In this study, we show that complement components are deposited in significant amounts in the retina in EAU and that inhibition of the AP of complement can both reduce complement deposition and significantly reduce EAU. The inhibitory effect of CRIg appears to be mediated via inhibition of macrophage inducible nitric oxide synthase (iNOS) expression and production of nitric oxide (NO) presumably via reduced complement activation.

The complement receptor of the Ig superfamily (CRIg, also a member of B7 family‐related proteins termed V‐set and Ig domain‐containing 4, VSIG4 20 ) is a receptor for the β‐chain of multimers C3b, iC3b, and C3c 21 , 22 and is expressed in a subset of tissue‐resident macrophages 20 , 21 , 23 . Binding of C3b, iC3b, and C3c to CRIg promotes the clearance of opsonised particles ( e.g. pathogens or apoptotic cells) coated with these complement fragments by macrophages 21 , 23 . In addition, CRIg can also selectively inhibit AP complement activation 24 , 25 by abrogating the interaction of C3 and C5 with their convertases C3bBb and C3bBbC3b of the AP 24 . A soluble form of CRIg ( i.e. CRIg fusion protein, CRIg‐Fc), composed of the extracellular portion of murine CRIg and the Fc portion of murine IgG1, has been shown to attenuate pathology in a number of settings through selective suppression of AP‐mediated complement activation 25 , 26 . CRIg‐Fc has a high binding affinity for the dimeric C3b 2 subunit as compared with the monomeric C3b subunit 25 . It therefore selectively suppresses the AP by blocking C5 binding to its convertase C3bBbC3b of the AP, but does not influence the binding of C5 to the convertase C3bC4b of the CP 24 .

EAU is a long‐established model of endogenous posterior uveoretinitis that closely resembles the human disease clinically and pathologically 11 - 13 . The disease represents a T‐cell‐driven autoimmune response to retinal antigens 11 , 14 , in which both Th1 and Th17 T cells are involved 15 , 16 . Complement has also been shown to be involved in EAU. Mice deficient in complement C3 are less susceptible to EAU 17 , whereas mice deficient in the decay‐accelerating factor develop greater EAU than their wild‐type controls 18 . Furthermore, EAU can be suppressed by introducing the soluble complement activation inhibitor (sCrry) 17 , recombinant decay‐accelerating factor 18 , or complement C5 monoclonal antibody 19 . The contribution of complement activation via the AP to the pathology of EAU, however, remains to be elucidated.

Complement proteins are synthesized primarily by hepatocytes in the liver and released into the plasma for tissue distribution. In the eye, a low degree of complement activation exists under physiological conditions 3 , which increases with age 4 , 5 . How complement activation is regulated in the retina in pathophysiological conditions is not well defined. Although plasma complement components can easily reach ocular tissues lacking a tight blood tissue barrier such as the sclera and choroid, the retina is relatively closed off to the immune system due to the blood–retinal barrier, yet retinal complement activation occurs even under normal aging conditions 5 . The fact that many complement components including C1q, C3, and the complement regulatory proteins complement factor H (CFH) and complement factor B (CFB) can be produced locally in the eye 4 , 6 - 10 suggests that the retina also has the ability to regulate complement activation. We have shown previously that inflammatory cytokines negatively regulate CFH 9 , but positively regulate CFB 4 production. Since CFH and CFB are exclusively involved in AP complement activation 1 , this suggests that the AP might be involved in modifying retinal inflammation. The aim of this study was therefore to investigate the role of the AP using the model of experimental autoimmune uveoretinitis (EAU).

Complement constitutes one of the main components of the innate immune system and is important for cellular integrity, tissue homeostasis and modifying the adaptive immune response. Complement can be activated through the classical pathway (CP), the mannose‐binding lectin pathway, and the alternative pathway (AP). The key difference between different pathways rests on how the enzymes, i.e. C3 and C5 convertases, are formed. The convertases of C3 and C5 of the CP and lectin pathway comprise the complement components C4bC2b and C4bC2bC3b, respectively, whereas in the AP they are composed of C3bBb (C3 convertase) and C3bBbC3b (C5 convertase) 1 . In addition to these three well‐known pathways, complement is also activated by a pathway that acts independently of C3 to bypass the C3 convertase and is mediated by direct thrombin action on the C5 convertase 2 .

