Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis[S]

Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI−/− cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1β, IL-6, and TNF-α lower IL-10 and transforming growth factor β]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI−/− versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI−/− macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation.


Analyses of efferocytosis and membrane ruffl ing
For efferocytosis assays, thymocytes were labeled with carboxyfl uorescein diacetate, succinimidyl ester (CFDA SE) green cell tracer (Invitrogen) and apoptosis was induced with 600 rad irradiation. Apoptotic cells were added onto cultured phagocytes in vitro or injected into mice in vivo for 2 h. The phagocytes were stained with CMTPX red cell tracer (Invitrogen). The efferocytosis of apoptotic cells was then visualized using fl uorescence microscopy and quantitated by fl ow cytometry ( 12,13 ). For analysis of membrane ruffl ing, phagocytes were incubated for 20 min with or without apoptotic thymocytes. After washing away apoptotic thymocytes, the cells were then fi xed, permeabilized, stained with rhodamine phalloidin (Cytoskeleton, Inc.), and counterstained with Hoechst.

Analyses of lesion necrosis, cell death, and efferocytosis
Necrosis was quantitated by hematoxylin and eosin (H and E) stain. For analysis of cell death and efferocytosis, proximal aortic cryosections were dual stained with terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and macrophage antibody; nuclei were counterstained with Hoechst. The free versus macrophage-associated TUNEL stain in the sections were quantitated and normalized to total lesion area as described ( 13 ).

Phospholipid-SR-BI binding assay
PS (Avanti) was loaded onto nitrocellulose membrane-C (HyBond) ( 14 ) and incubated with glutathione S-transferase (GST)-tagged SR-BI (Novus) or GST alone. The bound protein was detected by anti-SR-BI (Novus) or anti-GST (Novus) and secondary antibody (Sigma). For a cell-based assay, apoptosis was induced in SR-BI Ϫ / Ϫ macrophages, and exposure of PS was analyzed by annexin V directly or by incubation with GST-SR-BI protein or GST alone. SR-BI was detected using anti-SR-BI antibody.

Immunoprecipitation, Western blotting, and Rac1/RhoA-GTP activity assay
Macrophage lysates were incubated with antibody against either SR-BI or Src, and then with magnetic beads (Invitrogen). The magnetic beads were boiled with sample buffer at 70°C for 5 min, and the supernatant was used for detecting Src or SR-BI by immunoblotting. For Western blot, primary antibodies specifi c for SR-BI (Novus), total Akt, pAkt, PI3K p85, HMGB1, Src, pSrc, Na + /K + -ATPase (Cell Signaling), Rac1 (Upstate), RhoA (Cytoskeleton), Lamp-1 (Millipore), and ␤ -actin (Sigma) were applied. To assess activation of Rac1/RhoA, aliquots of the cell important determinants of infl ammation and atherosclerotic lesion development ( 7 ). Intimal macrophages undergo apoptosis due to lipid toxicity, oxidative stress, and signaling from bioactive molecules. Failure of apoptotic cell clearance promotes secondary necrosis, with release of proteolytic enzymes and other active molecules into the surrounding tissue causing infl ammation and plaque destabilization ( 7,8 ). Interestingly, SR-BI binds phosphatidylserine (PS) and oxidized phospholipids; both serve as "eat me" signals on apoptotic cells ( 9 ). However, macrophages have a variety of receptors to recognize different apoptotic cells and ligands for phagocytosis ( 7 ), and whether macrophage SR-BI mediates efferocytosis in the setting of atherosclerosis remains unknown.
In this study, we examined the hypothesis that macrophage SR-BI defi ciency impairs efferocytosis of apoptotic cells in atherosclerotic lesions, promoting lesion infl ammation and necrosis. To accomplish this, we performed BM transplantation studies using both ApoE Ϫ / Ϫ and LDLR Ϫ / Ϫ recipient mice. Our in vitro studies show that macrophage SR-BI functions as a PS receptor for recognition and clearance of apoptotic cells. Importantly, hematopoietic SR-BI deletion caused marked accumulation of noninternalized apoptotic cells in atherosclerotic lesions, resulting in increased necrosis. In addition, hematopoietic SR-BI deletion increased serum infl ammatory cytokine levels and enhanced expression of proinfl ammatory genes in macrophage foam cells. Finally, we determined that macrophage SR-BI mediates efferocytosis via a novel proto-oncogene tyrosine-protein kinase (Src)/phosphoinositide 3-kinase (PI3K)/Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling pathway. Taken together, our results show that macrophage SR-BI protects against atherosclerotic lesion necrosis and progression by regulating efferocyte signaling and macrophage survival.

