Cd47-Sirpα interaction and IL-10 constraininflammation-induced macrophage phagocytosis ofhealthy self-cellsZhen Biana,b, Lei Shia, Ya-Lan Guoa, Zhiyuan Lva, Cong Tanga, Shuo Niua, Alexandra Tremblaya,Mahathi Venkataramania, Courtney Culpeppera, Limin Lib, Zhen Zhoub, Ahmed Mansoura, Yongliang Zhangc,Andrew Gewirtzd, Koby Kiddera,e, Ke Zenb, and Yuan Liua,d,1

aProgram of Immunology and Cell Biology, Department of Biology, Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA 30302;bState Key Laboratory of Pharmaceutical Biotechnology, Nanjing Advanced Institute for Life Sciences, Nanjing University, Nanjing, Jiangsu 210093,China; cDepartment of Microbiology and Immunology, Yong Loo Lin School of Medicine, Life Science Institute (LSI) Immunology Programme, NationalUniversity of Singapore, Singapore 117456; dCenter for Inflammation, Immunity and Infection, Georgia State University, Atlanta, GA 30303; andeDepartment of Cell Biology, Rutgers University, New Brunswick, NJ 08901

Edited by Jason G. Cyster, University of California, San Francisco, CA, and approved July 11, 2016 (received for review October 28, 2015)

Rapid clearance of adoptively transferred Cd47-null (Cd47−/−) cells incongeneic WT mice suggests a critical self-recognition mechanism,in which CD47 is the ubiquitous marker of self, and its interactionwith macrophage signal regulatory protein α (SIRPα) triggers inhib-itory signaling through SIRPα cytoplasmic immunoreceptor tyrosine-based inhibition motifs and tyrosine phosphatase SHP-1/2. However,instead of displaying self-destruction phenotypes, Cd47−/− miceman-ifest no, or only mild, macrophage phagocytosis toward self-cellsexcept under the nonobese diabetic background. Studying ourrecently established Sirpα-KO (Sirpα−/−) mice, as well as Cd47−/−

mice, we reveal additional activation and inhibitory mechanismsbesides the CD47-SIRPα axis dominantly controlling macrophage be-havior. Sirpα−/− mice and Cd47−/− mice, although being normallyhealthy, develop severe anemia and splenomegaly under chronic co-litis, peritonitis, cytokine treatments, and CFA-/LPS-induced inflam-mation, owing to splenic macrophages phagocytizing self-red bloodcells. Ex vivo phagocytosis assays confirmed general inactivity ofmacrophages from Sirpα−/− or Cd47−/− mice toward healthy self-cells, whereas they aggressively attack toward bacteria, zymosan,apoptotic, and immune complex-bound cells; however, treating thesemacrophages with IL-17, LPS, IL-6, IL-1β, and TNFα, but not IFNγ,dramatically initiates potent phagocytosis toward self-cells, for whichonly the Cd47-Sirpα interaction restrains. Even for macrophages fromWT mice, phagocytosis toward Cd47−/− cells does not occur withoutphagocytic activation. Mechanistic studies suggest a PKC-Syk–medi-ated signaling pathway, towhich IL-10 conversely inhibits, is requiredfor activating macrophage self-targeting, followed by phagocytosisindependent of calreticulin. Moreover, we identified spleen red pulpto be one specific tissue that provides stimuli constantly activatingmacrophage phagocytosis albeit lacking in Cd47−/− or Sirpα−/− mice.

phagocytosis | macrophage | CD47 | SIRPα | cytokine

To maintain tissue integrity and homeostasis, tissue macro-phages and other immunological phagocytes must be pre-

vented from phagocytizing healthy self-cells. One essentialmechanism that prevents macrophages from doing so is CD47-signal regulatory protein α (SIRPα) interaction-mediated in-hibition (1–4). In this mechanism, CD47, a broadly expressed cellsurface protein that acts as a marker of self, along with SIRPα,the counter receptor of CD47 expressed on macrophages andother phagocytic leukocytes, serve as an inhibitory signalingregulator (5–7). On CD47 extracellular ligation, SIRPα increasestyrosine phosphorylation in the cytoplasmic domain immuno-receptor tyrosine-based inhibition motifs (ITIMs), leading to ac-tivation of the SH2-containing tyrosine phosphatase (SHP-1/2),which then mediates inhibitory signaling events through proteindephosphorylation. The end result of this CD47-SIRPα-SHPsignaling is inhibition of phagocytosis toward self-cells (8).

The CD47-SIRPα mechanism was first reported by Oldenborget al. (1), who had demonstrated in red blood cell (RBC)transfusion experiments that WT mice rapidly eliminate syngeneicCd47-null (Cd47−) RBCs through erythrophagocytosis in thespleen and that the lack of tyrosine phosphorylation in SIRPαITIMs was associated with this macrophage aggressiveness. Later,adoptive transfer/allograft experiments by others further demon-strated phagocytic clearance of platelets, lymphocytes, hematopoi-etic cells, and splenocytes in the absence of CD47-SIRPα–mediatedinhibition (9–11). In all of these cases, the phagocytosis occurredswiftly and completely eliminated donor cells in less than 24 h,without facilitation by antibodies or immune complexes. Inclinationthat this phagocytosis differed from that which is mediated by an-tibodies or complements was supported by using Rag-1−/− mice andC3−/− mice, in which lack of lymphocytes or complements had nothindered elimination of Cd47−RBCs (1, 10). In line with the role ofthe CD47-SIRPα interaction in inhibiting macrophage phagocyto-sis, cancer studies found that tumor cells are commonly associatedwith increases in CD47 expression as a way to evade immunologicaleradication (12–15), whereas perturbation of CD47-SIRPα inter-action provides opportunities for cancer eradication, especially in

Significance

The present study reveals that macrophage phagocytosis to-ward healthy self-cells is controlled by a two-tier mechanism: aforefront activation mechanism requiring the inflammatorycytokine-stimulated protein kinase C (PKC)-spleen tyrosine ki-nase (Syk) pathway, to which IL-10 conversely regulates, and thesubsequent self-target discrimination mechanism controlled bythe CD47-signal regulatory protein α (SIRPα)–mediated inhibi-tion. The findings significantly expand our understanding ofmacrophage phagocytic plasticity and behavior under differentconditions and also provide insights into strategies for enhancingtransplantation tolerance and macrophage-based cancer eradica-tion, especially for cancers toward which therapeutic antibodiesare yet unavailable.

Author contributions: K.Z. and Y.L. designed research; Z.B., L.S., L.L., and Z.Z. performedresearch; Y.-L.G., Z.L., C.T., S.N., A.T., M.V., C.C., A.M., Y.Z., A.G., and K.K. contributed newreagents/analytic tools; Z.B., L.S., Z.Z., and Y.L. analyzed data; and Z.B., K.Z., and Y.L.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The complete transcript profiling data of red pulp macrophages fromSirpα−/− mice and WT mice have been deposited in the Gene Expression Omnibus (GEO)database, www.ncbi.nlm.nih.gov/geo (accession no. GSE78191).1To whom correspondence should be addressed. Email: yliu@gsu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1521069113/-/DCSupplemental.