The effects of CRIg‐Fc on macrophage iNOS gene expression and NO production. BMDM were stimulated with 100 ng/mL of LPS for 24 h in the presence of different concentrations of CRIg‐Fc or 100 μg/mL of IgG1control protein. iNOS gene expression was quantified by qRT‐PCR (A) and NO production was quantified by Griess method (B). (A) iNOS gene fold change versus macrophages without LPS stimulation. (B) NO's production in the supernatants of different macrophages. * p <0.05, compared with PBS‐treated cells, Dunnett's multiple comparison test, n =5. The experiment was repeated twice.

NO produced by infiltrating macrophages are one of the important mediators of retinal damage in EAU 29 , 30 . When stimulated with LPS, BM‐derived macrophages (BMDM) expressed high levels of iNOS gene (Fig. 7A ) and produced large amounts of NO (Fig. 7B ). In vitro CRIg‐Fc treatment dose‐dependently suppressed iNOS gene expression (Fig. 7A ) as well as NO production induced by LPS in BMDM (Fig. 7B ). Control protein (anti‐gp120, mouse IgG1) showed no effect on either iNOS gene expression (Fig. 7B ) or NO production (Fig. 7B ).

The effects of CRIg‐Fc on cytokine production. (A) Spleen cells from PBS‐treated EAU and CRIg‐Fc‐treated EAU mice were stimulated with 25 μg/mL of pIRBP for 48 h. Cytokines were analyzed by cytometric bead assay (CBA). * p <0.05, ** p <0.01, n =6, Student's t ‐test. (B) Spleen cells from PBS‐treated EAU mice were stimulated with 25 μg/mL of pIRBP in the presence of different concentrations of CRIg‐Fc for 48 h. Cytokines were analyzed by CBA assay. * p <0.05, ** p <0.01, n =6, Dunnett's multiple comparison test.

Splenocytes from day 25 p.i. EAU mice produced significant amounts of IFN‐γ, IL‐6, IL‐17A, and TNF‐α but little IL‐4, IL‐10, IL‐12, and IL‐21 upon stimulation with pIRBP in vitro (Fig. 6A ). Treatment with CRIg‐Fc in vivo (treatment from day 1 to day 22 p.i.) led to a marked reduction in in vitro INF‐γ, IL‐6, IL‐17A, and TNF‐α production compared with cells from PBS‐treated EAU mice (all cells were stimulated in vitro with 25 μg/mL pIRBP, Fig. 6A ). In addition, in vitro treatment of CRIg‐Fc also significantly reduced the production of pIRBP‐induced IFN‐γ, IL‐2, IL‐6, and IL‐17A in cells of PBS‐treated EAU mice (Fig. 6B ). The production of IL‐10, however, was slightly increased by the same concentration of CRIg‐Fc (Fig. 6B ). Interestingly, the production of in vitro pIRBP‐induced TNF‐α was not affected by CRIg‐Fc treatment (Fig. 6B ).

The effects of CRIg‐Fc on T‐cell proliferation. (A) Spleen cells from PBS‐treated EAU and CRIg‐Fc‐treated EAU and nonimmunized normal mice were stimulated with or without 25 μg/mL of pIRBP and analyzed by thymidine incorporation. * p <0.05, n =4. (B) spleen cells from PBS‐treated EAU mice were stimulated with 25 μg/mL of pIRBP or 2.5 μg/mL Con A in the presence of different concentrations of CRIg‐Fc. Cell proliferation was analyzed by thymidine incorporation. * p <0.05, ** p <0.01, n =6, Dunnett's multiple comparison test.

