Encyclopedia of Cancer DOI 10.1007/978-3-642-27841-9_7215-1 # Springer-Verlag Berlin Heidelberg 2015

Efferocytosis

Nikko Brix*, Anna Tiefenthaller and Kirsten Lauber Clinic for Radiotherapy and Radiation Oncology, LMU Munich, Munich, Germany

Keywords

Apoptosis; Necrosis; Phagocytes; ; Dendritic cells; Immune response; Inflammation; Immune tolerance; Autoimmunity; Cancer; “Find-me” signals; “Eat-me” signals; “Don’t-eat-me” signals

Definition

The term efferocytosis (from effere, Latin for “to take away”, “to carry to the grave”, “to bury”) has been defined as the process of apoptotic removal, which involves phagocyte recruitment, dying cell recognition, and engulfment.

Characteristics

The Efferocytic Process

Apoptotic Cell Death In higher multicellular organisms, the removal of dying cells is a common event: It is estimated that one million cells undergo per second in a human adult, and fundamental biological processes, such as embryogenesis, the resolution of inflammation, or homeostatic cell turnover involve apoptosis and subsequent clearance of dying cells. This is performed either by neighboring cells (when they are endowed with “amateur” phagocyte capacity) or by professional phagocytes, such as macrophages and immature dendritic cells (DCs), respectively. Macrophages and DCs serve as “undertakers” of dying cells with different tasks. Whereas macrophages can powerfully engulf and degrade huge amounts of dying cell material, DCs act as sentinels, which capture antigen and (cross-)present it to T cells, thus sculpting adaptive immune responses. The entire process of apoptosis is finely controlled, swift, and innoxious for the surrounding tissue. These characteristics starkly distinguish it from the events being observed during necrosis, which is considered to be a nonphysiological form of cell death where plasma rupture and the uncontrolled release of cytosolic danger signals occur. Importantly, apoptosis is an immunogenically silent event. It is morphologically characterized by cell shrinking, chromatin condensation, nuclear fragmentation, and plasma membrane blebbing. On the molecular level, a set of proteases termed commonly initiate and execute this form of programmed cell death. Besides, -independent forms of apoptosis have been reported. The integrity of organelles and the plasma membrane is preserved until late stages of apoptosis. Thus, the liberation of pro-inflammatory intracellular molecules, including heat shock proteins, high mobility group box 1 protein (HMGB1), S100 proteins, and uric acid, which may damage neighboring cells and induce inflammation, is prevented.

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Phagocyte Recruitment by Apoptotic Cells In order to “orchestrate” their own burial, apoptotic cells send out soluble chemotactic factors to trigger monocyte/ recruitment, which is a premise for efficient efferocytosis. These “find-me” signals comprise biomolecules of very different classes, such as nucleotides (mainly ATP and UTP), phospho- lipids (e.g. lysophosphatidylcholine and sphingosine-1-phosphate), and proteins (for instance, the ectodomain of the IL-6 receptor and soluble fractalkine), which are recognized by the corresponding phagocyte receptors.

Recognition of Apoptotic Cells by Phagocytes Upon attraction to the apoptotic site, phagocytes must precisely distinguish between healthy and dying cells. This substep of the efferocytic cascade is guided by the exposure of “eat-me” signals on the apoptotic cell surface. Translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane is the best-known and probably most important example. This phospholipid is recognized by bona fide PS receptors on the phagocytes (e.g. brain angiogenesis inhibitor 1 (BAI-1), T cell immunoglobulin mucin domain (TIM) family members 1, 3, and 4, and the stabilins 1 and 2) or indirectly via soluble bridging proteins, which bind to both PS and their respective phagocytic cell surface receptors. Examples of bridging proteins secreted by phagocytes are milk fat globule EGF factor 8 (MFG-E8) and developmental endothelial locus 1 (Del-1). Moreover, bridging proteins derived from apoptotic cells, such as annexin A1, or from interstitial body fluids (b2-glycoprotein, growth arrest specific gene 6 (GAS6), and protein S) have been described. Furthermore, additional “eat-me” signals apart from PS exist. Examples are the surface exposure of ICAM-3 on apoptotic cells being recognized by CD14 and altered sugars, which are detected by lectins on the phagocyte. Besides, the inactivation and/or the lack of “don’t-eat-me” signals (e.g. CD47 and CD46) contribute to efficient dying cell engulfment. These proteins are expressed on healthy cells and thus protect them from being accidentally ingested by phagocytes. Taken together, a plethora of different signals mediate the engulfment of apoptotic cells, and it is currently being unraveled which receptor- ligand axis dominates in which tissue or organ.