MATERIALS AND METHODS
A detailed description of all experimental methods is available online in the supplementary data .

Mice
SR-BI +/ Ϫ (1:1 mixed C57BL/6×S129 genetic background) were obtained from the Jackson Laboratory and backcrossed over 10 generations onto the C57BL/6 background. Animal protocols were performed according to the regulations of Vanderbilt University's Institutional Animal Care and Usage Committee. Mice were maintained on chow or a Western-type diet containing 21% milk fat and 0.15% cholesterol (Teklad).

Plasma membrane protein and phagosome preparation
Plasma membrane proteins were extracted using a plasma membrane protein extraction kit (BioVision). Phagosomes were isolated as described ( 15 ) using latex beads (Sigma).

RNA isolation and real-time RT-PCR
Total RNA was purifi ed using Aurum Total RNA kit (Bio-Rad). cDNA was synthesized with reverse transcriptase (Bio-Rad). Relative quantitation of target mRNA was performed using specifi c primers (supplementary Table), SYBR probe (Bio-Rad), and iTaqDNA polymerase (Bio-Rad) on an IQ5 thermocylcer (Bio-Rad) and normalized to ␤ -actin.

Statistical analysis
Data are presented as mean ± SEM. Differences between mean values were determined by Kruskal-Wallis test (Bunn's multiple comparison), one-way ANOVA (Bonferroni's post test), Mann-Whitney test, and Student's t -test using GraphPad PRISM.
These results demonstrate that macrophage SR-BI plays a critical role in efferocytosis of apoptotic cells.

Deletion of macrophage SR-BI enhances infl ammation
As defective phagocytosis of apoptotic cells results in secondary cellular necrosis and maladaptive infl ammation, we next examined the effects of SR-BI defi ciency on infl ammation in vitro and in vivo. Peritoneal macrophages were isolated from LDLR Ϫ / Ϫ mice transplanted with either WT or SR-BI Ϫ / Ϫ BM and fed either a chow or Westerntype diet for 16 weeks ( Fig. 2 ). Both WT and SR-BI Ϫ / Ϫ macrophages from mice on the Western diet had higher

Effects of hematopoietic SR-BI deletion on lesional efferocytosis, apoptosis, necrosis, collagen, and fi brous cap thickness
We next examined whether the increased lesional accumulation of dead cells resulting from deletion of SR-BI was due to defective efferocytosis. Staining of lesions for apoptosis, nuclei, and macrophages ( Fig. 3A-F ) enabled the quantitation of free versus macrophage-associated TUNEL stain, as previously described ( 13 ). The ratio of free to macrophage-associated dead cells was 17-fold progression and necrosis in SR-BI-defi cient lesions. One key feature of plaque instability is collagen and fi brous cap reduction or depletion. Dying macrophages are a major source of proteases that originate from the necrotic core and degrade the extracellular matrix and induce plaque with the effects on lesion active caspase 3 levels, the accumulation of free TUNEL-positive cells ( Fig. 3F ) was more pronounced in SR-BI Ϫ / Ϫ versus WT lesions (4.3-fold) and DKO versus ApoE Ϫ / Ϫ lesions (16.2-fold), indicating that defective efferocytosis is a major contributor to lesion rupture. Therefore, we analyzed the collagen content and fi brous cap thickness in atherosclerotic lesions by Masson's trichrome stain. We observed that hematopoietic SR-BI defi ciency caused 65.2 and 48.5% reductions in lesion collagen content and fi brous cap thickness in DKO → ApoE Ϫ / Ϫ mice compared with ApoE Ϫ / Ϫ → ApoE Ϫ / Ϫ mice ( Fig. 4E-G ), suggesting that hematopoietic SR-BI defi ciency contributes to unstable plaque formation.

