Krysko, Dmitri

Ravichandran, Kodi

Vandenabeele, Peter

Ghent University Academic Bibliography

COMMENTMacrophages regulate the clearanceof living cells by calreticulinDmitri V. Krysko 1,2, Kodi S. Ravichandran3,4,5 & Peter Vandenabeele 4,5,6Recently, Feng, Weissman and colleagues demonstrated that activated macrophages secretecalreticulin (CRT), which binds to the surface of viable target cells and marks them for removalby programmed cell phagocytosis (PrCP)1. CRT is one of the ‘eat me’ signals, which is oftenthought to localize in the lumen of the endoplasmic reticulum (ER), but was also found on theplasma membrane of apoptotic cancerous cells (Fig. 1), where it is believed to function as an ‘eatme’ signal2 and a damage-associated molecular pattern (DAMP) responsible for the immuno-genicity of apoptotic cancerous cells3. Remarkably, some viable cells also expose CRT but areapparently protected from engulfment. One of the mechanisms for this protection is related torepelling signals mediated by so-called ‘don’t eat me’ signals concurrently expressed on the cells,such as CD31, CD200 and CD474. Importantly, clearance of viable cells has been recentlyrecognized in a few contexts, and it has been intriguing, in part due to the lack of an obviousmechanism, and also for its relevance. Some examples of reported live cell clearance includesuicidal emperipolesis5, entosis6, phagoptosis7, and cell cannibalism8. However, the source andspeciﬁc role of CRT in the engulfment of viable cells and the binding moiety on the surface ofviable cells have not been known yet.Feng et al. showed that activated macrophages are a source of CRT bound on the surface ofneutrophils. In order to distinguish between efferocytosis9 of apoptotic neutrophils versus theuptake of CRT-marked ‘live’ or ‘aged’ neutrophils by PrCP, the authors performed a comparisonbetween wild type and Bcl-2-overexpressing neutrophils and conﬁrmed a 25-old report from thesame group that the eventual extent of uptake is not affected by blocking apoptosis in the targetcells10. However, although several mechanisms of CRT exposure on the surface of cellsundergoing immunogenic apoptosis have been described11, which mechanisms of CRT secretionare used by macrophages to enhance PrCP are currently unknown and many more interestingand challenging ﬁndings are expected. In this regard it has also been suggested the links betweenER calcium depletion and detection of ER-resident proteins, including CRT, in the extracellularspace12.What does the CRT bind to on the surface of neutrophils and possibly other viable cellsengulfed via PrCP? Since CRT is involved in the quality control of glycoproteins in the ER by alectin-like function, the authors asked whether during PrCP, the extracellular binding of CRT onneutrophils (and other living cells) may also involve sugar moieties. Feng et al. found that CRTselectively bound to asialoglycans displaying the Tri-antennary and multivalent type II chainepitopes (Tri/m-II). This speciﬁcity of CRT-binding was conﬁrmed on mature murine neu-trophils, as well as on the cell surface of a viable human acute myeloid leukemia cell line (HL60)and a human colon cancer cell line (SW620) by competitive binding with Phytohaemagglutinin-L (PHA-L), a lectin with the same speciﬁcity for Tri/m-II1. These ﬁndings demonstrate that thisCRT binding and speciﬁcity can be extended to other cell types than neutrophils and suggest thatPrCP could be a general phenomenon that may occur during different biological processes suchas inﬂammation resolution and anti-cancer surveillance (Fig. 1).DOI: 10.1038/s41467-018-06807-9 OPEN1 Department of Basic Medical Sciences, Ghent University, Ghent 9000, Belgium. 2National Research Lobachevsky State University of Nizhni Novgorod,Nizhny Novgorod 603950, Russia. 3 The Center for Cell Clearance and the Department of Microbiology, Immunology, and Cancer Biology, University ofVirginia, Charlottesville 22908-0734 VA, USA. 4 Inﬂammation Research Center, VIB, Ghent 9052, Belgium. 5Department of Biomedical Molecular Biology,Ghent University, Ghent 9052, Belgium. 