Gene Deficiency in Activating Fcγ Receptors Influences the Macrophage Phenotypic Balance and Reduces Atherosclerosis in Mice

Immunity contributes to arterial inflammation during atherosclerosis. Oxidized low-density lipoproteins induce an autoimmune response characterized by specific antibodies and immune complexes in atherosclerotic patients. We hypothesize that specific Fcγ receptors for IgG constant region participate in atherogenesis by regulating the inflammatory state of lesional macrophages. In vivo we examined the role of activating Fcγ receptors in atherosclerosis progression using bone marrow transplantation from mice deficient in γ-chain (the common signaling subunit of activating Fcγ receptors) to hyperlipidemic mice. Hematopoietic deficiency of Fcγ receptors significantly reduced atherosclerotic lesion size, which was associated with decreased number of macrophages and T lymphocytes, and increased T regulatory cell function. Lesions of Fcγ receptor deficient mice exhibited increased plaque stability, as evidenced by higher collagen and smooth muscle cell content and decreased apoptosis. These effects were independent of changes in serum lipids and antibody response to oxidized low-density lipoproteins. Activating Fcγ receptor deficiency reduced pro-inflammatory gene expression, nuclear factor-κB activity, and M1 macrophages at the lesion site, while increasing anti-inflammatory genes and M2 macrophages. The decreased inflammation in the lesions was mirrored by a reduced number of classical inflammatory monocytes in blood. In vitro, lack of activating Fcγ receptors attenuated foam cell formation, oxidative stress and pro-inflammatory gene expression, and increased M2-associated genes in murine macrophages. Our study demonstrates that activating Fcγ receptors influence the macrophage phenotypic balance in the artery wall of atherosclerotic mice and suggests that modulation of Fcγ receptor-mediated inflammatory responses could effectively suppress atherosclerosis.


Introduction
Atherosclerosis is considered not only merely a lipid disorder, but also a chronic inflammatory disease of the arterial wall [1]. Innate (monocyte-derived macrophages) and adaptive (T lymphocytes) immune response both contribute to the arterial inflammation that characterizes atherosclerosis, a dynamic process also involving different cell types such as endothelial cells and vascular smooth muscle cells (SMC). Early atherosclerotic lesions are characterized by the presence of foam cells in the intima as a result of macrophage infiltration and lipid internalization. Foam cells secrete pro-inflammatory cytokines thereby amplifying the initial inflammatory response. Sustained inflammation then results in lesion progression, degradation of the extracellular matrix causing plaque destabilization and rupture [2].
Diversity and plasticity are hallmarks of monocyte/macrophage system, which are reflected in plaque formation and progression [3]. Two major monocyte subsets termed classical (Ly6C high ) and non-classical (Ly6C low ) function as control switches of the immune system and maintain the balance between pro-and antiinflammatory activities [4]. Different macrophage subtypes (M1, classically activated and pro-inflammatory; M2, anti-inflammatory and tissue repair) also participate in inflammatory processes during atherogenesis, being M1 the predominant phenotype in human and murine atheromata. M1 macrophage markers include inducible nitric oxide synthase (NOS), interleukin (IL)-12, tumor necrosis factor-a (TNF-a), interferon-c (IFN-c), and arginase (Arg) 2, whereas enhanced expression of IL-4, IL-10, Arg1, mannose receptor and galactose type C-type lectin associate with M2 phenotype [3][4][5].
Antigen-antibody immune complexes (IC) are implicated in many autoimmune and inflammatory diseases. Autoimmunity is also an independent risk factor of atherosclerosis and cardiovascular disease associated mortality [6]. Autoantibodies against oxidized low-density lipoproteins (oxLDL) have been found in blood and lesions of atherosclerotic patients and are considered cardiovascular biomarkers [7,8]. IgG autoantibodies have a pro-atherogenic effect through the formation of oxLDL-containing IC and cell activation via specific IgG Fc receptors (FccR) [6,9]. In mice, four FccR classes have been identified (FccRI/CD64, FccRII/CD32, FccRIII/CD16, and FccRIV/CD16-2) differing by their distinct affinity for IgG subclasses, cellular distributions, and effector functions [10,11]. Activating FccR (I, III and IV) associate with the common c-chain that contains the intracellular tyrosine-based activating motif, and upon ligand binding they stimulate phagocytosis, oxidative burst, cytokine release, and antibody-dependent cell-mediated cytotoxicity. In contrast, engagement of inhibitory FccRIIB abrogates inflammatory responses and instead regulates alternative signaling cascades [10,11].
It has been previously proven that bone marrow transplantation using knockout mice is a useful approach to study the contribution of hematopoietic cells to atherogenesis, and that hematopoieticoriginating macrophages are the major type of cells recruited to the vascular wall during atherosclerotic lesion formation [22]. Accordingly, the present study investigates the effect of activating FccR deficiency within bone marrow-derived cells on the development of atherosclerotic plaques in the Apolipoprotein E knockout (ApoE 2/2 ) mouse model. For this purpose, we used donor marrow from double deficient mice in ApoE and c-chain [16], the common subunit necessary for assembly, cell-surface localization, and functionality of activating FccR (types I, III and IV). We therefore hypothesize that functional deficiency in activating FccR attenuates atherosclerosis progression in mice by reducing the inflammatory state of monocytes/macrophages.