To further understand the mechanism of CRIg‐Fc‐mediated inhibition of retinal inflammation, the proliferation of T cells from EAU mice treated with or without CRIg‐Fc was evaluated. Without in vitro IRBP stimulation, splenocytes from PBS‐treated EAU mice showed low levels of spontaneous proliferation (500 CPM on 3 H incorporation, Fig. 5A ). Cells from CRIg‐Fc treated (days 1–22 p.i.) EAU mice had the same levels of 3 H incorporation as the cells of nonimmunized normal mice (around 200 CPM, Fig. 5A ), indicating the lack of proliferation. After IRBP peptide (25 μg/mL) stimulation, splenocytes from PBS‐treated EAU mice proliferated massively as compared with cells from nonimmunized normal mice (Fig. 5A ). However, the level of cell proliferation in CRIg‐Fc‐treated EAU mice was significantly lower than that of PBS‐treated EAU mice (Fig. 5A ). Splenocytes from day 18 to day 24 p.i. CRIg‐Fc‐treated EAU mice showed similar results (data not shown).

Retinal C3d deposition and CFB expression after CRIg‐Fc treatment. (A–G) Retinal sections from control IgG1‐treated EAU (A, C, E) and CRIg‐Fc‐treated EAU (B, D, F) mice were stained for C3d and PI and observed by confocal microscopy. Ch, choroid; GL, ganglion layer; ONL, outer nuclear layer; and CB, ciliary body. (G) Isotype control staining. (H) CRIg‐Fc on CFB gene expression in EAU mice. CFB gene expression in EAU mice treated with control IgG1 or CRIg‐Fc was expressed as fold changes against nonimmunized normal mice. N =5, Student's t ‐test. qRT‐PCR was performed twice.

In addition to reduced retinal inflammation (Fig. 3G ), complement C3d deposition in the photoreceptor/RPE layer (Fig. 4A and B ), the ganglion cell layer (Fig. 4C and D ), and the ciliary body (Fig. 4E and F ) was also markedly reduced by CRIg‐Fc treatment, indicating decreased AP‐mediated complement activation. Furthermore, quantitative real‐time PCR (qRT‐PCR) analysis revealed that the 59‐fold increase in CFB expression in isotype‐IgG1 EAU mice was restored to the essentially normal values by treatment with CRIg‐Fc (Fig. 4H ). There was also a 50% reduction in CFB gene expression in RPE/choroid/sclera tissue of CRIg‐Fc‐treated mice as compared with that of isotype IgG1‐treated EAU mice, although the reduction did not reach statistical significance (Fig. 4H ).

The effects of CRIg‐Fc on clinical and histological scores of EAU. (A–F) Mice were treated i.p. with 4 mg/kg of CRIg‐Fc from day 1 to day 22 p.i. and clinical and histological investigations were carried out at day 25 p.i. (A) A control (PBS treated) EAU mouse fundus shows severe vasculitis and retinal infiltration. (B) A CRIg‐Fc‐treated mouse fundus shows mild inflammation around the optic nerve and the inferior retina. (C) Clinical score. (D) An H&E section from a PBS‐treated EAU mouse, showing severe retinal and vitreous infiltration, retinal fold (arrow), and granuloma (asterisk). (E) An H&E section from a CRIg‐Fc‐treated EAU mouse, showing a few infiltrates in the retina and vitreous, and a small retinal fold. Many of the photoreceptor outer segments are largely intact (double‐headed arrows). Ch, choroid; Re, retina; and Vi, vitreous. (F) Retinal infiltration and structural scores in PBS‐treated EAU and CRIg‐Fc‐treated EAU mice. (G) Clinical score of EAU in mice treated with CRIg‐Fc or control mouse IgG1 (anti‐gp120) from day 18 to day 24 p.i. (H) Clinical score of EAU in mice treated with PBS or CRIg‐Fc from day 1 to day 10 p.i. Mann–Whitney test was used to compare the difference between CRIg‐Fc‐treated group and PBS/IgG1‐treated group.

