© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12271

APMIS

Review Article

Autophagy mediates neutrophil responses to bacterial infection
 ABDERRAHMAN CHARGUI1,2 and MICHELE VERONIQUE EL MAY1 Laboratory of Histology, Embryology and Cell Biology, Faculty of Medicine, Tunis, Tunisia, 2Higher School of Agriculture, Kef, Tunisia

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Chargui A, El May MV. Autophagy mediates neutrophil responses to bacterial infection. APMIS 2014. Neutrophils constitute the first line of cellular defense against pathogens and autophagy is a fundamental cellular homeostasis pathway that operates with the intracellular degradation/recycling system. Induction of the autophagic process in neutrophils, in response to invading pathogens, constitutes a crucial mechanism in innate immunity. Exploration of autophagy has greatly progressed and diverse strategies have been reported for studying this molecular process in different biological systems; especially in infectious and inflammatory diseases. Furthermore, the role of autophagy in neutrophils, during pathogenic infection, continues to be of interest, due to the role of the cell in immunity function, its recruitment to the site of infection and its implication in inflammatory diseases. This review focuses on the known role of autophagy in neutrophils defence against pathogenic infections. A more detailed discussion will concern the recent findings highlighting the role of autophagy in inflammation and cell death in infected neutrophils. Key words: Infected-neutrophil; autophagy; inflammation. Abderrahman Chargui, Mich
ele Veronique El May, Laboratory of Histology, Embryology and Cell Biology, Faculty of Medicine, Tunis, Tunisia. e-mails: [email protected]; [email protected]

Neutrophils constitute the first line of cellular defense against pathogens and one of the essential components of human innate immune system recruited to the site of infection. They represent the host’s most effective and numerous front-line defenders (1). Neutrophils employ several antimicrobial strategies including exocytosis-mediated release of antimicrobial molecules into the extracellular medium (2), phagocytosis (3), and neutrophil extracellular traps (NETs) formation (3). Recently, autophagy has been described as a novel antimicrobial mechanism of neutrophils. Autophagy is an intracellular catabolic process by which cellular components and pathogens are degraded through the lysosomal machinery. Induction of the autophagic process, in neutrophils, in infection conditions, constitutes a crucial mechanism in innate immunity (4). Conserved from yeast to humans, autophagy is fundamental to eukaryotic cell homeostasis (5, 6). Autophagy functions in diverse cellular processes such as growth and development, cancer, inflammation (7–9), and is Received 16 December 2013. Accepted 13 February 2014

implicated in both cell survival and death, depending on the cell type and stress conditions. Consequently, autophagy, as a process, has been associated not only with disease progression but also with its prevention (10, 11). Intriguingly, while certain bacteria and viruses can subvert and manipulate autophagic pathways during establishment of infection, autophagy plays a protective role against intracellular replication of several pathogens including adherent invasive Escherichia coli (AIEC) (12). The efficiency of pathogen killing by neutrophils through degranulation and phagocytosis relies on the timely activation of neutrophil autophagic process (13). Infected neutrophils contain multivesicular bodies, which are formed by the autophagic pathway, and are able to fuse with the phagosome which contains the pathogens (14). Sequential mobilization of neutrophil lysosomes/endosomes and the release of their cargoes into the autophagic-vesicles are necessary events to mediate the intracellular pathogen killing. However, some pathogenic bacteria escape from the phagosome into the cytoplasm and induce the autophagic pathway (15). 1