Ethics Statement
The housing and care of animals and all the procedures carried out in this study were strictly in accordance with the Directive 2010/63/EU of the European Parliament and were approved by the Institutional Animal Care and Use Committees of Autonoma University and IIS-Fundacion Jimenez Diaz.

Mouse Sacrifice and Tissue Preparation
Mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (15 mg/kg). The adequacy of anesthesia and animal safety were monitored by standard methods (pedal withdrawal, palpebral reflexes, respiration, and skin/mucous membrane color) prior to all experimental procedures. Blood was obtained through retro-orbital puncture. Hearts were perfused with sterile saline via the left ventricle at physiological pressure. Spleens and thoraco-abdominal aortas were preserved in liquid N 2 and processed for mRNA analysis [25].

Biochemistry and Flow Cytometry
Total serum cholesterol and triglyceride concentrations were measured using standard enzymatic methods. Total serum immunoglobulins were measured by sandwich ELISA using specific antibodies recognizing mouse IgM, IgG, IgG 1 , IgG 2a/c and IgG 3 (BD Biosciences, San Diego, CA). Specific anti-oxLDL immunoglobulins were determined by ELISA using microtitration plates coated with native LDL and oxLDL (1 mg/mL), as previously described [20]. FACS analysis of mouse blood leukocytes was performed with the following fluorochromeconjugated antibodies and appropriated isotype controls: CD45-FITC, CD3e-PE-Cy7, CD4-PE, CD8a-FITC and CD19-PE form BD Biosciences; CD115-PE and Ly6C-APC from eBioscience (San Diego, CA). Data were acquired using a BD FACS Canto II Flow Cytometer and analyzed with BD FACSDiva software (BD Biosciences).

Isolation of Murine Macrophages
Peritoneal macrophages were harvested by peritoneal lavage of anesthetized mice (ApoE 2/2 , c 2/2 ApoE 2/2 and WT), then plated and allowed to adhere for 2 h. After extensive washing with PBS to eliminate non-attached cells, macrophages were lysed and processed for RNA and protein expression analysis. For foam cell counting, peritoneal macrophages on coverslips were incubated for 6 h in DMEM containing oxLDL alone (25 mg/mL) and oxLDL-containing IC (IgG1 and IgG2 subclasses, 1:0.5 ratio of oxLDL to IgG), then fixed with 4% paraformaldehyde and stained with oil-red-O/hematoxylin. The total cell and foam cell (.8 lipid droplets) numbers were counted, and the percentage of foam cells was calculated. Bone marrow-derived cells were differentiated for 7 days in DMEM containing 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine (Life Technologies, Rockville, MD), and supplemented with 10% L929-cell conditioned medium as a source of macrophage colony stimulating factor. Differentiated macrophages were serum-starved for 12 h before addition of soluble IC (75 mg/mL) for different time periods. The oxLDL-IC and soluble IC used in this study were prepared as previously described [16,20,29].

