Phagocytosis comes from Ancient Greek phagein (φαγεῖν) and kytos (κύτος) meaning “to eat” and “cell”. It is the process allowing cells to engulf particles larger than 200 nm. The term was coined by Carl Friedrich Wilhelm Claus to describe the set of cells capable of absorbing and degrading citrus thorns inserted into starfish larvae observed in 1882 by ÉlieMechnikov.
In human cells, phagocytosis role extends beyond immunity, for example it prevents severe anemia by enabling clearance of erythroblast nuclei (Kawane et al., 2001), it prevents toxic iron deposits in the kidney by removing senescent erythrocytes (Theurl et al., 2016), it enables correct synaptogenesis by pruning the synapses that are superfluous or inactive to optimize neuronal communication (Petanjek et al., 2011) and prevents permanent brain damage by clearing cellular debris during brain ischemia (Morizawa et al., 2017), it clears the lumen of seminiferous tubules from apoptotic cells for efficiency spermatogenesis (Shaha et al., 2010), and it even prevents blindness by clearing the shed outer segments of photoreceptor cells in the retina (Nandrot et al., 2000). Underlying this plethora of roles, we still find common cellular mechanisms.
At the cellular level, phagocytosis is a three step process: first, receptors present at the cell surface recognize the foreign particle; second, a local remodeling of the cytoskeleton and plasma membrane is induced by a signaling cascade, and forms the phagocytic cup around the foreign particle; third, the phagocytic cup closes and forms an intracellular compartment called a phagosome (Desjardins et al., 2005) (Fig.2).
Fig.2 Phagocytosis – General overview
Phagocytosis is a three step process: first, receptors present at the cell surface recognize the foreign particle (in orange);
second, a local remodeling of the cytoskeleton and plasma membrane is induced by a signaling cascade, and forms the phagocytic cup around the foreign particle; third, the phagocytic cup closes and forms an intracellular compartment called a phagosome,
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In D. discoideum, phagocytosis, like for numerous unicellular organisms, is mainly used for feeding purposes.
D. discoideum cells mainly phagocytose bacteria, and the same general steps are conserved. Only internalization of D. discoideum lectin (Discoidin I) coated bacteria differs from the typical course of phagocytosis and phagosome maturation. Discoidin-coating of extracellular bacteria protects them from IC killing (Dinh et al., 2018). When fluorescent Discoidin-coated E. coli were fed to D. discoideum, the fluorescent signal within the cells persisted on average 25 min, whereas it persisted 4 min with fluorescent non-coated E. coli. The results indicated that E. coli survived longer within the phagosomes when coated by Discoidin I. The authors hypothesized that Lectin-induced modified bacterial internalization can be distinguished from phagocytosis and has a different maturation process. One explanation could be that during starvation, it might be beneficial to store bacteria as food and delay their IC killing, to provide germinating spores with a readily available food source.
Binding and recognition of particle
Human phagocytic cells exhibit at the plasma membrane, endosomal membranes and in the cytosol, specific pattern recognition receptors (PRR). These PRRs recognize either damage-associated molecular pattern (DAMP) molecules or microbial-associated molecular pattern (MAMP) molecules (Tang et al., 2012) (Fig.3).
More specifically MAMPs ranges from components of the peptidoglycan cell wall to nucleic acids. Four types of PRRs directly bind them: toll-like receptors (TLRs), integrins, scavenger receptors, C-type lectins (Iwasaki et al., 2015). Another set of receptors, called opsonic receptors, recognize host-derived opsonins, from the Greek opsōneîn “to prepare for eating”, bound to foreign microorganisms. Opsonic receptors include FcγR (antibodies), CR3 (complement), Integrin alpha-5/beta-1 receptor (fibronectin), and Integrin alpha-v/beta-3 and alpha-v/beta-5 receptors (lactadherin) (Liu et al., 2013, Freeman et al., 2014) (Fig.3). Heterologous expression of lectins, scavenger receptors, integrins and FcγR in non-phagocytic cells is sufficient to trigger phagocytosis;
TLRs on the other hand do not (Flannagan et al., 2012). Although TLRs do not directly trigger phagocytosis, they play two important roles. First, they stimulate phagocytosis: incubation of macrophages with the TLRs ligands increases bacterial phagocytosis and conversely presence of TLRs antagonists reduces the phagocytic rate of the macrophages (Doyle et al., 2004). Second, TLRs activation induces a signaling cascade triggering inflammation, another innate immune system mechanism. In short, upon binding to MAMPs, TLRs recruit adaptor molecules possessing a Toll–interleukin 1 receptor (TIR) domains such as MyD88, Mal, TRIF, TRAM, and SARM. In turn, TIR adaptors activation leads to stimulation of mitogen activated protein kinases (MAPKs) and NF-κB, eventually upregulating phagocytosis and inflammation-related genes (Cen et al., 2018). Not all plasma membrane located PRRs are direct phagocytic receptors, but all do trigger a signaling cascade leading directly or indirectly to phagocytosis.
