Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer

Abstract

Apoptosis is an evolutionarily conserved and tightly regulated cell death modality. It serves important roles in physiology by sculpting complex tissues during embryogenesis and by removing effete cells that have reached advanced age or whose genomes have been irreparably damaged. Apoptosis culminates in the rapid and decisive removal of cell corpses by efferocytosis, a term used to distinguish the engulfment of apoptotic cells from other phagocytic processes. Over the past decades, the molecular and cell biological events associated with efferocytosis have been rigorously studied, and many eat-me signals and receptors have been identified. The externalization of phosphatidylserine (PS) is arguably the most emblematic eat-me signal that is in turn bound by a large number of serum proteins and opsonins that facilitate efferocytosis. Under physiological conditions, externalized PS functions as a dominant and evolutionarily conserved immunosuppressive signal that promotes tolerance and prevents local and systemic immune activation. Pathologically, the innate immunosuppressive effect of externalized PS has been hijacked by numerous viruses, microorganisms, and parasites to facilitate infection, and in many cases, establish infection latency. PS is also profoundly dysregulated in the tumor microenvironment and antagonizes the development of tumor immunity. In this review, we discuss the biology of PS with respect to its role as a global immunosuppressive signal and how PS is exploited to drive diverse pathological processes such as infection and cancer. Finally, we outline the rationale that agents targeting PS could have significant value in cancer and infectious disease therapeutics.

Figure 1

Molecular structure of PS and major biosynthetic and degradative pathways: (a) PS is comprised of a glycerol backbone esterified at the sn-1 and sn-2 carbons of the glycerol moiety with two fatty acyl chains of variable length and saturation, as well as a phosphate linkage at the sn-3 position covalently linked to serine (a). In eukaryotic cells, PS is synthesized from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by PSS1 and PSS2, respectively, and can be catabolized by phosphatidylserine decarboxylase (PSD) to generate PE. (b) During apoptosis and cell stress, PS is externalized to the outer surface of the plasma membrane, where it can be detected by fluorophores such as FITC-annexin V or GFP-lactadherin 3 (green). Red staining indicates Rhod-2AM that monitors intracellular Ca2+ levels, which are elevated during apoptosis and during cell stress

Figure 2

Models for the different forms of PS externalization: As noted in the text, PS can be externalized under a variety of physiological and patho-physiological conditions that include platelet activation (a) and caspase-dependent apoptosis (b). (a) Activated platelets promote a Ca²⁺-TMEM16-mediated externalization of PS that serves as a nucleation scaffold for the recruitment of hemostasis factors that initiate blood clotting (indicated by the solid black line in a). (b) Apoptotic cells externalize PS via the caspase 3/7-mediated cleavage of Xkr8 that serves as an eat-me signal for various PS receptors (TAMs, TIMs, and αvβ5 and αvβ3 integrins). Recent studies suggest that during apoptosis, the surface density of the PS may reach a critical threshold that clusters and activates PS receptors. Why PS externalized on apoptotic cells (Xkr8-dependent) serves as a signal for efferocytosis, while PS expressed on stressed and activated cells (TMEM16-dependent) has not been completely elucidated

Figure 3

The PS receptor Mertk acts as an inhibitory receptor to promote homeostasis and tissue tolerance: Mertk, a member of the TAM family of PS receptors, interacts with externalized PS on apoptotic cells via its bridging molecule Gas6 to drive efferocytosis and tissue tolerance. Once engaged, Mertk transmits an inhibitory signal that inhibits NF-kB and the production of inflammatory cytokines from TLRs. Efficient efferocytosis also produces the production of tolerogenic factors such as IL-10 and TGF-β that tolerize the local microenvironment in favor of M2 macrophages, immature DCs, and Tregs. When Mertk is targeted by knockout, or inhibited by therapeutics, TLR-induced activation of inflammatory cytokines proceeds unabated in the absence of dampening signals, leading to an immunogenic environment characterized by the production of M1 macrophages, antigen presenting mature DCs and CD8⁺ T cells as discussed throughout the text

Figure 4

Modalities of PS exposure on infectious agents: (a). Silent Phagocytosis Clearance of PS-exposing particles (e.g., apoptotic remnants), via silent phagocytosis of monocytes/macrophages. (b). Energy loss Diminished energy reserves cause breakdown of membrane asymmetry and lead to PS exposure on infected monocytes (e.g., HCV). Enveloped Virions budding from infected cells expose PS. (c) Direct exposure of PS on HIV-1, Ebola, Variola and other highly pathogenic enveloped viruses. (1) Replication and budding from PS-rich surfaces (e.g., golgi apparatus, endoplasmic reticulum) allows PS exposure on newborn virions. (2) Clusters of Enteroviruses are packed and released non-lytically in PS-exposing lipid vesicles elevating their infectivity. (d) Bystander exposure of PS (Trypanosoma brucei) T. brucei evolutive forms do not expose PS. Parasites are engulfed as bystanders together with PS-exposing apoptotic cell remnants. (e) Trojan horse (Leishmania) (1) Upon infection of the host PS-exposing Leishmania promastigotes are engulfed primarily by neutrophils. (2) ‘Apoptotic', PS-exposing Leishmania promastigotes induce release of TGF-β by neutrophils silencing their leishmanicidal response at the side of the sand-fly bite. (3) Infected PS-exposing Neutrophils are silently phagocytosed by monocytes enabling intracellular replication. Hiding of promastigotes in ‘apoptotic' neutrophils not only delivers viable Leishmania into macrophages but also delays the immune response against the parasite until the first line neutrophilic response is resolved. (4) PS-exposing Leishmania promastigotes can actively invade into monocytes for intracellular replication. (5) Persistent infection and intracellular replication in the amastigote form within professional phagocytes

Figure 5

Apoptotic death versus apoptotic mimicry during leishmanial infection and establishment. Metacyclic promastigotes accumulate in the sand-fly hindgut. The infective inoculum contains live parasites together with morphologically and biochemically characterized apoptotic parasites. The presence of apoptotic and viable parasites is necessary for the establishment of the infection. Live parasites infect host cells, whereas dead parasites downregulate the production of nitric oxide (left panel). In the mammalian host, amastigote forms disseminate the disease and expose PS at their surface without any other phenotype of apoptotic death (center panel). PS recognition by the macrophage leads to an active anti-inflammatory response, mainly characterized by TGF-β and IL-10 production. This generates a feedback effect leading to increased macrophage deactivation and parasite proliferation. Susceptible mice strains upregulate PS exposure on intracellular amastigotes by a mechanism yet to be defined (right panel)

Figure 6

PS targeting antibodies selectively target the tumor microenvironment. Localization of near-infrared (NIR)-labeled Bavituximab F(ab)₂ to orthotopically implanted PC3 prostate tumor in male SCID mice. Animals were injected with 25 μg NIR-PGN650 (a) or NIR-control IgG F(ab')₂ (b). Fluorescent imaging was conducted 24 h following injection of NIR-labeled antibodies. Anti-PS antibodies specifically localize to tumor blood vessels (c)

Figure 7

Antibody-mediated blockade of the PS signaling pathway in the tumor microenvironment. As described in the text, PS is highly dysregulated in the tumor microenvironment by the combined action of a oxidative stress and immature tumor vasculature, the secretion of PS-positive tumor exosomes, and the high apoptotic index of proliferating tumors. PS-targeting antibodies are thought to bind externalized PS and interfere with the inhibitory functions of PS in the tumor microenvironment by inhibiting PS binding to PS receptors and by Fcγ-mediated ADCC. The net effect is to activate immunogenic signals in the tumor microenvironment