mRNA Expression Analysis
Total RNA from aorta, spleen and macrophages was extracted with TRIzol reagent (Invitrogen) as described [25,28]. Expression of target genes was analyzed by real-time quantitative PCR (Applied Biosystem, Foster City, CA) and normalized to housekeeping 18S transcripts.

Statistical Analysis
Data are presented as mean6SEM. Differences across experimental groups were analyzed for statistical significance by unpaired Student's t test or by one-way ANOVA followed by post-hoc Bonferroni pairwise comparison using GraphPad Prism 5 (GraphPad Software, La Joya, CA). Values of P,0.05 were considered significant.

Reconstitution with c 2/2 ApoE 2/2 Bone Marrow Reduces Atherosclerotic Lesion Size
To examine whether an activating FccR-dependent mechanism in macrophages accounts for increased atherosclerosis in vivo, we performed bone marrow transplantation experiments. Irradiated ApoE 2/2 mice were reconstituted with bone marrow derived from either c 2/2 ApoE 2/2 double deficient mice (cARA group) or control ApoE 2/2 donor mice (ARA group), and after recovering were fed on atherogenic diet for 10 weeks. Chimerism in cARA group was confirmed by genomic PCR analysis of peripheral blood ( Figure 1A). Successful engraftment of marrow occurred in more than 90% of animals. After recovery, over 90% of blood cells were from donor origin and blood immune cell composition was similar between genotypes. Furthermore, survival of both chimera groups remained similar along the study (log-rank test, P.0.05; Figure 1B).
The size and extent of atherosclerotic lesion was quantified by morphometry after oil-red-O/hematoxylin staining. Aortic lesions in chimeric ApoE 2/2 recipient mice receiving bone marrow cells from c 2/2 ApoE 2/2 donors were 2.5-fold smaller by area (P,0.0001) than mice transplanted with ApoE 2/2 bone marrow ( Figure 1, C-E). Moreover, quantification of lipid content in plaques (oil-red-O stained area) revealed a reduced lipid accumulation in cARA compared with ARA mice (% of plaque area: 16.163.1 versus 50.1612.3; P = 0.002; not shown). Both groups had comparable body weight and total cholesterol and triglyceride levels at the end of the study period (Table 1). These findings suggest that FccRs in bone marrow-derived cells are critical for lesion development in this model.

Attenuated Inflammation and Enhanced Stability in Atherosclerotic Plaques of FccR Deficient Mice
Immunohistochemical analysis of cross-sections adjacent to sections of maximum plaque area showed a significantly decreased accumulation of monocytes/macrophages (Moma2 + ) and T lymphocytes (CD3 + and CD4 + ) at the atherosclerotic lesions of cARA mice (Figure 2, A-C), while the number of T regulatory cells (Foxp3 + ) was increased ( Figure 2D). Regression analysis showed correlation of lesion area with Moma2, CD3, CD4 and To assess plaque vulnerability, aortic lesions were analyzed for collagen (picrosirius red staining) and SMC (a-actin immunofluorescence), which are important stabilizing components of the plaque. Atherosclerotic plaques in cARA mice exhibited a modest, but significant increase in total collagen and SMC content ( Figure 3A) as compared with those of control ARA mice (% of total plaque area: picrosirius red, 20.961.9 versus 12.263.6, P = 0.04; a-actin, 18.562.1 versus 12.861.4, P = 0.03). Interest-ingly, the relative ratio of collagen to lipids (picrosirius red/oil-red-O) and SMC to macrophages (a-actin/Moma2) did markedly increase in the cARA group ( Figure 3B). The higher collagen and SMC content and the lower lipid and leukocyte content in lesions of FccR deficient mice are consistent with a more stable plaque phenotype. There was also a significant reduction in lesional apoptosis (Tunel staining) in cARA mice, whereas the number of proliferating cells (PCNA staining) was not different between the two groups ( Figure 3, C and D).