6Methusalem Program, Ghent University, Ghent 9052, Belgium. Correspondence and requests for materials shouldbe addressed to D.V.K. (email: Dmitri.Krysko@ugent.be)NATURE COMMUNICATIONS |          (2018) 9:4644 | DOI: 10.1038/s41467-018-06807-9 | www.nature.com/naturecommunications 11234567890():,;Neuraminidase is a glycoside hydrolase that removes sialicacids from the terminal positions of glycans and exposes thecryptic Tri/m-II, leading to increased CRT binding. Havingidentiﬁed the asialoglycan epitope involved in CRT binding, theauthors then examined whether CRT-binding and PrCP could bemodulated by neuraminidase treatment1. They found that thistreatment signiﬁcantly promoted PrCP of different types of viablehuman cancer cells by both human and mouse macrophages. In acomplementary approach, Feng et al. found that knocking out theneuraminidase-4 gene (neu-4) in a cancer cell line, signiﬁcantlydecreased PrCP. To test the potential usefulness of these obser-vations to clinical settings, the authors mined transcriptomedatabases of cancers and this revealed higher expression of genespromoting the removal of sialic acids (such as neuraminidases)correlated with an improved survival, while higher expression ofthe genes enhancing sialic acid expression (such as sialyl trans-ferases) correlated with a worse outcome. Also, during normaland pathological haematopoiesis the level of CRT or PHA-Lbinding correlated inversely with cellular half-life, with highestbinding in blast cells and lowest in multipotent progenitors,leukemia stem cells and haematopoeitic stem cells. These studiesindicate that regulation of asialoglycan levels on the cell surfaceand the subsequent PrCP due to the CRT mediated recognitioncould be important during homeostasis and inﬂammation reso-lution and as well as immunosurveilance.This work by Feng et al. reveals several new pieces of infor-mation and as all good studies, also raises a number of questions.First, previous studies have suggested that CRT on the surface ofapoptotic cells functions as a (DAMP responsible for theimmunogenicity of apoptotic cancerous cells3,13,14. This currentwork places CRT in a new context, i.e. in the clearance of ‘viable’cancer cells with CRT acting as an ‘eat me’ signal. Whethermodulation of the asialoglycan epitope by neuraminidase treat-ment also modulate the uptake of apoptotic cells, whether thiswill modulate their immunogenicity is a question that could alsobe relevant for tumor immunotherapy. In this regard, Feng et al.showed that neuraminidase treatment leads to a decrease intumor engraftment and growth in mice, and that in humans,stronger expression of genes enhancing sialic acids expressioncorrelates with a worse outcome1. Future studies addressing theanti-tumor responses could be useful. This raises also the ques-tion whether other phagocytic cells, such dendritic cells, cansecrete CRT to decorate their preys, and whether engulfment ofviable cancer cells would lead to antigen presentation. Second, thecurrent work suggesting the modulation of asialoglycoproteins onthe surface of viable cells and the section of CRT from macro-phages, begin to provide mechanistic insights. Although notfocus of this work, whether different types of tissue macrophagesas well as their differentiation (such as M1-like versus M2-likesignatures) might alter CRT secretion, or whether the PrCP israther a feature of tumor associated macrophages remain to beseen. Finally, beyond tumor immunosurveillance, an intriguingobservation is the interaction between viral neurominidases (suchas Inﬂuenza virus) and the host cells. Indeed Inﬂuenza neuro-minidases have been shown to enhance phagocytosis ofInﬂuenza-infected cells through desialylation15. Future investi-gations will lead to a better understanding of the molecularmechanisms of the clearance of viable cells during cancer andviral infections with relevance to anti-tumor therapies as well asnormal hematopoietic development.Received: 10 July 2018 Accepted: 20 September 2018References1. Feng, M. et al. Programmed cell removal by calerticulin in tissue homeostasisand cancer. Nat. Commun. 9, 3194 (2018).2. Gardai, S. J. et al. Cell-surface calreticulin initiates clearance of viable orapoptotic cells through trans-activation of LRP on the phagocyte. Cell 123,321–334 (2005).3. Galluzzi, L., Buque, A., Kepp, O., Zitvogel, L. & Kroemer, G. Immunogeniccell death in cancer and infectious disease. Nat. Rev. Immunol. 