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Free intracellular bacteria are eventually ubiquitinated, and then recognized by the xenophagy machinery, an autophagic control of intracellular pathogens, through recruitment of p62 (also known as SQSTM1) and LC3 (light chain 3) (16). Indeed, autophagy involves the sequestration of regions of the cytosol within double-membrane –bound compartments and delivery of the contents to the lysosomes for degradation. Autophagy has been shown to be an important regulator in many critical biological processes, such as cellular response to starvation, cell death, cancer, neurodegenerative diseases (17–20), and more recently, host defence (21–23). Rapidly accumulating evidence has shown that autophagy is a component of innate immunity and is involved in host defence resulting in elimination of pathogens (24–26). Several pathogens, including bacteria, have evolved to block the autophagic responses (13, 27, 28). Previously, Mitroulis et al. evidenced that autophagy was implicated in human neutrophils in both a phagocytosis-independent and phagocytosis-dependent initiation manner (29). Neutrophils play a pivotal role in bacterial clearance, and autophagy is one of the main degradation pathways which connects to phagocytosis and degrades its contents (30). On the other hand, autophagy could be responsible for the detection and elimination of invasive pathogens, including intracellular bacteria, viruses and parasites (31). However, some pathogenic bacteria can escape from the autophagic vesicles and subvert the autophagic process (12, 13, 16, 28). In this review, we firstly identify the autophagic process and present the different levels of its implication in neutrophil defences against pathogenic bacteria. Secondly, we discuss recent findings highlighting the role of autophagy in inflammation and cell death in infected neutrophils.

AUTOPHAGY PROCESS AND ROLE Autophagy is a process in which cytosol and organelles are sequestered within double-membrane vesicles that deliver their contents to the lysosome/ vacuole for enzymatic degradation and recycling of the resulting macromolecules (32). It plays an important role in the cellular response to stress, and is involved in various developmental pathways and functions, such as tumor suppression, resistance to pathogens and extension of lifespan (33–35). Conversely, autophagy defects may be associated with cancers, cardiomyopathies, neurodegenerative and inflammatory diseases (36–38). Substantial progress has been made in identifying the autophagy genes (Atg) necessary for the

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execution of autophagy and in understanding its molecular basis. Under nutrient-rich conditions, the serine–threonine kinase mammalian target of rapamycin (mTOR) phosphorylates UNC-51-like kinase (ULK)/Atg1 kinase and then inhibits autophagy; whereas during periods of nutrient deprivation, mTOR dissociates from the ULK complex and activates autophagy (39). Although autophagy is nonspecific under starvation, some selective types of autophagy utilize cargo receptors such as p62, NBR1 (neighbor of BRCA1 gene 1) and Alfy (Autophagy-linked FYVE protein) which are three scaffold proteins with an ubiquitin-associated domain for binding of the ubiquitinated cargo and an LC3 interaction region for binding of the autophagosomal protein LC3 (40). Formation of the autophagosome is a complex process and neither the mechanism of vesicle formation nor the donor membrane origin is known. The final breakdown of the sequestered cargo relies on well-characterized lysosomal proteases. By contrast, the way the integrity of the lysosome membrane is maintained during degradation is unknown. Technically, the autophagic process is revealed by several methods, such as electronic microscopy (EM) to visualize the phagophore, double membranes (autophagosome) and simple membranes (autolysosomes) vesicles; immuno-fluorescence and western blot to detect the LC3 (LC3-I and LC3-II). Another useful method for assaying autophagy activation, both in vitro and in vivo (41), is the change in subcellular distribution of LC3, going from a diffuse (LC3I) to punctuated (LC3-II, autophagosomes) staining, as evidenced by immunohistochemistry or transfection of the green fluorescent protein-LC3 fusion protein. However, the accumulation of autophagosomes could either indicate increased autophagic initiation or decreased autophagic degradation. In this latter case, when autophagic flux is blocked, the degradation of LC3-II cannot occur and the LC3-positivity increases as a result of the accumulation of autophagosomes. To know whether the accumulation of autophagosomes is due to increased or defective autophagy, several assays have been used to monitor autophagic flux, including co-treatment with lysosomal inhibitors E64d and pepstatin A, or bafilomycin A1 followed by analysis by western blot of autophagy substrates LC3-II and p62 (42, 43). In recent years, research on the autophagic process has greatly increased and is gaining prominence in the fields of biology and medicine. Several markers of the autophagic process were discovered and various strategies have been reported for studying this molecular process in different biological systems both in physiological and pathological and/