Inflammatory Mediator Expression and Immunoglobulin Isotype Distribution in Transplanted Mice
Lesions of cARA mice showed significantly less protein expression of macrophage chemokine CCL2 than controls ( Figure 4A). Likewise, Southwestern histochemistry showed an attenuated activity of NF-kB, the transcription factor that regulates many pro-inflammatory and pro-atherosclerotic genes    Figure 4D).
Changes in the adaptive immune response were assessed by measuring serum antibody titers to oxLDL epitopes. Total antibody isotypes (IgM and IgG) and IgG subclasses (1, 2a/c and 3) were not different between the experimental groups ( Figure 5, A and B). Interestingly, oxLDL-specific IgG response, but not IgM, was increased in cARA mice relative to ARA controls ( Figure 5C). Determination of anti-oxLDL IgG subclasses revealed significant differences in IgG 1 and IgG 2a/c levels in cARA mice ( Figure 5D). However, the relative ratio of IgG 2a/c (Th1 response) to IgG 1 (Th2 response) did not change between the groups (1.260.1 versus 1.360.1; P.0.05), thus indicating no apparent Th1/Th2 bias.

FccR Deficiency Affects Monocyte/macrophage Activation State in Atherosclerotic Mice
Our findings thus far indicate that FccR deficiency reduced the accumulation of inflammatory cells in atherosclerotic plaque. We then examined whether FccR deficiency influenced the leukocyte count or subset frequency in peripheral blood using flow cytometry ( Table 2). Blood counts for total leukocyte number and subgroups (B cells, T cells and monocytes) revealed no differences between ARA and cARA mice. Interestingly, analysis of monocyte subset distribution (Ly6C high and Ly6C low ) of CD115 + -gated monocytes revealed decreased percentages of Ly6C high and increased percentages of Ly6C low subsets in cARA group. This resulted in a significant decrease in the ratio of Ly6C high :Ly6C low monocytes in FccR deficient mice compared with controls (Table 2), thus indicating a reduced activation state of circulating monocytes.
To further examine the macrophage phenotype associated with FccR deficiency in atherosclerotic lesions, the gene expression of Arg2 and Arg1 isoforms was analyzed to distinguish between the pro-inflammatory classical M1 and the anti-inflammatory alternative M2 response, respectively. Immunofluorescence and gene expression analysis ( Figure 6) revealed that both macrophage markers are present in atherosclerotic lesions, being Arg2 (M1) most abundantly expressed in ARA mice, and Arg1 (M2) the predominant marker in cARA. Furthermore, calculation of Arg2/Arg1 ratio (average per group: ARA, 2.260.1; cARA, 0.560.1, P,0.0001) and regression analysis ( Figure 6C) demonstrated that higher lesion size correlated with the dominance of M1 over the M2 macrophage phenotype marker.