Apoptotic Cell Engulfment The signaling mechanisms that are initiated upon ligation of phagocytic “eat-me” signal receptors are far from being fully understood. Yet, it is known that the ingestion of apoptotic cells requires massive modifications in the phagocyte’s actin cytoskeleton. These are regulated by small GTPases of the RHO family, such as RHOA, RAC, and CDC42. RHOA activation has been described to inhibit apoptotic cell engulfment via binding and thereby activating its downstream effector Rho-associated coiled-coil containing protein kinase (ROCK). ROCK activation may in turn alter the phosphorylation status of myosin light chain and thus the phagocytic actin cytoskeleton structure and contractility. Inversely, RHOA inhibition was shown to promote dying cell engulfment. In contrast to RHOA, RAC activation positively affects apoptotic cell engulfment. For instance, the PS receptor BAI-1 is known to activate RAC by recruiting the adaptor protein engulfment and cell motility 1 (ELMO1) and its binding partner dedicator of cytokinesis 180 (DOCK180).

The Post-Phagocytic Immune Response Apoptotic cell death itself is not only immunogenically silent, it also shapes the post-phagocytic immune response. Unlike the uptake of necrotic cell material by macrophages, apoptotic cell engulfment induces the secretion of anti-inflammatory cytokines including interleukin-10 (IL-10), transforming growth factor b (TGF-b), and prostaglandin E2 (PGE2). The removal of apoptotic cell material by DCs that have been educated in this milieu leads to tolerogenic (cross-)presentation of antigens in the draining lymph nodes.

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Among the “tolerate-me” signals, which are involved in the immunomodulatory processes being initiated upon apoptotic cell clearance, exposure of PS is of crucial importance. However, since PS exposure is also observed during necrosis after plasma membrane rupture, it is evident that several other biomolecules might contribute to sculpting the immunological outcome of dying cell . Moreover, the cause of apoptotic cell death, the predominating dying cell type, and the quantity of apoptotic cells naturally modify the post-phagocytic immune response. The latter point is of particular importance: Although efferocytosis is performed rapidly under physiological conditions, excessive apoptotic cell death and/or inefficient clearance may overwhelm the phagocytic capacity: For instance, accumulating, uncleared apoptotic cells can be observed in the context of autoimmune diseases or after tumor radio(chemo)therapy as discussed below. As a result, these apoptotic cells may transit into secondary necrosis culminating in plasma membrane rupture and liberation of pro-inflammatory intracellular components. Considering their disparate origin, the soluble mediators released from primary and secondary necrotic cells encode for different immunological outcomes but commonly induce pro-inflammatory processes, which may atten- uate the anti-inflammatory milieu at a formerly apoptotic site.

Diseases Caused by Malfunctioning Efferocytosis The fact that apoptotic cells are rarely seen in homeostatic tissues testifies the efficiency of dying cell removal by professional and “amateur” phagocytes. In case of malfunctioning dying cell clearance, insufficient immunological quiescence causes recruitment of inflammatory cells into the tissue. Accumu- lation of this cell debris might initiate immunological reactions against self-antigens resulting in different types of chronic inflammatory diseases.