SR-BI protein interacts with PS and localizes in phagosomes
The observation that the combined deletion of macrophage SR-BI and ApoE impairs efferocytosis beyond the single deletion of either gene suggests that SR-BI mediates efferocytosis through pathways that are both dependent and independent of interaction with ApoE. Therefore, we examined the possibility that macrophage SR-BI interacts with apoptotic cell PS to initiate efferocytosis. We fi rst compared the binding of GST-tagged SR-BI to PS in nonapoptotic control or apoptotic SR-BI Ϫ / Ϫ macrophages (supplementary Fig. 6A). Both annexin V-and GST-tagged SR-BI bound to the surface of apoptotic SR-BI Ϫ / Ϫ macrophages and not on nonapoptotic cells, suggesting that SR-BI interacts with membrane PS of apoptotic cells. Using a direct phospholipid SR-BI binding assay, we confi rmed that SR-BI is a PS receptor. In contrast to GST protein, GST-tagged SR-BI interacted with nitrocellulose membrane PS in a dose-dependent fashion (supplementary Fig. 6B). The interaction with PS was specifi c, as GSTtagged SR-BI interacted poorly with other phospholipids, including phosphatidylcholine, phosphatidylglycerol, sphingomyelin, and phosphatidylethanolamine (data not shown). Together, these data indicate that SR-BI functions directly as a receptor for apoptotic cells by interacting with apoptotic cell PS. Consistent with SR-BI directly mediating the phagocytosis of apoptotic cells, SR-BI protein was enriched in phagosomes (supplementary Fig. 6C) of WT macrophages exposed to latex beads.