FccR Deficiency Prevents Foam Cell Formation and Inflammatory Responses in Macrophages
Studies in peritoneal macrophages were further performed to assess lipid accumulation and foam cell formation in atherosclerotic mice. Quantification of oil-red-O staining in resident peritoneal cells from mice on high-fat diet revealed an increased number of foam cells in ApoE 2/2 as compared with WT littermates (% macrophage foam cells: WT, 26.161.9; ApoE 2/ 2 , 61.866.4; P = 0.01; n = 6) and a decrease in FccR deficient mice (% macrophage foam cells: 33.065.3, P = 0.01 versus ApoE 2/2 ; n = 6). In other experiments, peritoneal macrophages from ApoE 2/2 and c 2/2 ApoE 2/2 mice on chow diet were incubated for 6 h in the presence of oxLDL alone and oxLDLcontaining IC. As shown in Figure 7A, foam cell formation was similarly induced by oxLDL in both cell types. By contrast, foam cell numbers after oxLDL-IC incubation were significantly reduced in peritoneal cells from c 2/2 ApoE 2/2 mice versus ApoE 2/2 mice ( Figure 7A), despite having equivalent expression levels of scavenger receptors (SR-A and CD36; Figure 7B). These results confirm the involvement of FccR in oxLDL-IC uptake and foam cell formation.
Peritoneal macrophages from FccR deficient mice exhibited a reduced expression of pro-inflammatory genes (CCL2, TNF-a and Arg2) and increased expression of M2 response genes (IL-10, TGF-b and Arg1) ( Figure 7C). Accordingly, analysis of Arg2 and Arg1 protein expression revealed that the relative ratio of M1 to M2 markers was higher in peritoneal macrophages from ApoE 2/2 mice as compared with WT littermates, and was significantly reduced by FccR deficiency ( Figure 7D).
Further experiments were performed in bone-marrow derived mouse macrophages. Treatment with soluble IC resulted in a significant polarization of ApoE 2/2 macrophages to the M1-like phenotype, as evidenced by a predominant Arg2 levels ( Figure 8A) with concomitant increase in other classical activation markers, including pro-inflammatory cytokines (TNF-a, IL-6 and IFN-c; Figure 8A) and chemokines (CCL2 and CCL5; Figure 8C). In contrast, IC failed to prime c 2/2 ApoE 2/2 macrophages to the pro-inflammatory phenotype, as evidenced by reduced expression levels of M1 markers (Figure 8, A and C) and increased M2 response genes ( Figure 8B). Furthermore, macrophages from FccR deficient mice exhibited reduced oxidative stress in response to IC stimulation, as measured by NADPH oxidase-dependent superoxide production ( Figure 8D).