Having shown that AP‐mediated complement activation is likely to be involved in EAU and CRIg expression is lost at peak of disease, we then went on to test whether the administration of exogenous CRIg (CRIg‐Fc) would alter the progress of retinal inflammation. When CRIg‐Fc was administered (i.p.) daily from day 1 to day 22 p.i., the severity of retinal inflammation was significantly reduced (Fig. 3A–F ). Pathological investigation showed that retinal infiltration and structure damage were markedly improved by CRIg‐Fc treatment. In control PBS‐treated EAU retina, massive areas of inflammatory cell infiltration, retinal folds, and granulomas were frequently seen (Fig. 3D ), whereas in CRIg‐Fc‐treated EAU mouse retina, only mild foci of infiltration were seen, and retinal structure was largely preserved (Fig. 3E ). On average, there was a 54% reduction in the inflammatory cell infiltration score and a 58% reduction in the structural damage score in CRIg‐Fc‐treated mice as compared with PBS‐treated EAU mice ( p <0.05) (Fig. 3A–F ). When CRIg‐Fc was injected after T‐cell priming and the initiation of EAU ( i.e. from day 18 to day 24 p.i.), retinal inflammation was also significantly reduced (Fig. 3G ). However, when CRIg‐Fc was injected only at the T‐cell priming stage, i.e. from day 1 to day 10 p.i. no significant reduction in EAU severity was observed (Fig. 3H ).

CRIg expression in normal and EAU mice. (A and B) Spleen cells from normal and day 25 p.i. EAU mice were stained for F4/80 and CRIg and analyzed by flow cytometry. (A) Histogram of CRIg expression in F4/80 + macrophages. (B) Percentage of CRIg + macrophage in normal and day 25 p.i. EAU mice. N =5 mice. (C–F) F4/80 and CRIg expression in EAU mouse retina. Retinal sections from a normal mouse (C), a day 25 p.i. (D and E) and a day 35 p.i. (F) EAU mouse retinas were stained for F4/80, CRIg, and PI and observed by confocal microscopy. Images shown in (E) and (F) are areas of retinal ganglion layer and the vitreous. Ch, choroid; SC, sclera; GL, ganglion layer; and Vi, vitreous.

There was no significant change in the number of CRIg + cells among spleen F4/80 + macrophages in day 25 p.i. EAU mice as compared with nonimmunized normal mice (Fig. 2A, B ). In the normal mouse eye, CRIg was expressed by a proportion of resident choroidal macrophages with some low‐level coexpression with F4/80 macrophages (Fig. 2C ) 5 . However, in peak‐stage EAU (day 25 p.i.), CRIg was not detected in any F4/80 + macrophages in the choroid or sclera (Fig. 2D ), or in infiltrating macrophages in the inflamed retina and vitreous (Fig. 2E ). This is similar to data in mouse autoimmune myocarditis 20 . In EAU, inflammation peaks at days 21–28 p.i. 27 and the severity decreases after this time, but persists as a low‐grade chronic inflammation (Xu et al. unpublished data) 28 . Interestingly, as the severity of disease decreased many CRIg + F4/80 + macrophages was detected (day 35 p.i. EAU, Fig. 2F and day 60 p.i. EAU, data not shown) in the retina, suggesting that CRIg + macrophages may be involved in the resolution of inflammation.

CFB was detected at the apical portion of the RPE cells in normal mouse retina (Fig. 1B‐i ) 4 . The expression of CFB in RPE cells increased significantly in the retinas of mice with early stage EAU (day 18 p.i.) (p.i., post‐immunization) (Fig. 1B‐ii ). As disease progressed, CFB expression further extended from the RPE layer to photoreceptors (Fig. 1B ). Infiltrating cells also expressed CFB (arrows in Fig. 1B‐iii ). Real‐time RT‐PCR analysis revealed a 61‐fold increase in CFB mRNA expression in the retina of day 25 p.i. EAU mice as compared with that of noninflamed normal mice (Fig. 1C ). The expression of CFB mRNA in RPE/choroid/sclera tissue also increased significantly in day 25 p.i. EAU mice (5.68‐fold) (Fig. 1C ). These results suggest that a high level of AP‐mediated complement activation is likely to be present in the retina in EAU and may contribute to EAU pathology. Isotype control staining did not reveal any positivity (Fig. 1B‐iv ).

