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Phagocytic Integrins: Activation and Signaling

Phagocytic integrins are endowed with the ability to engulf and dispose of particles of different natures. Evolutionarily conserved from worms to humans, they are involved in pathogen elimination and apoptotic and tumoral cell clearance. Research in the field of integrin-mediated phagocytosis has shed light on the molecular events controlling integrin activation and their effector functions. However, there are still some aspects of the regulation of the phagocytic process that need to be clarified. Here, we have revised the molecular events controlling phagocytic integrin activation and the downstream signaling driving particle engulfment, and we have focused particularly on αMβ2/CR3, αXβ2/CR4, and a brief mention of αVβ5/αVβ3integrins.

Integrins are characterized by requiring activation to be functional. This review has focused on the main events determining β 2 integrin activation and downstream signaling in relation to cytoskeletal remodeling and particle engulfment, and it makes a special mention of the main differences between other phagocytic integrins, especially those involved in apoptotic cell clearance.

INTEGRIN STRUCTURE AND ACTIVATION
Phagocytic integrins are heterodimeric (α and β subunit) receptors. Subunits are divided into ectodomains, a transmembrane helix, and short cytoplasmic tails. The α-subunit ectodomains contain Mg 2+ -binding metal-ion-dependent adhesive sites (MIDAS) and Adjacent to MIDAS (AdMIDAS), which binds inhibitory Ca 2+ or activating Mn 2+ (46,47). Ligand binding can occur either at the αI-domain (α-subunit) in α X , α M , and α 2 or at the α/β-chain interface in integrins without the αI domain ( Figure 1A, Table 1). Integrins are tightly regulated by conformational changes, a hallmark of which is cytoplasmic tail separation (48). Integrin conformations are described according to the state of the headpiece (open/closed; H + /H − ) and leg ectodomains (extended/bent; E + /E − ) (49). Resting integrins remain in an inactive/"bent" (E − H − ) conformation with the lowest free energy (−4.0 kcal/mol for α 5 β 1 ) with respect to fully activated integrins (50). E − H − is characterized by a closed ligand-binding site and clasped membrane proximal regions (51). In activated integrins (E + H + ), the hybrid domain (β-subunit) swings away from the α-chain, and the membrane proximal regions unclasp. This correlates with the rearrangement of the MIDAS and opening of the ligand binding site (51).
Structural and mutational studies have investigated models of integrin activation to explore whether integrin extension or leg separation occurs first. Mutations and deletions of the CDloop (β-subunit terminal domain) have been proposed to keep integrins from extending and have shown no impact on α V β 3 and α IIb β 3 activation (52); there is little proof that mutations in this region affects β 2 integrins (53), strongly indicating that releasing these constraints is not enough to induce activation.
Structural studies (54) have demonstrated that α X β 2 follows the "switch-blade" model of activation, where leg separation occurs first, releasing constraints of the bent conformation and opening of the ligand-binding site resulting in an intermediate/low affinity conformation E + H − (55). The E + H − conformation has a free energy between 1.6 and 0.5 kcal/mol, meaning the high affinity conformation is thermodynamically favored (50,56). Mutations in the EGF3 repeat of the β 2 -subunit have also been shown to induce a high affinity conformation through destabilizing the thermodynamically favorable bent conformation and facilitating leg separation (57). It is noteworthy that an E − H + conformation has been described for α L β 2 and α M β 2 , allowing integrins to bind ICAM in cis, which may regulate neutrophil function (58); however, the specifics of how this activation takes place remain unknown.
Integrin activity is regulated by changes in affinity and aggregation, with the latter affecting receptor avidity. Cytoplasmic proteins bind to α-or β-subunits causing tail separation, stabilizing their high affinity conformation (48,59). This can be triggered either through signaling from other receptors ("inside-out" signaling, Figure 1B), direct ligandbinding, or experimentally, using Mn 2+ ("outside-in" signaling, Figure 1C), which triggers downstream signaling pathways (60).