Discussion
The present study demonstrates a key role for macrophage FccR in initiation and progression of vascular lesions in experimental atherosclerosis. Our bone marrow transplantation studies show that hematopoietic gene deficiency in c-chain, the common signaling subunit of activating FccR (types I, III and IV), is sufficient to limit atheroma plaque formation in mice. The underlying mechanisms are independent of serum lipids and Th1/ Th2 changes. Instead, our data indicate that FccR deficiency reduces circulating inflammatory monocytes, impairs foam cell formation and skews the distribution of macrophage population within the plaque towards the anti-inflammatory phenotype.
Inflammation is recognized as a major contributor to atherogenesis through adverse effects on lipoprotein metabolism and arterial wall biology. Accumulating evidence indicates that both humoral and cell-mediated pro-atherogenic immunity against altered self-antigens generated by hypercholesterolemia (such as oxLDL particles) play a predominant role in the atherogenic process [6]. Clinical studies have demonstrated a positive association between the degree of established atherosclerosis and antibody levels to oxLDL, mainly IgM and IgG isotypes [7,8]. High production of IgM and IgG anti-oxLDL antibodies has also been observed in hypercholesterolemic mice [30,31]. It has been proposed that atheroprotective IgM contributes to maintain low plasma levels of oxLDL and blocks the uptake of oxLDL by lesional macrophages, thereby preventing the oxLDL accumulation and foam cell formation [32,33]. On the other hand, IgG antibodies are pro-atherogenic through the formation of oxLDLcontaining IC and induction of FccR-mediated inflammatory responses in resident and infiltrating cells [6,34]. In fact, FccR is the most important macrophage receptor involved in the clearance of oxLDL-IC [9]. In the current study, despite the difference in atherosclerosis severity, the IgM anti-oxLDL response remained unchanged in mice reconstituted with c 2/2 ApoE 2/2 bone marrow. Interestingly, the IgG antibodies to oxLDL increased, probably due to impaired phagocytic activity in FccR deficient cells. In mouse models, severe hypercholesterolemia is associated with an antibody switch from Th1 (IgG 2a ) to Th2 (IgG 1 ) response [30], whereas B cell depletion reduces the production of anti-oxLDL antibodies and limits atherosclerosis development [35]. As the concentrations of both IgG subclasses were enhanced in the serum of c 2/2 ApoE 2/2 transplanted mice, we can consider that, instead of a polyclonal activation of B cells, the two Th1-and Th2type T cells are indeed involved in the antibody response against oxLDL. Although this antibody pattern suggests a more pronounced Th1 and Th2 immune response in FccR deficient mice, our analysis of the Th1/Th2 ratio did not substantiate systemic changes in Th cell composition. Furthermore, flow cytometry analysis revealed similar content of blood leukocytes and subgroups (B cells, T cells and monocytes) in both groups of transplanted mice. Interestingly, lack of FccR declined the number of circulating Ly6C high monocytes, the classical inflammatory subset predominant in hypercholesterolemic mice [36] that is preferentially adhered to activated endothelium, accumulated in lesions, and locally differentiated into macrophages [4,37]. Our observations confirm these previous reports highlighting the prominent role of classical monocytes in atherogenesis and as source for lesional macrophages [36,37], and suggest that a reduced monocyte activation state is involved in the atheroprotective effect of FccR deficiency.
The coordinate expression of activating and inhibitory FccR ensures the homeostasis of IC-driven inflammatory responses. In fact, gene deficiency in activating FccR is associated with diminished inflammation in response to IC, while severe inflammation was observed following deletion of inhibitory FccRIIB [11,38]. In atherosclerotic mice, FccRIII deficiency caused a reduction in aortic lesion size [17], whereas FccRIIB deletion aggravates the atherosclerotic process [18]. Our early study demonstrated that mice lacking c-chain associated FccR developed less atherosclerotic plaques characterized by a reduction in leukocyte content and inflammatory mediators [16]. Recently,  Ng et al. [19] reported an increased generation of circulating T regulatory cells with a concomitant decrease in Th17 cells in cchain deficient mice. The current data complement these previous immunological changes and also directly address the functional role of FccR activation on resident and recruited monocytes/ macrophages during atherosclerosis promotion. We cannot, however, rule out that FccR on other cells also modulate plaque development and progression, since bone marrow transplantation reconstitutes more than monocytes/macrophages [22]. In fact, hematopoietic stem cells are able to differentiate into endothelial and vascular SMC progenitors [39], and bone marrow cells accumulated in atherosclerotic lesion differentiate into vascular SMC and foam cells thereby contributing to vascular remodeling [40]. Moreover, different FccR are found expressed on endothelial and SMC, which demonstrate pro-atherogenic response to FccR stimulation in vitro [16,28,41]. Thus, further studies will be needed to demonstrate conclusively the relative contribution of FccR on both resident vascular and infiltrating cells in this process. The presence of oxLDL antibodies and FccR in atheroma plaques has been clearly demonstrated in patients [12,42]. In mice, adoptive transfer studies of CD4 + T cells specific to oxLDL have shown that Th1 cell responses play an important role in promoting