C3d deposition and CFB expression in EAU. Normal mouse and day 25 p.i. EAU mouse eye sections were stained for C3d (A) or CFB (B) and PI and observed by confocal microscopy. (A‐i) C3d low‐level deposition in Bruch's membrane (arrows) of a normal mouse eye. (A‐ii–iv) C3d deposition in a day 25 p.i. EAU mouse. (B‐i) CFB expression in the retina of a normal mouse. (B‐ii) CFB expression in the retina of a day 18 p.i. EAU mouse. (B‐iii) CFB expression in the retina of a day 25 p.i. EAU mouse retina. (B‐iv) Isotype control staining for CFB. Ch, choroid; CB, ciliary body; ONL, outer nuclear layer; INL, inner nuclear layer; GL, ganglion layer; Vi, vitreous; and RPE, retinal pigment epithelia. (C) CFB gene expression in the retina, RPE/choroid/sclera of normal and EAU mice. qRT‐PCR were performed as described in Materials and methods section. Data were presented as fold changes in CFB gene expression in the retina and RPE/choroid/sclera in EAU mice against normal mice. N =5 mice, Student's t ‐test. The experiment was performed twice.

Discussion

Although complement activation is beneficial in clearing infection and is essential for tissue homeostasis, unregulated complement activation may contribute to the pathogenesis of autoimmune disease. The data reported in this article using EAU as a model disease support this view. During inflammation, complement‐mediated damage is well recognised. Complement activation may amplify the inflammatory response not only by the formation of the membrane attack complex (C5b‐9), but also by releasing a variety of complement fragments, particularly the anaphylatoxins C3a and C5a. The anaphylatoxin molecules C3a and C5a enhance vascular permeability (i.e. breakdown of blood–retinal barrier in the retina), promote T‐cell costimulatory and survival signals 31, 32, recruit immune cells, activate mononuclear phagocyte, and release inflammatory mediators 33, 34. A more recent study has shown that in the presence of IFN‐γ, C5a is able to induce macrophage NO production and contributes to retinal damage in EAU 19. The C5a/C5aR pathway has also been shown to negatively regulate Th17‐ and Treg‐cell differentiation via reduction in TGF‐β secretion 35, 36. Dendritic cells deficient in C5aR produce high levels of TGF‐β which promotes Treg production, or in the presence of IL‐6 and IL‐23, promotes the induction of Th17 cells and IL‐17‐associated inflammatory disease 35. In addition, C5a also promotes γδ T‐cell IL‐17A production and blocking of C5a with a neutralizing antibody suppresses T‐cell IL‐17 production 36. Control of complement activation in EAU is likely, therefore, to have beneficial action at multiple levels.

Since complement can be activated through a number of pathways, it is important to identify which pathways are active in EAU. Our study suggests that the AP‐mediated complement activation contributed significantly to EAU pathology. What causes excessive AP complement activation in EAU is not known. AP complement activation occurs spontaneously at low levels in a “tick‐over” manner in physiological conditions. The process can be amplified under certain pathological conditions where other factors such as factor B, factor D, and properdin are preferentially generated in situ, allowing the full operation of the amplification loop. TNF‐α is one of the main inflammatory cytokines present at high levels in EAU 37, 38 and we have previously shown that TNF‐α downregulates CFH production 9, and upregulates CFB production 4. In this study, CFB was found massively upregulated in EAU retina (Fig. 1), which may contribute to uncontrolled AP complement activation. In addition, during EAU, Ig may be increased both systemically and locally, which may result in increased C3b 2 –IgG complex, i.e. the precursor of the AP amplification loop 39, further enhancing AP complement activation. However, further studies are required for the full understanding of the mechanism.