Macrophage SR-BI regulates efferocytosis via Src/PI3K/Rac1 signaling
Efferocytosis is precisely regulated in prokaryotic and eukaryotic cells with evolutionarily overlapping signaling pathways leading to Rac1 activation and cytoskeletal rearrangements for engulfment. Therefore, we examined Rac1 activation in WT and SR-BI Ϫ / Ϫ macrophages and the effects of pharmacological activation of Rac1 on membrane ruffl ing and efferocytosis ( Fig. 5 ). Upon incubation with apoptotic cells, activation of Rac1, detected as Rac1-GTP, was markedly increased in WT cells compared with view that Rac1 is a mediator of efferocytosis from convergent signaling pathways ( 16 ). As CN02 likely activates multiple signaling pathways, we also examined the effects of the specifi c Rac1 inhibitor, NSC23766, on efferocytosis in WT versus SR-BI Ϫ / Ϫ macrophages ( Fig. 5E, F ). NSC23766 markedly decreased the levels of active Rac1 in WT cells ( Fig. 5G ) and reduced WT efferocytosis of apoptotic thymocytes to levels similar to SR-BI Ϫ / Ϫ cells ( Fig. 5E, F ). In addition, NSC23766 did not affect SR-BI Ϫ / Ϫ macrophage efferocytosis, suggesting that Rac1 activation in efferocytosis is downstream of SR-BI. We also examined potential involvement of efferocytosis-related factors in SR-BI-defi cient macrophages; we did not fi nd signifi cant changes of several relevant genes or proteins, including Mertk , C1qa , SR-BI Ϫ / Ϫ cells ( Fig. 5A ). Consistent with defective efferocytosis and decreased Rac1-GTP in SR-BI Ϫ / Ϫ macrophages, incubation of SR-BI Ϫ / Ϫ macrophages with apoptotic thymocytes did not increase the number of cells with ruffl ed membranes ( Fig. 5C, D ). In contrast, membrane ruffl es markedly increased in WT macrophages exposed to apoptotic bodies. Activation of Rac1 with CN02 increased the levels of Rac1-GTP in both WT and SR-BI Ϫ / Ϫ macrophages ( Fig. 5A ). Interestingly, CN02 treatment corrected the defective efferocytosis and membrane ruffl ing in SR-BI Ϫ / Ϫ macrophages exposed to apoptotic thymocytes ( Fig. 5B-D ). The fi nding that pharmacological activation of Rac1 rescues efferocytosis in the absence of SR-BI supports the  phosphorylation must act upstream of PI3K/Rac1 activation, as inhibition of Src phosphorylation with PP2 decreased PI3K p85, pAkt, and Rac1-GTP levels in WT macrophages ( Fig. 6D ). Rac1 was confi rmed to be a downstream target of PI3K, as inhibition of PI3K by Wortmannin decreased Rac1-GTP levels in WT cells ( Fig. 6F ). In contrast to observations with Sertoli cell SR-BI ( 19 ) or macrophage LDL receptor-related protein 1 (LRP1), macrophage SR-BI was not associated with the engulfment adaptor PTB domain (GULP) ( Fig. 7A ). Importantly, coimmunoprecipitation of SR-BI and Src demonstrated that the two proteins are directly associated in WT macrophages ( Fig. 7B ), suggesting that SR-BI plays a direct role in activation of Src in macrophages. Several receptors cause Src membrane targeting and subsequent autophosphorylation ( 20 ). Therefore, we examined the effects of SR-BI expression on plasma membrane recruitment of activated Src by transfecting WT or SR-BI Ϫ / Ϫ macrophages with pCMV6-mSR-BI plasmid. In both cell types incubated with apoptotic cells, plasma membrane pSrc levels increased in a stepwise fashion over time with increasing levels of SR-BI expression ( Fig. 7C ), demonstrating that SR-BI activates Src in macrophages.
Recent studies have shown that the proinfl ammatory necrotic cell marker, HMGB1, binds cytoplasmic Src, preventing its association with transmembrane receptors ( 21 ). Compared with WT cells isolated from recipient LDLR Ϫ / Ϫ mice fed a Western diet, SR-BI Ϫ / Ϫ macrophages had 2-and 1.7-fold higher HMGB1 mRNA and serum protein levels, respectively (supplementary Fig. 8A, C). In addition, HMGB1 protein levels were increased in SR-BI Ϫ / Ϫ cells compared with WT macrophages with and without free cholesterol enrichment (supplementary Fig. 8B). Consistent with SR-BI regulating HMGB1 levels, transfection of SR-BI Ϫ / Ϫ macrophages with SR-BI markedly decreased HMGB1 protein (supplementary Fig. 8D). Interestingly, knockdown of HMGB1 expression partially restored efferocytosis in SR-BI Ϫ / Ϫ macrophages, suggesting that SR-BI regulation of HMGB1 also impacts Src activation and efferocytosis via other receptors (supplementary Fig. 8E).

