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Home , Inflammation, Macrophage, Phagocytosis, Splenocyte

Cd47-Sirpα interaction and IL-10 constrain -induced of healthy self-cells

Zhen 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 and , 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 and Immunology, Yong Loo Lin School of Medicine, Life Science Institute (LSI) Immunology Programme, National University of Singapore, Singapore 117456; dCenter for Inflammation, and , Georgia State University, Atlanta, GA 30303; and eDepartment 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 in The CD47-SIRPα mechanism was first reported by Oldenborg congeneic WT mice suggests a critical self-recognition mechanism, et al. (1), who had demonstrated in red cell (RBC) in which CD47 is the ubiquitous marker of self, and its interaction transfusion experiments that WT mice rapidly eliminate syngeneic − with macrophage signal regulatory α (SIRPα) triggers inhib- Cd47-null (Cd47 ) RBCs through erythrophagocytosis in the itory signaling through SIRPα cytoplasmic immunoreceptor tyrosine- and that the lack of tyrosine phosphorylation in SIRPα based inhibition motifs and tyrosine phosphatase SHP-1/2. However, ITIMs was associated with this macrophage aggressiveness. Later, − − instead of displaying self-destruction phenotypes, Cd47 / mice man- adoptive transfer/allograft experiments by others further demon- ifest no, or only mild, macrophage phagocytosis toward self-cells strated phagocytic clearance of , , hematopoi- except under the nonobese diabetic background. Studying our etic cells, and in the absence of CD47-SIRPα–mediated − − − − recently established Sirpα-KO (Sirpα / ) mice, as well as Cd47 / inhibition (9–11). In all of these cases, the phagocytosis occurred mice, we reveal additional activation and inhibitory mechanisms swiftly and completely eliminated donor cells in less than 24 h, besides the CD47-SIRPα axis dominantly controlling macrophage be- without facilitation by or immune complexes. Inclination −/− −/− havior. Sirpα mice and Cd47 mice, although being normally that this phagocytosis differed from that which is mediated by an- − − healthy, develop severe anemia and splenomegaly under chronic co- tibodies or complements was supported by using Rag-1 / mice and − − litis, , treatments, and CFA-/LPS-induced inflam- C3 / mice, in which lack of lymphocytes or complements had not − mation, owing to splenic phagocytizing self-red blood hindered elimination of Cd47 RBCs(1,10).Inlinewiththeroleof cells. Ex vivo phagocytosis assays confirmed general inactivity of the CD47-SIRPα interaction in inhibiting macrophage phagocyto- α−/− −/− macrophages from Sirp or Cd47 mice toward healthy self- sis, studies found that tumor cells are commonly associated cells, whereas they aggressively attack toward , zymosan, with increases in CD47 expression as a way to evade immunological apoptotic, and -bound cells; however, treating these eradication (12–15), whereas perturbation of CD47-SIRPα inter- β α γ macrophages with IL-17, LPS, IL-6, IL-1 ,andTNF , but not IFN , action provides opportunities for cancer eradication, especially in dramatically initiates potent phagocytosis toward self-cells, for which only the Cd47-Sirpα interaction restrains. Even for macrophages from − − Significance WT mice, phagocytosis toward Cd47 / cells does not occur without phagocytic activation. Mechanistic studies suggest a PKC-Syk–medi- ated signaling pathway, to which IL-10 conversely inhibits, is required The present study reveals that macrophage phagocytosis to- for activating macrophage self-targeting, followed by phagocytosis ward healthy self-cells is controlled by a two-tier mechanism: a independent of . Moreover, we identified spleen red forefront activation mechanism requiring the inflammatory to be one specific tissue that provides stimuli constantly activating cytokine-stimulated protein C (PKC)-spleen tyrosine ki- − − − − macrophage phagocytosis albeit lacking in Cd47 / or Sirpα / mice. nase (Syk) pathway, to which IL-10 conversely regulates, and the subsequent self-target discrimination mechanism controlled by α α – phagocytosis | macrophage | CD47 | SIRPα | cytokine the CD47-signal regulatory protein (SIRP ) mediated inhibi- tion. The findings significantly expand our understanding of macrophage phagocytic plasticity and behavior under different o maintain tissue integrity and , tissue macro- conditions and also provide insights into strategies for enhancing Tphages and other immunological must be pre- transplantation tolerance and macrophage-based cancer eradica- vented from phagocytizing healthy self-cells. One essential tion, especially for toward which therapeutic antibodies mechanism that prevents macrophages from doing so is CD47- are yet unavailable. signal regulatory protein α (SIRPα) interaction-mediated in- hibition (1–4). In this mechanism, CD47, a broadly expressed cell Author contributions: K.Z. and Y.L. designed research; Z.B., L.S., L.L., and Z.Z. performed surface protein that acts as a marker of self, along with SIRPα, research; 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 new the counter receptor of CD47 expressed on macrophages and reagents/analytic tools; Z.B., L.S., Z.Z., and Y.L. analyzed data; and Z.B., K.Z., and Y.L. wrote the paper. other phagocytic leukocytes, serve as an inhibitory signaling The authors declare no conflict of interest. regulator (5–7). On CD47 extracellular ligation, SIRPα increases tyrosine phosphorylation in the cytoplasmic domain immuno- This article is a PNAS Direct Submission. Data deposition: The complete transcript profiling data of macrophages from − − receptor tyrosine-based inhibition motifs (ITIMs), leading to ac- Sirpα / mice and WT mice have been deposited in the Gene Expression Omnibus (GEO) tivation of the SH2-containing tyrosine phosphatase (SHP-1/2), database, www.ncbi.nlm.nih.gov/geo (accession no. GSE78191). which then mediates inhibitory signaling events through protein 1To whom correspondence should be addressed. Email: [email protected]. α dephosphorylation. The end result of this CD47-SIRP -SHP This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. signaling is inhibition of phagocytosis toward self-cells (8). 1073/pnas.1521069113/-/DCSupplemental.