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or infection conditions. In fact, autophagy is a critical cellular process orchestrating the lysosomal degradation of cellular components in order to maintain cellular homeostasis and to respond to cellular stress (44). Over the last 12 years autophagy has emerged as an essential section of innate and adaptive immune responses where it plays roles in antigen presentation, production of bactericidal peptides, direct and indirect killing of intracellular and extracellular pathogens and in the regulation of inflammatory responses (13, 45–47). Autophagy of pathogens, named xenophagy, has emerged as a powerful system of eliminating intracellular bacteria (34, 48). However, there is a complex interplay between the host autophagic mechanisms and pathogens. Numerous microorganisms have evolved strategies to escape or subvert host autophagy to survive and establish a persistent infection (13, 49, 50). Autophagy has been implicated in AIEC infection in cultured macrophages and neutrophils in vitro (12, 13). However, the biological significance of autophagy in AIEC infection in vivo and its role in neutrophil–AIEC interaction is still unknown. Indeed, identification of mechanisms or virulence factors exploiting autophagy may provide a new strategy for therapeutic intervention in infectious diseases such as Crohn’s disease.

AUTOPHAGY IN NEUTROPHIL DEFENSES AGAINST PATHOGENS For a long time, neutrophils have been recognized only as professional phagocytic cells that are able to phagocytize and destroy infectious agents (51). Certainly, they are basic anti-infectious agents in host defense but can mediate tissue destruction (52). However, it is now clear that the role of neutrophils go far beyond phagocytosis and pathogen killing. Neutrophils are essential cells for immunity that are absolutely required to build and modulate the innate response (53). Neutrophils can be rapidly and efficiently mobilized against inflammation and they constitute the first line of host defense against pathogens. It has long been known that neutrophils use two strategies to kill invading pathogens: Firstly, the engulfment of microbes and secondly, the secretion of antimicrobial substances. A decade ago, a novel third function was identified, that is the formation of NETs, whereby neutrophils kill extracellular pathogens while minimizing damage to the host cells (54). Following successive steps of activation (adhesion, chemotaxis and migration), neutrophils can phagocytize pathogens and mobilize their microbicidal effector molecules, including reactive oxygen species (ROS), antibiotic proteins,

© 2014 APMIS. Published by John Wiley & Sons Ltd

proteinases and NETs. All of these means of cell signaling activate autophagy in infected neutrophils. Furthermore, invading pathogens, which evade from phagocytic mechanism, induce an autophagic process known as xenophagy; ‘an autophagic control of intracellular pathogens’ (31). Additionally, other pathogens such as measles virus strains, adenovirus B and D, human herpes virus 6, Neisseria bacteria and several strains of group A streptococcus induce xenophagy through an autophagic receptor, such as cluster of differentiation 46 (CD46) and Toll-like receptor (TLR) (29, 55). Therefore, upon microorganism identification, a cell surface pathogen receptor can directly trigger autophagy. Once in intracellular compartment, bacteria will be ubiquitinated and recruited to phagophore membrane via LC3 and p62 for degradation by lysosomal enzymes (Fig. 1). However, another pathogenic bacterium, such as AIEC, impairs autophagy and induces inflammation and cell death in infected neutrophils (13). Autophagy and extracellular traps formation