DISCUSSION
Our studies examined the hypothesis that macrophage SR-BI plays a critical role in mediating efferocytosis of apoptotic cells in atherosclerotic lesions. Our fi ndings demonstrate that macrophage SR-BI binds PS on apoptotic cells and mediates efferocytosis via a Src/PI3K/Rac1 pathway. Hematopoietic SR-BI deletion resulted in dramatically increased atherosclerotic lesion necrosis and noninternalized dead cells with reduced collagen content and fi brous cap thickness, supporting a critical role for macrophage SR-BI in mediating efferocytosis in vivo. Combined deletion of macrophage SR-BI and ApoE exacerbated the impairment in efferocytosis and the accumulation of lesion-free dead cells beyond the single deletion of either gene, suggesting that SR-BI mediates efferocytosis by C1qc , Gas6 , Mfge8 , Thbs1 , annexin A , CD36 and CD68 , and Arg-1 and iNos (supplementary Fig. 7A, B).
Studies have shown that Src activation induces Rac1 activity ( 17 ) and that PI3K activity is important for phagosome formation via the Fc ␥ receptor ( 18 ). Therefore, we examined the possibility that macrophage SR-BI mediates efferocytosis by activating Rac1 via a Src/PI3K pathway ( Fig. 6 ). Compared with SR-BI Ϫ / Ϫ macrophages, incubation of WT macrophages with apoptotic thymocytes markedly enhanced the activation of Src as evidenced by increased phosphorylation of tyrosine 416 in the kinase catalytic domain of Src ( Fig. 6A ). SR-BI also induced activation of PI3K, as shown by increased PI3K p85 and pAkt levels in WT versus SR-BI Ϫ / Ϫ macrophages exposed to apoptotic cells ( Fig. 6A ). Following activation of Src, WT macrophage Rac1 activity was strikingly induced, but RhoA activity, which has been shown to inhibit efferocytosis, was not altered ( Fig. 6A ). Both Src and PI3K activation were critical to SR-BI-mediated efferocytosis, as inhibition with PP2 or Wortmannin, respectively, decreased efferocytosis in WT macrophages but had no effect in SR-BI Ϫ / Ϫ cells ( Fig. 6C, D ). In addition, incubation with necrotic cells did not induce activation of Src, PI3K, and Akt in WT cells ( Fig. 6B ), demonstrating that the signaling is specifi c for efferocytosis of apoptotic cells. SR-BI-induced Src resulted in increased expression of the proinfl ammatory markers IL-1 ␤ , IL-6, MCP-1, MMP-9, and TNF-␣ , and decreased expression of the anti-infl ammatory cytokines IL-10 and TGF-␤ ( Fig. 2 ). These observations clearly reveal that hematopoietic cell SR-BI deletion elicits maladaptive infl ammatory responses that are associated with defective efferocytosis and contribute to atherosclerosis development. Other studies have shown that SR-BI-null mice develop a systemic autoimmune response characterized by autoantibodies and deposition of renal immune complexes ( 25 ). SR-BI defi ciency also enhances lymphocyte proliferation ( 25 ). However, we determined that plaque lymphocyte content was not affected by hematopoietic SR-BI defi ciency (supplementary Fig. 9), but that the live macrophage content was decreased (supplementary Fig. 3C), suggesting that dying macrophages are the key infl ammatory cells impacting atherogenesis in SR-BI Ϫ / Ϫ lesions.
Our studies suggest that ApoE also protects against atherosclerosis by controlling efferocytosis, as deletion of hematopoietic ApoE in LDLR Ϫ / Ϫ mice caused lesional accumulation of free dead cells ( Fig. 3 ). In vitro studies have shown that phagocyte and apoptotic cell ApoE enhance efferocytosis ( 26,27 ), and ApoE binds PS (data not shown). Furthermore, ApoE binds SR-BI to stimulate selective cholesteryl ester uptake and cholesterol effl ux, suggesting a functional interaction ( 28 ). However, our results show that combined deletion of hematopoietic SR-BI and ApoE in LDLR Ϫ / Ϫ mice promotes the accumulation of lesion-free apoptotic cells, necrosis, and development of atherosclerosis beyond deletion of either gene alone ( Figs. 3,4 ), supporting independent anti-atherogenic roles. In addition, receptor-mediated efferocytosis is enhanced by PS bridging molecules ( 29 ) making it likely that SR-BI-mediated phagocytosis is made more effi cient by other proteins, such as ␤ 2-glycoprotein 1 and MFGE8 ( 29 ).

Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling
Two overlapping signaling pathways have evolved to mediate efferocytosis in Caenorhabditis elegans and mammals: CED-1 (LRP1)/CED-6 (GULP)/CED-10 (Rac) and CED-2 (CrkII)/CED-5 (DOCK180)/CED-12 (ELMO)/CED-10 (Rac). Both pathways converge at Rac1 activation ( 30,31 ). Osada and colleagues ( 32 ) have shown that the association of GULP with SR-BI is required for activation of MAPK and Rac1 in nonprofessional phagocytic Sertoli cells . In contrast, we found that macrophage SR-BI does not associate with GULP ( Fig. 7A ), showing that macrophage SR-BI efferocytosis signaling is uniquely different from the Sertoli cell SR-BI pathway. We report that signaling molecules downstream of SR-BI, including Src and PI3K, play critical roles in SR-BI-mediated induction of Rac1 activation. Indeed, incubation of SR-BI-expressing macrophages with apoptotic cells induces SR-BI/Src interaction, Src membrane recruitment, and Src phosphorylation ( Fig. 7B, C ). Importantly, inhibition of Src decreased efferocytosis and Rac1 activation in WT cells. The mechanisms for Src activation vary among different receptors and cell types. In processes that are both dependent and independent of its interaction with ApoE.

