Fcα Receptor Type I and Its Association with Atherosclerosis Development

Objectives Atherosclerosis is a chronic inflammatory disease characterized by lipid accumulation and local inflammation, which are regulated by the immune system. The immunological aspects of this disease are unclear. Immunoglobulin A regulates many cell responses through interactions with Fcα receptor type I (FcαRI). Anti-FcαRI antibody inhibits activating receptors by inducing an inhibitory immunoreceptor tyrosine-based activation motif configuration. However, the role of FcαRI in atherosclerosis development is unclear. Here, we investigated the utility of FcαRI targeting to induce inhibitory immunoreceptor tyrosine-based activation motif signaling in atherosclerosis treatment. Materials ApoE-/- transgenic mice expressing the FcαRIR209L/FcRγ chimeric protein (FcαRIR209L/FcRγApoE-/- mice) were generated. We prepared an FcαRIR209L/FcRγ transfectant (I3D) from a mouse macrophage cell line (RAW264.7). Methods Anti-FcαRI or control antibody was used to investigate a high-fat-diet-induced FcαRIR209L/FcRγApoE-/- mouse model of atherosclerosis. The antibody was also used to assess macrophage foam cell formation via Oil Red O staining and mitogen-activated protein kinase signaling via immunoblotting in the FcαRIR209L/FcRγ-expressing RAW264.7 macrophage cell line I3D. Results Targeting of monovalent FcαRI induced inhibitory effects in the FcαRIR209L/FcRγApoE-/- mouse model of atherosclerosis by inhibiting macrophage infiltration. FcαRI targeting using the anti-FcαRI antibody also reduced mitogen-activated protein kinase signaling and foam cell formation, leading to decreased interleukin (IL)-1b and monocyte chemoattractant protein (MCP)-1. Conclusions We demonstrated that targeting monovalent FcαRI suppresses atherosclerosis development. These findings can support the future clinical exploration of FcαRI targeting for atherosclerosis treatment.