17, 97–111(2017).4. Chao, M. P., Majeti, R. & Weissman, I. L. Programmed cell removal: a newobstacle in the road to developing cancer. Nat. Rev. Cancer 12, 58–67 (2011).MacrophageViable cellsApoptoticcellsCRTViablecellAsialoglycoproteinsSialic acidsNeuraminidase Cell deathinducers  PhagocytosisPSCD47MacrophageCD91PS receptorsmCRTa bCD47Fig. 1 Mechanisms of recognition of viable (a) and apoptotic (b) cells. a A soluble calreticulin (sCRT) secreted by activated macrophages is capable ofbinding to asialoglycans on viable target cells, depending on their developmental or damage state, which in turn, marks them for uptake. Neuraminidase-4is one enzyme that could be involved in the generation of cell surface asialoglycans that bind sCRT. The cell-surface mCRT on the target cells can nowinteract with CD91, which is also known as low-density lipoprotein receptor-related protein 1 (LRP1). b CRT and CD91 have also been linked toimmunogenic apoptotic cell death. On the surface of apoptotic cells expose phosphatidylserine (PS) and mCRT that function as “eat me” signals. ThemCRT expression is particularly linked to cell death induced by agents that are known to induce immunogenic forms of apoptotic cell death. The expressionof “don’t eat me” signals (e.g., CD47) on the apoptotic cells is either reduced or its location modiﬁed. Many membrane-bound receptors for PS have beenreported, including those that bind PS directly or indirectly via bridging molecules. mCRT can bind CD91 on the phagocyte. The authors thank Servier forthe ﬁgures that were produced using Servier Medical Art (www.servier.com)COMMENT NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06807-92 NATURE COMMUNICATIONS |          (2018) 9:4644 | DOI: 10.1038/s41467-018-06807-9 | www.nature.com/naturecommunications5. Sierro, F. et al. Suicidal emperipolesis: a process leading to cell-in-cellstructures, T cell clearance and immune homeostasis. Curr. Mol. Med. 15,819–827 (2015).6. Krishna, S. & Overhotzer, M. Mechanisms and consequences of entosis. CellMol. Life Sci. 73, 2379–2386 (2016).7. Metayer, L. E., Vilalta, A., Burke, G. A. A. & Brown, G. C. Anti-CD47antibodies induce phagocytosis of live, malignant B cells by macrophages viathe Fc domain, resulting in cell death by phagoptosis. Oncotarget 8,60892–60903 (2017).8. Lozupone, F. & Fais, S. Cancer cell cannibalism: a primeval option to survive.Curr. Mol. Med. 15, 836–841 (2015).9. Henson, P. M. Cell removal: efferocytosis. Annu. Rev. Cell Dev. Biol. 33,127–144 (2017).10. Lagasse, E. & Weissman, I. L. bcl-2 inhibits apoptosis of neutrophils but nottheir engulfment by macrophages. J. Exp. Med. 179, 1047–1052 (1994).11. Garg, A. D. et al. A novel pathway combining calreticulin exposure andATP secretion in immunogenic cancer cell death. EMBO J. 31, 1062–1079(2012).12. Peters, L. R. & Raghavan, M. Endoplasmic reticulum calcium depletionimpacts chaperone secretion, innate immunity, and phagocytic uptake of cells.J. Immunol. 187, 919–931 (2011).13. Obeid, M. et al. Calreticulin exposure dictates the immunogenicity of cancercell death. Nat. Med. 13, 54–61 (2007).14. Krysko, D. V. et al. Immunogenic cell death and DAMPs in cancer therapy.Nat. Rev. Cancer 12, 860–875 (2012).15. Watanabe, Y., Shiratsuchi, A., Shimizu, K., Takizawa, T. & Nakanishi, Y.Stimulation of phagocytosis of inﬂuenza virus-infected cells through surfacedesialylation of macrophages by viral neuraminidase. Microbiol. Immunol. 48,875–881 (2004).Author contributionsD.V.K., K.S.R., and P.V. wrote and edited the manuscript.Additional informationCompeting interests: The authors declare no competing interests.Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional afﬁliations.Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is not included in thearticle’s Creative Commons license and your intended use is not permitted by statutoryregulation or exceeds the permitted use, you will need to obtain permission directly fromthe copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.© The Author(s) 2018NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06807-9 COMMENTNATURE COMMUNICATIONS |          (2018) 9:4644 | DOI: 10.1038/s41467-018-06807-9 | www.nature.com/naturecommunications 3

Macrophages regulate the clearance of living cells by calreticulin

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Macrophages regulate the clearance of living cells by calreticulin

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Ghent University Academic Bibliography