CD36, but not G2A, modulates efferocytosis, inflammation, and fibrosis following bleomycin-induced lung injury.

Macrophage G2A and CD36 lipid receptors are thought to mediate efferocytosis following tissue injury and thereby prevent excessive inflammation that could compromise tissue repair. To test this, we subjected mice lacking G2A or CD36 receptor to bleomycin-induced lung injury and measured efferocytosis, inflammation, and fibrosis. Loss of CD36 (but not G2A) delayed clearance of apoptotic alveolar cells (mean 78% increase in apoptotic cells 7 days postinjury), potentiated inflammation (mean 56% increase in lung neutrophils and 75% increase in lung KC levels 7 days postinjury, 51% increase in lung macrophages 14 days postinjury), and reduced lung fibrosis (mean 41% and 29% reduction 14 and 21 days postinjury, respectively). Reduced fibrosis in CD36−/− mice was associated with lower levels of profibrotic TH2 cytokines (IL-9, IL-13, IL-4), decreased expression of the M2 macrophage marker Arginase-1, and reduced interstitial myofibroblasts. G2A, on the other hand, was required for optimal clearance of apoptotic neutrophils during zymosan-induced peritoneal inflammation (50.3% increase in apoptotic neutrophils and 30.6% increase in total neutrophils 24 h following zymosan administration in G2A−/− mice). Thus, CD36 is required for timely removal of apoptotic cells in the context of lung injury and modulates subsequent inflammatory and fibrotic processes relevant to fibrotic lung disease.


LPC ESI-MS/MS analysis
LPC concentration in BAL fl uid was measured by ESI-MS/MS as previously described ( 24,25 ). Briefl y, total lipids were extracted, dried under nitrogen, and resuspended in methanol: chloroform (2:1 v/v). Samples were subsequently measured by direct injection using an API-4000 Q Trap Quadrupole mass spectrometer.

Cytokine analysis of BAL fl uid
BAL fl uid concentrations of cytokines were measured using the Bio-Plex Pro Mouse Cytokine 23-plex assay (Bio-Rad) and a Bio-Plex 200 system (Bio-Rad) according to manufacturer's instructions. TGF-␤ activity in 250 l BAL fl uid samples was measured as previously described using a reporter assay in mink lung epithelial cells (MLEC) stably transfected with an expression construct containing a truncated plasminogen activator inhibitor-1 (PAI-1) promoter containing a TGF-␤ response element fused to the fi refl y luciferase gene ( 26 ).

Hydroxyproline analysis of lung homogenates
Lungs were homogenized in 2 ml PBS, and 1 ml of lung homogenate was desiccated by baking at 110°C in a 4 ml glass vial. Dried lung homogenates were subsequently hydrolyzed in 1 ml 6N HCL at 110°C for 16 h. After cooling, 25 l of hydrolyzed lung homogenate was transferred to a 5 ml glass tube, combined with 1 ml Chloramine T solution (1.4% Chloramine T, 10% 1-propanol, 0.5M sodium acetate), and incubated for 20 min at room temperature. One milliliter of Erlich's solution (1M p -dimethylaminobenzaldehyde, 70% 1-propanol, 20% perchloric acid) was then added and incubated for 15 min at 65°C. The solution (200 l) was then transferred to a clear 96-well plate, and absorbance was measured at 550 nm. The amount of hydroxyproline was determined against a hydroxyproline standard curve.