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conjunction with therapeutic anticancer antibodies (4, 9, 16–21).Conversely, the lack of a compatible CD47 to ligate the recipientSIRPα is considered to be associated with tissue rejection inxenotransplantation (22–25).Although these studies are exciting, major obstacles still remain

and hinder further understandings of the CD47-SIRPα mecha-nism, as well as how to control macrophage behavior in diseasetherapies. Resolution of at least two puzzles remains paramount:first, why macrophages do not attack self in mice deficient of Cd47or Sirpα? Despite that the Cd47-Sirpα–mediated inhibition ismissing, mice deficient of Cd47 (26), Sirpα (established in thisstudy), or the Sirpα cytoplasmic domain (27) are relatively healthy,manifesting no or only minor phenotypes (9, 18, 28) that suggestmacrophage phagocytosis toward self-cells. Adoptive transfer ofCd47-null RBCs into these mutant mice also failed to inducerapid elimination (1) (also shown in this study). A case of Cd47deficiency-associated lethal anemia in nonobese diabetic (NOD)mice had been reported, but this is associated with autoimmunityand increased antibody binding to RBC-mediated elimination(20). Having worked with WT and Cd47−/− hematopoietic chi-mera, Wang et al. (11) suggested that Cd47 expression onnonhematopoietic cells is required for macrophages to developself-discrimination and that the lack of Cd47 expression inCd47−/− mice confers macrophage tolerance. However, as thestudy had shown that this “trained” tolerance is not applied toCd47− RBCs (11), it thus has not explained why mice deficientof Cd47 or Sirpα display incapability to clear endogenous ortransferred RBCs. Hence, is there another mechanism(s) besidesCD47-SIRPα that controls macrophage phagocytosis toward self?Second, the CD47-SIRPα mechanism serves as a “safety valve”against undesirable phagocytosis only when macrophages are ini-tiated to phagocytose toward healthy self-cells. Unlike macrophagephagocytosis toward other targets, such as microbial pathogens,immune complex-labeled targets, or apoptotic cells, on whichspecific “eat-me” signals are displayed and induce phagocytosis(29–31), healthy self-cells usually do not attract macrophageattacking. The CD47-SIRPα mechanism alone, however, providesno explanation for how and when macrophages are triggered toattack healthy self-cells, nor does it describe how this phagocyticprocess is carried out. Thus, the core question remains: what isthe mechanism that initiates macrophages to phagocytose healthyself-cells?In this study, we established a strain of Sirpα KO (Sirpα−/−)

mice. Further characterization of macrophage phagocytosis inSirpα−/− and Cd47−/− mice found that, although these mutantmice generally display no defects, acute anemia associated withpotent macrophage phagocytosis toward self-RBCs exhibits un-der inflammatory conditions or can be induced by treatment withinflammatory factors. Additional ex vivo and in vivo studies col-lectively suggest that macrophage phagocytosis toward self is dy-namically controlled by concomitant, yet critical, mechanisms thatdetermine macrophage phagocytic activation; it is governed byproinflammatory and antiinflammatory tissue environmentswhile coupled with subsequent target selection via CD47-SIRPα–mediated inhibition.

ResultsAcute Anemia in Mice Deficient of Cd47-Sirpα–Mediated InhibitionUnder Inflammatory Conditions. A strain of Sirpα KO (Sirpα−/−)mice was established by targeted inactivation of the Sirpα gene inembryonic cells (Fig. 1A). PCR genotyping and immunoblot[Western blot (WB)] confirmed disruption of the Sirpα gene anddepletion of Sirpα protein expression. Similar to Cd47−/− mice,Sirpα−/− mice appeared healthy under the standard specific path-ogen-free (SPF) housing conditions, having displayed no tissue/organ damage suggestive of enhanced macrophage phagocytosistoward self.

However, when inducing colitis with low-dose dextran sodiumsulfate (DSS, 1–2%), Sirpα−/− mice displayed not only severecolitis but also acute anemia; the latter was associated with en-hanced macrophage erythrophagocytosis in the spleen. As shownin Fig. 1B (SI Appendix, Fig. S1), compared with WT littermates,Sirpα−/− mice developed more severe colitis, demonstrating fasterbody weight loss, worse diarrhea/clinical scores, and enhancedpolymorphonuclear leukocytes (PMN) infiltration into intestinesunder DSS treatment. Given that Sirpα-mediated inhibitory sig-naling negatively regulates leukocyte inflammatory response, itwas not surprising that Sirpα deficiency exacerbates DSS-inducedcolitis. Similar results have been observed in our previous studyusing mice with truncated Sirpα cytoplasmic domain (32). Strik-ingly, colitic Sirpα−/− mice, but not WT mice, had also developedsignificant splenomegaly and acute anemia (Fig. 1C). Pale-coloredabdominal cavities were seen in colitic Sirpα−/− mice (Fig. 1C),which were confirmed as anemia by peripheral hemoglobin re-duction (40–50%; Fig. 1D). Dissection of their enlarged spleensrevealed an extensive expansion of red pulp, to a point that haddisrupted the white pulp structure. Spleen hemoglobin assaysconfirmed splenomegaly associated with increased RBC trapping,suggestive of aggressive erythrophagocytosis by red pulp macro-phages (Fig. 1E). Together, these results suggest that active colitisinduces macrophage-mediated RBC destruction in Sirpα−/− mice.Further analyses revealed that the anemia developed in colitic

Sirpα−/− mice was associated with IL-17. As shown in Fig. 1F, theanemia progressed slowly at the initial phase during colitis butwas abruptly aggravated after days 7–8 when IL-17 started toarise in the serum. As demonstrated by us previously, IL-17 ishighly induced at the postacute/chronic phase of DSS-inducedcolitis (33, 34). To test whether IL-17 played a role, we performedthree experiments. First, Sirpα−/− mice under DSS-induced colitiswere given an anti–IL-17 neutralization antibody. As shown inFig. 1G, giving anti–IL-17 antibody on day 6 and 8 when IL-17arises during DSS-induced colitis largely ameliorated anemiaand splenomegaly in Sirpα−/− mice. Second, healthy Sirpα−/− micewere administrated with colitis serum samples that contained highlevels of IL-17. As shown in Fig. 1H, injections of colitis seruminto Sirpα−/− mice directly induced acute anemia. The effect wasconfirmed to be specific as control serum from healthy mice orcolitis serum mixed with anti–IL-17 antibody failed to cause ane-mia. Third, a recombinant IL-17A was administrated into healthySirpα−/−mice. As shown in Fig. 1, I and J, injections of IL-17A alone(2×, 10 μg/kg, i.v.) directly induced acute anemia and splenomegalyin Sirpα−/− mice (SI Appendix, Fig. S2). Notably, IL-17 injectionalso induced acute anemia and splenomegaly in Cd47−/− mice. Asreported previously, Cd47−/− mice are resistant to low-dose DSS-induced colitis and are defective for IL-17 induction in vivo (34, 35).This explains why low-dose DSS treatment does not induce anemiain Cd47−/− mice, as anemia is secondary to the colitic condition andcolitis-induced IL-17 (SI Appendix, Fig. S3).In addition to colitis, other inflammatory conditions and cy-

tokines, such as zymosan-induced peritonitis, LPS, and Freund’scomplete adjuvant (CFA)-induced inflammation, also inducedanemia and splenomegaly in mice lacking Sirpα or Cd47. Asshown in Fig. 1K, repetitively inducing peritonitis by zymosan(3×, every other day) led to severe anemia and splenomegaly inSirpα−/− and Cd47−/− mice, albeit this condition, per se, is shorttermed and self-resolving. Administration of IL-6 (2×, 10 μg/kg, i.v.),the signature cytokine associated with zymosan-induced perito-nitis, also induced the same result. Injection of low-dose LPS(0.25 mg/kg, i.p.), or CFA (s.c.), in Sirpα−/− and Cd47−/− miceinduced anemia and splenomegaly as well.