Introduction
Atherosclerosis is a chronic disease with a multifactorial etiology that ultimately leads to the development of rupture-prone plaques and atherothrombotic events 1) .Clinical trials and animal experiments have supported the notion that advanced plaques share common properties including augmented lipidrich necrotic core and macrophage accumulation 2) .Macrophages often accumulate in various regions of vulnerable plaques and are the main source of cytokines 3) .During the last decade, oxidized lowdensity lipoprotein (ox-LDL) and its interactions with monocytes/macrophages were considered the primary atherogenic components in dyslipidemia 4) .The concentration of ox-LDL is markedly elevated in atherosclerotic lesions and reaches cytotoxic levels and subsequent inflammatory events 3) .Mitogen-activated protein kinase (MAPK)-induced phosphorylation events play important roles in macrophage Yuya DESAKI 1) , Yutaka KANAMARU 1) , Renato MONTEIRO 2) , Yusuke SUZUKI 1) 1) Department of Nephrology, Juntendo University Faculty of Medicine, Tokyo, Japan 2) Center for Research on Inflammation, Université de Paris, Paris, France Objectives: Atherosclerosis is a chronic inflammatory disease characterized by lipid accumulation and local inflammation, which are regulated by the immune system.The immunological aspects of this disease are unclear.Immunoglobulin A regulates many cell responses through interactions with Fcα receptor type I (FcαRI).Anti-FcαRI antibody inhibits activating receptors by inducing an inhibitory immunoreceptor tyrosine-based activation motif configuration.However, the role of FcαRI in atherosclerosis development is unclear.Here, we investigated the utility of FcαRI targeting to induce inhibitory immunoreceptor tyrosine-migration in plaques 5) .Intracellular MAPK signaling cascades are involved in the pathogenesis of cardiac and vascular diseases.In macrophages, an interaction between CD36 and ox-LDL induces the phosphorylation of Lyn, one of several Src-family tyrosine kinases in immune cells, and subsequent activation of extracellular signal-regulated kinase (ERK) and p38 mediates the uptake of ox-LDL 6) , leading to inhibition of MAPK signaling and attenuation of foam cell formation 7) .Ox-LDL induces vascular smooth muscle cell proliferation and inflammation with the foam cell formation 8) .Since MAPK signaling pathway affects foam cell aggregation and inflammatory responses, inhibition of nuclear factor-κB and MAPK signaling attenuates atherosclerosis 9) .
Fcα receptor type I (FcαRI; CD89) is the only Fc receptor specific to immunoglobulin A (IgA) expressed on myeloid cells, including macrophages, monocytes, dendritic cells, Kupffer cells, neutrophils, and eosinophils 10) .FcαRI is expressed in the presence or absence of a physical association with the FcγR adaptor, which contains an immunoreceptor tyrosine-based activation motif (ITAM).FcαRI, a unique member of the FcR family, exerts a dual role in immune cell inhibition and activation 10,11) .It is known that an inhibitory signal is generated when monomeric IgA (mIgA) binds to two FcαRI, while an active signal is required the binding of IgA-immune complexes to FcαRI.Serum IgA is generated mainly as a monomeric form (about 85% to 90% of total serum IgA) by bone marrow plasma cells.Therefore, we think that an inhibitory signal of FcαRI is dominant at least in circulation.An inhibitory ITAM (ITAMi) configuration induced by monovalent targeting of FcαRI (anti-FcαRI Fab fragment) initiates the recruitment of Src homology domain 2-containing proteintyrosine phosphatase-1 (SHP-1), which has inhibitory potential 12) .This step leads to the deactivation of the inflammatory reaction, thereby preventing autoimmune processes.Previous studies demonstrated the involvement of FcγR signal activation through the ITAM-containing FcγR adaptor in diseases 5,13) .The anti-FcαRI fragment antigenbinding (Fab) region negatively regulates the magnitude of the innate immune response and has been used as an anti-inflammatory drug to treat kidney diseases 14) .Furthermore, the inhibitory signal induced by anti-FcαRI Fab in the FcαRIR209L/ FcRγ chimeric receptor is more potent than that in wild-type FcαRI, which is expressed in the presence or absence of a physical association with the FcγR adaptor.
FcαRI targeting can halt disease progression and lupus activation by selectively inhibiting cytokine production, leukocyte recruitment, and renal inflammation 15) .FcαRI-mediated inhibition can suppress several inflammatory diseases in mice, including asthma and glomerulonephritis.Intravenous mIgA and anti-FcαR monovalent antibodies are promising tools for immunotherapy 16) .In this study, we aimed to evaluate whether FcαRI targeting can prevent atherosclerosis.

Animals
The mice were bred and maintained in the mouse facilities of the Research Institute for Diseases of Old Age (Juntendo University School of Medicine, Tokyo, Japan).All experiments were conducted in accordance with national guidelines and were approved by a local ethics committee(Juntendo University School of Medicine Animal Experiment Committee; the approval number is 270258).

Animal study protocol
Ten male FcαRIR209L/FcRγ transgenic (Tg) ApoE −/− mice (12-week-old) with a C57BL/6J background were used.The animals were fed a diet containing 15% cocoa butter and 0.25% cholesterol, which was obtained from the Animal Center of Juntendo University.After anesthesia (40 mg/ kg pentobarbital sodium intraperitoneally), a constrictive silastic tube (0.30 mm), inserted via the caudal vein, was used to elicit plaque formation 17) .The mice were divided into two groups (n = 5 per group): group 1, FcαRIR209L/FcRγ Tg ApoE −/− mice were administered 20 µg of control Fab in 200 µL of saline once daily via the caudal vein for three months; group 2, FcαRIR209L/FcRγ Tg ApoE −/− mice were administered 20 µg of A77 (anti-FcαRI antibody) Fab in 200 µL of saline once daily via the caudal vein for three months.Serum samples and aorta tissues were collected at the end of the study.