Effects of macrophage SR-BI on atherosclerotic lesion development, efferocytosis, and necrosis
Similar to previous studies by us and others ( 5, 6 ), we found that hematopoietic cell defi ciency of SR-BI accelerates atherosclerosis development in ApoE Ϫ / Ϫ and LDLR Ϫ / Ϫ mice. Defective efferocytosis is believed to accelerate atherosclerosis and promote plaque vulnerability by causing increased infl ammation and necrosis in atherosclerotic lesions ( 8,22 ). Here we demonstrate, for the fi rst time, that SR-BI is localized in phagosomes of WT macrophages and can directly bind PS in vitro and at the surface of apoptotic macrophages (supplementary Fig. 6), and that the efferocytosis of apoptotic cells by SR-BI-defi cient macrophages is decreased both in vitro and in vivo ( Fig. 1 ). Similarly, Kawasaki et al. ( 19 ) demonstrated that Sertoli cell SR-BI binds PS liposomes. We clearly show that hematopoietic SR-BI defi ciency leads to a marked accumulation of lesionfree apoptotic cells and enlarged necrotic core formation, which are hallmarks of defective efferocytosis and lesion progression in vivo. Hence, our studies suggest that a major atheroprotective mechanism mediated by macrophage SR-BI is the efferocytosis of apoptotic cells.
Several studies led to our hypothesis that macrophage SR-BI expression plays a critical role by mediating lesion efferocytosis. Rat Sertoli cells can mediate phagocytosis of apoptotic spermatogenic cells via SR-BI ( 19,23 ), and transgenic expression of human SR-BI in HEK-293 cells promotes engulfment of apoptotic thymocytes ( 24 ). Our current results show, for the fi rst time, that SR-BI plays a critical role in mediating efferocytosis of apoptotic cells in macrophages, which are professional phagocytes. Importantly, the number of free dead cells was markedly increased in atherosclerotic lesions containing SR-BI Ϫ / Ϫ macrophages ( Fig. 3 ). Both enhanced apoptosis and defective efferocytosis contribute to formation of the necrotic core. We observed that SR-BI defi ciency also led to increased macrophage apoptosis in atherosclerotic lesions (supplementary Fig. 5), but our data suggests that SR-BI plays a major role in the efferocytosis of apoptotic cells in lesions containing DKO cells, as the effects on active caspase 3 levels were modest compared with the accumulation of free dead cells. If apoptotic cells are not cleared quickly, they undergo secondary necrosis, release infl ammatory cytokines, and elicit immune responses ( 7 ). The physiological relevance of macrophage SR-BI-mediated efferocytosis in atherosclerosis is supported by the tremendous increase in plaque necrosis. After only 8 weeks on a Western diet, plaque necrosis was increased 4-fold and the ratio of free to macrophage-associated TUNEL stain was up 14-fold in recipients of DKO marrow compared with controls ( Figs. 3, 4 ). A key feature of vulnerable plaque is collagen degradation. We demonstrated that hematopoietic SR-BI defi ciency dramatically reduced the collagen and fi brous cap thickness in lesions of DKO → ApoE Ϫ / Ϫ mice compared with ApoE Ϫ / Ϫ → ApoE Ϫ / Ϫ mice ( Fig. 4 ).
Consistent with this concept, hematopoietic SR-BI defi ciency endothelial cells, SR-BI-induced activation of Src resulting in Rac1 activation requires association of the PDZK1 adaptor protein with the PDZ binding domain in the C terminal of SR-BI ( 33 ). However, THP-1 macrophages do not express PDZK1 ( 34 ), and we did not detect PDZK1 mRNA in peritoneal macrophages (data not shown). Thus, interaction with the PDZK1 adaptor protein is likely not relevant for macrophage SR-BI efferocytosis and suggests that macrophage and endothelial cell SR-BI activate Src via different mechanisms. PI3K activity is downstream of Src and critical to macrophage SR-BI-mediated efferocytosis, as inhibition of Src activity reduced pAkt and PI3K p85 levels, and inhibition of PI3K activity impaired efferocytosis in WT cells ( Fig. 6 ). The PI3K products, pAkt and phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P 3 ], promote effi cient efferocytosis ( 18 ) by mediating phagocyte survival ( 35 ) and PI(3,4,5)P 3 and phagosome synthesis.
Macrophages have an abundance of efferocytic receptor pathways, and studies have shown that deletion of a single efferocytic receptor or bridging molecule (i.e., MFGE8, C1q) ( 7, 36 ) is suffi cient to promote accumulation of free apoptotic cells in atherosclerotic lesions. These observations suggest that the environment of atherosclerotic lesions is proinfl ammatory and cytotoxic to the extent that multiple efferocytic pathways are required. Ligand interaction with both LRP1 ( 37 ) and Mertk ( 29 ) is associated with suppression of phagocyte toll-like receptor 4 infl ammatory signaling. We also found that SR-BIdefi cient phagocytes have a proinfl ammatory phenotype, which likely increases phagocyte death, further impairing efferocytosis. In addition, studies suggest that there is cross talk among efferocytic pathways making it likely that deletion of a critical player impacts phagocytosis via other receptors. Mertk-induced signaling enhances Rac1 activation and phagocytosis via ␣ v ␤ 5 integrin ( 38,39 ). In addition, ligand interaction with LRP1 ( 40 ) regulates the internalization of phagocytic integrin ␣ M ␤ 2 ( 41 ). Because our results show that pharmacological activation of Rac1 corrects the defective efferocytosis in the absence of SR-BI ( Fig. 5A,  B ), and specifi c inhibition of Rac1 signifi cantly reduces efferocytosis in WT macrophages, but not in SR-BI-null macrophages ( Fig. 5E-G ), we propose that SR-BI-induced Src/PI3K/Rac1 signaling likely stimulates phagocytosis via other receptors as well. Consistent with this possibility, transfection of phagocytes with increasing amounts of SR-BI increased plasma membrane levels of pSrc Tyr416 in a stepwise dose response ( Fig. 7C ) and rescued defective efferocytosis from SR-BI-defi cient macrophages ( Fig. 7D ). In addition, studies have shown that cytoplasmic HMGB1 interacts with Src, preventing association with plasma membrane receptors, which results in decreased Rac1 activation ( 21 ). These results raise the possibility that the effects of SR-BI on HMGB1 expression may impact other efferocytic pathways (supplementary Fig. 8).
In summary, macrophage SR-BI plays a crucial role in mediating efferocytosis of apoptotic cells in atherosclerosis, reducing plaque necrosis and infl ammation. SR-BI interacts directly with apoptotic cell PS and induces efferocytosis signaling via Src/PI3K/Rac1 (supplementary Fig. 10).
Besides directly mediating efferocytosis, SR-BI may also impact other efferocytic pathways by controlling Src activity via regulating HMGB1 levels, as well as by direct activation. Thus, macrophage SR-BI defi ciency causes severely impaired lesion efferocytosis leading to secondary necrosis and a maladaptive infl ammatory response (supplementary Fig. 10). Increased plaque necrosis due to defective efferocytosis may be a critical prelude to plaque rupture and myocardial infarction; hence, the signaling pathways involved in macrophage SR-BI-mediated efferocytosis provide attractive targets for plaque stabilization and the prevention of cardiovascular events.