Talin1 and Kindlin-3
Talin1 and Kindlin-3 are the best-characterized integrin activators. Both belong to the FERM family but interact with distinct NPXY motifs in the cytoplasmic tails of β 1 , β 2 , and β 3 , and they thus contribute differently to activation (74). Although Talin-binding is required for efficient β 5 activation during adhesion, it is dispensable for phagocytosis (75). α V β 5 requires an unknown mediator that recognizes a YEMAS motif proximal to the NPXY. A candidate could be the FERM family FRMD5, as it promotes β 5 -Kindlin-2 interaction and induces ROCK activation during adhesion (76), yet there is no information of its relevance in phagocytosis.
Hematopoietic cell-specific Kindlin-3 is mutated in LADIII, causing β 1 /β 2 /β 3 activation defects (85,86) and preventing neutrophils adhesion to iC3b and ICAM-1 (87). Kindlin-3 binds to the membrane-distal NPKF sequence in the β 2 subunit tail without excluding Talin1 binding ( Figure 1B) (87). Studies of their individual contributions to activation revealed that Kindlin-3 is not sufficient to induce the high-affinity state of α L β 2, whereas Talin1 promotes full activation (88). Whether binding of Talin1 and Kindlin-3 is sequential or simultaneous and their exact contribution to integrin activation remains to be explored. The signaling events directing Kindlin-3 to integrins also remain elusive, as in T cells, Kindlin-3 localization at immune synapses depends on Rap1 and Mst-1/RapL signaling (89), whereas no such interaction has been described for phagocytic cells.
Besides RIAM, Rap1 effectors RapL and RGS14 (Regulator of G-Protein Signalling-14) have been proposed to regulate α M β 2 activation by inside-out signaling ( Figure 1B). The former is proposed to interact with α M -subunit inducing integrin tail separation and integrin activation (99); however, RapL has only been shown to interact with a GFFKR motif in α L cytoplasmic tail, and there is no direct evidence that it plays a role in α M β 2 activation (100). For RGS14, the integrin activation mechanism is unknown but seems to be dependent on Talin1-binding to β 2 (101).
Recently, a direct interaction between Rap1-GTP and Talin1 was described at Talin1 F0 and F1 subdomains (102)(103)(104)(105). Synergistic interaction between this region and an F1 lipidinteracting helix facilitates relocation of Talin1 and its integrinactivating function ( Figure 1B) (105,106). This pathway could be relevant for fast cell responses, as disruption in mice impaired platelet aggregation, neutrophil adhesion, extravasation, and phagocytosis but had no effect on macrophage adhesion and migration (104).

OUTSIDE-IN SIGNALING
Outside-in signaling during phagocytosis initiates upon ligand interaction, stabilizing the active conformation, separating integrin tails, allowing for the binding of actin cytoskeletal linkers (Talin1 and/or Kindlin-3), and reorganizing cytoskeletal constraints, as described in the picket-fence model (2). This generates the force needed to drive membrane extension and particle engulfment/internalization (Figure 1C). Regulators have been described in focal complex-like formations at the phagocytic cup (107).

CLUSTERING AND TYROSINE KINASES
One of the earliest events in outside-in signaling could be ligand-induced clustering, a process requiring Talin1 and/or Kindlin-3 (74, 108). Kindlin-3-induced clustering is reported to activate Src family kinases (SFKs) (109,110) by the exclusion of tyrosine phosphatases such as CD45 (68). Size exclusion of these membrane-bound phosphatases with large extracellular domains seems to be a common feature of integrin-mediated close-contact immune processes, such as Dectin-1 and FcγRIII phagocytosis and immune synapse formation (68,111,112). This process does not exclude SFKs but favors their activation due to removing the inhibitory effect of these phosphatases (109,110). However, there are as of yet only indirect evidences (109,110) that phosphatases such as CD45 are excluded during integrin-mediated phagocytosis.
A requirement for SFK activation has been described for β 1 , β 2 , and β 3 integrins (109,114,116). Hck, Fgr, and Lyn are the representative SFKs in myeloid cells. Hck co-localized with α M β 2 at phagocytic cups of complement-opsonized zymosan (117,118), and the Hck knockout phenocopied the α M knockout (119). However, in U937 macrophage-like cells, Hck and Fgr siRNA, unlike Lyn, had no effect on particle internalization (120), and genetic restitution of Fgr-deficient cells inhibited adhesion, spreading, and Syk activation (121). In contrast, the Hck −/− Fgr −/− Lyn −/− triple knockout showed no inhibition in CR3mediated phagocytosis (122), which may point to compensatory roles of other ubiquitously expressed SFKs. Despite the research into outside-in activation of SFKs, the exact mechanism and individual contribution of each SFK have yet to be dissected.
SFK activity precedes activation of tyrosine kinases Syk and FAK family member Pyk2. Syk is necessary for phagocytosis of iC3b-opsonized beads/zymosan and localizes at phagocytic cups (107,123), whereas Pyk2 contributes to clearance of complement-opsonized bacteria (124). Clustering of β 2 integrins results in Syk activation (125), which in turn triggers Pyk2 signaling (126). Pharmacological inhibition of Syk and FAK kinases points to non-redundant functions during phagocytosis and to a possible sequential activation (107).
PI(3,4)P 2 recruits and induces Vinculin activation through disrupting an auto-inhibitory interaction (135). This is dependent on Syk activity and, to a lesser extent, on FAK/Pyk2 and is upstream from ROCK activation (107). In focal complexes, RIAM contributes to Vinculin binding to Talin1, as RIAM-Talin1 interaction unmasks a Vinculin binding site in Talin1 (77). Afterwards, Vinculin binding to F-actin and α-actinin favors filament bundling and force generation (136,137).