E5434–E5443 | PNAS | Published online August 30, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1521069113 Downloaded by guest on September 26, 2021 conjunction with therapeutic anticancer antibodies (4, 9, 16–21). However, when inducing with low-dose dextran sodium PNAS PLUS − − Conversely, the lack of a compatible CD47 to ligate the recipient sulfate (DSS, 1–2%), Sirpα / mice displayed not only severe SIRPα is considered to be associated with tissue rejection in colitis but also anemia; the latter was associated with en- (22–25). hanced macrophage erythrophagocytosis in the spleen. As shown Although these studies are exciting, major obstacles still remain in Fig. 1B (SI , Fig. S1), compared with WT littermates, − − and hinder further understandings of the CD47-SIRPα mecha- Sirpα / mice developed more severe colitis, demonstrating faster nism, as well as how to control macrophage behavior in body weight loss, worse diarrhea/clinical scores, and enhanced therapies. Resolution of at least two puzzles remains paramount: polymorphonuclear leukocytes (PMN) infiltration into intestines first, why macrophages do not attack self in mice deficient of Cd47 under DSS treatment. Given that Sirpα-mediated inhibitory sig- or Sirpα? Despite that the Cd47-Sirpα–mediated inhibition is naling negatively regulates leukocyte inflammatory response, it missing, mice deficient of Cd47 (26), Sirpα (established in this was not surprising that Sirpα deficiency exacerbates DSS-induced study), or the Sirpα cytoplasmic domain (27) are relatively healthy, colitis. Similar results have been observed in our previous study manifesting no or only minor phenotypes (9, 18, 28) that suggest using mice with truncated Sirpα cytoplasmic domain (32). Strik- −/− macrophage phagocytosis toward self-cells. Adoptive transfer of ingly, colitic Sirpα mice, but not WT mice, had also developed Cd47-null RBCs into these mutant mice also failed to induce significant splenomegaly and acute anemia (Fig. 1C). Pale-colored −/− rapid elimination (1) (also shown in this study). A case of Cd47 abdominal cavities were seen in colitic Sirpα mice (Fig. 1C), deficiency-associated lethal anemia in nonobese diabetic (NOD) which were confirmed as anemia by peripheral re- mice had been reported, but this is associated with duction (40–50%; Fig. 1D). of their enlarged and increased binding to RBC-mediated elimination revealed an extensive expansion of red pulp, to a point that had − − (20). Having worked with WT and Cd47 / hematopoietic chi- disrupted the structure. Spleen hemoglobin assays mera, Wang et al. (11) suggested that Cd47 expression on confirmed splenomegaly associated with increased RBC trapping, nonhematopoietic cells is required for macrophages to develop suggestive of aggressive erythrophagocytosis by red pulp macro- phages (Fig. 1E). Together, these results suggest that active colitis self-discrimination and that the lack of Cd47 expression in − − − − α / Cd47 / mice confers macrophage tolerance. However, as the induces macrophage-mediated RBC destruction in Sirp mice. Further analyses revealed that the anemia developed in colitic study had shown that this “trained” tolerance is not applied to − − − α / F Cd47 RBCs (11), it thus has not explained why mice deficient Sirp mice was associated with IL-17. As shown in Fig. 1 , the α anemia progressed slowly at the initial phase during colitis but of Cd47 or Sirp display incapability to clear endogenous or – transferred RBCs. Hence, is there another mechanism(s) besides was abruptly aggravated after days 7 8 when IL-17 started to CD47-SIRPα that controls macrophage phagocytosis toward self? arise in the serum. As demonstrated by us previously, IL-17 is Second, the CD47-SIRPα mechanism serves as a “safety valve” highly induced at the postacute/chronic phase of DSS-induced colitis (33, 34). To test whether IL-17 played a role, we performed against undesirable phagocytosis only when macrophages are ini- α−/− tiated to phagocytose toward healthy self-cells. Unlike macrophage three experiments. First, Sirp mice under DSS-induced colitis were given an anti–IL-17 neutralization antibody. As shown in phagocytosis toward other targets, such as microbial , Fig. 1G, giving anti–IL-17 antibody on day 6 and 8 when IL-17 immune complex-labeled targets, or apoptotic cells, on which arises during DSS-induced colitis largely ameliorated anemia specific “eat-me” signals are displayed and induce phagocytosis − − − − and splenomegaly in Sirpα / mice. Second, healthy Sirpα / mice INFLAMMATION (29–31), healthy self-cells usually do not attract macrophage IMMUNOLOGY AND were administrated with colitis serum samples that contained high attacking. The CD47-SIRPα mechanism alone, however, provides levels of IL-17. As shown in Fig. 1H, injections of colitis serum no explanation for how and when macrophages are triggered to −/− into Sirpα mice directly induced acute anemia. The effect was attack healthy self-cells, nor does it describe how this phagocytic confirmed to be specific as control serum from healthy mice or process is carried out. Thus, the core question remains: what is colitis serum mixed with anti–IL-17 antibody failed to ane- the mechanism that initiates macrophages to phagocytose healthy mia. Third, a recombinant IL-17A was administrated into healthy self-cells? α−/− I J α α−/− Sirp mice.AsshowninFig.1, and , injections of IL-17A alone In this study, we established a of Sirp KO (Sirp ) (2×,10μg/kg, i.v.) directly induced acute anemia and splenomegaly mice. Further characterization of macrophage phagocytosis in −/− − − − − in Sirpα mice (SI Appendix,Fig.S2). Notably, IL-17 injection α / / − − Sirp and Cd47 mice found that, although these mutant also induced acute anemia and splenomegaly in Cd47 / mice. As − − mice generally display no defects, acute anemia associated with reported previously, Cd47 / mice are resistant to low-dose DSS- potent macrophage phagocytosis toward self-RBCs exhibits un- induced colitis and are defective for IL-17 induction in vivo (34, 35). der inflammatory conditions or can be induced by treatment with This explains why low-dose DSS treatment does not induce anemia − − inflammatory factors. Additional ex vivo and in vivo studies col- in Cd47 / mice, as anemia is secondary to the colitic condition and lectively suggest that macrophage phagocytosis toward self is dy- colitis-induced IL-17 (SI Appendix,Fig.S3). namically controlled by concomitant, yet critical, mechanisms that In addition to colitis, other inflammatory conditions and cy- determine macrophage phagocytic activation; it is governed by tokines, such as zymosan-induced peritonitis, LPS, and Freund’s proinflammatory and antiinflammatory tissue environments complete adjuvant (CFA)-induced inflammation, also induced α– while coupled with subsequent target selection via CD47-SIRP anemia and splenomegaly in mice lacking Sirpα or Cd47. As mediated inhibition. shown in Fig. 1K, repetitively inducing peritonitis by zymosan (3×, every other day) led to severe anemia and splenomegaly in − − − − Results Sirpα / and Cd47 / mice, albeit this condition, per se, is short Acute Anemia in Mice Deficient of Cd47-Sirpα–Mediated Inhibition termed and self-resolving. Administration of IL-6 (2×,10μg/kg, i.v.), −/− Under Inflammatory Conditions. A strain of Sirpα KO (Sirpα ) the signature cytokine associated with zymosan-induced perito- mice was established by targeted inactivation of the Sirpα gene in nitis, also induced the same result. Injection of low-dose LPS − − − − embryonic cells (Fig. 1A). PCR genotyping and immunoblot (0.25 mg/kg, i.p.), or CFA (s.c.), in Sirpα / and Cd47 / mice [Western blot (WB)] confirmed disruption of the Sirpα gene and − − induced anemia and splenomegaly as well. depletion of Sirpα protein expression. Similar to Cd47 / mice, − − Sirpα / mice appeared healthy under the standard specific path- In Vivo Assessment of Macrophage Phagocytosis by Adoptive ogen-free (SPF) housing conditions, having displayed no tissue/ Transfer. RBC adoptive transfer experiments were performed + organ damage suggestive of enhanced macrophage phagocytosis to further assess macrophage phagocytosis in vivo. Cd47 or − − − toward self. Cd47 RBCs isolated from WT or Cd47 / mice, respectively,

