Inhibition Of Efferocytosis Research Articles

MerTK-mediated efferocytosis promotes immune tolerance and tumor progression in osteosarcoma through enhancing M2 polarization and PD-L1 expression

ABSTRACT The poor progress of immunotherapy on osteosarcoma patients requires deeper delineation of immune tolerance mechanisms in the osteosarcoma microenvironment and a new therapeutic strategy. Clearance of apoptotic cells by phagocytes, a process termed “efferocytosis,” is ubiquitous in tumors and mediates the suppression of innate immune inflammatory response. Considering the massive infiltrated macrophages in osteosarcoma, efferocytosis probably serves as a potential target, but is rarely studied in osteosarcoma. Here, we verified M2 polarization and PD-L1 expression of macrophages following efferocytosis. Pharmacological inhibition and genetic knockdown were used to explore the underlying pathway. Moreover, tumor progression and immune landscape were evaluated following inhibition of efferocytosis in osteosarcoma model. Our study indicated that efferocytosis promoted PD-L1 expression and M2 polarization of macrophages. Ëfferocytosis was mediated by MerTK receptor in osteosarcoma and regulated the phenotypes of macrophages through the p38/STAT3 pathway. By establishing the murine osteosarcoma model, we emphasized that inhibition of MerTK suppressed tumor growth and enhanced the T cell cytotoxic function by increasing the infiltration of CD8+ T cells and decreasing their exhaustion. Our findings demonstrate that MerTK-mediated efferocytosis promotes osteosarcoma progression by enhancing M2 polarization of macrophages and PD-L1-induced immune tolerance, which were regulated through the p38/STAT3 pathway.

Inhibition of Efferocytosis by Extracellular CIRP-Induced Neutrophil Extracellular Traps.

Phagocytic clearance of apoptotic cells by the macrophages (efferocytosis) is impaired in sepsis, but its mechanism is poorly understood. Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel damage-associated molecular pattern that fuels inflammation. We identify that eCIRP-induced neutrophil extracellular traps (NETs) impair efferocytosis through a novel mechanism. Coculture of macrophages and apoptotic thymocytes in the presence of recombinant murine CIRP (rmCIRP)-induced NETs significantly inhibited efferocytosis. Efferocytosis was significantly inhibited in the presence of rmCIRP-treated wild-type (WT), but not PAD4-/- neutrophils. Efferocytosis in the peritoneal cavity of rmCIRP-injected PAD4-/- mice was higher than WT mice. Milk fat globule-EGF-factor VIII (MFG-E8), an opsonin, increased macrophage efferocytosis, whereas the inhibition of efferocytosis by NETs was not rescued upon addition of MFG-E8, indicating disruption of MFG-E8's receptor(s) αvβ3 or αvβ5 integrin by the NETs. We identified neutrophil elastase in the NETs significantly inhibited efferocytosis by cleaving macrophage surface integrins αvβ3 and αvβ5 Using a preclinical model of sepsis, we found that CIRP-/- mice exhibited significantly increased rate of efferocytosis in the peritoneal cavity compared with WT mice. We discovered a novel role of eCIRP-induced NETs to inhibit efferocytosis by the neutrophil elastase-dependent decrease of αvβ3/αvβ5 integrins in macrophages. Targeting eCIRP ameliorates sepsis by enhancing efferocytosis.

Activation of ROS/MAPKs/NF-κB/NLRP3 and inhibition of efferocytosis in osteoclast-mediated diabetic osteoporosis.

Diabetes mellitus (DM) affects bone metabolism and leads to osteoporosis; however, its pathogenetic mechanisms remain unknown. We found that high glucose (HG) conditions induced the production of reactive oxygen species (ROS) and the expression of proteins related to MAPKs [phosphorylated (p)-ERK, p-JNK, and p-p38], NF-κB (NF-κB, p-IκB, and IKK), and NACHT-LRR-PYD domains-containing protein 3 (NALP3) (NLRP3) [apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), caspase-1, IL-18, IL-1β, and NLRP3] in osteoclasts (OCs) in vitro. Further analysis showed that in HG-induced OCs, ROS is an upstream signal for MAPKs, NF-κB, and the NLRP3 inflammasome. Moreover, MAPKs mediated the activation of NF-κB and NLRP3, whereas NF-κB up-regulated the NLRP3 inflammasome response. Interestingly, HG inducement enhanced the bone resorption of OCs but inhibited their efferocytosis, whereas insulin and lipoxin A4 (4) treatment reversed this phenomenon. In streptozotocin-induced diabetic rats in vivo, the numbers and the bone-resorption capacity of OCs as well as the serum levels of TRACP-5b were significantly increased, and the expression of MAPK-, NF-κB-, and NLRP3 inflammasome-related proteins in the proximal tibia were also significantly elevated; however, treatment with insulin and LXA4 reversed this elevation. Together, these results demonstrated that the activation of ROS/MAPKs/NF-κB/NLRP3 and the inhibition of efferocytosis in OCs are the main causes of osteoporosis in DM.-An, Y., Zhang, H., Wang, C., Jiao, F., Xu, H., Wang, X., Luan, W., Ma, F., Ni, L., Tang, X., Liu, M., Guo, W., Yu, L. Activation of ROS/MAPKs/NF-κB/NLRP3 and inhibition of efferocytosis in osteoclast-mediated diabetic osteoporosis.

