’s membrane. MMT = macrophage to myofibroblast transition. TGFB = transforming growth factor β. a-SMA = alpha smooth muscle actin. C3a = complement C3a.

The suppressor of cytokine signaling protein 3 (SOCS3) critically controls immune cell activation, although its role in macrophage polarization and function remains controversial. Using experimental autoimmune uveoretinitis (EAU) as a model, we show that inflammation-mediated retinal degeneration is exaggerated and retinal angiogenesis is accelerated in mice with SOCS3 deficiency in myeloid cells (LysMSOCS3). At the acute stage of EAU, the population of infiltrating neutrophils was increased and the population of macrophages decreased in LysMSOCS3 mice compared with that in wild-type (WT) mice. Real-time RT-PCR showed that the expression of tumor necrosis factor-α, IL-1β, interferon-γ, granulocyte-macrophage colony-stimulating factor, and arginase-1 was significantly higher in the LysMSOCS3 EAU retina in contrast to the WT EAU retina. The percentage of arginase-1 infiltrating cells was significantly higher in the LysMSOCS3 EAU retina than that in the WT EAU retina. In addition, bone marrow-derived macrophages and neutrophils from the LysMSOCS3 mice express significantly higher levels of chemokine (C-C motif) ligand 2 and arginase-1 compared with those from WT mice. Inhibition of arginase using an l-arginine analog amino-2-borono-6-hexanoic suppressed inflammation-induced retinal angiogenesis without affecting the severity of inflammation. Our results suggest that SOCS3 critically controls the phenotype and function of macrophages and neutrophils under inflammatory conditions and loss of SOCS3 promotes the angiogenic phenotype of the cells through up-regulation of arginase-1.

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Aging-associated chronic oxidative stress and inflammation are known to be involved in various diseases, e.g., age-related macular degeneration (AMD). Previously, we reported the presence of dry AMD-like signs, such as elevated oxidative stress, dysfunctional mitophagy and the accumulation of detrimental oxidized materials in the retinal pigment epithelial (RPE) cells of nuclear factor erythroid 2-related factor 2, and a peroxisome proliferator-activated receptor gamma coactivator 1-alpha (NFE2L2/PGC1α) double knockout (dKO) mouse model. Here, we investigated the dynamics of inflammatory markers in one-year-old NFE2L2/PGC1α dKO mice. Immunohistochemical analysis revealed an increase in levels of Toll-like receptors 3 and 9, while those of NOD-like receptor 3 were decreased in NFE2L2/PGC1α dKO retinal specimens as compared to wild type animals. Further analysis showed a trend towards an increase in complement component C5a independent of component C3, observed to be tightly regulated by complement factor H. Interestingly, we found that thrombin, a serine protease enzyme, was involved in enhancing the terminal pathway producing C5a, independent of C3. We also detected an increase in primary acute phase C-reactive protein and receptor for advanced glycation end products in NFE2L2/PGC1α dKO retina. Our main data show C5 and thrombin upregulation together with decreased C3 levels in this dry AMD-like model. In general, the retina strives to mount an orchestrated inflammatory response while attempting to maintain tissue homeostasis and resolve inflammation.

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PurposeAging‐associated chronic oxidative stress and inflammation are known to be involved in various diseases, e.g. age‐related macular degeneration (AMD). Previously, we reported elevated oxidative stress, dysfunctional mitophagy and the accumulation of detrimental oxidized materials in the pigment epithelial (RPE) cells of dry AMD‐like nuclear factor erythroid 2‐related factor 2, and peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (NFE2L2/PGC1α) in a double knockout (dKO) mouse model. In this study, we show that mitochondrial‐derived oxidative stress leads to a buildup of complement component C5a, whereas the complement component C3a remains unaffected.MethodsImmunohistochemical stained retina samples from one‐year‐old NRF2/PGC1α ‐/‐ and age‐matched wild‐type mice were used for confocal microscopy analysis.ResultsImmunohistochemical analysis of pathogen recognition receptors (PRRs) revealed an increase in levels of Toll‐like receptors 3 (TLR3) and 9 (TLR9), while those of NOD‐like receptor 3 (NLRP3) were decreased in NFE2L2/PGC1α ‐/‐ retina. Further analysis showed a significant increase in complement component C5a independent of component C3, tightly regulated by complement factor H (CFH). Interestingly, we found that thrombin, a serine protease enzyme, was involved in enhancing the terminal pathway producing C5a, independent of C3. We also detect an increase in primary acute phase C‐reactive protein (CRP) and receptor for advanced glycation end products (RAGE) in NFE2L2/PGC1α ‐/‐ retina.ConclusionsOur study reveals previously unknown thrombin mediated C3 independent activation of C5a in the dry AMD like retina of NRF2/PGC1α double knockout mice model.

Uncontrolled inflammation in the retina can lead to fibrovascular membrane formation, which can cause severe structural damage to the retina thus leading to blindness. The pathogenesis of retinal fibrosis is not completely understood. Recent research has implicated the Hedgehog pathway transcription factor Gli1 in the development of fibrosis. This study aims to elucidate the role of Gli1 in the development of fibrovascular membranes in the retina utilising fluorescent reporter mice. Gli1CreERT2 mice were crossed with ROSA26TdTomato mice to produce Tomato x Gli1 crossbreeds. 6‐7 week old Heterozygous Tom x Gli1 mice received 100 mg/kg Tamoxifen daily (I.P.) for 5 days. Mice were monitored for 80 days to determine the duration Gli1‐Tomato expression. Eyes and other organs were collected for immunofluorescent staining. Tom x Gli1 mice received 4–5 laser burns per eye to induce choroidal neovascularisation and were monitored over 21 days to assess the expression of Gli1 in the injured retina using fundus imaging and immunofluorescent staining. Expression of Gli1‐Tomato was detected as far 80 days in the eye, predominantly in the choroid, sclera, cornea and ciliary body. Immunofluorescent staining displayed co‐expression of Gli1 with CD31+, NG2+, and Collagen 4+ blood vessels. Fundus imaging of CNV lesions displayed expression of Gli1‐tomato around the lesion. Immunostains of cryosections showed co‐expression of Gli1 with blood vessel markers in choroidal lesions. Gli1+ cells are expressed throughout the eye, most notably with blood vessels, and may contribute to the development of choroidal lesions in CNV.