In Vivo Assessment of Macrophage Phagocytosis by AdoptiveTransfer. RBC adoptive transfer experiments were performedto further assess macrophage phagocytosis in vivo. Cd47+ orCd47− RBCs isolated from WT or Cd47−/− mice, respectively,

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were labeled with carboxyfluorescein succinimidyl ester (CFSE),followed by transfer into different recipient mice and assessment oftheir clearance. As shown in Fig. 2 A–C, WT recipients swiftlycleared Cd47−RBCs in a few hours (t1/2 < 5 h), but retained Cd47+

RBC for over a month (t1/2 ∼ 3 wk), a time length matching thenormal life span of RBCs in the circulation. This result is consis-tent with a previous report (1), suggesting the absence of CD47-SIRPα–mediated inhibition is associated with erythrophagocytosisin the spleen. However, this manner of rapid clearance did notoccur in Sirpα−/− or Cd47−/− mice, which retained both Cd47+

RBCs and Cd47− RBCs for extended time periods. Adoptivetransfer of splenocytes produced the same results; the only rapidclearance found was in WT recipients to which Cd47− splenocyteswere transferred (SI Appendix, Fig. S4). Thus, different from WTmice, Sirpα−/− and Cd47−/− mice do not eliminate RBCs becauseof merely missing the CD47-SIRPα–mediated inhibition.However, treating Sirpα−/− and Cd47−/− mice with IL-17, or

inducing inflammatory conditions in these mice, instantly in-duced accelerated RBC clearance. As shown in Fig. 2, D and E,Sirpα−/− mice with 1% DSS for 8 d (when IL-17 started to arise)or who were directly injected with IL-17A displayed rapid clear-ance of transferred Cd47+ and Cd47− RBCs (∼50%, 24 h). ThatSirpα−/− mice cleared both Cd47+ and Cd47− RBCs was predict-able, as macrophages in these mice experience no inhibition by

the Cd47-Sirpα mechanism. Following this line, the RBC clear-ance in Sirpα−/− mice under DSS or IL-17A treatment was indeedmuch more extensive than the clearance of CFSE-labeled RBCshad shown, as Sirpα−/− macrophages in these mice would phago-cytose not only the transferred RBCs but also endogenous RBCsconcomitantly. Based on the CFSE-RBC clearance rate, the es-timated total RBCs phagocytosed in DSS/IL-17A–treated Sirpα−/−mice should be more than 5 × 109 in 24 h (nearly 50% of pe-ripheral RBCs without considering increased erythrogenesis; Fig.2 D and E). Comparably, WT mice cleared only the transferredCd47− RBCs (∼1 × 109). Accelerated RBC clearance was alsoobserved in Cd47−/− mice under IL-17A treatment, and only to-ward Cd47− RBCs. As expected, no enhanced clearance was ob-served in Cd47−/− mice treated by DSS, as DSS induced no colitisor IL-17. Interestingly, we observed that macrophage phagocytosiscan reach a plateau. As shown, mutant mice treated with one doseof IL-17A for 1 d rapidly cleared the transferred RBCs (Fig. 2E),whereas the same mice treated with IL-17A for multiple times anda longer period (d1 and d3) displayed only weak clearance oftransferred RBCs (<5%, 24 h; SI Appendix, Fig. S5). As the latterrecipient mice had already developed acute anemia and spleno-megaly by the time RBC transfer was performed, the result sug-gests a capacity saturation of macrophages as per observationsalso stated by others (28).

Fig. 1. Inflammatory conditions induce acute anemia in mice deficient of the Cd47-Sirpα–mediated inhibition. (A) Generation of Sirpα KO mice. PCR gen-otyping shows that WT allele produces a DNA fragment of 228 bp, whereas mutated allele produces a fragment of 502 bp. Western blot (WB) confirmeddepletion of Sirpα (∼120 kDa) protein in bone marrow leukocytes from Sirpα−/− mice. (B) Inducing colitis in mice by 1% or 2% DSS. Note that the coliticprogression in Sirpα−/− mice induced by 1% DSS was comparable to that in WT induced with 2% DSS. (C and D) Acute anemia and splenomegaly developed inSirpα−/− mice with colitis. WT mice and Sirpα−/− mice treated with DSS (2% DSS for WT and 1% DSS for Sirpα−/−) for 10 d were analyzed for peritoneal cavities(C) and peripheral blood (D). (E) H&E staining of spleens from WT and Sirpα−/− mice treated with 2% and 1% DSS for 10 d. (F) Time-course anemia devel-opment in Sirpα−/− mice during DSS-induced colitis and the correlation with IL-17 in serum. (G) IL-17 neutralization ameliorates anemia and splenomegaly inSirpα−/− mice under colitis. An anti–IL-17 antibody (10 μg, i.v.) was given on days 6 and 8 (arrows) during DSS-induced colitis. (H) IL-17–containing colitic seruminduces acute anemia in Sirpα−/− mice. Serum samples collected from healthy (ctl.) and colitic WT mice (2% DSS, 10 d) were administrated to healthy Sirpα−/−

mice (i.v., 3×, arrows) with or without anti–IL-17 Ab. (I and J) IL-17A directly induces acute anemia and splenomegaly in Sirpα−/− mice and Cd47−/− mice.Healthy mice were given recombinant IL-17A (10 μg/kg, i.v.) on days 1 and 3 (arrows, J); anemia and splenomegaly were analyzed on day 5 (I) or in a timecourse manner (J). (K) Acute anemia and splenomegaly in Sirpα−/− and Cd47−/− mice under zymosan-peritonitis (3×, every other day), IL-6 administration(2×, 10 μg/kg, i.v.), LPS (0.25 mg/kg, i.p.), and CFA (1×, s.c.) administrations. Error bars are ±SEM **P < 0.01, ***P < 0.001 vs. control or the beginning timepoint. Data presented in each panel represent at least three independent experiments with n ≥ 4, if applicable.