Abstract 558: MERTK-specific antibodies that have therapeutic antitumor activity in mice disrupt the integrity of the retinal pigmented epithelium in cynomolgus monkeys

Abstract MERTK, a member of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases, is a pleiotropic immune modulator that controls efferocytosis. Engagement of MERTK with its ligand GAS6, found anchored to phosphatidylserine exposed on the outer membrane of apoptotic cells, triggers MERTK phosphorylation and signaling events that culminate in the removal of apoptotic debris. Recent studies have highlighted the expression of MERTK on tumor-associated macrophages, and Mertk-deficient mice show reduced tumor cell growth accompanied by inflammatory cytokine production and alterations in macrophage activation. Thus, MERTK has emerged as a promising therapeutic target for augmenting innate antitumor immune responses. MERTK is also expressed in retinal pigmented epithelium (RPE) cells of the eye where it mediates phagocytosis of photoreceptor outer segment fragments. Mutations in MERTK that disrupt its expression or kinase activity lead to marked retinal degeneration and blindness in mice, rats, and humans. Due to known differences in blood-retinal permeability, we explored whether therapeutic antibodies targeting MERTK could inhibit macrophage-mediated efferocytosis and promote antitumor activity while sparing RPE toxicity. A diverse panel of high-affinity antibodies was developed to explore MERTK blockade in vitro and in vivo. Multiple antibodies disrupted MERTK-GAS6 binding and blocked human and murine macrophage-mediated efferocytosis. Two antibodies targeting distinct GAS6 binding epitopes were selected for further characterization. Both antibodies demonstrated antitumor activity in murine CT26 and MC38 syngeneic colorectal cancer models and led to alterations in immune cell-related gene expression. To investigate potential effects on RPE biology with MERTK antibodies, a multi-dose, 4-week cynomolgus monkey study with several in-life and post-mortem ophthalmologic endpoints was designed. While no abnormal ophthalmic or electroretinography (ERG) findings were detected, all animals treated with either MERTK antibody at all doses showed histological abnormalities of the retina, including vacuolation of the outer segments of photoreceptors, displacement of RPE cells, and single cell necrosis of the outer nuclear layer. These data suggest that inhibition of efferocytosis by antibody-mediated blockade of MERTK can promote immune activation and inhibit tumor growth in vivo; however, retinal toxicity consistent with histological observations made in Mertk mutant animals is an on-target effect. As several therapeutics that block MERTK function are currently in preclinical development, a thorough evaluation of retinal toxicity is warranted. Citation Format: Kerry F. White, Matthew Rausch, Jing Hua, Katherine H. Walsh, Christine E. Miller, Christopher C. Wells, Devapregasan Moodley, Benjamin H. Lee, Scott C. Chappel, Pamela M. Holland, Jonathan A. Hill. MERTK-specific antibodies that have therapeutic antitumor activity in mice disrupt the integrity of the retinal pigmented epithelium in cynomolgus monkeys [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 558.