Bian et al. PNAS | Published online August 30, 2016 | E5435 Downloaded by guest on September 26, 2021 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) confirmed − − depletion of Sirpα (∼120 kDa) protein in marrow leukocytes from Sirpα / mice. (B) Inducing colitis in mice by 1% or 2% DSS. Note that the colitic progression 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 in Sirpα−/− 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 in − − Sirpα / 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 serum − − − − induces 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 time course manner (J). (K) Acute anemia and splenomegaly in Sirpα−/− and Cd47−/− mice under zymosan-peritonitis (3×, every other day), IL-6 administration (2×,10μg/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 time point. Data presented in each panel represent at least three independent experiments with n ≥ 4, if applicable.

were labeled with carboxyfluorescein succinimidyl ester (CFSE), the Cd47-Sirpα mechanism. Following this line, the RBC clear- − − followed by transfer into different recipient mice and assessment of ance in Sirpα / mice under DSS or IL-17A treatment was indeed their clearance. As shown in Fig. 2 A–C, WT recipients swiftly much more extensive than the clearance of CFSE-labeled RBCs − + − − cleared Cd47 RBCs in a few hours (t1/2 < 5 h), but retained Cd47 had shown, as Sirpα / macrophages in these mice would phago- RBC for over a month (t1/2 ∼ 3 wk), a time length matching the cytose not only the transferred RBCs but also endogenous RBCs normal life span of RBCs in the circulation. This result is consis- concomitantly. Based on the CFSE-RBC clearance rate, the es- − − tent with a previous report (1), suggesting the absence of CD47- timated total RBCs phagocytosed in DSS/IL-17A–treated Sirpα / SIRPα–mediated inhibition is associated with erythrophagocytosis mice should be more than 5 × 109 in 24 h (nearly 50% of pe- in the spleen. However, this manner of rapid clearance did not ripheral RBCs without considering increased erythrogenesis; Fig. − − − − + occur in Sirpα / or Cd47 / mice, which retained both Cd47 2 D and E). Comparably, WT mice cleared only the transferred − − RBCs and Cd47 RBCs for extended time periods. Adoptive Cd47 RBCs (∼1 × 109). Accelerated RBC clearance was also − − transfer of splenocytes produced the same results; the only rapid observed in Cd47 / mice under IL-17A treatment, and only to- − − clearance found was in WT recipients to which Cd47 splenocytes ward Cd47 RBCs. As expected, no enhanced clearance was ob- − − were transferred (SI Appendix,Fig.S4). Thus, different from WT served in Cd47 / mice treated by DSS, as DSS induced no colitis − − − − mice, Sirpα / and Cd47 / mice do not eliminate RBCs because or IL-17. Interestingly, we observed that macrophage phagocytosis of merely missing the CD47-SIRPα–mediated inhibition. can reach a plateau. As shown, mutant mice treated with one dose − − − − However, treating Sirpα / and Cd47 / mice with IL-17, or of IL-17A for 1 d rapidly cleared the transferred RBCs (Fig. 2E), inducing inflammatory conditions in these mice, instantly in- whereas the same mice treated with IL-17A for multiple times and duced accelerated RBC clearance. As shown in Fig. 2, D and E, a longer period (d1 and d3) displayed only weak clearance of − − Sirpα / mice with 1% DSS for 8 d (when IL-17 started to arise) transferred RBCs (<5%, 24 h; SI Appendix,Fig.S5). As the latter or who were directly injected with IL-17A displayed rapid clear- recipient mice had already developed acute anemia and spleno- + − ance of transferred Cd47 and Cd47 RBCs (∼50%, 24 h). That megaly by the time RBC transfer was performed, the result sug- − − + − Sirpα / mice cleared both Cd47 and Cd47 RBCs was predict- gests a capacity saturation of macrophages as per observations able, as macrophages in these mice experience no inhibition by also stated by others (28).

E5436 | www.pnas.org/cgi/doi/10.1073/pnas.1521069113 Bian et al. Downloaded by guest on September 26, 2021 PNAS PLUS

+ − 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-labeled + RBCs. (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 mice under 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 by determination 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 selectively − − − + shown. 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.