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Ex Vivo Studies of Macrophage Phagocytosis. To understand thefunctional differences of macrophages in Sirpα−/− or Cd47−/−

mice vs. in WT mice, and how macrophages in Sirpα−/− or Cd47−/−

mice changed their phagocytic characteristics and became eryth-rophagocytic under inflammatory conditions, we examined mac-rophage phagocytosis ex vivo. Three different tissue macrophagesincluding splenic macrophages, peritoneal macrophages (PEMs),and bone marrow-derived macrophages (BMDMs) were tested.As shown in Fig. 3 A and B, consistent with in vivo studies, splenicmacrophages freshly isolated fromWTmice directly phagocytosedCd47− RBCs, whereas those from Sirpα−/− or Cd47−/− mice dis-played no phagocytosis. To our surprise, none of the PEMs orBMDMs from any mice displayed phagocytosis toward RBCs,irrespective of Cd47 expression. The fact that PEMs and BMDMsfrom WT mice failed to phagocytose even Cd47− RBCs was sur-prising, as splenic macrophages derived from the same mice haddisplayed direct phagocytosis. Further FACS analysis of WTsplenic macrophages after Cd47− RBC phagocytosis revealed thatthe phagocytic macrophages were F4/80+ red pulp macrophages,whereas other macrophages, such as F4/80−Cd169+ metallophilicmacrophages and F4/80−Cd209b+ marginal zone macrophages(1, 36, 37), displayed no phagocytosis (Fig. 3C). Testing macro-phage phagocytosis toward splenocytes obtained the same re-sults. In summary, among various macrophages tested in theseexperiments, only red pulp macrophages freshly isolated fromWT mice were phagocytic toward self-cells, whereas other splenicmacrophages, PEMs and BMDMs, and red pulp macrophagesfrom mutant mice were all incapable of phagocytosis irrespectiveof the Cd47 expression on target cells.Meanwhile, testing the same macrophages for phagocytosis

toward other targets that express the classical eat-me signalsrevealed that all macrophages, irrespective of their origins andphagocytic behavior toward self-cells, were potent phagocytestoward Escherichia coli, zymosan, apoptotic cells, antibody, orcomplement-bound targets (SI Appendix, Fig. S6).

Macrophage Phagocytic Plasticity.Despite that most macrophages,with the exception of WT red pulp macrophages, had displayedincapability to directly phagocytize self-cells, treating these mac-

rophages with LPS or IL-17 dramatically changed their phagocyticbehavior. As shown in Fig. 3D, treating splenic macrophages fromSirpα−/− or Cd47−/−mice with LPS or IL-17A rapidly induced theirphagocytosis toward RBCs. The same treatments also convertedall PEMs and BMDMs, including those previously incapable ofphagocytosis from WT and mutant mice, to potent phagocytestoward self (Fig. 3E and SI Appendix, Fig. S7). Strikingly, in allcases, whether phagocytosis occurred was governed by thepresence of Cd47-Sirpα–mediated inhibition. As shown, thetreated Sirpα+ macrophages, either from WT mice or Cd47−/−

mice, phagocytosed only Cd47− RBCs, whereas Sirpα− macro-phages from Sirpα−/− mice phagocytosed both Cd47+ RBCs andCd47− RBCs indiscriminately. Further testing with additionalcytokines found that IL-6, TNFα, and IL-1β, but not IFNγ, havethe ability to induce macrophage phagocytosis toward self, pro-viding a lack of the Cd47-Sirpα–mediated inhibition (Fig. 3E andSI Appendix, Fig. S7). Testing different cell types as the phagocytictargets found that macrophages activated by LPS or cytokineswere also capable of phagocytizing murine splenocytes, B16 mel-anoma cells, and EL4 lymphoblasts in a Cd47-Sirpα–controllablemanner (Fig. 3 F and G) and human RBCs and human HT29 co-lonic epithelial cells on which the expressed CD47 was incompatiblefor murine Sirpα (SI Appendix, Fig. S8). Moreover, we found thatthioglycollate, a reagent commonly used to elicit macrophages in theperitoneum, activates PEM phagocytosis toward RBCs (Fig. 3H),explaining the seemingly discrepant results reported by others (28).Converse to phagocytic activation, IL-10 strongly inhibits mac-

rophage phagocytosis. As shown in Fig. 4A, IL-10 dose-dependentlyinhibited LPS-, IL-17–, IL-6–, TNFα-, and IL-1β–induced acti-vation of PEM phagocytosis toward RBCs. Interestingly, WT redpulp macrophages also displayed remarkable phagocytic plasticityex vivo. As shown in Fig. 4B, these macrophages, which directlyphagocytized Cd47− RBCs immediately following isolation, com-pletely lost this capacity after 2 d of culturing, but maintainedphagocytosis toward E. coli, zymosan, apoptotic cells, and anti-body- or complement-bound targets (Fig. 4C). To test whetherthese macrophages could be revived to target RBCs, we treatedthese macrophages with LPS and IL-17A, which dramaticallyrekindled the “culture-retarded” WT spleen macrophages for

Fig. 2. Macrophage phagocytosis in vivo assessed by adoptive transfer experiments. (A–C) Clearance of adoptively transferred Cd47+ or Cd47− RBC in re-cipient mice. (A) FACS analyses of CFSE-RBCs in peripheral blood at 30 min (initial time point) and 18 h after transfer. (B) Time course clearance of CFSE-labeledRBCs. (C) The half-time (t1/2) of RBC clearance. Error bars are ±SEM. **P < 0.01, ***P < 0.001 vs. WT mice clearance of Cd47+ RBCs. (D) RBC clearance in miceunder DSS-induced colitis. Mice treated with DSS (1% for Sirpα−/− mice, 2% for WT and Cd47−/− mice) for 8 d (d8) were transferred with CFSE-RBCs followed bydetermination of RBC clearance after 24 h. FACS data of Cd47+ RBCs and Cd47− RBCs in different mice at 30 min and 24 h after transfer were selectivelyshown. Total RBC phagocytosis was calculated based on the rates of CFSE-RBC clearance and the fact that Sirpα−/− mice eliminate both Cd47− RBC and Cd47+

RBC, whereas WT mice and Cd47−/− mice eliminate only Cd47− RBCs. (E ) RBC clearance in mice treated with IL-17A. Mice treated once with IL-17A (10 μg/kg,i.v.) were transferred with CFSE-labeled Cd47+ or Cd47− RBCs a day later. Error bars are ±SEM. **P < 0.01, ***P < 0.001 vs. control or the initial time point.Data presented in each panel represent at least three independent experiments with n ≥ 4, if applicable.

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potent phagocytosis toward Cd47− RBCs. Again, this rejuvenatedphagocytosis was subject to the control of the Cd47-Sirpα mech-anism, as it completely avoided Cd47+ RBCs.Moreover, the expression of Sirpα alone appears to convey

inhibition in phagocytosis. As shown in Fig. 4D, as well as otherfigures, LPS/cytokine-treated Sirpα− macrophages consistentlydisplayed much more potent phagocytosis toward self-cells thanthe same-treated WT macrophages. Real-time recording showedthat the treated Sirpα− PEMs displayed an extraordinary capa-bility to grab and uptake RBCs, resulting in rapid phagocytosis ofmultiple RBCs in a short time period during which the same-treated WT PEMs phagocytized only one to two RBCs (SI Ap-pendix, Movies S1–S4). Analysis of Sirpα in WT macrophagesafter LPS or IL-17A treatment revealed a level of Sirpα ITIMphosphorylation and SHP-1 association even in the absence ofextracellular CD47 ligation; however, cell surface ligation byCd47+ RBCs led to further increased Sirpα ITIM phosphorylationand SHP-1 association (Fig. 4E). These results suggest that Sirpαexpression alone conveys partial inhibition, whereas stronger in-hibitory signaling triggered by Cd47 ligation is needed for effec-tively blocking macrophage phagocytosis toward self.