Lung TUNEL, immunohistochemical, and immunofl uorescence analysis
Lungs were fi lled with 0.8 ml of 1:1 PBS:OCT (Tissue-Tek) solution, excised from the animal, and subsequently placed in tissue blocks, covered in OCT, fl ash-frozen in liquid nitrogen, and stored at Ϫ 80 ° C. For six randomly chosen male WT, G2A Ϫ / Ϫ , and CD36 Ϫ / Ϫ mice, 8 m lung cryosections were stained with anti-F4/80 Alexa555 antibody followed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) as previously described ( 24 ). Quantitation of apoptotic cells was performed by enumerating the number of TUNEL + cells per microscopic fi eld in color images captured on an Olympus BX60 fl uorescence microscope. For each mouse, eight random 100× microscopic fi elds physiological and pathophysiological contexts ( 6 ), despite the fact that a large body of in vitro data point to CD36 as an important mediator of apoptotic cell recognition and uptake ( 3,17,18 ). Importantly, there is no in vivo evidence supporting a requirement for either CD36 or G2A receptor in efferocytosis using an animal disease model in which tissue injury and clearance of apoptotic cells was monitored in conjunction with subsequent infl ammatory and fi brotic repair processes. Furthermore, previous studies of CD36-defi cient (CD36 Ϫ / Ϫ ) mice in certain disease models have yielded results that were unexpected based on prior in vitro data ( 19 ). In addition, functions have been ascribed to CD36 that could infl uence infl ammation and fibrosis following tissue injury independently of its effects on efferocytosis. For example, CD36 can modify infl ammation as a coreceptor for various Toll-like receptors (TLR) ( 20 ) and has also been postulated to facilitate tissue fi brosis following injury by promoting activation of the profi brotic cytokine, transforming growth factor-␤ (TGF-␤ ), through direct interaction with thrombospondin-1 (TSP-1) ( 21,22 ). However, little is known regarding the physiological relevance and functional interplay between these various properties of CD36 in vivo.
To address the need for in vivo studies to complement the signifi cant body of in vitro data supporting a key role for CD36 and G2A receptors in efferocytosis, we used wildtype (WT), G2A-defi cient (G2A Ϫ / Ϫ ) and CD36 Ϫ / Ϫ mice to directly test whether G2A and/or CD36 receptors are required for the clearance of apoptotic cells following tissue injury in vivo and to monitor their subsequent effects on infl ammation and fi brosis using the established bleomycin model of lung injury ( 23 ). The results of this study demonstrate that CD36 (but not G2A) is required for apoptotic cell clearance in the context of bleomycin-induced lung injury and that CD36 modulates subsequent infl ammatory and fi brotic responses in a manner that points to this receptor as a potential therapeutic target in fi brotic pulmonary disease.

Animals
G2A-suffi cient (G2A +/+ ) and G2A Ϫ / Ϫ mice were derived by intercrossing mice heterozygous for the G2A mutant allele (G2A +/ Ϫ ) that were backcrossed greater than 10 generations onto the C57BL/6J background. CD36 Ϫ / Ϫ mice were generously provided by Dr. Maria Febbraio (Cleveland Clinic Foundation) and were backcrossed more than six generations onto the C57BL/6J background. All mice were maintained under pathogen-free conditions, and all studies were conducted in conformity with Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals and with approval from the Animal Care Committee of the University of Alabama at Birmingham.

Bleomycin-induced lung injury
Six-to eight-week-old male WT, G2A Ϫ / Ϫ , and CD36 Ϫ / Ϫ mice were challenged with a single intratracheal instillation of 5 units of bleomycin sulfate (Calbiochem) per kilogram of body weight. Bronchoalveolar lavage (BAL) samples were recovered from mice at various time points following bleomycin administration epithelial and endothelial cell apoptosis is associated with infl ammatory cell recruitment, followed by apoptotic cell clearance, resolution of infl ammation, and the orchestration of tissue repair mechanisms characterized by excessive fi brosis ( 23 ). We measured the kinetics of apoptotic alveolar cell clearance following bleomycin-induced lung injury in WT, G2A Ϫ / Ϫ , and CD36 Ϫ / Ϫ mice. Prior to bleomycin instillation (5 units/kg body weight), the lungs of WT, G2A Ϫ / Ϫ , and CD36 Ϫ / Ϫ mice contained comparable numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive (TUNEL + ) apoptotic cells ( Fig. 1A ). An approximately 13-fold increase in TUNEL + apoptotic cells was observed in the lungs of WT, G2A Ϫ / Ϫ , and CD36 Ϫ / Ϫ mice two days following bleomycin instillation, and no signifi cant effect of G2A defi ciency or CD36 defi ciency on the extent of bleomycin-induced apoptosis was detected at this time point ( Fig. 1A ). However, CD36 defi ciency signifi cantly delayed the subsequent clearance of these apoptotic cells (mean 78% increase in apoptotic cells seven days postinjury in CD36 Ϫ / Ϫ mice) ( Fig. 1A, B ).
(each fi eld containing a major airway around which TUNEL + apoptotic cells were consistently concentrated) were quantifi ed. Trichrome staining was performed as previously described ( 25 ). For assessment of ␣ smooth muscle cell actin ( ␣ SMA) expressing myofi broblasts in lungs of control and bleomycin-treated mice, three 8 m lung cryosections (spaced approximately 200 m apart) from each animal (fi ve for each experimental group) were stained with Cy3-conjugated anti-␣ SMA antibody (Sigma-Aldrich; #C6198). Fluorescence intensity in images taken from three random 100× microscopic fi elds of each lung cryosection (each fi eld containing a major airway) was quantifi ed using the histogram function of Adobe Photoshop CS5.1 (Adobe Systems, San Jose, CA). The average fl uorescence intensity value was measured for each fi eld, excluding the ␣ SMA highly expressing airway smooth muscle cells, in order to selectively quantify interstitial myofi broblasts.

