*5.3.1 LC3 assisted phagocytosis*

The phagosome may be changed to either promote or hinder maturation, affecting the degradation of phagosome material after fusion with lysosomes. During the LAP, one such shift occurs. A subset of proteins necessary for canonical autophagy in LAP, in combination with LAP-specific regulators, allows the LC3 family to be combined with the LAP family. LC3 family proteins facilitate autophagosomes in autophagy, as LC33 depletion also inhibits lysosome fusion [34]. FYCO1, a RAB7 effector protein, interacts with LC3 and PI3P to promote autophagosome-lysosome fusion [35]. Similarly, phagosome LC3 promotes phagosome-lysosome fusion to form a phagolysosome. The rubicon homologue PLEKHM1 interacts with both RAB7 and LC3 and can promote phagosome and endosome trafficking [48, 99]. These processes are most likely shared by autophagia and phagocytosis, both of which are involved in lysosomal targeting and fusion. To date, it has not been thought that the ability to conjugate LC3 with the phagosome directly affects the lysosome's functional potential: impaired maturation and degradation seen in LAP-deficient cells and animals has not been linked to direct lysosomal defects [49, 50].

#### **5.4 Liver X receptors**

Other pathways with roles in efferocytosis include the liver X receptors (LXRs) and the PPARs, nuclear receptor families that control genes involved in lipid metabolism and transport [100–103]. By engulfing a dying cell, a phagocyte can effectively double its lipid content, requiring an appropriate transcriptional response which is mediated by these nuclear receptors. LXR deficiency results in their inability to clear the apoptotic thymocytes in macrophages, but does not alter the swallowing of inert beads or other substrates [104]. Similarly, mice that are deficient in LXRs in many tissues accumulate dead cells due to failure of in vivo efferocytosis [104]. Pharmacologic LXR agonists increase efferocytosis and clearance by increasing the expression of receptors, including MERTK, both in cell culture and in vivo136. In addition, during efferocytosis, the existence of LXRs induces the expression of ABCA1 cholesterol transporters, which emit excess cholesterol [105], and genes involved in mitochondrial lipid oxidation, enabling excess lipids to catabolize effectively [105, 106]. Efferocytosis is caused similarly to LXRs by the PPAR family. In response to apoptotic cell engulfment, PPARγ expression occurs and remains elevated until efferocytosis [107] has been resolved. In combination with activation of LXRs, PPARγ activation has been demonstrated to enhance efferocytosis by macrophages [104]. In fact, inhibition of PPARγ by a pharmaceutical antagonist resulted in impaired efferocytosis [107].

#### **5.5 Resolution of phagocytosis**

After the phagosome has fused with lysosomes and its cargo has been degraded, a resolution mechanism restores homeostasis within the phagocyte, allowing for further phagocytosis. Since dying cells are not the only phagocytic cargo, the effect of phagocytosis can differ depending on what cargo is internalised. After lysosomal degradation in the form of efferocytosis, some of the components of the cell corpse can be recaptured and recycled for use by the phagocytic cell. Carbohydrates, amino acids, lipids and nucleotides are recycled to replenish cellular stores and can potentially be used as building blocks and sources of energy by the phagocytic cell [108]. The internalisation and degradation of cargo can also influence the activation of signalling pathways; for example, DNA that escapes degradation can activate the DNA sensing cGAS-STING pathway and contribute to the production of type I interferons, as discussed earlier. These events may lead to the loss of the immune tolerance of the apoptotic corpse and can potentially result in autoimmunity [109]. As described earlier, LAP facilitates rapid maturation and generation of phagolysosomes and efficient degradation of cells and promotes the production of antiinflammatory cytokines, including IL-10, a method that promotes immune silence. On the other hand, LAP disruption decreases phagosome maturation, contributing to the activation of inflammatory signalling pathways and to the production of proinflammatory mediators for IL-1β and [110–113]. The exact mechanism that leads to this switch in cytokine production has not been well known in the absence of LAP. Furthermore, failure in LAP-deficient phagocytes to effectively degrade the cell corpse can result in the leakage of phagosome contents, such as DNA, into the cytosol, which in turn induces the production of type I interferon via STING [114] sensing. STING-dependent interferon expression by tumour-related macrophages in LAP-deficient mice in several cancer models promoted T-cell-mediated anticancer immunity [114]. Following efferocytosis, the phagocyte must restore the functions of cytoskeletal components such as actin and microtubules to ensure that its phagocytic capacity is maximised for additional [115–118]. This restoration of function has not been well studied in contrast with the upstream processes of

*Efferocytosis: An Interface between Apoptosis and Pathophysiology DOI: http://dx.doi.org/10.5772/intechopen.97819*

phagocytosis. However, such details can be inferred from studies investigating the effectors such as RAB27A in the events leading to phagocytosis. RAB27A negatively affects phagocytosis by prolonging the actin coating of nascent phagosomes, resulting in impaired transition to the stage of phagosome sealing [119]. Therefore, RAB27A and related effectors may be essential for the restoration of actin and other cytoskeletal networks following the completion of phagocytosis. It is evident, however, that cytoskeleton restoration after phagocytosis is an ATP-dependent process and may involve multiple components [120]. An additional function of recovery is the recycling or continued expression of membrane receptors to accept subsequent cargo. Impaired recycling results in reduced membrane expression of receptors such as TREM2 and TLR4, which recognize a variety of amyloids and pathogen products [121, 122] respectively, and can also reduce the membrane expression of dying cell recognizing receptors. This decrease in the available surface receptors can impair the endocytic and phagocytic capacity of the phagocyte cell, which means that these restorative events are necessary to ensure that the phagocyte will continue to recognize and clear phagocyte cargo.