Impact of Klebsiella pneumoniae on efferocytosis of polymorphonuclear cells

Abstract Efficient clearance of invading pathogen and the infiltrating cells by phagocytic cells are two key events that are required for successful elimination of a pathological insult. Clearance of the pathogen is usually achieved by phagocytosis and dead cells are cleared by a process called efferocytosis. While there is substantial information on various strategies employed by a number of pathogens to subvert phagocytosis, our knowledge about inhibition of efferocytosis by microbes is extremely limited. In that regard, we observed that, polymorphonuclear cells (PMNs) infected with Klebsiella pneumoniae (KPn), an opportunistic pathogen and a frequent cause of nosocomial infections leading to sepsis, were efferocytosed less than their uninfected counterparts. This inhibition did not correlate with the infection induced effect on phosphatidylserine (PS) exposure, a well-studied “eat me” signal recognized by efferocytic cells or p53 expression, as a marker of apoptosis in PMNs. We further observed that bacterial replication is required for the full inhibitory effect on efferocytosis since heat-killed KPn were not as effective in inhibiting efferocytosis of PMNs. Next we examined the possible role of LPS in diminishing effecrocytosis, given that KPn is a Gram-negative bacterium. By utilizing cells deficient in TLR-4 (a receptor for LPS) and direct stimulation with LPS we determined that LPS is able to directly inhibit efferocytosis of PMNs. To our knowledge, this study is the first to show a modulatory effect of KPn infection and LPS on PMNs leading to inhibition of their efferocytic uptake by macrophages. This could have implications in acute and chronic airway inflammatory diseases such as pneumonia, sepsis, cystic fibrosis, and COPD.

Abstract A126: Taking out the trash: Efferocytosis in the tumor microenvironment

Abstract We have examined the malignant tumor microenvironment (TME) from a perspective that is often noted but overlooked: tissue repair. It has long been observed that tumors resemble ‘wounds that do not heal,’ but the reasons remain obscured. We found that macrophages in the tumor microenvironment orchestrate wound healing and immune tolerance in response to tumor cell death, a feature common to all solid tumors. We used genetic and pharmacological methods to block macrophage-mediated engulfment of dying tumor cells, a process referred to as efferocytosis. We found that MerTK-mediated efferocytosis of dying tumor cells induces macrophage-mediated production of Th2-like cytokines, including IL-4, IL-13, IL-10, and TGF-beta. Blockade of the receptor tyrosine kinase MerTK through pharmacological tyrosine kinase inhibition or through genetic MerTK abalation blocked macrophage-mediated efferocytosis in the tumor microenvironment, impairing M2-like macrophage polarization and expression of Th2-like cytokines in the tumor microenvironment. Because cell death is a major histological feature of the breast during post-partum involution of milk-producing breast epithelia, we studied the impact of MerTK-mediated efferocytosis in a model of post-partum breast cancer (ppBC). Currently, ppBC accounts for nearly 25% of all breast cancers in young (pre-menopausal) women. In contrast to breast cancers diagnosed during pregnancy, which correlate with a favorable prognosis, ppBCs are highly aggressive, metastatic, and life-threatening, even when corrected for molecular breast cancer subtype and for the age of the woman at diagnosis. We used genetic and pharmacological methods to block MerTK-mediated efferocytosis in ppBCs, demonstrating that efferocytosis increased ppBC metastasis by nearly 10-fold, but that genetic or pharmacological MerTK inhibition markedly decreased metastasis in ppBCS. Similarly, targeted inhibition of efferocytosis-induced cytokines, including TGF-beta, substantially reduced metastasis of efferocytosis-competent ppBCs. Collagen deposition, TReg expansion, and Th2 cytokine upregulation were found to be dependent on efferocytosis-induced TGF-beta signaling. These findings in ppBC, in which widespread tumor cell death was initiated by physiological post-partum involution, prompted our interest in the impact of therapeutically-induced cell death and subsequent efferocytosis on pro-malignant leukocyte behaviors in the tumor microenvironment. As many as 70-80% of primary breast tumors treated with cytotoxic chemotherapy show a partial response in the pre-surgical (neoadjuvant) setting, and lack of a pathological complete response (pCR) is a strong predictor of tumor recurrence following surgical tumor excision. We tested the hypothesis that efferocytosis, the immune system's inherent response to cell death, results in tumor repair (increased ‘wound healing’) in response to cytotoxic treatment. In support of this hypothesis, a single dose of doxorubicin or lapatinib induced a transient wave tumor cell death within 24 hours, followed at post-treatment day 4 with increased M2 macrophages and TRegs within the tumor microenvironment, and increased expression of Th2-like cytokines. Importantly, increased metastases were seen in tumor-bearing mice treated once with doxorubicin over what was seen in untreated mice. However, targeted inhibition of efferocytosis using a MerTK kinase inhibitor (TKIs) in combination with doxorubicin blocked expression of Th2-like cytokines, limited the number of M2-like macrophages in the tumor microenvironment, and prevented therapy-enhanced tumor metastasis. These findings support continued investigation into the role of MerTK and efferocytosis in therapeutic tumor responses. Note:This abstract was not presented at the conference. Citation Format: Rebecca S. Cook. Taking out the trash: Efferocytosis in the tumor microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A126.