Recent studies have revealed that autophagic activity is required for the release of NETs, representing a distinct form of active neutrophil death, namely NETosis. Indeed, NETs formation is beneficial during host defense against invading pathogens. Neutrophils contribute to pathogen clearance by expressing and/or producing NETs, which are structures of chromatin that capture extracellular bacteria and fungi (55). Furthermore, NETs also express antibacterial factors such as a-defensin, myeloperoxidase (MPO) and calprotectin (56, 57). NETs are formed in response to a variety of proinflammatory stimuli, such as lipopolysaccharides (LPS), interleukin 8 (IL-8) and tumor necrosis factor (TNF), as well as to various microorganisms and pathogens (58–60). The expression of NETs depends on the production of ROS, which can be stimulated by various agents such as bacteria, fungi, protozoa and also some soluble molecules (61–63). NETs are released during a novel form of cell death. This cell death type (NETosis) is different from apoptosis and necrosis and depends on the enzymatic ROS production by NADPH oxidase (64). During this phenomenon, the nucleus decondenses and intracellular membranes disintegrate allowing the mixing of nuclear and cytoplasmic components. Ultimately, the plasma membrane ruptures to release NETs, structures that contain chromatin and granule proteins. NETs attack and kill a variety of microbes such as parasites, fungi and bacteria (65–67). In fact, NETs neutralize pathogens with antimicrobial proteins including

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Fig. 1. Degradation of bacteria by Xenophagy in infected neutrophil. When pathogenic bacteria enter in neutrophil will be ubiquitined and fixed to autophagy adaptor ‘p62’. The complex stimulates autophagy, which begins with the formation of isolation membranes, termed a ‘phagophore’ that sequesters it. The phagophore expands into a double-membrane completed vesicle, an ‘autophagosome’, and subsequently, the autophagosome rapidly fuses with a lysosome to become an ‘autolysosome’ where the content is finally degraded through the action of hydrolytic enzymes. UB, Ubiquitin; p62, also know SQSTM1; and LC3, light chain 3.

neutrophil elastase and histones proteins that are bound to the genomic DNA. Whereas parasites (68) and bacteria (69) are probably killed by these histones contained in NETs, in a previous study Urban et al., found that purified histones did affect Candida albicans in vitro (70). Moreover, the overall composition and regulation of NETs has not been explored. Recently, Remijsen’s team has shown that NETs formation needs both autophagy and ROS generation, and inhibition of either autophagy or NADPH oxidase activity prevents NETs formation and leads to neutrophil cell death by apoptosis (71). Furthermore, Li’s group showed that histone hypercitrullination catalyzed by peptidylarginine deiminase 4 is essential for chromatin decondensation during NETs formation (72). In contrast, the molecular actors of NETs composition and regulation may be dependent on the type of infection agent, which induces NETosis death in infected neutrophils. However, NETs release from cells not undergoing NETosis has also been reported (73). The localization of several neutrophil enzymes with pro-inflammatory function, like elastase, MPO or proteinase 3, in NETs and the increasing evidence for the implication of NETs in non-infectious diseases (74–76), suggest that the formation of these structures plays a role in the amplification of the inflammatory responses, which are characterized by the autophagic activity (77). Autophagy constitutes a critical cellular mechanism for the preservation of cell integrity, while it is implicated in the regulation of innate immune functions (9). Other data suggest 4

that autophagy is required for NETosis (71). These studies suggested that this type of death occurring only in neutrophils is probably regulated by intracellular ROS levels. Indeed, several studies showed that ROS mediates autophagy process (78–80), and neutrophils are a high producer of ROS. NarniMancinelli’s group showed that bacterial killing involves activation of a high number of monocytes and neutrophils, producing higher levels of ROS during the secondary infection than during the primary one (81). ROS are probably inducing a rapid pH increase in the primary vesicles of phagocytosis, as well as augmented levels of cellular autophagy, both allowing for Listeria monocytogenes clearance. Recently, Mitroulis’ group showed the implication of autophagy in NETs formation in activated neutrophils (82). Here, we can say that the relationship between autophagy and NETs formation is the fate of infected neutrophils and is mediated by the intracellular ROS levels. However, ROS are insufficient to induce NETosis, though they are clearly necessary for NETs formation (64). We therefore imagine that the interaction between ROS production and histone hypercitrullination promotes the collapse of both nuclear and granular membranes and mediates intracellular chromatin decondensation, while inhibiting the apoptotic process and induces NETosis, which engaged autophagic cell death. In a recent study, Itakura et al. demonstrate a pivotal role of the mTOR pathway in coordinating intracellular signaling events following neutrophil activation leading to NETosis (83). Their data prove that © 2014 APMIS. Published by John Wiley & Sons Ltd