Zymosan-induced peritonitis
Mice were injected intraperitoneally with 1 mg of Zymosan A (Sigma-Aldrich) in PBS. At indicated time points (0, 6, 24, and 48 h), mice were euthanized and peritonea were fl ushed with 5 ml HBSS. Peritoneal exudate cells were counted using a hemocytometer, stained with anti-Gr-1 FITC and anti-CD115 APC antibodies, washed, and subsequently stained with Annexin-V PE and 7-AAD according to the manufacturers protocol (BD Biosciences). Cells were analyzed using a FACS Calibur fl ow cytometer. Neutrophils and macrophages were identifi ed as Gr-1 + CD115 and Gr-1 + CD115 + , respectively. Apoptotic and necrotic cells were identifi ed as Annexin-V + 7-AAD Ϫ and Annexin-V + 7-AAD + , respectively. Data were acquired from a total of 10 5 peritoneal cells from each zymosan-injected animal. For each genotype (WT, G2A Ϫ / Ϫ , and

Statistical analysis
Statistical analysis was performed using SigmaStat (Systat Software, Inc.). Student t -test was used for single comparisons and one-way ANOVA (ANOVA) was used for multiple comparisons. For all statistical analyses, P < 0.05 was considered signifi cant.

Loss of CD36, but not G2A, delays apoptotic cell clearance following bleomycin-induced lung injury
We addressed the requirement for G2A and CD36 receptors using a physiologically relevant in vivo model of lung injury in which apoptosis of resident cells was induced and the kinetics of their subsequent clearance measured over time. Intratracheal bleomycin instillation is a widely employed model for the study of pulmonary infl ammation and fi brosis associated with injury, in which initial alveolar Ϫ / Ϫ

Reduced lung fi brosis in bleomycin-treated CD36 ؊ / ؊ mice
The signifi cantly greater bronchoalveolar neutrophil and macrophage numbers in bleomycin-treated CD36 Ϫ / Ϫ G2A defi ciency, on the other hand, had no such effect ( Fig. 1A, B ), despite robust increases in bronchoalveolar lavage (BAL) fl uid LPC concentrations beginning two days following bleomycin instillation (coincident with the induction of apoptosis) ( Fig. 2 ).

Increased bronchoalveolar neutrophil and macrophage numbers following bleomycin-induced lung injury in
The chemotactic function of G2A has been postulated to contribute to the recruitment of monocytes and T cells into sites of infl ammation in response to locally generated LPC ( 11,16,27 ). G2A defi ciency may thus modulate the induction and/or resolution of infl ammation following bleomycin-induced lung injury. Furthermore, defective apoptotic cell clearance in CD36 Ϫ / Ϫ mice ( Fig. 1A, B ) could result in the amplifi cation and/or protraction of infl ammation ( 28,29 ). To address these issues, we measured bronchoalveolar infl ammatory cell composition in WT, G2A Ϫ / Ϫ , and CD36 Ϫ / Ϫ mice 2 days following bleomycin instillation ( Fig. 3 ). G2A defi ciency had no signifi cant impact on neutrophil, monocyte, CD4 + T cell, CD8 + T cell, or B cell numbers at later time points and did not affect bronchoalveolar macrophage numbers ( Fig. 3 ). Taken together, these data demonstrate that G2A signaling is redundant for normal apoptotic cell clearance following bleomycin-induced lung injury and does not modify the initiation or resolution of infl ammation in this model. CD36 defi ciency, however, resulted in signifi cant increases Ϫ / Ϫ mice.
Data are average ± SD of 5 animals for each time point (* P < 0.05 relative to day 0 control).

