Abstract

Introduction

The innate immune system is a complex system, through which macrophages, dendritic cells, and neutrophils respond to pathogens. The way by which neutrophils and macrophages engulf and respond to foreign pathogens is a subject of great interest in cell biology [1]. However, the manners by which the cells survive, and a pathogen persists inside of the cell are subjects of continued interest in chronic inflammation. Autophagy is a process, wherein phagosomes digest and degrade cellular refuse and allow for the recycling of macromolecules [2, 3]. Autophagy becomes truly necessary during cellular survival, particularly in conditions of starvation or stress [4]. Similarly, the intimate role of autophagy with cell survival is of great interest in cell biology, yet its relevance to leukocytes has remained under studied. An involvement of membrane dynamics in autophagy is shared with other key leukocyte functions, such as phagocytosis. Hence, it seems likely that these two functions—autophagy and leukocytic engulfment—could be related by similar underlying mechanisms, a concept advanced by Mitroulis et al. [5].

Autophagy is a process wherein phagosomes digest and degrade cellular refuse and allow for the recycling of macromolecules. Autophagy becomes truly necessary during cellular survival, particularly in conditions of starvation or stress. The double membranes of phagosomes sequester cellular debris derived from various other cellular components, such as the plasma membrane and the Golgi body or as proteins and lipids are reused/recycled, in a specific or fixed location within the cell (Fig. 1). Furthermore, autophagy has been linked to the development of leukemia [6]. Its role in this pathology has been somewhat under studied.

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Figure 1.

Autophagy principles.

The formation of a double membranous intracellular structure that upon fusing with a lysosome, ensures degradation of abnormal, excess cytoplasmic content. This strategy is also used to engulf and eliminate invading pathogens. Atg (autophagy-related genes) and light chain 3 (LC3) are proteins needed in the initial formation of the spherical structure. mTOR is part of the nutrient sensor system: when enough nutrients are available outside of the cell, there is no need for autophagy (i.e., mTOR negatively affects autophagy). This process is reversed during starvation.

In approaching autophagy in leukocytes, it is pertinent to examine relationships that already exist in the cell, which could possibly mimic or act similarly in function to autophagy, such as cell survival. A prominent protein in the cell-survival mechanism is PLD and its enzymatic reaction product, PA. The rapid generation of PA and the protein–protein interactions of PLD are the two main ways through which PLD exerts its control over cellular functions [7,–9]. The mammalian PLD2 isoform can lead to cytoskeleton organization through a variety of effectors, such as S6K, actin, Rac2, and WASp [10, 11], while up-regulating leukocyte chemotaxis [12, 13]. PLD2 has actually two different enzymatic activities—lipase and guanine nucleotide exchange factor—the latter acts upon small GTPases of the cellular motility machinery, such as Rac and Rho [14,–16]. PLD is under expression control by mTOR [17], and mTOR is a key component of autophagy [18]. Furthermore, a link between PLD2 expression and β-catenin (the latter also being important in autophagy) has been established [19].

Based on this previous study, we propose that PLD2 and its reaction product PA are at the center of a cell-signaling network that interacts with PKC, mTOR, and β-catenin-Wnt. We further propose that this interaction is needed during autophagy and contributes to cell survival in leukocytes and that disruption of such an interaction could lead to inadequate leukocyte functionality (Fig. 2).

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Figure 2.

Proposed model for autophagy that implicates PLD.

The formation of the double membrane of the autophagosome is benefited by the presence of phospholipids, such as PA, which confer negative curvature and vesicularization, and as such, PA is a proautophagy signal. How PA would come about this is the subject of this predictive model. Likewise, PKC is also proautophagy, as PKC phosphorylates and is a positive regulator of PLD. mTOR is a known inhibitor of autophagy, and it also specifically represses expression of PLD2, a major contributor of the intracellular PA pool. β-Catenin has a dual role when it comes to the involvement of PLD. First, β-catenin initially up-regulates PLD2 expression. Second, if there is excessive autophagy or an aberrant signal leading to it, then PLD would provide the means to degrade the phagosomal center, one of which is the Dvl, and thus, the Wnt pathway. Taking all this into consideration, we propose the existence of a PLD-PA-PKC-β-catenin-Wnt pathway that regulates autophagy and cell survival in leukocytes and that disruptions in this pathway can lead to inadequate leukocyte functionality.

A FOUR-PRONGED ROLE OF PLD IN AUTOPHAGY

The proposal stated above, which could account for the formation and maintenance of the autophagosomes, is dissected into four different categories (Fig. 2), as follows:

• PLD-generated PA participates in the formation of the initial autophagosome.

• There is interplay between phospholipids and a PLD2-PKC interaction that leads to enhanced autophagy.

• mTOR down-regulates PLD gene expression and suppresses autophagy.

• The Wnt/β-catenin pathway interacts with PLD in a dual context.

We will elaborate further on these four possibilities now and present the rationale of why PLD is important to autophagy in uncompromised and fully functioning leukocytes.

The Wnt/β-catenin pathway interacts with PLD in a dual context

We propose that PLD2 is a downstream effector in the Wnt pathway with PLD2 inhibiting Wnt, and Wnt is an integral protein to the Wnt/β-catenin signaling pathway, which regulates gene expression and actin anchoring [35, 36], whereas the disruption of this pathway leads to senescence [19, 37]. Wnt acts by binding a frizzled receptor on the surface of the cell, which alters its conformation to a Dvl, which is directly responsible for controlling the amount of β-catenin that is generated and reaches the nucleus. Wnt/β-catenin controls several transcription factors, and as reported in ref. [19], β-catenin is able to up-regulate PLD2 gene expression in some cases. Such elevated PLD2 expression could result in an enhanced capability of the cell to initiate autophagy and delay senescence, as indicated in Fig. 2. However, a second scenario should be considered that could impact leukocyte homeostasis, which is overstimulation of the autophagy pathway, and that leads to impaired leukocyte physiology via enhanced or up-regulated autophagy (Fig. 3). To this, PLD2 would serve as a canonical negative-feedback mechanism, which would bring down β-catenin levels and prevent overstimulation of the autophagy pathway. Following PLD2-mediated up-regulation of autophagosome formation and autophagy completion, PLD2 or components involved in autophagy could down-regulate Wnt via degradation of the Dvl [38].