The protective effect of CRIg‐Fc in EAU is not limited to its direct action on AP complement activation and subsequent reduction in the release of anaphylatoxins. In addition to its function as a complement receptor 22, CRIg is also a B7 family‐related protein known as B7 family‐related proteins VSIG4 20. A previous study has shown that CRIg (VSIG4) is a potent negative regulator of T‐cell responses 20, and VSIG4‐Ig fusion protein inhibits cytotoxic T‐ and B‐cell responses to viral antigen 20. In this study, CRIg‐Fc suppressed T‐cell proliferation both in vivo and in vitro. However, as we used a mixed population of splenocytes, whether the reduced cell proliferation is a direct effect of CRIg‐Fc on T cells or an indirect effect through other APC remains to be elucidated. In addition, CRIg‐Fc also reduced inflammatory cytokines IFN‐γ, TNF‐α, IL‐6, and IL‐17 production in T cells (Fig. 6), and NO production in macrophages (Fig. 7), further supporting the negative immune regulation roles of CRIg 20. In vivo treatment of mice with CRIg‐Fc at the disease priming stage (i.e. days 1–10 p.i.) did not affect disease progression, suggesting that CRIg‐Fc has no effect or very limited effect on antigen presentation and T‐cell activation in EAU.

EAU is traditionally recognized to be a Th1/Th17 CD4 T‐cell‐mediated disease 40, there is, however, increasing recognitions of the central role of macrophages both as mediators of disease 38, 41 and as suppressors of inflammation 42. Although CRIg mRNA is expressed in mature dendritic cells, neutrophils as well as tissue macrophages 20, CRIg protein has been detected in only a certain subset of resident macrophages 20, 21, and the expression of CRIg declines once the macrophages are activated 20, 21. We found that CRIg was expressed in a subset of choroidal macrophage in normal mouse eyes (Fig. 2C) 5, but was completely absent on retinal inflammatory macrophages in peak stage EAU; remarkably, CRIg expression on macrophage returned and in increase amounts in the resolving stages of EAU (Fig. 2F). Whether this change in expression was due to reprogramming of resident macrophages or represented de novo recruitment of macrophages at different stages of disease is unclear. What is clear is that CRIg+ macrophages may belong to the “suppressive” variety of macrophage and may play important roles in tissue homeostasis. They may also be involved in the resolution of inflammation probably by promoting the clearance of apoptotic cells 21, 23.

One of the homeostatic roles of the choroidal CRIg+ macrophage might be to prevent tissue overt complement activation. When the tissue is inflamed (such as in EAU), tissue‐resident CRIg+ macrophages are quickly consumed or negatively regulated by inflammatory cytokines, and the newly recruited macrophages do not express CRIg. The lack of CRIg molecules allows complement activation proceeding uncontrolled in EAU. When exogenously administering the soluble form of CRIg i.e. CRIg‐FC, complement activation is blocked resulting in reduced C3a/C5a production, which may indirectly affect inflammatory cytokine production. It is also possible that CRIg‐Fc may inhibit pro‐inflammatory CRIg− macrophages and suppress NO, TNF‐α, and other mediators including complement components (such as CFB) production. The effect of CRIg‐Fc on Th1/Th17 cytokine production observed in this study may be indirectly resulted from the suppression of the pro‐inflammatory macrophage activation, or C5a production (as a result of reduced complement activation). Further mechanistic studies on the suppressive effect of CRIg‐Fc on macrophages and dendritic cells, the possible unknown receptors for CRIg‐Fc, and the signalling pathways will be important to understand the immune regulation roles of CRIg and such experiments are undergoing in the investigators' laboratory.

In summary, in this study we show that the AP complement activation plays detrimental roles in retinal pathology. Blocking AP‐mediated complement activation with CRIg‐Fc reduces retinal inflammation. CRIg‐Fc not only selectively blocks the AP complement activation, but also suppresses inflammatory macrophage function and reduces disease severity in EAU. CRIg‐Fc could be a good candidate for uveitis therapy.