Rheumatic Diseases The term “rheumatism” summarizes a wide range of autoimmune diseases affecting different organs. Their common pathogenesis is associated to autoimmune mechanisms – immune reactions against the body’s own tissues. From an immunological point of view, defective efferocytosis after tissue destruction leads to accu- mulation of cell corpses, which progress into secondary necrosis and release their intracellular contents (e.g., DNA, nucleosomes, and histones) upon rupture. The immune system generates antibodies against these intracellular antigens, which in complex with their cognate antigens can stimulate an exacerbation of the inflammatory cascade. The immune reaction against own tissue structures and intracellular components results in general symptoms such as fever, pain, fatigue and progressive organ destruction when not adequately treated. Systemic erythematosus (SLE) is a systemic that results from genetic and environmental stimuli, including ultraviolet light. As autoimmunity targets diverse intracellular compo- nents, several parts of the body are affected, especially inner organs (e.g., lungs, kidneys, liver, and the central nervous system) and the skin (butterfly rash). Although the etiopathogenesis of SLE is not completely understood, it is considered to involve multifactorial events and genetic predispositions. Alterations in regulators of apoptosis and efferocytosis, including C-reactive protein (CRP), pentraxins, and the complement system (especially C1q), lead to increased occurrence of secondary necrotic cell debris. The accumulation of secondary necrotic cell debris in the germinal centers of the lymph nodes favors complement-mediated immune complex formation. Blood phagocytes subsequently engulf these strongly pro-inflammatory structures, and the inflammatory reaction is further fueled by inappropriate cytokine release and production of inflammatory proteins, such as CRP. Clinically, these chronic inflammatory processes become manifest in fever, fatigue, and immune-mediated progressive organ destruction.

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Even though defective apoptotic cell clearance has not yet been proven to contribute directly to the onset of , there exists some indirect evidence that this disease of progressive joint destruction could be caused by compromised efferocytosis as well. The extracellular debris located in inflamed joints contains – among other factors – histones and HMGB1, which both can inhibit efferocytosis. Eliminating these factors reportedly results in improved phagocytosis and could therefore be an interesting strategy for antirheumatic therapies.

Atherosclerosis as one of the widest-spread cardiovascular diseases is considered to be a chronic inflammatory disorder, which is linked – at least in part – to lacking engulfment of apoptotic macrophages by their healthy counterparts. Tissue-resident macrophages undergo apoptotic cell death after excessive uptake of oxidized lipids. They remain in the vessel wall and transit into secondary necrosis due to defective clearing mechanisms. These secondary necrotic cells then stimulate chronic inflammation, further impairing phagocytosis and forming atherosclerotic plaques. In addition, malfunctioning or dead macrophages lack the ability of producing antiphlogistic cytokines (e.g., TGF-b, IL-10) and therefore do not contribute to atheroprotection.

Chronic Lung Diseases Several chronic lung diseases are related to apoptosis and defective cell clearance, e.g., chronic obstruc- tive pulmonary disease (COPD), , pulmonary fibrosis, cystic fibrosis (CF), and acute respiratory distress syndrome (ARDS). In all of these diseases, phagocytosis is reduced, mainly by neutrophil- produced reactive oxygen species (ROS) and subsequent activation of the efferocytosis inhibitor RHOA. Today, the common treatment of these diseases includes corticosteroid therapy. The mechanisms underlying corticosteroid action involve the inhibition of pro-inflammatory cytokine production and enhanced induction of apoptosis as well as efferocytosis.

Allograft Tolerance Allotransplant patients are commonly treated with immunosuppressive agents in order to suppress allograft rejection. In mouse models, apoptotic cells appear to be a major contributor to immunogenic tolerance after graft transplantation. Hence, the question arises, if classic immunosuppressive treatment could be complemented by apoptotic cell transfer in order to exploit the apoptosis-dependent immunotolerance inducing effects.

Infection and Microbes In case of tissue infection by microbes, pathogen-associated molecular patterns (PAMPs, e.g., nucleic acids, lipopolysaccharides, bacterial endotoxins), and danger signals derived from the host (e.g., ATP, HMGB1, heat shock proteins, uric acid, mitochondrial DNA) induce local and to some extent also systemic inflammation. This involves invasion of immune cells – neutrophils in the early phase, mono- cytes and macrophages later on. After pathogen engulfment, neutrophils locally undergo a specific form of apoptosis, the so-called phagocytosis-induced cell death (PICD), thus stimulating their internalization by macrophages and ensuring a second round of degradation of the internalized pathogens. Accordingly, efferocytosis appears to be a fundamental, well-established, and conserved mechanism of pathogen defense in mammals. However, in some cases, pathogens hijack this way of phagocytic clearance for infection. For Chlamydia pneumonia, it has been reported that efferocytosis of apoptotic, infected neutrophils, and subsequent lysosomal escape lead to macrophage infection – a phenomenon known as Trojan-horse strategy. Alternatively, exposure of PS is utilized as a means of apoptotic mimicry by certain viruses (vaccinia virus, HIV, and others) and parasites (Leishmania and Trypanosoma species). They

Page 4 of 7 Encyclopedia of Cancer DOI 10.1007/978-3-642-27841-9_7215-1 # Springer-Verlag Berlin Heidelberg 2015 expose PS on their in order to be engulfed by macrophages and exploit the macrophages’ tolerogenic, post-efferocytic state for pathogen spread and immune escape. Apart from the disorders mentioned above, there are many more, which can be linked to defects or disturbances in efferocytosis. Consequently, the complexity of cell death, dying cell clearance, and subsequent immune reactions require further research in order to unravel the mechanisms of pathogenesis and to develop novel treatment approaches.