atherosclerosis [43]. Our study demonstrates that reconstitution with c-chain deficient marrow in mice with developing atherosclerosis significantly attenuates further progression without affecting plasma lipid levels. First, the decrease in atheroma size correlates with a reduced number of T cells and macrophage foam cells and higher T regulatory cell content in aortic lesions. This indicates a deficient pro-atherogenic immune activation in the artery and also a key role of T regulatory cell function in FccR deficient mice, consistent with previous studies of the protective effect of T regulatory cells in atherosclerosis progression [19,44]. Secondly, hematopoietic FccR deficiency resulted in the development of a lipid-poor collagen-rich atherosclerotic lesion characterized by higher collagen/lipid and SMC/macrophage ratios and low apoptosis, thereby suggesting a more stable plaque phenotype. Because most acute complications of atherosclerosis are caused by the rupture of an unstable lipidrich collagen-poor lesion [1], strategies to modulate activating FccR may have a beneficial role in slowing lesion progression.
In parallel with the reduction in leukocyte infiltration, hematopoietic FccR deficiency leads to reduced local expression of genes involved in leukocyte activation and migration during atherogenesis [45,46], including adhesion molecule ICAM-1, chemokine CCL2, pro-atherogenic Th1 cytokines TNF-a and IFN-c, and Th2 cytokine IL-4. However, and consistent with the IgG subclass ratios, the relative proportion of Th1/Th2 cytokine expression in aorta and spleen remained unmodified, thus discarding either lesional or systemic Th1/Th2 imbalance.
Transcription factor NF-kB is a major regulator of immunoinflammatory responses that controls cytokine gene expression and has a pivotal role in atherogenesis [46]. We have earlier demonstrated NF-kB activation and target gene expression in experimental atherosclerosis [25] and also in cultured cells after FccR cross-linking [16,20,29]. Keeping in line with this, our study shows that FccR deficiency blunted the activity of NF-kB in cells of the intima and media, thus indicating that NF-kB's effect on inflammation and lipid handling can be regulated by IC in vascular and infiltrating cells. These data do not exclude a contribution of systemic inflammatory factors, such as serum cytokines and C-reactive protein previously shown to be increased in atherosclerosis [45,47], but suggest that the atherogenic effects of FccR are mainly mediated via inflamed cells localized in plaques.
Besides effects on the plaque macrophage content, FccR deficiency had a dramatic effect on the inflammatory state of these cells, with a general decrease and increase, respectively, in markers of M1 (classically activated) and M2 (alternatively activated or tissue repair) macrophages. Although this classification scheme has been considered to be overly simplistic, it has been a useful start with which to broadly characterize the macrophage heterogeneity known to occur in many tissues including atherosclerotic plaques [3]. Previous works have evaluated the phenotype of macrophages associated with progression of atherosclerosis in mice. While early lesions are infiltrated by reparative M2 macrophages, the M1 sub-type appeared and prevailed during plaque progression [48]. These M1 cells accumulated in damaged vessel wall are able to secrete many inflammatory cytokines that amplify Th1 immune response, affect SMC function and proliferation, and also contribute to lipoprotein oxidation through the production of reactive oxygen species [4,49]. In our work, the reduction in plaque lipid content and lesion size was accompanied by a decrease in infiltrating macrophages and pro-inflammatory mediators, along with a lower ratio of M1/M2, as determined by differential expression of Arg isoforms. Macrophage Arg converts arginine to ornithine and urea and competes directly for substrate with NOS, and the two isoforms (mitochondrial Arg2 and cytosolic Arg1) have been evaluated as surrogate markers of M1 and M2 phenotypes during atherosclerotic progression [48,50]. Indeed, classical M1 macrophages in the lesions express Arg2 and inducible NOS, but not Arg1, while alternatively activated M2 macrophages exclusively express Arg1. In our paper, the prevalence of M2 marker (Arg1) in aortic lesions from FccR deficient mice suggests that these smaller lesions contain macrophages of anti-inflammatory and tissue repair phenotype and consequently may be more stable. Concurrently, the pattern of pro-and anti-inflammatory cytokine expression in isolated macrophages strongly indicates that FccR deficient macrophages resemble features of anti-inflammatory M2-like macrophages. It has been previously reported that IC-stimulated macrophages more closely resemble M1 than M2 in terms of cytokine profile and functionality [51]. Furthermore, selective cross-linking of inhibitory FccRII/CD32, but not activating FccR (CD64 and CD16) accounts for the induction of CCL1 chemokine in human monocytes and is linked to M2 activation program [52]. An additional mechanistic study [53] revealed the involvement of activating FccR and NF-kB pathway in the polarization of human monocytes toward pro-inflammatory M1 macrophages by Creactive protein. In line with this, our data indicate that the proatherogenic role of activating FccR is related to the proinflammatory and pro-oxidative action on macrophages.
Collectively, our results document a role for activating FccR in controlling the macrophage phenotypic balance in the artery wall of atherosclerotic mice. We therefore propose that strategies to modulate FccR activating/inhibitory balance and effector func-