Ex Vivo Studies of Macrophage Phagocytosis. To understand the rophages with LPS or IL-17 dramatically changed their phagocytic − − − − functional differences of macrophages in Sirpα / or Cd47 / behavior. As shown in Fig. 3D, treating splenic macrophages from − − − − − − − − mice vs. in WT mice, and how macrophages in Sirpα / or Cd47 / Sirpα / or Cd47 / mice with LPS or IL-17A rapidly induced their mice changed their phagocytic characteristics and became eryth- phagocytosis toward RBCs. The same treatments also converted rophagocytic under inflammatory conditions, we examined mac- all PEMs and BMDMs, including those previously incapable of rophage phagocytosis ex vivo. Three different tissue macrophages phagocytosis from WT and mutant mice, to potent phagocytes

including splenic macrophages, peritoneal macrophages (PEMs), toward self (Fig. 3E and SI Appendix, Fig. S7). Strikingly, in all INFLAMMATION and -derived macrophages (BMDMs) were tested. cases, whether phagocytosis occurred was governed by the IMMUNOLOGY AND As shown in Fig. 3 A and B, consistent with in vivo studies, splenic presence of Cd47-Sirpα–mediated inhibition. As shown, the + − − macrophages freshly isolated from WT mice directly phagocytosed treated Sirpα macrophages, either from WT mice or Cd47 / − − − − − − − Cd47 RBCs, whereas those from Sirpα / or Cd47 / mice dis- mice, phagocytosed only Cd47 RBCs, whereas Sirpα macro- − − + played no phagocytosis. To our surprise, none of the PEMs or phages from Sirpα / mice phagocytosed both Cd47 RBCs and − BMDMs from any mice displayed phagocytosis toward RBCs, Cd47 RBCs indiscriminately. Further testing with additional irrespective of Cd47 expression. The fact that PEMs and BMDMs found that IL-6, TNFα, and IL-1β, but not IFNγ, have − from WT mice failed to phagocytose even Cd47 RBCs was sur- the ability to induce macrophage phagocytosis toward self, pro- prising, as splenic macrophages derived from the same mice had viding a lack of the Cd47-Sirpα–mediated inhibition (Fig. 3E and displayed direct phagocytosis. Further FACS analysis of WT SI Appendix, Fig. S7). Testing different cell types as the phagocytic − splenic macrophages after Cd47 RBC phagocytosis revealed that targets found that macrophages activated by LPS or cytokines + the phagocytic macrophages were F4/80 red pulp macrophages, were also capable of phagocytizing murine splenocytes, B16 mel- − + whereas other macrophages, such as F4/80 Cd169 metallophilic anoma cells, and EL4 lymphoblasts in a Cd47-Sirpα–controllable − + macrophages and F4/80 Cd209b marginal zone macrophages manner (Fig. 3 F and G) and human RBCs and human HT29 co- (1, 36, 37), displayed no phagocytosis (Fig. 3C). Testing macro- lonic epithelial cells on which the expressed CD47 was incompatible phage phagocytosis toward splenocytes obtained the same re- for murine Sirpα (SI Appendix,Fig.S8). Moreover, we found that sults. In summary, among various macrophages tested in these thioglycollate, a reagent commonly used to elicit macrophages in the experiments, only red pulp macrophages freshly isolated from , activates PEM phagocytosis toward RBCs (Fig. 3H), WT mice were phagocytic toward self-cells, whereas other splenic explaining the seemingly discrepant results reported by others (28). macrophages, PEMs and BMDMs, and red pulp macrophages Converse to phagocytic activation, IL-10 strongly inhibits mac- from mutant mice were all incapable of phagocytosis irrespective rophage phagocytosis. As shown in Fig. 4A, IL-10 dose-dependently of the Cd47 expression on target cells. inhibited LPS-, IL-17–, IL-6–, TNFα-, and IL-1β–induced acti- Meanwhile, testing the same macrophages for phagocytosis vation of PEM phagocytosis toward RBCs. Interestingly, WT red toward other targets that express the classical eat-me signals pulp macrophages also displayed remarkable phagocytic plasticity revealed that all macrophages, irrespective of their origins and ex vivo. As shown in Fig. 4B, these macrophages, which directly − phagocytic behavior toward self-cells, were potent phagocytes phagocytized Cd47 RBCs immediately following isolation, com- toward , zymosan, apoptotic cells, antibody, or pletely lost this capacity after 2 d of culturing, but maintained complement-bound targets (SI Appendix, Fig. S6). phagocytosis toward E. coli, zymosan, apoptotic cells, and anti- body- or complement-bound targets (Fig. 4C). To test whether Macrophage Phagocytic Plasticity. Despite that most macrophages, these macrophages could be revived to target RBCs, we treated with the exception of WT red pulp macrophages, had displayed these macrophages with LPS and IL-17A, which dramatically incapability to directly phagocytize self-cells, treating these mac- rekindled the “culture-retarded” WT spleen macrophages for

Bian et al. PNAS | Published online August 30, 2016 | E5437 Downloaded by guest on September 26, 2021 Fig. 3. Assaying macrophage phagocytosis ex vivo. (A and B) Macrophage phagocytosis toward RBCs. Freshly isolated splenic macrophages (MØ) and PEM + − and 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 toward RBC 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 bars are ±SEM. **P < 0.01, ***P < 0.001 vs. no treatment controls. (H) Thioglycollate activates PEM phagocytosis toward RBCs. PEM lavaged without (ctl.) or with Brewer 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 three independent experiments with n ≥ 4, if applicable.