In D. discoideum opsonin-independent phagocytosis has been described: five integrin-like protein (Sib family), and three scavenger-like receptors (Lmp family) have been identified and characterized (Cornillon et al., 2006, Sattler et al., 2018) (Fig.4). The Sib family, which comprises 5 members (SibA-E), shows similarity with integrin β. Among them, SibA and SibC are involved directly in adhesion to substrate, beads and bacteria.
Moreover, all Sib proteins interact directly, via their membrane-proximal NPxY motif, with TalinA, an actin-binding protein (Cornillon et al., 2006, Cornillon et al., 2008). Both features make SibA and SibC good phagocytic receptors candidates. However, we should take into consideration that TalinA is mostly found at the posterior cortex during migration, whereas a recent study would suggest that TalinB, the second homolog of Talin in D. discoideum, is restricted to the leading edge of migrating cells. Compared to TalinA, TalinB contains a I/LWEQ domain and a villin headpiece domain in its C-terminal actin-binding region which seem to play a role in both TalinA and B segregation (Tsujioka et al., 2019). Phagocytic receptors binding TalinB could be better phagocytic receptor candidates as phagocytosis compares to a directed migration over a surface. SibA and C binding affinity to TalinB have not been measured yet. Concerning the Lmp family, which comprises 3
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members (LmpA-C), they are homologs of LIMP-2/CD36 scavenger receptors. LmpA and C are found in endosomes and lysosomes similar to LIMP-2, and LmpB is present at the cell surface like CD36 (Sattler et al., 2018). LmpA genetic inactivation induces a severe defect in phagocytosis for beads as well as Gram+ and Gram- bacteria, whereas LmpB inactivation specifically inhibits uptake of mycobacteria and B. subtilis. Most likely LmpA affects actin dynamic as lmpA KO cells have a massive decrease of motility, and LmpB regulates phagocytosis triggering as binding to bacteria is not affected (Sattler et al., 2018). LmpB is an interesting PRR candidate, specific for Gram+ bacteria, and LmpA play a more global role in phagocytosis, motility and even phagosome maturation, which is heavily perturbed in lmpA KO cells.
Fig.3 Phagocytosis initiation in macrophages
1) In the case of opsonin-dependent phagocytosis: antibodies (Ab) and C3bi, opsonizing the bacteria, are bound respectively by FcR and CR3. In Ab bound-state, the FcR is quickly phosphorylated, Syk recognizes the phosphorylated immunoreceptor tyrosine-based activation motifs.
Activation of Syk, further stimulates RAC, which in turn activates the actin-branching Arp 2/3 complex.
In the case of C3bi binding to CR3, activation of CR3 stimulates PI3K activity, leading to PIP2
formation with binds WASP relieving inhibition of Arp 2/3.
2) In the case of opsonin-independent phagocytosis: the MAMPs are directly recognized by the receptors. Both integrins and scavenger receptors stimulate Src kinases after binding. Src kinases activates Rho, which will release the WASP inhibition on Arp2/3 to allow actin-branching. The represented scavenger is class A member MARCO which binds bacterial CpG DNA.
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Interestingly in D. discoideum no C-type lectins, nor TLR enchased in the plasma membrane have been characterized so far (Fig.4). Concerning TLRs, two cytosolic proteins in D. discoideum contain toll/interleukin 1 receptor (TIR) domains: TirA and TirB. TIR domains are a hallmark of TLRs, but neither TirA nor TirB contain both TLR canonical domains: leucine-rich repeats (LRR) and transmembrane domains. Despite not displaying all the features of a TLR, TirA still plays a role in efficient IC killing: tirA mutant cells exposed to an avirulent strain of Legionella exhibit a growth defect (Chen et al., 2007).
D. discoideum cells, unlike in human cells, use a single G-protein coupled receptor (GPCR), fAR1, to induce phagocytosis following binding to folate and lipopolysaccharide (LPS) (Pan et al., 2016, Pan et al., 2018) (Fig.4). Folate and LPS are two classical MAMPS: Folate is secreted by many bacterial species such as K.
pneumoniae and is a strong chemoattractant for D. discoideum and LPS are a component on the bacterial outer membrane. In human cells, LPS binds TLR4 and folate binds folate receptors, which are cell surface glycosylphosphatidylinositol-anchored glycoproteins. In contrast, D. discoideum cells use a single receptor for the two MAMPS. In addition, fAR1 is also different from other chemoattractant GPCRs: it belongs to the class C GPCR family and exhibits a VFT extracellular domain for sensing multiple ligands (Pan et al., 2018). The main features of fAR1 is that it binds folate and LPS and also mediates engulfment of K. pneumoniae, as far1 KO cells exhibit a specific phagocytosis defect (Pan et al., 2016). fAR1 is the only known GPCR bridging chemotaxis and phagocytosis.