Signaling Mechanisms Regulating Macrophage Phagocytosis TowardSelf. Multiple pharmacological inhibitors were tested for theireffects on LPS and cytokine-induced activation of macrophagephagocytosis toward self. As shown in Fig. 5A, treating macro-

phages with inhibitors against MAP kinases, p38 (SB203580)and MEK (PD98059); Src family tyrosine kinases, PP1 and PP2;JAK 1 (JAK inhibitor I); JAK3 (JAK3 inhibitor I); phospholipaseC (PLC) (U73122); Btk (LFM-13); or NF-κB (JSH-23) diminishedphagocytic activation induced by certain, but not all stimuli. Forexample, inhibition of JAKs blocked IL-6 and IL-17, but not LPSand TNFα, for activation of PEM phagocytosis. These results werecomprehensible; different stimuli trigger distinctive downstreammolecules, especially at the proximal signaling region. In com-parison, inhibition of PI3K by LY294002 and inhibition of Syk byPiceatannol or R406 inhibited phagocytic activation by all stimuli.As shown, LY294002 (20 μM) partially inhibited PEM phago-cytic activation, whereas Syk inhibitors Piceatannol (100 μM) andR406 (400 nM) nearly completely eliminated PEM activation in-duced by all stimuli. Fig. 5B shows Piceatannol and R406 dose-dependently inhibited LPS-induced activation of PEM phagocytosistoward RBC. As shown in Fig. 5C, testing Syk kinase activity bythe phosphorylation at Y519/520 (SykPY) in PEMs found increasesin SykPY (thus Syk activity) after LPS/cytokine stimulation, sup-porting a key role of Syk in macrophage phagocytic activation.Conversely, IL-10, the phagocytic suppressive cytokine, counter-repressed Syk phosphorylation induced by activation factors. Al-though Syk was suggested to activate myosin-II (38), this appearedto not be the case in macrophage phagocytic activation; however,myosin-II was required for the later phagocytosis process in a way

Fig. 3. Assaying macrophage phagocytosis ex vivo. (A and B) Macrophage phagocytosis toward RBCs. Freshly isolated splenic macrophages (MØ) and PEMand in vitro-derived BMDM were tested for phagocytosis toward CFSE-labeled Cd47+ or Cd47− RBCs. (C) Only red pulp macrophages are RBC phagocytes.(D) Activation of splenic MØ from Sirpα−/− and Cd47−/− mice for phagocytosis toward RBCs by LPS and IL-17A. (E) Activation of PEM for phagocytosis towardRBC by LPS, IL-6, IL-1β, IL-17, and TNFα, but not IFN-γ. (F and G) LPS and IL-17A–activated PEM phagocytosis toward splenocytes (F), B16, and EL4 (G). Error barsare ±SEM. **P < 0.01, ***P < 0.001 vs. no treatment controls. (H) Thioglycollate activates PEM phagocytosis toward RBCs. PEM lavaged without (ctl.) or withBrewer thioglycollate (3%, i.p.) elicitation was tested. Error bars are ±SEM. ***P < 0.001 vs. control PEM. Data presented in each panel represent at least threeindependent experiments with n ≥ 4, if applicable.

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similar as in Fc-mediated phagocytosis (38, 39) (SI Appendix, Fig.S9). Depletion of calcium and magnesium or the presence ofEDTA hindered macrophage phagocytic activation, possiblythrough affecting macrophage adhesion or the activation of thespecific phagocytic receptor.Moreover, we found that phorbol 12-myristate 13-acetate

(PMA), the PKC activator, dramatically activates macrophagesfor phagocytosis toward self. As shown in Fig. 5D, even at lownanomolar concentrations, PMA treatment instantly initiatedPEM to phagocytose RBCs. In addition, PKC appears to beupstream of Syk in the phagocytic activation pathway (Fig. 5E).Inhibition of Syk by Piceatannol and R406 eliminated the PMA-induced PEM phagocytosis, whereas PMA treatment failed torescue Syk inhibition-repressed PEM phagocytic activation byLPS (Fig. 5F). That PKC induces Syk activation in macrophageswas consistent with previous reports (40–42). Interestingly, PMA-induced phagocytosis disregards the Cd47-Sirpα–mediatedinhibition. As shown, PMA-treated WT PEMs aggressively phago-cytosed both Cd47− and Cd47+ RBCs, a target indiscriminationsimilar to macrophages treated by pervanadate to abolish SHPsignaling (Fig. 5G). Because PMA treatment neither reducedSirpα expression nor affected Sirpα ITIM phosphorylation andSHP-1 association (Fig. 5H), this effect of PMA suggests an in-terference of the signaling pathway downstream of the Sirpα-ITIM-SHP axis.

What Is the Phagocytic Receptor on Macrophages for UptakingHealthy Self-Cells? Given that calreticulin (CRT) interactionwith LDL receptor-related protein (LRP1) has been suggested tomediate phagocytosis of apoptotic cells and viable cells (43–45),we examined these proteins. As shown in Fig. 6A, neither the in-hibitory antibodies against CRT or LRP1 (46) nor LRP1 receptor-associated protein (RAP), which inhibits CRT-LRP1 binding(43, 47, 48), showed inhibition on LPS/cytokine–activated PEMphagocytosis toward RBCs and splenocytes. However, these samereagents significantly inhibited phagocytosis toward apoptotic cells(Fig. 6B) (more data are shown in SI Appendix, Figs. S10 and S11).Although a previous study (46) had suggested that increase of CRTon the macrophage surface is associated with phagocytosis towardtumor cells, assessment of CRT and LRP1 levels on PEMs found

no convincing correlation of these protein expressions with PEMphagocytic activation. As shown in Fig. 6C, except for that LPStreatment induced an increase in CRT (consistent with ref. 46),none of the other phagocytic activation factors induced elevation ofCRT or LRP1 on PEMs. Conversely, CRT and LRP1 expressionon PEMs were even reduced (∼50%) after PMA treatment, whichpotently activates phagocytosis toward self. Protein coimmunopre-cipitation also failed to detect alteration of CRT-LRP1 association,or LRP1 tyrosine phosphorylation, in PEMs after phagocytic acti-vation, thus ruling out LRP1 to be a direct target downstream ofSyk (SI Appendix, Fig. S12). Collectively, these results suggest thatCRT and LRP1 are unlikely to be the receptor-ligand pair that isactivated by phagocytic stimuli and mediates phagocytosis towardhealthy self-cells.We also tested other known phagocytic receptors for their roles

in activated macrophage phagocytosis toward self. As shown inFig. 6D, antibody inhibition of scavenger receptor A (anti–SR-A)or Fc receptors (anti-Cd16/Cd32) (49), or inhibitors against scav-enger receptor B (BLT1) (50), dectin (laminarin) (51), and com-plement (heparin) (52) mediated phagocytosis, failed to affectLPS-induced PEM phagocytosis toward RBCs. Interestingly, an-tibody against integrin Cd11b (Cd11b/Cd18) impeded phagocy-tosis. It is possible that anti-Cd11b antibody affected macrophageadhesion and membrane spreading, through which inhibitedphagocytosis toward RBCs. The role of CD11b in phagocytosisof RBCs has also been reported for dendritic cells (53).