− potent phagocytosis toward Cd47 RBCs. Again, this rejuvenated phages with inhibitors against MAP , p38 (SB203580) phagocytosis was subject to the control of the Cd47-Sirpα mech- and MEK (PD98059); Src family tyrosine kinases, PP1 and PP2; + anism, as it completely avoided Cd47 RBCs. JAK 1 (JAK inhibitor I); JAK3 (JAK3 inhibitor I); phospholipase Moreover, the expression of Sirpα alone appears to convey C (PLC) (U73122); Btk (LFM-13); or NF-κB (JSH-23) diminished inhibition in phagocytosis. As shown in Fig. 4D, as well as other − phagocytic activation induced by certain, but not all stimuli. For figures, LPS/cytokine-treated Sirpα macrophages consistently example, inhibition of JAKs blocked IL-6 and IL-17, but not LPS displayed much more potent phagocytosis toward self-cells than and TNFα, for activation of PEM phagocytosis. These results were the same-treated WT macrophages. Real-time recording showed comprehensible; different stimuli trigger distinctive downstream α− that the treated Sirp PEMs displayed an extraordinary capa- molecules, especially at the proximal signaling region. In com- bility to grab and uptake RBCs, resulting in rapid phagocytosis of parison, inhibition of PI3K by LY294002 and inhibition of Syk by multiple RBCs in a short time period during which the same- Piceatannol or R406 inhibited phagocytic activation by all stimuli. treated WT PEMs phagocytized only one to two RBCs (SI Ap- μ pendix – α As shown, LY294002 (20 M) partially inhibited PEM phago- , Movies S1 S4). Analysis of Sirp in WT macrophages cytic activation, whereas Syk inhibitors Piceatannol (100 μM) and after LPS or IL-17A treatment revealed a level of Sirpα ITIM R406 (400 nM) nearly completely eliminated PEM activation in- phosphorylation and SHP-1 association even in the absence of duced by all stimuli. Fig. 5B shows Piceatannol and R406 dose- extracellular CD47 ligation; however, cell surface ligation by + α dependently inhibited LPS-induced activation of PEM phagocytosis Cd47 RBCs led to further increased Sirp ITIM phosphorylation C and SHP-1 association (Fig. 4E). These results suggest that Sirpα toward RBC. As shown in Fig. 5 , testing Syk kinase activity by the phosphorylation at Y519/520 (SykPY) in PEMs found increases expression alone conveys partial inhibition, whereas stronger in- PY hibitory signaling triggered by Cd47 ligation is needed for effec- in Syk (thus Syk activity) after LPS/cytokine stimulation, sup- tively blocking macrophage phagocytosis toward self. porting a key role of Syk in macrophage phagocytic activation. Conversely, IL-10, the phagocytic suppressive cytokine, counter- Signaling Mechanisms Regulating Macrophage Phagocytosis Toward repressed Syk phosphorylation induced by activation factors. Al- Self. Multiple pharmacological inhibitors were tested for their though Syk was suggested to activate -II (38), this appeared effects on LPS and cytokine-induced activation of macrophage to not be the case in macrophage phagocytic activation; however, phagocytosis toward self. As shown in Fig. 5A, treating macro- myosin-II was required for the later phagocytosis process in a way

E5438 | www.pnas.org/cgi/doi/10.1073/pnas.1521069113 Bian et al. Downloaded by guest on September 26, 2021 similar as in Fc-mediated phagocytosis (38, 39) (SI Appendix, Fig. no convincing correlation of these protein expressions with PEM PNAS PLUS S9). Depletion of calcium and magnesium or the presence of phagocytic activation. As shown in Fig. 6C, except for that LPS EDTA hindered macrophage phagocytic activation, possibly treatment induced an increase in CRT (consistent with ref. 46), through affecting macrophage or the activation of the none of the other phagocytic activation factors induced elevation of specific phagocytic receptor. CRT or LRP1 on PEMs. Conversely, CRT and LRP1 expression Moreover, we found that phorbol 12-myristate 13-acetate on PEMs were even reduced (∼50%) after PMA treatment, which (PMA), the PKC activator, dramatically activates macrophages potently activates phagocytosis toward self. Protein coimmunopre- for phagocytosis toward self. As shown in Fig. 5D, even at low cipitation also failed to detect alteration of CRT-LRP1 association, nanomolar concentrations, PMA treatment instantly initiated or LRP1 tyrosine phosphorylation, in PEMs after phagocytic acti- PEM to phagocytose RBCs. In addition, PKC appears to be vation, thus ruling out LRP1 to be a direct target downstream of upstream of Syk in the phagocytic activation pathway (Fig. 5E). Syk (SI Appendix,Fig.S12). Collectively, these results suggest that Inhibition of Syk by Piceatannol and R406 eliminated the PMA- CRT and LRP1 are unlikely to be the receptor-ligand pair that is induced PEM phagocytosis, whereas PMA treatment failed to activated by phagocytic stimuli and mediates phagocytosis toward rescue Syk inhibition-repressed PEM phagocytic activation by healthy self-cells. LPS (Fig. 5F). That PKC induces Syk activation in macrophages We also tested other known phagocytic receptors for their roles was consistent with previous reports (40–42). Interestingly, PMA- in activated macrophage phagocytosis toward self. As shown in induced phagocytosis disregards the Cd47-Sirpα–mediated Fig. 6D, antibody inhibition of scavenger receptor A (anti–SR-A) inhibition. As shown, PMA-treated WT PEMs aggressively phago- or Fc receptors (anti-Cd16/Cd32) (49), or inhibitors against scav- − + cytosed both Cd47 and Cd47 RBCs, a target indiscrimination enger receptor B (BLT1) (50), dectin (laminarin) (51), and com- similar to macrophages treated by pervanadate to abolish SHP plement () (52) mediated phagocytosis, failed to affect signaling (Fig. 5G). Because PMA treatment neither reduced LPS-induced PEM phagocytosis toward RBCs. Interestingly, an- Sirpα expression nor affected Sirpα ITIM phosphorylation and tibody against Cd11b (Cd11b/Cd18) impeded phagocy- SHP-1 association (Fig. 5H), this effect of PMA suggests an in- tosis. It is possible that anti-Cd11b antibody affected macrophage terference of the signaling pathway downstream of the Sirpα- adhesion and spreading, through which inhibited ITIM-SHP axis. phagocytosis toward RBCs. The role of CD11b in phagocytosis of RBCs has also been reported for dendritic cells (53). What Is the Phagocytic Receptor on Macrophages for Uptaking Healthy Self-Cells? Given that calreticulin (CRT) interaction Different Spleen Environments in WT Mice and Mutant Mice. From with LDL receptor-related protein (LRP1) has been suggested to our data, it is possible that all macrophages have the capability to mediate phagocytosis of apoptotic cells and viable cells (43–45), phagocytize healthy self-cells; however, initiating the capacity is we examined these . As shown in Fig. 6A,neitherthein- dependent on the presence of activating stimuli. The fact that − − − − hibitory antibodies against CRT or LRP1 (46) nor LRP1 receptor- red pulp macrophages in WT mice, but not Sirpα / or Cd47 / − associated protein (RAP), which inhibits CRT-LRP1 binding mice, directly phagocytize Cd47 RBCs without requiring fur- (43, 47, 48), showed inhibition on LPS/cytokine–activated PEM ther activation suggests that the WT spleen provides constant