reduced in bronchoalveolar cells from CD36
Ϫ / Ϫ mice 14 days following bleomycin-induced lung injury ( Fig. 5C ). The reduced levels of TH2 cytokines and active TGF-␤ observed in bleomycin treated CD36 Ϫ / Ϫ mice may collectively contribute to the less fi brotic milieu in these animals. As ␣ SMA-positive myofi broblasts are major cellular mediators of fi brosis following bleomycin-induced injury ( 33,34 ) and because their differentiation is regulated to a significant extent by TH2 cytokines ( 35,36 ), we assessed lung interstitial ␣ SMA expression to determine whether the reduced fi brosis in CD36 Ϫ / Ϫ mice was associated with decreased numbers of myofi broblasts. Signifi cant reductions in ␣ SMA expressing interstitial myofi broblasts (independent of airway smooth muscle staining) were observed in the lungs of CD36 Ϫ / Ϫ mice compared with their WT counterparts 14 days following intratracheal bleomycin administration ( Fig. 6 ). mice ( Fig. 3 ), indicative of increased infl ammation, and their reduced levels of profi brotic TH2 cytokines, which are known to promote alternative profi brotic M2 macrophage activation ( 30 ) ( Fig. 4B, C ), suggested that lung fi brosis may be reduced in these animals. Indeed, defective apoptotic cell clearance can prevent the polarization of macrophages to a profi brotic M2 phenotype, thus impairing the orchestration of fi brotic repair processes ( 32 ). Furthermore, a direct profi brotic role for CD36 following bleomycin-induced lung injury has been proposed through its interaction with thrombospondin-1 (TSP-1) and resulting induction of active TGF-␤ ( 21,22 ). We therefore measured the hydroxyproline content of lungs, a quantitative measure of fi brosis ( 23 ), from WT, G2A Ϫ / Ϫ , and CD36 Ϫ / Ϫ mice 14 and 21 days following bleomycin instillation. While lung hydroxyproline content was unaffected by G2A defi ciency, CD36 Ϫ / Ϫ mice had signifi cant reductions in hydroxyproline, indicative of reduced fi brosis ( Fig. 5A ). Consistent with this observation, trichrome-stained sections of lung from the same CD36 Ϫ / Ϫ mice exhibited less extensive areas of collagen deposition ( Fig. 5B ). Finally, consistent with reduced M2 macrophage polarization, expression of the M2 macrophage marker, Arginase-1 (ARG-1), was signifi cantly  Ϫ / Ϫ mice at the indicated time points following intratracheal bleomycin (5 units/kg body weight) administration. Data are average ± SD of fi ve animals for each genotype at each time point.
To accurately identify and quantify apoptotic versus viable neutrophils in peritoneal exudates, we employed a fl ow cytometric approach similar to that employed for the examination of BAL cells in supplementary Fig. I in combination with Annexin-V staining. Robust neutrophilic infl ammation was elicited within 6 h of intraperitoneal zymosan injection, which was almost completely resolved by 48 h in WT, G2A Ϫ / Ϫ , and CD36 Ϫ / Ϫ mice ( Fig. 7A ). In G2A Ϫ / Ϫ mice, however, the kinetics of neutrophil clearance was signifi cantly attenuated (mean 30.6% increase in peritoneal neutrophils 24 h following zymosan injection) ( Fig. 7A ), and this was associated with increases of both apoptotic (Annexin-V + 7AAD Ϫ , mean 50.3% increase) and postapoptotic/necrotic (Annexin-V + 7AAD + , mean 42.9% increase) neutrophils ( Fig. 7B ). In CD36 Ϫ / Ϫ mice, on the other hand, we did not detect any impairment of neutrophil clearance ( Fig. 7 ). Interestingly, numbers of postapoptotic/necrotic (but not apoptotic) neutrophils were signifi cantly reduced in CD36 Ϫ / Ϫ mice 6 h (but not at any other time point) following intraperitoneal zymosan injection (mean 55% decrease) ( Fig. 7B ), suggesting either a reduced rate of postapoptotic neutrophil necrosis or an increased clearance of postapoptotic/necrotic neutrophils in the absence of CD36.