Different Spleen Environments in WT Mice and Mutant Mice. Fromour data, it is possible that all macrophages have the capability tophagocytize healthy self-cells; however, initiating the capacity isdependent on the presence of activating stimuli. The fact thatred pulp macrophages in WT mice, but not Sirpα−/− or Cd47−/−

mice, directly phagocytize Cd47− RBCs without requiring fur-ther activation suggests that the WT spleen provides constantstimulation supporting macrophage phagocytosis toward self,whereas the spleens in mutant mice do not. The observationsthat red pulp macrophages isolated from WT mice quickly di-minished the phagocytic capacity, which was then rekindled byextrinsic stimuli, support this notion. In addition, adoptive transferof nonphagocytic Sirpα− BMDMs into WT mice instantly induced

Fig. 4. (A) IL-10 inhibits macrophage phagocytosis toward self. PEMs were treated with LPS and activating cytokines along with various concentrations of IL-10 before testing phagocytosis toward RBCs. (B) Phagocytic plasticity of WT red pulp macrophages. The phagocytic capacity toward Cd47− RBCs displayed byfreshly isolated WT splenic macrophages was lost after 2 d (d2) of in vitro culturing. Treatments of cultured macrophages with LPS and IL-17A re-elicited theirphagocytosis toward RBCs. (C) Phagocytosis toward Cd47− RBCs, E. coli, zymosan, apoptotic cells, and antibody or complement-bound hRBCs. Error bars are±SEM. ***P < 0.001 vs. freshly isolated splenic macrophages. (D) Microscopic images of RBC phagocytosis by LPS- and IL-17A–treated splenic macrophages.(E) Sirpα ITIM phosphorylation and SHP-1 association under LPS and IL-17A treatments with or without Cd47 ligation. WT PEMs treated with LPS or IL-17Awere further treated with Cd47+ RBCs or Cd47-AP (10 min, 37 °C) followed by cell lysis, Sirpα immunoprecipitation, and WB detection of Sirpα phosphorylation(SirpαpY) and SHP-1 association. Data presented in each panel represent at least three independent experiments with n ≥ 4, if applicable.

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RBC loss (10–30%, 48 h) and splenomegaly (Fig. 7A). Cotransferwith CSFE-labeled Cd47+ RBCs confirmed that Sirpα− macro-phages mediated erythrophagocytosis in WT mice (Fig. 7B). Tissueanalyses indicated that the adoptively transferred Sirpα− macro-phages were distributed in red pulp of the WT spleen (Fig. 7C).Further analyses of spleens cells found that those from Sirpα−/−

and Cd47−/−mice produce relatively higher levels of IL-10, but lesserIL-17 and IL-6 compared with those from WT mice (Fig. 7D and SIAppendix, Fig. S13). Mutant mouse spleens were also associated witha deficit of Cd11c+ dendritic cells (DCs), especially the major mi-gratory and antigen-presenting Cd8−DCs and Cd4+ Th lymphocytes[Fig. 7E and previous reports (54, 55)]. Other leukocytes, includingnatural killer (NK) cells, NKT cells, Cd8 T cells, B cells, and thetotal Cd11b+ myeloid cells demonstrated no reduction. Interest-ingly, red pulp macrophages are even increased in Sirpα−/− andCd47−/−mice (SI Appendix, Table S1 and Fig. S14). Therefore, it ispossible that the deficiency of Cd11c+Cd8− DCs and Cd4+ Th cellsand the increase of IL-10 collectively cause inactivity of eryth-rophagocytosis in the spleens of Sirpα−/− and Cd47−/− mice. Tran-

scription profiles revealed that red pulp macrophages in Sirpα−/−mice, compared with that in WT mice, express reduced levels offunctional-stimulating molecules, such as IL-6, IL-17, IL-1β, NF-κB,and Cd40, but higher levels of suppressive molecules such as TGFβ(Fig. 7F) (more transcript data are shown in SI Appendix, Fig. S15and Tables S2 and S3). In another experiment, we examined micethat were housed under a germ-free (GF) condition and mice de-ficient of MyD88 (MyD88−/−), both being associated with a re-duction of Cd11c+Cd8−DCs and Cd4+ Th cells in the spleen as well(Fig. 7G) (56–59). These mice manifested attenuated clearance ofCd47− RBCs in RBC adoptive transfer experiments (Fig. 7H).

DiscussionThe present study reveals that multilayered mechanisms governmacrophage phagocytic behavior toward healthy self-cells. In ad-dition to the previously identified CD47-SIRPα–mediated mech-anism that prevents phagocytosis, there are additional mechanismsat the forefront level that determine macrophage propensity toeither phagocytose toward, or be restrained from, the surrounding

Fig. 5. Role of Syk in phagocytic activation. (A) Testing cell signaling inhibitors on LPS/cytokine-induced macrophage phagocytic activation. PEMs weretreated with LPS and cytokines in the presence of various inhibitors. After washing, inhibitor-free macrophages were tested for phagocytosis toward RBCs.(B) Syk inhibitors Piceatannol and R406 dose-dependently inhibited LPS-induced PEM phagocytic activation. (C) Syk activity is regulated by phagocytic stimuliand IL-10. PEMs treated with LPS and activating cytokines, or together with IL-10, were tested for total Syk and phosphorylated Syk (SykpY, specific at Y519/520).(D) PMA-induced macrophages phagocytosis toward RBCs. WT and Sirpα−/− PEMs were treated with PMA (37 °C, 30 min) before testing phagocytosis towardRBCs. (E) Depiction of PKC-Syk–mediated macrophage phagocytic activation toward self. (F) Syk is downstream of PKC. (Left) Inhibition of Syk by Piceatannoland R406 prevented PMA-induced phagocytic activation. (Right) PMA treatment failed to rescue LPS-mediated phagocytic activation-suppressed by Syk in-hibition. Error bars are ±SEM. ***P < 0.001 vs. the respective controls. (G) Inhibition of SHP by pervanadate eliminates Cd47-dependent phagocytic recognition.LPS-treated WT PEMs were further briefly treated with pervanadate before testing for phagocytosis toward RBCs. Error bars are ±SEM. ***P < 0.001 vs. therespective controls. (H) PMA treatment does not affect Sirpα expression or Cd47 ligation-induced Sirpα phosphorylation and SHP-1 association. Data presentedin each panel represent at least three independent experiments with n ≥ 4, if applicable.