phagocytosis toward RBCs and splenocytes. However, these same stimulation supporting macrophage phagocytosis toward self, INFLAMMATION reagents significantly inhibited phagocytosis toward apoptotic cells whereas the spleens in mutant mice do not. The observations IMMUNOLOGY AND (Fig. 6B) (more data are shown in SI Appendix, Figs. S10 and S11). that red pulp macrophages isolated from WT mice quickly di- Although a previous study (46) had suggested that increase of CRT minished the phagocytic capacity, which was then rekindled by on the macrophage surface is associated with phagocytosis toward extrinsic stimuli, support this notion. In addition, adoptive transfer − tumor cells, assessment of CRT and LRP1 levels on PEMs found of 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 by freshly 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 their phagocytosis 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-17A + were 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.

Bian et al. PNAS | Published online August 30, 2016 | E5439 Downloaded by guest on September 26, 2021 − − RBC loss (10–30%, 48 h) and splenomegaly (Fig. 7A). Cotransfer scription profiles revealed that red pulp macrophages in Sirpα / + − with CSFE-labeled Cd47 RBCs confirmed that Sirpα macro- mice, compared with that in WT mice, express reduced levels of phages mediated erythrophagocytosis in WT mice (Fig. 7B). Tissue functional-stimulating molecules, such as IL-6, IL-17, IL-1β,NF-κB, − analyses indicated that the adoptively transferred Sirpα macro- and Cd40, but higher levels of suppressive molecules such as TGFβ phages were distributed in red pulp of the WT spleen (Fig. 7C). (Fig. 7F) (more transcript data are shown in SI Appendix,Fig.S15 − − Further analyses of spleens cells found that those from Sirpα / and Tables S2 and S3). In another experiment, we examined mice − − and Cd47 / mice produce relatively higher levels of IL-10, but lesser that were housed under a germ-free (GF) condition and mice de- − − IL-17 and IL-6 compared with those from WT mice (Fig. 7D and SI ficient of MyD88 (MyD88 / ), both being associated with a re- + − + Appendix,Fig.S13). Mutant mouse spleens were also associated with duction of Cd11c Cd8 DCs and Cd4 Th cells in the spleen as well + a deficit of Cd11c dendritic cells (DCs), especially the major mi- (Fig. 7G)(56–59). These mice manifested attenuated clearance of − + − gratory and -presenting Cd8 DCs and Cd4 Th lymphocytes Cd47 RBCs in RBC adoptive transfer experiments (Fig. 7H). [Fig. 7E and previous reports (54, 55)]. Other leukocytes, including natural killer (NK) cells, NKT cells, Cd8 T cells, B cells, and the Discussion + total Cd11b myeloid cells demonstrated no reduction. Interest- The present study reveals that multilayered mechanisms govern − − ingly, red pulp macrophages are even increased in Sirpα / and macrophage phagocytic behavior toward healthy self-cells. In ad- − − Cd47 / mice (SI Appendix, Table S1 and Fig. S14). Therefore, it is dition to the previously identified CD47-SIRPα–mediated mech- + − + possible that the deficiency of Cd11c Cd8 DCs and Cd4 Th cells anism that prevents phagocytosis, there are additional mechanisms and the increase of IL-10 collectively cause inactivity of eryth- at the forefront level that determine macrophage propensity to − − − − rophagocytosis in the spleens of Sirpα / and Cd47 / mice. Tran- either 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 were treated 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 stimuli and 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 toward RBCs. (E) Depiction of PKC-Syk–mediated macrophage phagocytic activation toward self. (F) Syk is downstream of PKC. (Left) Inhibition of Syk by Piceatannol and 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. the respective controls. (H) PMA treatment does not affect Sirpα expression or Cd47 ligation-induced Sirpα phosphorylation and SHP-1 association. Data presented in each panel represent at least three independent experiments with n ≥ 4, if applicable.

E5440 | www.pnas.org/cgi/doi/10.1073/pnas.1521069113 Bian et al. Downloaded by guest on September 26, 2021 shown by our data, not only macrophages from mutant mice PNAS PLUS but also PEM and BMDM from WT mice displayed phagocytic inactivity toward even Cd47-null RBCs in the absence of stim- ulation. WT red pulp macrophages, although displaying direct phagocytosis toward Cd47-null RBC in vivo, were unable to main- tain this aggressiveness shortly after isolation. It is as if the “not attack-self” is a default mode for all macrophages, whereas “attack-self” represents an exceptional, hyper-phagocytic status for which special activating mechanisms are needed. This idea that macrophages are generally set to not attack self-cells is con- sistent with the fact that, in most nonlymphoid tissues, suppressive cytokines IL-10 and TGFβ tend to dominate (e.g., in peritoneum) and repress phagocytosis toward self. The spleen red pulp appears to be an exceptional tissue, constantly providing stimuli sustaining the phagocytic capacity toward self. Lack of such a stimulating