DISCUSSION
A role for CD36 in apoptotic cell clearance in vivo has hitherto only been demonstrated in a skin punch wound healing assay using CD36 Ϫ / Ϫ mice ( 6 ). The fact that CD36 was required for optimal apoptotic cell clearance following bleomycin-induced lung injury ( Fig. 1 ) is therefore an important fi nding, particularly as other receptors in addition to CD36 are likely involved and may have been predicted to obscure any effects of CD36 defi ciency (i.e., make CD36 functionally redundant in this model) ( 2 ). Indeed, similar effects on efferocytosis and lung infl ammation were recently reported for the MER tyrosine kinase receptor in mice following bleomycin-induced lung injury ( 39 ). The modulatory effects of CD36 on subsequent infl ammatory and fi brotic events also reveal the importance of this receptor in regulating processes that are pathophysiologically relevant to idiopathic pulmonary fi brosis (IPF). Notably, the reduced rate of apoptotic cell clearance in the lungs of bleomycin-treated CD36 Ϫ / Ϫ mice was associated with an increase in neutrophils (on day 7 postintratracheal bleomycin administration) and macrophages (on day 14) that could not be explained by increased initial neutrophil and monocyte infi ltration (on day 2) ( Fig. 3 ). However, multiplex analysis of BAL fl uid cytokine levels did reveal signifi cantly higher levels of the neutrophilic chemokine KC (CXCL1) in bleomycin-treated CD36 Ϫ / Ϫ mice 7 and 14 days postbleomycin treatment ( Fig. 4A ). KC (CXCL1) is a potent chemokine for neutrophils whose receptor was shown to be important for neutrophilic recruitment into the airways of bleomycin-treated mice ( 40 ). While the main source of this increased bronchoalveolar CXCL1 in CD36 Ϫ / Ϫ mice remains to be determined, it is possible that Delayed clearance of neutrophils in zymosan-treated G2A ؊ / ؊ mice Frasch et al. reported that antibody-mediated G2A blockade or genetic deletion of G2A both signifi cantly delay the clearance of neutrophils in the murine zymosaninduced peritoneal infl ammation model ( 37,38 ). In the latter, more recent study, both viable and apoptotic neutrophils were identifi ed and enumerated by morphological examination in peritoneal exudate cytospin preparations from wild-type and G2A Ϫ / Ϫ animals; both viable and apoptotic neutrophil populations were found to be signifi cantly increased 18-48 h following intraperitoneal zymosan injection ( 38 ). Thus, we considered why G2A defi ciency did not affect bronchoalveolar neutrophil numbers following bleomycin instillation in our present study ( Fig. 3 ). Furthermore, we reasoned that in addition to elevated production of the neutrophilic chemokine KC (CXCL1) ( Fig.  4A ), defective neutrophil clearance may have contributed to the increased neutrophil numbers observed following bleomycin-induced lung injury in CD36 Ϫ / Ϫ mice ( Fig. 3 ). To address these issues, we measured the kinetics of neutrophil clearance following intraperitoneal zymosan injection in autoimmunity ( 44,45 ), it is interesting that no evidence for an increased predisposition of G2A Ϫ / Ϫ mice to autoimmunity compared with their age, gender, and genetically matched WT counterparts has been reported ( 46 ). Nevertheless, in this present study, we were able to provide important confi rmatory data supporting a key role for G2A in apoptotic neutrophil clearance in the zymosan-induced peritoneal infl ammation model ( Fig. 7 ) as originally reported by Frasch et al. ( 37,38 ). This, in conjunction with data from the bleomycin-induced lung injury model, further underscores the potential degree of context-dependent functional redundancy between these receptors in terms of their requirement for efferocytosis in vivo. Indeed, while it is widely accepted that the in vivo relevance of individual receptors is likely to be determined to a signifi cant extent by the context, tissue, and cell-type in which apoptosis occurs, published comparative studies similar to ours employing in vivo models that directly address this issue are lacking. Impaired apoptotic cell clearance in CD36 Ϫ / Ϫ mice was associated with augmented lung infl ammation following bleomycin-induced injury ( Fig. 3 ) and subsequently a reduced fi brotic response ( Fig. 5A ). Several