Cd47-Sirpα interaction and IL-10 constrain inflammation-induced macrophage phagocytosis 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 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, National University of Singapore, Singapore 117456; dCenter for Inflammation, Immunity and Infection, 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 blood 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 protein α (SIRPα) triggers inhib- Cd47-null (Cd47 ) RBCs through erythrophagocytosis in the itory signaling through SIRPα cytoplasmic immunoreceptor tyrosine- spleen 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 platelets, lymphocytes, hematopoi- except under the nonobese diabetic background. Studying our etic cells, and splenocytes 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 antibodies 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, peritonitis, cytokine treatments, and CFA-/LPS-induced inflam- C3 / mice, in which lack of lymphocytes or complements had not − mation, owing to splenic macrophages 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, cancer studies found that tumor cells are commonly associated cells, whereas they aggressively attack toward bacteria, zymosan, with increases in CD47 expression as a way to evade immunological apoptotic, and immune complex-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 calreticulin. Moreover, we identified spleen red pulp forefront activation mechanism requiring the inflammatory to be one specific tissue that provides stimuli constantly activating cytokine-stimulated protein kinase 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 homeostasis, tissue macro- conditions and also provide insights into strategies for enhancing Tphages and other immunological phagocytes must be pre- transplantation tolerance and macrophage-based cancer eradica- vented from phagocytizing healthy self-cells. One essential tion, especially for cancers 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 red pulp 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 colitis 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 acute anemia; the latter was associated with en- xenotransplantation (22–25). hanced macrophage erythrophagocytosis in the spleen. As shown Although these studies are exciting, major obstacles still remain in Fig. 1B (SI Appendix, 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 disease 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 hemoglobin re- mice had been reported, but this is associated with autoimmunity duction (40–50%; Fig. 1D). Dissection