Fig. 6. (A) Blocking CRT or LRP1 failed to inhibit LPS/cytokine-induced PEM phagocytosis toward RBCs. LPS/cytokine-activated PEM phagocytosis toward RBCs 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-associated protein (RAP; 20 μg/mL), all dialyzed free of sodium azide (note: sodium azide potently inhibits phagocytosis toward self-cells even at low concen- trations). (B) CRT-LRP1 controls macrophage phagocytosis toward apoptotic cells. PEM (unstimulated) phagocytosis toward apoptotic B16 cells was tested in 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) Macrophage cell 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 by FACS. Increased and decreased expressions were marked by arrows. (D)Ex- ploring other phagocytic receptors. Activated PEM phagocytosis toward RBCs was tested in the presence of antibodies against SR-A, 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 control without 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. INFLAMMATION IMMUNOLOGY AND

self-cells. Indeed, the CD47-SIRPα–mediated inhibition is rele- vant and indispensable only when the phagocytosis toward “self” is initiated in a tissue environment. Remarkably, multiple inflam- matory conditions and proinflammatory cytokines/factors (e.g., LPS, IL-1β,IL-6,IL-17,andTNFα) 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 studies − and also previously (1), WT mice rapidly eliminate Cd47 RBC −/− −/− α − − − − through erythrophagocytosis, whereas Sirp or Cd47 mice Fig. 7. Sirpα / or Cd47 / mice, but not WT mice, are deficient of macrophage cannot. We found that the lack of phagocytic stimulation is at- stimulation in the spleen. (A–C) /macrophages (2 × 107) derived from − − tributed to the latent macrophage behavior in mutant mice, as bone marrow cells of WT or Sirpα / mice were labeled with CMTMR and − − inflammatory stimulations that activate the PKC-Syk pathway transferred into three strains of recipient mice. (A)OnlySirpα / macrophages instantly elicit phagocytosis. In particular, these mutant mice, in WT mice displayed phagocytosis toward RBCs, resulting in anemia and although generally manifesting no macrophage-mediated destruc- splenomegaly. Error bars are ±SEM. *P < 0.05, **P < 0.01 vs. control by transferring WT monocytes/macrophages into WT mice. (B) Cotransfer of CFSE- tion, have developed acute anemia under inflammatory conditions + − − labeled Cd47 RBCs along with Sirpα / /macrophages into WT mice due to splenic macrophages directly phagocytizing self-RBCs. Ex confirmed rapid RBC clearance. Error bars are ±SEM. ***P < 0.001 vs. the initial vivo treatments of macrophages isolated from these mice with LPS α−/− −/− time point. (C) Distribution of CMTMR-labeled Sirp macrophages in spleen or cytokines, or transfer of the nonphagocytic Sirpα macro- red pulp (RP) but not white pulp (WP). (D) Higher levels of IL-10 and lesser IL-17 phages into WT mice, have instantly induced erythrophagocytosis. produced by spleen cells and in serum from Sirpα−/− and Cd47−/− mice. (E) However, in these experiments, we failed to observe macrophages Decreases in Cd11c+Cd8− DC and Cd4+ helper (Th) lymphocytes in the spleen of − − − − displaying tolerance or “split tolerance” suggested previously by Sirpα / and Cd47 / mice. (F) Transcription profiling of red pulp macrophages −/− + others (11); instead, phagocytically activated macrophages derived from WT and Sirpα mice. F4/80 red pulp macrophages were affinity isolated α before mRNA isolation and profiling. The red-colored molecules (most being from mice without Cd47 or Sirp expression displayed the same, α−/− direct phagocytosis toward RBCs, splenocytes, and other cells as activating) are expressed at higher levels in WT than in Sirp red pulp macrophages, whereas the blue-colored molecules (most being suppressive) are macrophages from WT mice, providing an absence of Cd47-Sirpα + − − − expressed oppositely. (G) Reduction of Cd11c Cd8 DCs in the spleens of α / − − inhibition. Another similarity of macrophages from Sirp ,or MyD88 / mice and germ-free (GF)–conditioned mice. (H) Attenuated clearance −/− − − − Cd47 , mice and WT mice is that they all need phagocytic ac- of Cd47 RBC in MyD88 / mice and GF mice. Data presented in each panel tivation to gain direct phagocytosis toward healthy self-cells. As represent at least three independent experiments with n ≥ 4, if applicable.

Bian et al. PNAS | Published online August 30, 2016 | E5441 Downloaded by guest on September 26, 2021 environment in the spleen becomes a “compensation” mechanism mechanism that controls the receptor-mediated internalization of − − − − that maintains Sirpα / or Cd47 / mice to be healthy despite the healthy self-cells after the phagocytic recognition. absence of Cd47-Sirpα–mediated inhibition. Although the CD47-SIRPα mechanism may be dispensable under Materials and Methods normal conditions, it becomes extremely important under in- Mice. All experiments using animals and procedures of animal care and flammatory conditions and infection, during which host macrophages handling were carried out following protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Georgia State University. WT would gain phagocytosis toward healthy self-cells (as suggested by this −/− −/− −/− (C57BL/6J) and Cd47 (B6.129-CD47tm1Fpl/J) mice were from The Jackson study). The finding that Sirpα and Cd47 mice rapidly develop − − Laboratory. Sirpα KO (Sirpα / ) was established by replacing the exons 2–4 anemia under inflammatory challenges suggests that lack of these and their flanking regions with a neomycin resistant cassette in the Sirpα proteins may significantly reduce the threshold for anemia under in- gene (collaboration with Chen Dong at the facility of University of Texas MD flammatory conditions. Inflammation-associated anemia (also called Anderson Cancer Center, Houston). All mice were housed in an SPF facility. “anemia of inflammation” or “anemia of chronic disease”)isamong DSS-induced colitis and zymosan-induced peritonitis were described previously the most frequent complications observed in hospitalized patients. (32–34). Mouse peripheral blood hemoglobin levels were determined by lysis Reported previously by us and others (60, 61), SIRPα expression in of 10 μL whole blood in 1 mL water followed by OD reading at 540 nm.