Foam cell formation and Oil Red O staining
I3D cells (1 × 10 6 cells/well) were seeded into 6-well plates and stimulated with ox-LDL (100 µg/mL for 24 h; Yiyuan, Guangzhou, China) in the presence or absence of A77 Fab (100 µg/mL for 12 h).Subsequently, the cells were washed with phosphate-buffered saline (PBS) and stained with Oil Red O (Sigma Aldrich Chemicals).Stained (red) foam cells were imaged under a microscope at 40× magnification.

Immunohistochemical staining
For light microscopy, the sections of mouse aorta tissues were sectioned at 3 μm, paraffin-embedded, and stained using the periodic acid-Schiff reagent.For immunohistochemical staining, frozen mouse aorta tissues were sectioned at 3 μm, fixed in -20°C acetone, and blocked by incubation in a blocking solution (PBS [pH 7.2] containing 2.0% bovine serum albumin, 2% fetal calf serum, and 0.2% fish gelatin at room temperature) for 60 min.Histological features were graded, and F4/80+ cells were counted blindly.A minimum of 10 equatorially sectioned aortas were assessed per animal.The results are expressed as the number of cells per high-power field, which was quantified using a KS-400 version 4.0 image analysis system (KS-400; Carl Zeiss Vision, Oberkochen, Germany).

Enzyme-linked immunosorbent assay (ELISA)
Blood samples were collected from each mouse from the retro-orbital venous plexus under general anesthesia by inhalation of ether at the end of the study.Interleukin (IL)-1b and monocyte chemoattractant protein (MCP)-1 levels were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol.

Data presentation
All experiments were repeated more than three times, and representative results are shown.Data are expressed as the mean ± 2 standard error.Statistical analyses were performed using the Student's unpaired t-test (specifically, for immunoblotting determination, we compared the results with those of each respective control) and analysis of variance.p < 0.05 was considered to indicate statistical significance.

Monovalent targeting of FcαRI decreases ox-LDL-induced foam cell formation in FcαRIR209L/ FcRγ (I3D) cells
FcαRI-FcRγ ITAMi function can be triggered in the absence of co-aggregation.Therefore, we predicted that monovalent targeting, in addition to inhibiting co-expressed ITAM-bearing receptors, affects the responses of receptors involved in different signaling pathways 11) .We analyzed the effect of anti-FcαRI Fab A77 pretreatment on the foaming response of FcαRIR209L/FcRγ (I3D) to ox-LDL.Oil Red O staining showed that A77 Fab, but not the Ctrl Fab, markedly inhibited ox-LDL-induced foam cell formation (Figure 2).

Ox-LDL-mediated MAPK signaling in FcαRIR209L/FcRγ (I3D) cells
Next, we analyzed the effect of anti-FcαRI (A77 Fab) pretreatment on MAPK in response to ox-LDL in I3D cells.Key events in ox-LDL-mediated signaling, such as JNK, p38, and p42-p44 ERK MAPK phosphorylation, as evaluated by immunoblotting using phospho-specific antibodies, are shown in Figure 2. Phosphorylation was strongly inhibited in I3D cells after preincubation with A77 Fab but not after incubation with Ctrl Fab (Figure 3).

Oil Red O staining of the aorta of wild-type
ApoE-deficient/FcαRIR209L/FcRγ phenotype mice fed a high-fat diet for three months FcαRIR209L/FcRγ Tg ApoE −/− mice were administered 20 µg of Ctrl Fab in 200 µL of saline once daily via the caudal vein for three months; in group 2, FcαRIR209L/FcRγ Tg ApoE −/− mice were administered 20 µg of Ctrl Fab in 200 µL of saline once daily via the caudal vein for three months.Staining the mouse aortas using Oil Red O showed that staining levels were lower in the A77 Fab treatment group than in the Ctrl Fab treatment group (Figure 4).