G protein α12 (Gα12) is a negative regulator of kidney injury molecule-1-mediated efferocytosis.

Kidney injury molecule-1 (KIM-1) is a receptor for the "eat me" signal, phosphatidylserine, on apoptotic cells. The specific upregulation of KIM-1 by injured tubular epithelial cells (TECs) enables them to clear apoptotic cells (also known as efferocytosis), thereby protecting from acute kidney injury. Recently, we uncovered that KIM-1 binds directly to the α-subunit of heterotrimeric G12 protein (Gα12) and inhibits its activation by reactive oxygen species during renal ischemia-reperfusion injury (Ismail OZ, Zhang X, Wei J, Haig A, Denker BM, Suri RS, Sener A, Gunaratnam L. Am J Pathol 185: 1207-1215, 2015). Here, we investigated the role that Gα12 plays in KIM-1-mediated efferocytosis by TECs. We showed that KIM-1 remains bound to Gα12 and suppresses its activity during phagocytosis. When we silenced Gα12 expression using small interefering RNA, KIM-1-mediated engulfment of apoptotic cells was increased significantly; in contrast overexpression of constitutively active Gα12 (QLGα12) resulted in inhibition of efferocytosis. Inhibition of RhoA, a key effector of Gα12, using a chemical inhibitor or expression of dominant-negative RhoA, had the same effect as inhibition of Gα12 on efferocytosis. Consistent with this, silencing Gα12 suppressed active RhoA in KIM-1-expressing cells. Finally, using primary TECs from Kim-1+/+ and Kim-1-/- mice, we confirmed that engulfment of apoptotic cells requires KIM-1 expression and that silencing Gα12 enhanced efferocytosis by primary TECs. Our data reveal a previously unknown role for Gα12 in regulating efferocytosis and that renal TECs require KIM-1 to mediate this process. These results may have therapeutic implications given the known harmful role of Gα12 in acute kidney injury.

Abstract 63: MerTK and Mfge8-Dependent Efferocytosis Orchestrates Postischemic Cardiac Healing Through VEGF-A and Il-10 Regulation

Inflammation orchestrates tissue remodeling after myocardial infarction (MI). Efferocytosis is mainly carried out by the membrane protein tyrosin kinase mertk (MerTK) and the milk fat globule epidermal growth factor (Mfge8) and may balance inflammation resolution after MI. We reasoned that efficient MerTK and Mfge8-dependent clearance of apoptotic debris by infiltrated myeloid cells is critical to the angiogenic and regenerative fine-tuning of the ischemic cardiac tissue. We first demonstrated that the left ventricle ejection fraction assessed by echocardiography and the number of capillaries were decreased by around 1.5-fold in MerTK -/-, Mfge8 -/- and double deficient mice when compared to wild-type animals (WT), 14 days after the onset of ischemia (p<0.01, n=10 per group). These effects were associated with an increased infarct size, fibrotic area as well as apoptotic cells number and were also observed in irradiated WT mice transplanted with bone marrow-derived cells isolated from MerTK -/-, Mfge8 -/- and double deficient animals. In cardiac tissue of WT CD45.1 recipient mice, the number of infiltrated CD45.2 MerTK -/-/Mfge8 -/- bone marrow-derived cells was similar to that of CD45.2 WT cells. In addition, the amount of Ly6Chigh and Ly6Clow monocytes as well as that of macrophages was unaffected in infarcted heart of MerTK -/-/Mfge8 -/- bone-marrow transplanted compared to WT mice. In contrast, deficient efferocytosis was related to an increased number of cardiac annexinV-positive microparticles and a downregulation of the pro-angiogenic growth factor VEGF-A and the anti-inflammatory cytokine IL-10 protein levels. Notably, FACS-sorted Ly6Clow monocytes from ischemic cardiac tissue of MerTK -/-/Mfge8 -/- mice expressed lower levels of VEGF-A when compared to WT cells. Hence, inhibition of efferocytosis leads to an adverse cardiac remodeling after MI through phenotypic alterations of phagocytic cells.