studies have reported a similar association between increased lung infl ammation and reduced fi brosis following lung injury ( 47 ). Moreover, increased macrophage infi ltration and reduced fi brosis was reported in the kidneys of CD36 Ϫ / Ϫ mice following unilateral ureteral obstruction ( 48 ). One could therefore speculate that an abnormal accumulation of apoptotic cells in the lungs of bleomycin-treated CD36 Ϫ / Ϫ mice may have prevented the timely resolution of infl ammation, leading to the impairment of subsequent fi brotic changes ( 4,35 ). Furthermore, defective apoptotic cell clearance can promote the polarization of macrophages to a an increase in CXCL1-mediated neutrophil recruitment, rather than a decrease in neutrophil clearance, may contribute to the augmented neutrophilic infl ammation observed in CD36 Ϫ / Ϫ mice following lung injury. Indeed, this is supported by our failure to detect a suppressive effect of CD36 defi ciency on neutrophil clearance in the zymosaninduced peritoneal infl ammation model ( Fig. 7 ). However, we cannot exclude the possibility that impaired clearance of neutrophils resulting from loss of CD36-mediated interaction with NADPH oxidase-generated oxidized phospholipids ( 37 ) could have contributed to increasing neutrophils in the lungs of bleomycin-treated CD36 Ϫ / Ϫ mice, despite the lack of a similar effect in the zymosan-induced peritoneal infl ammation model.
It was somewhat surprising that, despite the widely held notion that G2A is involved in the clearance of apoptotic cells ( 2,13,14 ), we found that G2A defi ciency had no signifi cant effect on apoptotic cell clearance in the bleomycin-induced lung injury model ( Fig. 1 ). Indeed, although an LPC-dependent chemotactic function of G2A has been described in monocytes/macrophages and T cells in vitro ( 11,16,27 ), we did not detect any signifi cant effect of G2A defi ciency on the frequency of bronchoalveolar monocytes, macrophages, T cells, or neutrophils following bleomycin-induced lung injury ( Fig. 3 ), despite demonstrable increases in local LPC production ( Fig. 2 ). In this regard, it may be noteworthy that published studies of G2A deficiency in mouse models of infl ammatory diseases associated with local generation of LPC, such as atherosclerosis and multiple sclerosis, have similarly failed to provide evidence that either the efferocytic or chemotactic roles for G2A are important in vivo (41)(42)(43). Moreover, considering that defective apoptotic cell clearance has been linked to breakage of immune tolerance and the development of lung injury. Indeed, the neutrophilic chemokine, CXCL1, was signifi cantly increased in the lungs of bleomycin-treated CD36 Ϫ / Ϫ mice compared with their WT and G2A Ϫ / Ϫ counterparts ( Fig. 4A ), and it is known that KC (CXCL1) production by resident tissue macrophages is controlled by signaling from TLRs ( 50 ). In addition, studies with peptides blocking CD36 interaction with TSP-1 suggest that CD36/TSP-1 signaling is important for the activation of TGF-␤ following bleomycin-induced lung injury ( 21,22 ). Consistent with this, decreased lung fi brosis in CD36 Ϫ / Ϫ mice following bleomycin treatment was associated with a reduction in bronchoalveolar levels of active TGF-␤ at day 7 ( Fig. 4C ). Although the moderate extent to which active TGF-␤ was reduced in CD36 Ϫ / Ϫ mice may be somewhat surprising considering the aforementioned peptide blocking studies ( 21,22 ), it is noteworthy that genetic deletion of TSP-1 did not reduce active TGF-␤ or attenuate lung fi brosis following bleomycin-induced lung injury either ( 51 ). Similarly to TSP-1 ( 51 ), therefore, our data show that global CD36 defi ciency, as opposed to selective disruption of CD36/TSP1 interaction, does not have a robust suppressive effect on levels of active TGF-␤ following bleomycin-induced lung injury ( Fig. 4C ). This in turn suggests that the reduced lung fi brosis in CD36 Ϫ / Ϫ mice may occur through the combined cumulative effects of reduced TH2 cytokines (IL-9, IL-13, IL-4, IL-5) and TGF-␤ ( Fig. 4B, C ). Indeed, polarization to a TH2-type response or inactivation of TH2-type cytokines (IL-4, IL-13, or IL-9) can promote or inhibit, respectively, pulmonary fi brosis in mice following bleomycin-induced