The Role of Efferocytosis in Cancer Remarkably, apoptosis and efferocytosis are also highly relevant with regard to our understanding of cancer development and treatment. On the one hand, resistance to apoptosis is known to facilitate tumor cell growth and has therefore been defined as a hallmark of cancer. For instance, overexpression of anti- apoptotic proteins, such as B-cell lymphoma 2 (BCL-2) and B-cell lymphoma extralarge (BCL-XL), may prevent apoptotic cell death. Similarly, loss of p53 – a key DNA damage sensor endowed with the capacity to induce apoptosis – is a very common mechanism of tumor cell death evasion, and there are many other examples of apoptosis resistance in tumors. However, on the other hand, recent findings suggest that the consequences of tumor cell apoptosis for a growing tumor may be more complex. Apoptotic cell death implies tumor cell removal and tolerogenic antigen (cross-)presentation by tumor-associated macrophages (TAMs) and intratumoral DCs, respec- tively. Astonishingly, this efferocytic process may be one of the fundamental elements in the progression of certain tumors as recently demonstrated in lymphomas: In order to enforce angiogenesis and to escape the immune system, a moderate level of tumor cell apoptosis might be beneficial for overall tumor growth by providing a pro-angiogenic, non-inflammatory milieu. In this context, TAMs may release tumor- protective factors, which activate angiogenesis, matrix remodeling, and wound healing. Importantly, these findings do not contradict the well-established concept of apoptosis resistance as a hallmark of cancer. Evading apoptosis may be of crucial importance to initiate and establish the malignant phenotype. However, in later stages of tumor development, which are often accompanied by intratumoral hypoxia, an increase in angiogenesis and an immunotolerogenic milieu induced by TAM-dependent efferocytic processes may be beneficial for overall tumor growth. Conversely, the growth of certain tumors might be impeded by altering the amount and especially the quality of tumor cell death: Novel cancer therapeutics may induce a delay in apoptotic cell clearance or block apoptotic signaling. For instance, masking the “eat-me” signal PS on apoptotic cells by annexin V has been shown to mediate antitumor immunity in vivo – presumably by retarding efferocytosis. Moreover, established cancer therapy approaches, such as radio- and chemotherapy, may be fine-tuned (and combined with additional therapeutics) in order to prevent a preponderance of apoptosis and subsequent tolerogenic immune responses. In summary, therapeutic induction of apoptosis should undoubtedly be considered as a key mechanism to induce tumor regression, but the ambivalent character of apoptosis with regard to efferocytosis and its immunological implications should also be taken into account.

Prospects Extensive research on the process of dying cell clearance within the last decades has demonstrated that efferocytosis is much more than a simple method of “waste disposal” to eliminate dead cell corpses. Not only apoptotic cell death, phagocyte recruitment, and apoptotic cell engulfment but especially the post- phagocytic immunological consequences require further investigation as numerous patients may profit from a better understanding of the basic principles underlying the efferocytic process. New approaches aiming at modifying the quality and quantity of cell death and modulating the removal of dying cells may

Page 5 of 7 Encyclopedia of Cancer DOI 10.1007/978-3-642-27841-9_7215-1 # Springer-Verlag Berlin Heidelberg 2015 strongly improve the treatment of several severe diseases as these therapeutic strategies would not only control symptoms but target the origin of disease.

Cross-References

▶ Angiogenesis ▶ Apoptosis ▶ Autoimmunity and Cancer ▶ BCL2 ▶ Cancer ▶ Dendritic Cells ▶ Hypoxia ▶ Inflammation ▶ Macrophages ▶ Prostaglandins ▶ Transforming Growth Factor-Beta ▶ Tumor-Associated Macrophages

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