Signaling via Macrophage G2A Enhances Efferocytosis of Dying Neutrophils by Augmentation of Rac Activity

Phosphatidylserine (PS) and oxidized PS species have been identified as key ligands on apoptotic cells important for their recognition and removal (efferocytosis) by phagocytes, a requisite step for resolution of inflammation. We have recently demonstrated that lysophosphatidylserine (lyso-PS) generated and retained on neutrophils following short term activation of the NADPH oxidase in vitro and in vivo enhanced their clearance via signaling through the macrophage G-protein-coupled receptor G2A. Here, we investigated the signaling pathway downstream of G2A. Lyso-PS, either made endogenously in apoptosing neutrophils or supplied exogenously in liposomes along with lyso-PS(neg) apoptotic cells, signaled to macrophages in a G2A-dependent manner for their enhanced production of prostaglandin E2 (PGE2) via a calcium-dependent cytosolic phospholipase A2/cyclooxygenase-mediated mechanism. Subsequent signaling by PGE2 via EP2 receptors activated macrophage adenylyl cyclase and protein kinase A. These events, in turn, culminated in enhanced activity of Rac1, resulting in an increase in both the numbers of macrophages efferocytosing apoptotic cells and the numbers of cells ingested per macrophage. These data were surprising in light of previous reports demonstrating that signaling by PGE2 and adenylyl cyclase activation are associated with macrophage deactivation and inhibition of apoptotic cell uptake. Further investigation revealed that the impact of this pathway, either the enhancement or inhibition of efferocytosis, was exquisitely sensitive to concentration effects of these intermediaries. Together, these data support the hypothesis that lyso-PS presented on the surface of activated and dying neutrophils provides a tightly controlled, proresolution signal for high capacity clearance of neutrophils in acute inflammation.

Urokinase-Type Plasminogen Activator Inhibits Efferocytosis of Neutrophils

Phagocytosis of apoptotic cells, also called efferocytosis, plays an essential role in the resolution of inflammation. Urokinase-type plasminogen activator (uPA) is a multifunctional protein that has been implicated in inflammatory conditions, including pneumonia and severe infection, which are often accompanied by the development of acute lung injury. However, the role of uPA in modulating efferocytosis of apoptotic neutrophils has not been defined. To characterize the role of uPA in regulation of efferocytosis and to delineate the underlying mechanisms involved in this process. In vitro and in vivo phagocytosis, immunoprecipitation, and Western blotting assays. The phagocytosis of apoptotic neutrophils by macrophages was significantly inhibited by uPA. Mutant uPA lacking the growth factor domain and catalytically inactive uPA had similar inhibitory effects on efferocytosis, as did wild-type uPA. In contrast, absence of the kringle domain abrogated the ability of uPA to diminish efferocytosis. Both the α(V)β₃ integrin and vitronectin seemed to be involved in the inhibition of efferocytosis by uPA. Incubation of macrophages with uPA also diminished activation of the small GTPase Rac-1, which normally occurs during ingestion of apoptotic neutrophils. Under in vivo conditions in the lungs, uPA decreased the uptake of apoptotic neutrophils by alveolar macrophages. Our data demonstrate a novel role for uPA in which it is able to diminish the uptake of apoptotic neutrophils by macrophages under both in vitro and in vivo conditions.

Macrophage phagocytosis of apoptotic neutrophils is critically regulated by the opposing actions of pro‐inflammatory and anti‐inflammatory agents: key role for TNF‐α

Apoptosis of inflammatory cells and their subsequent clearance (efferocytosis) by macrophages (Mphis) are key mechanisms orchestrating successful resolution of inflammation. Although the powerful proinflammatory agents lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-alpha) influence rates of inflammatory cell apoptosis, little is known about their effects on efferocytosis. We have demonstrated that LPS and TNF-alpha potently inhibit efferocytosis of neutrophils by monocyte-derived Mphis. Inhibition was both concentration and time dependent, although the effect of TNF-alpha was more rapid. We have found that soluble TNF receptor-I attenuated LPS inhibition of phagocytosis, indicating that TNF-alpha production is critical. Inhibition of efferocytosis by LPS was found to be positively associated with Mphi production of TNF-alpha, but negatively with interleukin-10 (IL-10) release. A critical role for IL-10 in the regulation of phagocytosis was suggested by 2 important findings: LPS inhibition was observed more rapidly in the presence of an anti-human IL-10 receptor-alpha antibody, and efferocytosis by IL-10-deficient Mphis was markedly reduced compared to wild-type Mphis. Furthermore, exogenous IL-10 and glucocorticoids reversed inhibitory effects of LPS on efferocytosis via suppression of TNF-alpha production. We suggest that efferocytosis is regulated in an autocrine manner by pro- and anti-inflammatory mediators, and the inflammatory milieu determines whether inflammation successfully resolves.