Fig.4 Phagocytosis initiation in Dictyostelium discoideum
Like in macrophages, MAMPs are directly recognized by their cognate receptors eventually triggering phagocytosis. SibA shares similarities with mammalian β integrin: notably an extracellular Von Willebrandt A domain, a glycine-rich transmembrane domain and a highly conserved cytosolic domain interacting with TalinA. LmpB is a scavenger receptor similar to the mammalian CD36 scavenger receptor. fAR1 is a GPCR, binding folate and LPS directly inducing phagocytosis.
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Phagocytic cup: formation
The membrane cytoskeleton of human phagocytes is very dynamic, even in its “resting” state before induction of phagocytosis. In FRAP experiments, locally photobleached GFP-tagged cytoskeleton components (actin, α-actinin, ezrin and myosins) are replaced by their fluorescent counterparts within seconds. Cytoskeleton core components have a turn-over of a few seconds (Fritzsche et al., 2013). The dynamic remodeling activity of the cytoskeleton relies on a competition between two polymerizing F-actin networks: an actin branching system relying on Arp2/3, and a mesh system relying on Rho/formin/myosin (Lomakin et al., 2015). For phagocytosis to happen, the balance needs to tilt towards Arp2/3 branched structures, as formin-polymerized actin immobilizes transmembrane proteins obstructing membrane ruffling and the diffusion of non-tethered membrane proteins and even lipids (Ostrowski et al., 2016).
Tilting the balance begins by activation of the Src-Family Kinase (SFK), after the binding of the microorganism to PRRs. Activation of the SFKs recruits locally Syk, which in turn phosphorylates adaptors, kinases, and lipases. The unstimulated Arp2/3 complex is weakly active and requires stimulation by nucleation promoting factors (NPFs) to branch a novel actin filament at a 70° angle from an existing one (Goley and Welch, 2006).
Members of the Wiskott-Aldrich syndrome protein (WASP) family are known to be major enhancers of the Arp2/3 complex activity (Politt and Insall, 2009). The WASP family contains two principal classes of proteins : WASPs and SCAR/WAVEs. Conformation change of WASPs proteins from inactive to active is triggered by binding to PIP2, a phospholipid, whereas the Scar/WAVE complex is activated by the Rho GTPase Rac. Once activated both classes of proteins enhance Arp2/3 branching activity (Fig.5).
Remodeling the cytoskeleton contributes to an increased diffusion of non-engaged PRRs, thus favoring the formation of novel focal points of adhesion to the target. Moreover, further activation of integrins via Rap GTPases, downstream of Syk activation, provides a linkage between the target and the actin cytoskeleton via talin, kindlin, vinculin, and other associated proteins. The integrins engaged and tethered to the actin cytoskeleton are in fact sufficient to form diffusional barriers for a majority of protein and even lipid rafts (Maxson et al., 2018). The diffusional barrier is necessary to delimits a perimeter for optimal phagocytosis.
Furthermore, if the target is too voluminous, presence of the diffusional barrier enables the maturation of the open tubular phagosomes while limiting a potential inflammation through the leakage of its content (Maxson et al., 2018).
In addition to the Integrin-based diffusion barrier, the protrusion is consolidated by Bin-Amphiphysin-Rvs167 (BAR) domain containing proteins. The BAR proteins are involved in stabilizing membrane curvature.
Additionally, they bind directly the WASP family proteins, including N-WASP and WAVE, which then activate the Arp2/3 complex (Hanawa-Suetsugu et al., 2019).
Concerning the phagocytosis machinery in D. discoideum, overall the protrusion extension mechanism is very similar to that used by mammalian cells. MAMPs binding to PRRs leads to the stimulation of the SCAR/WAVE complex by Rac homologs, notably Rac1. The SCAR/WAVE complex activates Arp2/3 leading to actin branching that drives the formation of the phagocytic cup (Rivero and Xiong, 2016) (Fig.6). Similarly, segregation of membrane proteins is visible in the phagocytic cup by a mechanism restricting lateral diffusion (Mercanti et al., 2006). Additionally, BAR proteins stabilize the phagocytic cup. Among the BAR proteins in D. discoideum, only one localizes specifically at the protrusive rim of the cup and regulates both Rac and Ras activity: RGBARG. RGBARG induces protrusion formation by activating RacG, whilst restricting expansion of the cup interior by locally inhibiting Ras activity (Buckley et al., 2019).