SIGNALING DURING PHAGOCYTOSIS OF APOPTOTIC CELLS
During apoptotic cell phagocytosis by mammalian α V β 5 /α V β 3 , a p130Cas-CrkII-Dock180-Elmo module induces Rac1 activation, which is responsible for cytoskeletal remodeling and phagosome formation (149,150). Other known signals include the activation of SFKs, as signals from the Mer-TK receptor recruit phosphorylated FAK to mammalian β 5 in a Src-dependent manner (151), and Syk and Pyk2 activation has been shown to occur for α V β 3 (152,153). There is also evidence that Rac-1 activation is dependent on RhoG and its GEF Trio (154,155), whereas RhoA inhibits engulfment (156), and the role of Cdc42 remains unclear (157)(158)(159).
An orthologous pathway using the CED-2-CED-5-CED10 module has been described for C. elegans INA-1, which activates the Rac ortholog and requires activation of SRC-1(Src-ortholog) (3). Similarly in Drosophila, severed axon clearance requires Src42A and Shark-the Src and Syk orthologs, respectively (160, 161)-pointing to an evolutionarily conserved pathway operating in apoptotic cell removal.

DISCUSSION AND FUTURE PERSPECTIVES
There are still critical gaps in the knowledge of phagocytic integrin signaling, specifically concerning proximal events and their hierarchy. There are several proposed alternative Talin1-recruitment mechanisms, but their contributions and significance are yet to be established. Rap1-Talin1 interaction is evolutionarily conserved and might constitute a mechanism for short-term adhesions (105), whereas Rap1-RIAM-Talin1 contacts would have a faster recruitment of effector proteins. In this line, it is yet to be established if RIAM is required for outsidein signaling, formation, and recycling of the focal adhesion-like complexes distributed in phagocytic cups (107).
Different F-actin nucleators/elongators are described to participate in CR3-mediated phagocytosis; however, their localization, recruitment, and relative contributions are unknown. The regulation of small GTPases, which control actin dynamics, remains obscure; there is scarce evidence of GEF and GAP spatiotemporal localization in phagocytic cups, and it is well established that GTPases negatively regulate each other, which also raises questions on signal termination and negative-feedback loops.
Many structural and signaling proteins required for phagocytic integrin function have potential post-translational modification-dependent functions, and, although there are several candidates, little work has been undertaken to establish Ser/Thr kinase and phosphatase recruitment and localization within the phagocytic cup.
Fine-grain elucidation of the molecular mechanisms involved in integrin-mediated phagocytosis will yield invaluable information on possible control points for phagocyte functions (antigenic capture, pathogen, tumor or apoptotic body elimination, etc.). Indeed, complementopsonized immune complexes and particles may be presented directly by subcapsular sinus macrophages to naïve B cells or conveyed to dendritic cells for B-cell presentation. This process requires cooperation between antigen-presenting cell α M β 2 /α X β 2 and B-cell CR1, CR2, and/or Fc receptors (162)(163)(164)(165). Manipulation of this pathway may inform new vaccine strategies (166).

AUTHOR CONTRIBUTIONS
AT-G and EL wrote the original draft. AT-G prepared the figures.