**2. Pathogen-induced phagocytosis**

The active role of the host cell in the process of pathogen internalization, involving cytoskeletal rearrangements after pathogen recognition, ultimately distinguishes nonprofessional phagocytosis from infection [7, 19, 57]. There may be a few exceptions to this rule, such as Rotaviruses, known to gain infectious entry into the cell using the zipper mechanism, described below [58]. Internalization of the pathogen is, however, only the initial stage in the bigger mechanism of phagocytosis. The pathogen-containing internalized vesicle, otherwise known as the early phagosome, requires subsequent fusion with lysosomes in order to achieve pathogen killing [59]. The early phagosome matures by fusion with internal endocytic vesicles [59], recruiting factors, such as Rab5 [60], a small GTPase important for the maturation of the phagosome, and early endosome antigen 1 (EEA1) [61]. Rab5 remains transiently expressed in the early phagosome, directing the fusion of early endosomes [62, 63]. The schematic in **Figure 1** depicts the process of endosome formation, maturation, and role of Rab proteins in phagocytosis. Rab5 has been extensively studied and understood in myeloid cells during professional phagocytosis and has also been shown to be constitutively expressed in nonmyeloid cells, including epithelial cells [64–66], fibroblasts [66], and smooth muscle cells [67], controlling the phagocytic processes. Rab5 is considered a master regulator of early endosome formation and trafficking to the early phagosome. Rab5 expressing early phagosomes initiates the process of pathogen killing or apoptotic recycling by creating a mildly acidic microenvironment (pH 6.1) within the phagosome and engaging in relatively low levels of hydrolysis [68]. Rab conversion is a term used to convey phagosome maturation beyond the early phagosome. Maturation involves the recruitment of Rab7, functionally replacing Rab5 in the phagosome [69]. Rab7, like Rab5, is a member of the GTPase family that manages the maturation of phagosomes and recruits other factors, such as the RAB7 interacting lysosomal protein (RILP), necessary for later phagosome fusion with lysosomes [70]. Formation of a late-stage phagosome also requires the recruitment of Lysosomal-Associated Membrane Process-1(LAMP-1), necessary for lysosomal fusion [27, 71] Rab7 functionally interacts with RILP [70, 72], resulting in lysosomal fusion with the late-stage phagosome. Consequently, the phagolysosome structure is formed, creating a more acidic environment (pH 5.5) and generating a cocktail of degradation enzymes and ROS in effort to kill invading pathogens or break down apoptotic bodies [57]. While the process leading to the formation of the phagolysosome is similar, the recognition of the pathogen by nonmyeloid cells and internalization can occur through one of several known pathways. These pathways,

#### **Figure 1.**

 *Internalization models for pathogen-induced phagocytosis. For nonprofessional phagocytes, phagocytosis is induced by the pathogen. Two primary models are proposed: 1) trigger phagocytosis, caused when type 3 / type 4 secretion systems (T 3 SS/T 4 SS) cause cytoskeletal rearrangement, resulting in "ruffles" of the host cell membrane that engulfs and internalizes the pathogen and 2) zipper phagocytosis where the pathogen engages with a receptor complementary to ligands expressed on the pathogen. Following cytoskeletal rearrangement, further receptors engage with the pathogen in a "zipper" or "ratchet" like fashion, engulfing the pathogen into the phagosome. This figure was created with BioRender.com .* 

including efferocytosis, zipper phagocytosis, trigger phagocytosis, and opsonization, are discussed in more detail below.

#### **2.1 Efferocytosis**

 Efferocytosis of apoptotic cells is the primary phagocytosis mechanism utilized by nonmyeloid cells. Recognition of apoptotic bodies is, therefore, critical for the clearance of apoptotic cells, and tissues have evolved ligand-receptor-based recognition as part of the initial engagement ultimately triggering efferocytosis of the apoptotic cell [ 7 , 73 , 74 ]. The primary component of this mechanism is the recognition of phosphatidylserine expressed in apoptotic cells [ 75 ]. During early apoptosis, phosphatidylserine molecules translocate to the cells' surface, anchoring to the membrane, where they act as an "eat-me" signal to localized phagocytes, both professional and nonprofessional [ 76 ]. Phosphatidylserine can be recognized by several receptors, including integrins αvβ3 and αvβ5 [ 9 , 29 , 34 ]. CD36 [ 29 , 34 ], CD91 [ 29 ], and even bio-specific phosphatidylserine receptors [ 16 , 77 ]. Other ligands have been proposed to induce receptor-mediated efferocytosis of apoptotic cells by neighboring nonprofessional phagocytes, including Apoptosis Inhibitor of Macrophage (AIM) recognition by Kidney Injury Molecule-1 (KIM-1) [ 78 ] and milk fat globule-epidermal growth factor 8 (MFG-E8) by integrin αvβ3 [ 79 ].