FcαRI targeting reduces leukocyte infiltration in A77 Fab-treated mice
To determine whether monovalent targeting of anti-FcαR has therapeutic implications for high-fat diet-induced atherosclerosis, we analyzed the effect of A77 Fab treatment in a high-fat-diet-induced FcαRIR209L/FcRγ Tg ApoE −/− mouse model of atherosclerosis.Control antibody-treated animals showed high CD11b+/F4/80+ macrophage infiltration into the aortic tissues (Figure 4).However, A77 Fab-treated mice showed decreased infiltration of aortic tissues by CD11b+/F4/80+ macrophages compared to that in control antibody-treated animals.Thus, A77 Fab treatment showed marked efficacy against atherosclerosis induced by a high-fat diet in FcαRIR209L/FcRγ Tg ApoE −/− mice(Figure 5).

FcαRI monomeric targeting blocks serum cytokine and chemokine production stimulated in atherosclerosis
To examine whether increased aortic macro- phage infiltration in FcαRIR209L/FcRγ ApoE −/− mice was correlated with serum cytokine and chemokine levels, we performed ELISA using serum isolated from the affected mice.At the end of the study, treatment with the control antibody significantly increased IL-1β and MCP-1 secretion.In contrast, the A77 Fab treatment decreased IL-1β and MCP-1 levels (Figure 5).

Discussion
In a previous study, RAW264.7 cells were stimulated with lipopolysaccharide or ox-LDL to mimic the development of atherosclerosis 7) .To assess the involvement of FcαRI/FcRγ in the inhibitory process, we generated a chimeric receptor plasmid by fusing the extracellular and R209L transmem-brane domains of FcαRI to the intracytoplasmic tail of human FcRγ 14) .We also generated transfectants expressing FcαRIR209L associated with FcRγ (I3D) in RAW 264.7 macrophages 14) .Several previous studies demonstrated that inhibitory signaling by myeloid FcαRI is a promising anti-inflammatory candidate for treating inflammatory diseases 10) .However, data supporting its inhibitory effects on atherosclerosis are lacking.
Although the presence of anti-ox-LDL IgG has been well-documented in clinical and animal studies, the role of FcγRs in the progression of atherosclerosis remains unclear.The role of activating FcγR in the progression of atherosclerosis using apoE-Fcγ-chain double-knockout mice was examined 19,20) .In apoE knockout mice, arterial lesion formation was significantly decreased in apoE-Fcγ-chain double-knockout mice.
We also conducted an additional in vivo experiment using a Tg mouse with an ApoE-deficient/ FcαRIR209L/FcRγ phenotype under high-fat diet feeding, which exhibited severe atherosclerotic lesions.The results showed that monovalent targeting of FcαRI in Tg mice with the ApoE-deficient/FcαRIR209L/FcRγ phenotype significantly diminished aortic lesions by an inhibitory ITAM (ITAMi) configuration induced using monovalent targeting of FcαRI through the inhibition of macrophage at the aortic lesion and decreased IL-1β and MCP-1 levels.
We observed that monovalent targeting of FcαRI was inhibited in an in vitro model of ox-LDLinduced foam cell formation.Ox-LDL-induced foam cell formation was markedly decreased in the A77 Fab-or A57 Fab-treated groups compared with that in the Ctrl Fab-treated group.The expression of phosphorylated ERK and p38 was also decreased in the A77 Fab-and A57 Fab-treated groups compared with that in the Ctrl Fab-treated group.Interestingly, phosphorylated JNK expression did not significantly differ between the A77 Fab-or A57 Fab-treated groups and the Ctrl Fabtreated group after stimulation with ox-LDL in FcαRIR209L/FcRγ chimeric receptor transfectant macrophages.In line with these findings, inhibitory signaling by myeloid FcαRI significantly decreased the phosphorylation levels of MAPK during atherosclerosis development.The anti-atherogenic properties of inhibitory signaling by myeloid FcαRI observed in I3D cells may be explained by its inhibitory effects on monocyte adhesion, oxidative stress, and the inflammatory response mediated by the ox-LDL/MAPK (ERK1/2/p38) signaling pathway, which was independent of JNK in macrophages.
A previous study demonstrated that IL-1β is upstream of the disrupted intestinal barrier function in a mouse model of Kawasaki disease vasculitis, which showed IgA vasculitis development and cardiac inflammation following genetic and pharmacological inhibition of IL-1β signaling 22) .Targeting mucosal barrier dysfunction and the IL-1β pathway may also apply to other IgA-related diseases, including IgA vasculitis, IgA nephropathy, and atherosclerosis.Elevated levels of circulating secretory IgA may promote atherosclerosis in Kawasaki disease.
Our study had some limitations.First, our in vivo physiological data are not sufficient.Unfortunately, this study did not check serum levels of cholesterol and ox-LDL in the mice.In our future study, we should check them and evaluate this point.However, we carefully reviewed optimal animal models in which the inhibitory effect of myeloid FcαRI in atherosclerosis development has been confirmed.Second, although inhibitory signaling by myeloid FcαRI showed multifunctional potential in vitro in both our and previous studies, the contribution of SH2-containing phosphatase SHP-1 recruitment should be evaluated.SHP-1 should be immunoprecipitated to demonstrate this association, and FcRγ co-immunoprecipitates in macrophages following treatment with anti-FcαRI Fab should be examined.
We demonstrated that monovalent targeting of FcαRI suppresses atherosclerosis development.These results indicate that the inhibitory signals of FcαRI require FcRγ single association.FcαRI is a complex receptor, and the balance in FcαRI targeting is associated with the development of atherosclerosis.These results demonstrate the potential of inhibitory signaling by myeloid FcαRI as a therapeutic approach for atherosclerosis.