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Figure 3.

β-Catenin/Wnt signaling with PLD involvement.

Initially, β-catenin induces PLD2 expression, through which the greater abundance of PLD2 begins autophagy through enzymatic activity and subsequent production of PA, which induces negative curvature in cell membranes. As indicated in the text, PKC also activates PLD2, strengthening activity and pools of PA available to help form the initial phagosome. With the production of PA and phagolysosomes, cell metabolites are recycled, and cell survival is enhanced, staving off senescence and keeping the leukocyte healthy and active. At the bottom of the scheme, a scenario is shown that depicts the outcome when excessive autophagy gives off a negative-feedback signal to that system. In particular, PLD would promote the degradation of the Dvl, and as such, the Wnt-β-catenin would be brought to a halt.

In these two scenarios described thus far, the role of PLD in the pathway would involve its own regulation by the pathway and its ability to regulate components of the same pathway. By ascertaining how the Wnt pathway partners with PLD2 during autophagy, we can begin to develop intermediate maps that can be used to elucidate further leukocyte autophagy regulation, and the components of autophagy that are associated with Wnt down-regulation can be examined to see where and when the Dvl is degraded and why PLD2 overexpression does not correct itself by degrading Dvl. This will allow the establishment of important targets to enhance (or possibly negate) autophagy in associated cell dysfunctions.

AUTOPHAGY, LEUKOCYTES, AND INTRACELLULAR PATHOGENS

Autophagy in leukocytes has been characterized as a unique process through which it also functions in the context of the innate immune response [39]. The involvement of mTOR during bacterial invasion and intracellular pathogen killing by neutrophils and macrophages has been shown [40]. In fact, several microbes have evolved sophisticated mechanisms to manipulate the mTOR pathway to their benefit and to the host's detriment [41, 42]. The intracellular killing of pathogens by neutrophils and macrophages and their contribution to the phagolysosome, as well as the subversion of the system by bacteria, are summarized in Fig. 4. Also, mTOR can disable turnover of metabolic waste through autophagy and disrupt PLD2-mediated survival signal generation. Its suppression of PLD2-mediated autophagy in leukocytes may lead to senescence and aberrant inflammation.

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Figure 4.

Intracellular killing by polymorphonuclear neutrophils (PMN) and macrophages and subversion of the system by bacteria.

Providing that PLD2 regulates the formation of the phagosome during autophagy, certain strains of bacteria subvert mTOR for their own benefit when mTOR is elevated so that autophagy (and presumably phagocytosis) is prevented. At later times and if the microbe has managed to survive, mTOR levels are low intracellularly, and any phagolysosomes formed would provide much-needed nutrients to warrant further survival of the microbes.

IF AUTOPHAGY DOES NOT FUNCTION WELL: SENESCENCE

One of the goals of cell survival is avoidance of senescence, which is the cessation of the cell cycle. An important aspect of failure to mount an efficient autophagy process is the deviation into cell senescence. The inability of PLD to stave off the effects of aging or even apoptosis stems from the accumulation of ceramide, which inhibits PLD and contributes to survival of a variety of cells [43,–45]. Leukocyte senescence has also been implicated as a cause of chronic inflammation, especially in patients with chronic obstructive pulmonary disease [46]. It stands to reason that the intimate association of PLD2 with cell survival and leukocyte physiology implicates a role for it in warding off senescence. The Wnt/β-catenin pathway up-regulates PLD2 expression, and Wnt down-regulation likens the probability of a cell entering senescence [47, 48]. Thus, senescence could be induced as a result of the Wnt-mediated loss of the survival signals of PLD2, as well as the loss of its ability to initiate and regulate autophagy.

An isoform of Wnt, Wnt5a, has been implicated to have anti-inflammatory effects on bronchial macrophages [49]. The Wnt pathway has many other proteins that interact with a variety of targets between the disheveled and β-catenin portions of the pathway. Previous studies have implied that the PLD proteins are controlled by Wnt signaling and that their expression is impacted by changes in the pathway [37]. We advance here the concept that PLD2 may have a key role in both Wnt-mediated changes in cell morphology and may also be involved in negatively regulating the Wnt pathway.

CONCLUSIONS

We have described several insights and perspectives on how autophagy, senescence, and phagocytosis could be linked to the Wnt pathway through PLD (which we have named “PLD-Wnt”). Wnt regulates PLD2 expression through β-catenin, and PLD2, in turn, regulates autophagy (and at the same time, phagocytosis, which has been shown to be intimately linked with autophagy), which keeps metabolic waste levels low and maintains cell survival and health. In cases where Wnt signaling is disrupted, PLD2 expression is down-regulated, leading to improper autophagy and cell deterioration. Aberrant autophagy could also be a starting point for a faulty innate immune response by phagocytes. Furthermore, Wnt can play a role in inflammation through overstimulation of PLD2, wherein autophagy provides a means for opportunistic pathogens to survive inside of the cell. Further study of this signaling PLD-Wnt pathway seems warranted.

ACKNOWLEDGMENTS

Footnotes

REFERENCES