macrophages is decreased following LPS stimulation, suggesting a + − dynamic for the CD47-SIRPα–mediated inhibition especially Adoptive Transfer Experiments. Cd47 RBCs and Cd47 RBCs were freshly col- −/− ∼ 9 on infection or activation of TLR. The expression of CD47 on cells lected from WT and Cd47 mice, respectively. After labeling with CFSE, 10 RBCs in 150 μL PBS were transfused (i.v.) into recipient mice. Blood samples can also be changed under different conditions (62–64). Also repor- α were collected 30 min after blood transfusion, and percentages of CFSE-labeled ted by us, alteration of clustering structures of SIRP on macrophages RBCs were determined by FACS. Blood samples were then collected at later or CD47 on tissue cells affects phagocytosis (65, 66). Moreover, data time points to assess the CFSE-RBC clearance. For -transfer experi- presented in this study show that CD47-SIRPα–mediated inhibi- ments, splenocytes (2 × 107) labeled by CFSE were transferred into recipient tion controls not only the phagocytic target selection, but also the mice followed by determination of clearance in peripheral blood and the phagocytic robustness once the target has been chosen. As shown in spleen by FACS. For monocyte/macrophage-transfer experiments, bone marrow − + this study, SIRPα macrophages, compared with SIRPα macro- cells were cultured in M-CSF (10 ng/mL) for 3–4 d to produce monocytes/ phages, are much more potent in phagocytosis toward self-cells once macrophages, which were then labeled with 5-(and-6)-(((4-chloromethyl)benzoyl) they have been activated by LPS or cytokines. Although both mutant amino) tetramethylrhodamine (CMTMR) and transferred into recipient mice. strains are deficient of CD47-SIRPα inhibition, more severe anemia α−/− −/− Macrophage Phagocytosis Assay. Splenic macrophages were obtained from ensued in Sirp animals than in Cd47 mice after their splenic D- and DNase I-digested spleen tissues and were enriched by macrophages were stimulated. Even without stimulation, the macro- plating at 37 °C for 2 h. Resident PEMs were obtained by directly lavaging the −/− phages in Sirpα mice were faster to clear RBCs than those in WT peritoneum using sterile PBS. Thioglycollate-eliciteds PEM were prepared by −/− or Cd47 mice. All these results suggest a SIRPα-ITIM–mediated i.p. injection of 1 mL 3% (wt/vol) Brewer thioglycollate, followed by lavage 4 d inhibition on general cell processes of macrophage phagocytosis later. BMDMs were prepared by culturing freshly isolated bone marrow cells in and are in concurrence with reports showing that the CD47-SIRPα the presence of M-CSF (10 ng/mL) for 5–6d(66).Totestphagocytosisexvivo, pathway also tempers Fc- and complement-mediated phagocytosis freshly isolated/prepared macrophages in 24-well plates were incubated with (19–21), as well as phagocytosis toward apoptotic cells (65). CSFE-labeled RBCs or other cells for 30 min (37 °C) in the absence or presence of inhibitory antibodies or reagents. After washing, macrophage phagocytosis The detailed mechanism by which macrophages directly phago- was analyzed microscopically or by FACS. To induce macrophage phagocytic cytize healthy self-cells is unknown, despite that the mechanism activation, macrophages were treated with LPS (20 ng/mL), TNF-α (20 ng/mL), that inhibits this phagocytosis via the CD47-SIRPα-SHP axis has and IL-1β (10 ng/mL) for 6–10 h; IL-6 (10 ng/mL), IL-17A (10 ng/mL), and IFN-γ been studied. Different from traditional phagocytosis aiming at (100 U/mL) for 12–24 h; or PMA (5-20 nM) for 30 min (all at 37 °C) before alien pathogens, immune complexes, debris, and dying self-cells, on phagocytosis assays. Phagocytic activation was also induced in the presence of which certain eat-me or non-self signals ensue phagocytosis, pharmacological inhibitors or varied concentrations of IL-10 to test their in- phagocytosis toward healthy self-cells is uncustomary. To date, the hibitory effects. Phagocytic indexes were expressed by the number of mac- molecules serving as the phagocytic ligands on healthy self-cells, rophages that ingested at least one target in 100 macrophages analyzed.

together with the phagocytic receptor on macrophages, remain + undefined. From the present study, it can be predicted that the Transcript Microarray Profiling. Red pulp microphages isolated by F4/80 se- lection were used for mRNA isolation. Microarrays were performed using specific phagocytic receptor on macrophages is either unexpressed whole Mouse Genome Microarray 4X44K v2 (Agilent Technologies). Briefly, or expressed but maintains inactivity until stimulation-induced ac- labeled cRNA was synthesized using the Quick Amp Labeling kit followed by tivation occurs. The rapid elicitation of phagocytosis by PMA (<30 hybridization using Agilent SureHyb. Slides were scanned by an Agilent DNA min) suggests the latter and that the PKC-Syk-mediated signaling Microarray Scanner, and the images were analyzed using Feature Extraction pathway likely activates this phagocytic receptor through an “inside- software 11.0.0.1 with a background correction. Normalization was carried out” mechanism. Along this line, it is possible that the CD47- out using GeneSpring GX 12.1 software. SIRPα–mediated SHP activity inhibitsthisphagocyticreceptor through protein dephosphorylation that counters the effect by Syk. Statistical Analysis. At least three independent experiments were performed ± The study has ruled out CRT-activated LRP1 and other known for each set of data, which were presented as the mean SEM. Statistical significance was assessed by Student t test for paired samples or one-way phagocytic receptors for mediating phagocytosis toward healthy ANOVA for the group number (k) > 2. self-cells. In particular, our data show that the CRT-LRP system controls phagocytosis toward apoptotic, but not healthy, self-cells. ACKNOWLEDGMENTS. We thank Dr. Chen Dong (MD Anderson Cancer Interestingly, antibody inhibition of CD11b/CD18 impedes acti- Center) for help in establishing the SIRPα KO mice, Dr. Jill Littrell for critical vated macrophage phagocytosis toward self; however, further stud- comments, and the Georgia State University Animal Resources Program for ies are required to delineate if CD11b/CD18 acts as the specific facilitating animal experiments. This work was supported, in part, by grants from National Institutes of Health (AI106839) and the American Cancer Soci- phagocytic receptor or functions as an integrin essential for ety (to Y.L.), the China Postdoctoral Science Foundation (2014M550284), and phagocytosis. Additional studies are also needed to define the a fellowship from the American Heart Association (15POST22810008) (to Z.B.).

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