Figure 2
Figure 2 Monovalent targeting of FcαRI decreases foam cell formation in FcαRIR209L/FcRγ (I3D) cells I3D cells were stimulated with oxidized low-density lipoprotein (ox-LDL) in the presence or absence of A77 Fab (A).Oil Red O staining indicated the presence of foam cells.I3D cells were stimulated with ox-LDL (100 µg/mL for 24 h) in the presence or absence of A77 Fab (100 µg/mL for 12 h).Stained (red) foam cells were imaged under a microscope at 40× magnification.In I3D cells stimulated with ox-LDL with A77 Fab, the number of macrophage foam cells was significantly reduced, as demonstrated by Oil Red O staining.The number of Oil Red O-positive cells was expressed as the percentage of total cells (B).Results were obtained from three independent experiments (p < 0.05).HPF, high-power field.

Figure 4
Figure 4 Oil red O staining of the aorta of wild-type ApoE-deficient/FcαRIR209L/FcRγ phenotype mice fed a high-fat diet for three months Wild-type ApoE-deficient/FcαRIR209L/FcRγ mice were generated, fed a high-fat diet for three months, and injected with A77 Fab or PBS Ctrl.Staining the mouse aortas with Oil Red O showed that the expression of A77 Fab (A) was lower than that of PBS Ctrl (B).Results were obtained from three independent experiments (p < 0.05).

Figure 5 FcαRI
Figure 5 FcαRI targeting reduces leukocyte infiltration and blocks serum cytokine and chemokine production stimulated in an atherosclerosis model Immunohistological analysis of aortic sections from each animal group using anti-mouse F4/80 Ab.The number of infiltrating macrophages is shown (A).Serum interleukin (IL)-1β (B) and monocyte chemoattractant protein-1 (MCP-1) (C) production in each group was measured using an enzyme-linked immunosorbent assay (ELISA).Anti-FcαRI Fab-injected group showed lower protein production compared to the control Fab-injected group (p < 0.05).HPF, high-power field.