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self-cells. Indeed, the CD47-SIRPα–mediated inhibition is rele-vant and indispensable only when the phagocytosis toward “self” isinitiated in a tissue environment. Remarkably, multiple inflam-matory conditions and proinflammatory cytokines/factors (e.g.,LPS, IL-1β, IL-6, IL-17, and TNFα) are found to activate mac-rophages for phagocytosis toward healthy self-cells. Conversely,IL-10 suppresses this phagocytosis.Different behavior of macrophages in WT mice vs. in Sirpα−/−

or Cd47−/− mice has long been observed. As shown in our studiesand also previously (1), WT mice rapidly eliminate Cd47− RBCthrough erythrophagocytosis, whereas Sirpα−/− or Cd47−/− micecannot. We found that the lack of phagocytic stimulation is at-tributed to the latent macrophage behavior in mutant mice, asinflammatory stimulations that activate the PKC-Syk pathwayinstantly elicit phagocytosis. In particular, these mutant mice,although generally manifesting no macrophage-mediated destruc-tion, have developed acute anemia under inflammatory conditionsdue to splenic macrophages directly phagocytizing self-RBCs. Exvivo treatments of macrophages isolated from these mice with LPSor cytokines, or transfer of the nonphagocytic Sirpα−/− macro-phages into WT mice, have instantly induced erythrophagocytosis.However, in these experiments, we failed to observe macrophagesdisplaying tolerance or “split tolerance” suggested previously byothers (11); instead, phagocytically activated macrophages derivedfrom mice without Cd47 or Sirpα expression displayed the same,direct phagocytosis toward RBCs, splenocytes, and other cells asmacrophages from WT mice, providing an absence of Cd47-Sirpαinhibition. Another similarity of macrophages from Sirpα−/−, orCd47−/−, mice and WT mice is that they all need phagocytic ac-tivation to gain direct phagocytosis toward healthy self-cells. As

shown by our data, not only macrophages from mutant micebut also PEM and BMDM from WT mice displayed phagocyticinactivity toward even Cd47-null RBCs in the absence of stim-ulation. WT red pulp macrophages, although displaying directphagocytosis toward Cd47-null RBC in vivo, were unable to main-tain this aggressiveness shortly after isolation. It is as if the “notattack-self” is a default mode for all macrophages, whereas“attack-self” represents an exceptional, hyper-phagocytic statusfor which special activating mechanisms are needed. This ideathat macrophages are generally set to not attack self-cells is con-sistent with the fact that, in most nonlymphoid tissues, suppressivecytokines IL-10 and TGFβ tend to dominate (e.g., in peritoneum)and repress phagocytosis toward self. The spleen red pulp appearsto be an exceptional tissue, constantly providing stimuli sustainingthe phagocytic capacity toward self. Lack of such a stimulating

Fig. 7. Sirpα−/− or Cd47−/−mice, but notWTmice, are deficient of macrophagestimulation in the spleen. (A–C) Monocytes/macrophages (2 × 107) derived frombone marrow cells of WT or Sirpα−/− mice were labeled with CMTMR andtransferred into three strains of recipient mice. (A) Only Sirpα−/− macrophagesin WT mice displayed phagocytosis toward RBCs, resulting in anemia andsplenomegaly. Error bars are ±SEM. *P < 0.05, **P < 0.01 vs. control bytransferring WT monocytes/macrophages into WT mice. (B) Cotransfer of CFSE-labeled Cd47+ RBCs along with Sirpα−/− monocyte/macrophages into WT miceconfirmed rapid RBC clearance. Error bars are ±SEM. ***P < 0.001 vs. the initialtime point. (C) Distribution of CMTMR-labeled Sirpα−/− macrophages in spleenred pulp (RP) but not white pulp (WP). (D) Higher levels of IL-10 and lesser IL-17produced by spleen cells and in serum from Sirpα−/− and Cd47−/− mice. (E)Decreases in Cd11c+Cd8− DC and Cd4+ helper (Th) lymphocytes in the spleen ofSirpα−/− and Cd47−/− mice. (F) Transcription profiling of red pulp macrophagesfromWT and Sirpα−/− mice. F4/80+ red pulp macrophages were affinity isolatedbefore mRNA isolation and profiling. The red-colored molecules (most beingactivating) are expressed at higher levels in WT than in Sirpα−/− red pulpmacrophages, whereas the blue-coloredmolecules (most being suppressive) areexpressed oppositely. (G) Reduction of Cd11c+Cd8− DCs in the spleens ofMyD88−/−mice and germ-free (GF)–conditioned mice. (H) Attenuated clearanceof Cd47− RBC in MyD88−/− mice and GF mice. Data presented in each panelrepresent at least three independent experiments with n ≥ 4, if applicable.

Fig. 6. (A) Blocking CRT or LRP1 failed to inhibit LPS/cytokine-induced PEMphagocytosis toward RBCs. LPS/cytokine-activated PEM phagocytosis towardRBCs was tested in the presence of anti-CRT antibody 1 (Abcam) and 2 (CST)(10 μg/mL each), anti-LRP1 (20 μg/mL), and the LRP1 receptor-associatedprotein (RAP; 20 μg/mL), all dialyzed free of sodium azide (note: sodiumazide potently inhibits phagocytosis toward self-cells even at low concen-trations). (B) CRT-LRP1 controls macrophage phagocytosis toward apoptoticcells. PEM (unstimulated) phagocytosis toward apoptotic B16 cells was testedin the presence of same antibodies and RAP as in A. Error bars are ±SEM.***P < 0.001 vs. phagocytosis in the presence of control IgG. (C) Macrophagecell surface CRT or LRP1. PEMs, with or without (ctl.) treatment with LPS,IL-17A, IL-6, or PMA, were labeled for cell surface LRP1 and CRT followed byFACS. Increased and decreased expressions were marked by arrows. (D) Ex-ploring other phagocytic receptors. Activated PEM phagocytosis toward RBCswas tested in the presence of antibodies against SR-A, Fc receptor Cd16/32(10 μg/mL of each), inhibitors against SR-B (BLT1, 5 μM), dectin (laminarin,100 μg/mL), complement (heparin, 40 U/mL), and antibody against Cd11b(10 μg/mL). Error bars are ±SEM. ***P < 0.001 vs. the respective controlwithout inhibition. More data can be seen in SI Appendix, Figs. S10 and S11.Data presented in each panel represent at least three independent experi-ments with n ≥ 4, if applicable.

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environment in the spleen becomes a “compensation” mechanismthat maintains Sirpα−/− or Cd47−/− mice to be healthy despite theabsence of Cd47-Sirpα–mediated inhibition.Although the CD47-SIRPα mechanism may be dispensable under