lung injury (52)(53)(54)(55). The profi brotic effects of these TH2 cytokines are mediated in part by their ability to promote the differentiation and proliferation of collagen-producing myofi broblasts ( 35 ). Thus, our fi nding that ␣ SMA-expressing myofi broblasts were signifi cantly reduced in the lungs of bleomycin-treated CD36 Ϫ / Ϫ mice compared with their WT counterparts ( Fig. 6 ) may be mechanistically relevant. Further studies are required to dissect the molecular and cellular pathways by which CD36 signaling regulates this cell type. In addition, other lipid-mediated mechanisms may account for the association between reduced efferocytosis and reduced fi brosis in CD36 Ϫ / Ϫ mice, including increased local generation of PGE2 by macrophages in response to increased numbers of apoptotic cells ( 56,57 ). Indeed, PGE2 produced as a result of the abnormal accumulation of apoptotic cells may act directly on myofi broblasts themselves to mediate antifi brotic effects ( 58,59 ). Finally, whether G2A and/or CD36 receptors are required or functionally redundant for efferocytosis may depend to a signifi cant degree on the types of lipids generated by the apoptotic cell as well as those associated with the local infl ammatory milieu. In addition to quantitative and qualitative differences in lipid composition of the apoptotic cell, the nature of the infl ammatory milieu (cytokine expression, involvement of TLR signaling, macrophage M1 versus M2 phenotype) may also be a major determinant of receptor function in efferocytosis. Characterization of lipid species generated in different models in vivo in conjunction with an accurate assessment of macrophage proinfl ammatory M1 phenotype, thus delaying transition to a fi brotic repair stage normally promoted by M2 macrophage polarization ( 32 ). Indeed, we found signifi cantly reduced expression of the M2 marker ARG-1 in BAL cells from CD36 Ϫ / Ϫ mice compared with their WT and G2A Ϫ / Ϫ counterparts ( Fig. 5C ). However, the reduced fi brosis in CD36 Ϫ / Ϫ mice may equally refl ect other recognized functions of CD36 independent of its role in efferocytosis. Indeed, it is unclear to what extent defective apoptotic cell clearance in CD36 Ϫ / Ϫ mice contributed to the proinfl ammatory and antifi brotic changes. Delineating the interrelationships between these processes will be diffi cult considering that the response to lung injury is complex, involving the coordinated action of multiple cytokines/chemokines, infl ammatory cells, and resident epithelial, endothelial, and fi broblastic cells ( 23 ). The effects of CD36 on lung infl ammation and fi brosis may thus be multifactorial, involving multiple cell types as well as multiple functional properties of CD36 (some possibly operational in the same cell type) ( 20,49 ). For example, CD36 has been identifi ed as a coreceptor for various Toll-like receptors (TLR) in macrophages promoting infl ammation in response to endogenous CD36 ligands ( 20 ). CD36 defi ciency could therefore infl uence infl ammatory processes independently of its impairment of apoptotic cell clearance following bleomycin-induced phenotype may therefore provide important mechanistic insights into this fundamental issue. Furthermore, as underscored by our fi ndings, a complete understanding of the relevance and functional redundancy between major receptors mediating "fi nd-me" and "eat-me" signals will necessitate the utilization of multiple in vivo models of tissue injury and infl ammation in addition to macrophagespecifi c genetic manipulation in order to separate their roles in efferocytosis from effects in other tissues. The cumulative data from such studies will also lead to a much better understanding of the extent to which deregulation of individual pathways contributes to the pathology of chronic infl ammatory diseases in which defective apoptotic cell clearance has been implicated as an etiological factor. In this regard, our fi ndings identify the CD36 receptor as a potential target for therapeutic intervention in fi brotic lung diseases, such as chronic obstructive pulmonary disease (COPD), and warrant further studies to delineate the key cellular and molecular mechanisms responsible among a potential pleiotropy of CD36 functions ( 20,49 ).