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Phagocytic cup: guiding and closure
In addition to PRRS, membrane lipids are actively involved in phagocytosis (Bohdanowicz et al., 2013).
Phosphoinositides (PIs) are phospholipids that can be phosphorylated at different positions (3, 4 or 5) of their inositol head group. PIP2 and PIP3 play essential functions in phagocytosis. PIP2, mostly localized to the inner plasma membrane leaflet and constitutes 1–2% of plasma membrane lipids. PIP2 recruits and binds actin-binding proteins driving local actin branching (Mu et al., 2018). Conversion of PIP2 to PIP3, by the action of the PI3 kinase at the tip of the protrusion recruits myosins which exert contractile activity and function as a purse string to facilitate phagosome closure (Ostrowski et al., 2016). Phagosome closure eventually needs dephosphorylation of PIP3 and disappearance of PIP2 mediated by respectively by OCRL and Inpp5B (Bohdanowicz et al., 2012).
Fig.5 Molecular mechanism of phagocytosis in macrophages
1) Pseudopod extension. The extension is driven by actin polymerization by the Arp 2/3 complex. Activation of the Arp 2/3 complex depends on both relieving WASP inhibition and activating the Scar/Wave complex. WASP inhibition is relieved by binding PIP2. Scar/Wave complex activation is induced by RAC. RAC is notably stimulated after the production of PIP3 which results from the phosphorylation of PIP2 by PI3K. Finally, membrane supply, to support the extension of the phagosome cup, are delivered via fusion in the actin free bottom of the phagocytic cup.
2) Phagosome closure. PIP3 and PIp2 are depleted at the tip of the pseudopod by OCRL and Inpp5B, reinstating WASP inhibition on Arp 2/3. OCRL is bound to the cytoskeleton by PADDL and the myosin located at the tip of the pseudopod generated the contractile force to close the phagosome.
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Additionally, PIP3 also recruits Rho GAPs that inactivate Rac1, terminating actin polymerization at the base of the phagocytic cup. Concomitant cleavage of PIP2, to DAG and IP3, by phospholipase C (PLC), also releases actin-binding proteins such as cofilin, WASp, and ezrin at the base of the phagocytic cup. The joint action of Rho GAPs and PLC, leading to the loss of actin at the base of the phagocytic cup, allows for fusion of vesicles with the plasma membrane (Niedergang and Chavrier, 2004). In a nutshell, PIP regulation is crucial to the protrusion formation, phagosome sealing and membrane fusion at the nascent phagosome base (Fig.5).
Interestingly PIP2 is sensitive to the membrane curvature. New evidence suggests that it could autonomously induce receptor-independent phagocytosis. Binding of a solid particle curves the membrane, which induces a local sorting of PIP2. PIP2 recruits and activate moesin in turn binding Syk and leading to receptor-independent phagocytosis (Mu et al., 2018). The authors hypothesized that this mechanism it may explain how phagocytes recognizes almost all variations of solid.
In D. discoideum, the role of phosphoinositides is also established. PIP2 accumulates near engaged PRRs and recruits NPFs and actin-binding proteins involved in membrane deformation in a similar fashion. PIP2 is also phosphorylated by PI3K and cleaved by PLC. The PIP2 decrease allows actin disassembly at the base of the phagocytic cup for vesicle fusion. Regarding phagosome closure, Dd5P4, the Dictyostelium homolog of human OCRL, dephosphorylates PIP3 into PIP2 which is an important step for the phagosome closure (Loovers et al., 2007). In conclusion, similar to mammalian phagocytic cells, PIP2 decrease allows actin disassembly and PIP3
dephosphorylation is also necessary for phagosome closure (Fig.6).
Phosphoinositides also plays a role in phagosome maturation in both human phagocytes and D. discoideum cells and will be subsequently described (Buckley et al., 2019, Luscher et al., 2019).
Fig.6 Molecular mechanism of phagocytosis in Dictyostelium discoideum
The phagocytic cup extension is, like in macrophages, driven by actin polymerization catalyzed by the Arp 2/3 complex. In a similar fashion activation of the Arp 2/3 complex requires to relieve WASP inhibition and promote the Scar/Wave complex activation. WASP inhibition is relieved by PIP2 binding. Scar/Wave complex activation is done by RAC1. RAC1 is stimulated after production of PIP3. PIP3 production is catalyzed by PI3K from PIP2. Membrane supply to support the extension are delivered via fusion in the actin free bottom of the phagocytic cup. At the tip of the pseudopod Dd5P4, the OCRL ortholog participates in reinstating WASP inhibition on Arp 2/3. Myosin located at the tip of the pseudopod generates the contractile for to close the phagosome. MyoVII is probably the main driving force.
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