normal conditions, it becomes extremely important under in-flammatory conditions and infection, during which host macrophageswould gain phagocytosis toward healthy self-cells (as suggested by thisstudy). The finding that Sirpα−/− and Cd47−/− mice rapidly developanemia under inflammatory challenges suggests that lack of theseproteins may significantly reduce the threshold for anemia under in-flammatory conditions. Inflammation-associated anemia (also called“anemia of inflammation” or “anemia of chronic disease”) is amongthe most frequent complications observed in hospitalized patients.Reported previously by us and others (60, 61), SIRPα expression inmacrophages is decreased following LPS stimulation, suggesting adynamic nature for the CD47-SIRPα–mediated inhibition especiallyon infection or activation of TLR. The expression of CD47 on cellscan also be changed under different conditions (62–64). Also repor-ted by us, alteration of clustering structures of SIRPα onmacrophagesor CD47 on tissue cells affects phagocytosis (65, 66). Moreover, datapresented in this study show that CD47-SIRPα–mediated inhibi-tion controls not only the phagocytic target selection, but also thephagocytic robustness once the target has been chosen. As shown inthis study, SIRPα− macrophages, compared with SIRPα+ macro-phages, are much more potent in phagocytosis toward self-cells oncethey have been activated by LPS or cytokines. Although both mutantstrains are deficient of CD47-SIRPα inhibition, more severe anemiaensued in Sirpα−/− animals than in Cd47−/− mice after their splenicmacrophages were stimulated. Even without stimulation, the macro-phages in Sirpα−/− mice were faster to clear RBCs than those in WTor Cd47−/− mice. All these results suggest a SIRPα-ITIM–mediatedinhibition on general cell processes of macrophage phagocytosisand are in concurrence with reports showing that the CD47-SIRPαpathway also tempers Fc- and complement-mediated phagocytosis(19–21), as well as phagocytosis toward apoptotic cells (65).The detailed mechanism by which macrophages directly phago-

cytize healthy self-cells is unknown, despite that the mechanismthat inhibits this phagocytosis via the CD47-SIRPα-SHP axis hasbeen studied. Different from traditional phagocytosis aiming atalien pathogens, immune complexes, debris, and dying self-cells, onwhich certain eat-me or non-self signals ensue phagocytosis,phagocytosis toward healthy self-cells is uncustomary. To date, themolecules serving as the phagocytic ligands on healthy self-cells,together with the phagocytic receptor on macrophages, remainundefined. From the present study, it can be predicted that thespecific phagocytic receptor on macrophages is either unexpressedor expressed but maintains inactivity until stimulation-induced ac-tivation occurs. The rapid elicitation of phagocytosis by PMA (<30min) suggests the latter and that the PKC-Syk-mediated signalingpathway likely activates this phagocytic receptor through an “inside-out” mechanism. Along this line, it is possible that the CD47-SIRPα–mediated SHP activity inhibits this phagocytic receptorthrough protein dephosphorylation that counters the effect by Syk.The study has ruled out CRT-activated LRP1 and other knownphagocytic receptors for mediating phagocytosis toward healthyself-cells. In particular, our data show that the CRT-LRP systemcontrols phagocytosis toward apoptotic, but not healthy, self-cells.Interestingly, antibody inhibition of CD11b/CD18 impedes acti-vated macrophage phagocytosis toward self; however, further stud-ies are required to delineate if CD11b/CD18 acts as the specificphagocytic receptor or functions as an integrin essential forphagocytosis. Additional studies are also needed to define the

mechanism that controls the receptor-mediated internalization ofhealthy self-cells after the phagocytic recognition.

Materials and MethodsMice. All experiments using animals and procedures of animal care andhandling were carried out following protocols approved by the InstitutionalAnimal Care and Use Committee (IACUC) of Georgia State University. WT(C57BL/6J) and Cd47−/− (B6.129-CD47tm1Fpl/J) mice were from The JacksonLaboratory. Sirpα KO (Sirpα−/−) was established by replacing the exons 2–4and their flanking regions with a neomycin resistant cassette in the Sirpαgene (collaboration with Chen Dong at the facility of University of Texas MDAnderson Cancer Center, Houston). All mice were housed in an SPF facility.DSS-induced colitis and zymosan-induced peritonitis were described previously(32–34). Mouse peripheral blood hemoglobin levels were determined by lysisof 10 μL whole blood in 1 mL water followed by OD reading at 540 nm.

Adoptive Transfer Experiments. Cd47+ RBCs and Cd47− RBCs were freshly col-lected from WT and Cd47−/− mice, respectively. After labeling with CFSE, ∼109

RBCs in 150 μL PBS were transfused (i.v.) into recipient mice. Blood sampleswere collected 30 min after blood transfusion, and percentages of CFSE-labeledRBCs were determined by FACS. Blood samples were then collected at latertime points to assess the CFSE-RBC clearance. For splenocyte-transfer experi-ments, splenocytes (2 × 107) labeled by CFSE were transferred into recipientmice followed by determination of clearance in peripheral blood and thespleen by FACS. For monocyte/macrophage-transfer experiments, bone marrowcells were cultured in M-CSF (10 ng/mL) for 3–4 d to produce monocytes/macrophages, which were then labeled with 5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR) and transferred into recipient mice.

Macrophage Phagocytosis Assay. Splenic macrophages were obtained fromcollagenase D- and DNase I-digested spleen tissues and were enriched byplating at 37 °C for 2 h. Resident PEMs were obtained by directly lavaging theperitoneum using sterile PBS. Thioglycollate-eliciteds PEM were prepared byi.p. injection of 1 mL 3% (wt/vol) Brewer thioglycollate, followed by lavage 4 dlater. BMDMs were prepared by culturing freshly isolated bone marrow cells inthe presence of M-CSF (10 ng/mL) for 5–6 d (66). To test phagocytosis ex vivo,freshly isolated/prepared macrophages in 24-well plates were incubated withCSFE-labeled RBCs or other cells for 30 min (37 °C) in the absence or presenceof inhibitory antibodies or reagents. After washing, macrophage phagocytosiswas analyzed microscopically or by FACS. To induce macrophage phagocyticactivation, macrophages were treated with LPS (20 ng/mL), TNF-α (20 ng/mL),and IL-1β (10 ng/mL) for 6–10 h; IL-6 (10 ng/mL), IL-17A (10 ng/mL), and IFN-γ(100 U/mL) for 12–24 h; or PMA (5-20 nM) for 30 min (all at 37 °C) beforephagocytosis assays. Phagocytic activation was also induced in the presence ofpharmacological inhibitors or varied concentrations of IL-10 to test their in-hibitory effects. Phagocytic indexes were expressed by the number of mac-rophages that ingested at least one target in 100 macrophages analyzed.

Transcript Microarray Profiling. Red pulp microphages isolated by F4/80+ se-lection were used for mRNA isolation. Microarrays were performed usingwhole Mouse Genome Microarray 4X44K v2 (Agilent Technologies). Briefly,labeled cRNA was synthesized using the Quick Amp Labeling kit followed byhybridization using Agilent SureHyb. Slides were scanned by an Agilent DNAMicroarray Scanner, and the images were analyzed using Feature Extractionsoftware 11.0.0.1 with a background correction. Normalization was carriedout using GeneSpring GX 12.1 software.

Statistical Analysis. At least three independent experiments were performedfor each set of data, which were presented as the mean ± SEM. Statisticalsignificance was assessed by Student t test for paired samples or one-wayANOVA for the group number (k) > 2.

ACKNOWLEDGMENTS. We thank Dr. Chen Dong (MD Anderson CancerCenter) for help in establishing the SIRPα KO mice, Dr. Jill Littrell for criticalcomments, and the Georgia State University Animal Resources Program forfacilitating animal experiments. This work was supported, in part, by grantsfrom National Institutes of Health (AI106839) and the American Cancer Soci-ety (to Y.L.), the China Postdoctoral Science Foundation (2014M550284), anda fellowship from the American Heart Association (15POST22810008) (to Z.B.).

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Bian et al. PNAS | Published online August 30, 2016 | E5443

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