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the pharmacological inhibition of the mTOR pathway accelerates the rate of NETs release following neutrophil stimulation with the bacteria-derived peptide, while autophagosome formation is enhanced by mTOR inhibitors. Previously, we found that AIEC stimulates NETs formation and induces autophagic death in infected neutrophils (13). Our result demonstrated that AIEC-infected neutrophils underwent NETosis. Interestingly, this process takes place after autophagy since it requires 6 h of infection whereas autophagy is visible at 1 h post infection with AIEC and reaches a plateau by 3 h. Therefore, we identified the link between autophagy and NETosis by studying trap formation in autophagy deficient PLB Sh-atg5 cells. Differentiated PLB Sh-atg5 cells infected with AIEC-LF82 for 6 h were unable to form extracellular Traps (13). Autophagy and antimicrobial secretions

Neutrophils have an important role in host defense against microbial infection. Recruitment of neutrophils from the bloodstream to a site of infection implicates their identification of inflammatory mediators, their binding to adhesion molecules of the vascular endothelium, and their transmigration across the endothelial barrier (84). Indeed, the ability of neutrophils to perform these tasks depends on a sophisticated mobilization mechanism that triggers the release of granule contents and the concomitant up-regulation of various receptors to the plasma membrane (85). Secretory processes are also important for the extravascular transmigration of neutrophils through tissues. At different steps of an infectious process, neutrophils progressively up-regulate receptors and release various effector molecules. Once the cells have reached the focus of infection, they are fully activated and are able to fight the infection by secreting ROS, antimicrobial peptides, and degradative enzymes (86). These substances can be preferentially targeted to phagosome compartments to achieve efficient killing and degradation of internalized microorganisms. A third antimicrobial mechanism of neutrophils was identified recently by Tan’s team, who showed that the transfer of neutrophil granule proteins from apoptotic neutrophils to macrophages enhances the intracellular killing of Mycobacterium tuberculosis (87). As well as this direct microbicidal function, infected neutrophils produce several types of inflammatory cytokines such as TNFa, IL-1b and IL-8 (88). Recently, we found in infected neutrophil-like (differenciated PLB985) cells an increased capacity to secrete IL-8. Our data showed that secretion of this pro-inflammatory cytokine was related to the

© 2014 APMIS. Published by John Wiley & Sons Ltd

ability of AIEC to adhere to cell (13). It was also established that normal neutrophils had the capacity to secrete pro-inflammatory cytokines, in patients with intestinal inflammation (89). In contrast, the neutrophil disorders are regularly associated with pathogenic infections, which induce autophagic responses in macrophages (90, 91) and in neutrophils (13). Indeed, infected neutrophils secrete several cytokines, which are able to stimulate the autophagic process (92). Furthermore, autophagy is very sensitive to bacteria and their toxins, such as LPS which induces autophagy in macrophages enhancing mycobacterium localization within autophagosomes (93). In our study, we showed the conversion of LC3-I to LC3-II in neutrophils treated with LPS (data not shown). However, the level of LC3-II expression was higher in AIEC infected neutrophils than in LPS treated neutrophils (13). In neutrophils, autophagy is induced in response to numerous different stimuli, including environmental and cellular stresses, such as nutrient deprivation, growth factor withdrawal and in response to various immune stimuli. However, the presence of sensitive autophagy receptor on cellular surface or into cytosol is essential for autophagy induction (15, 55, 94). In contrast, antimicrobial secretions which help neutrophils to kill pathogens destabilize both intra and extracellular homeostasis. This activates the autophagic process, which assures intracellular stabilization. The presence of antimicrobial products such as cathelicidin, a protein that has direct antimicrobial activity, facilitates the induction of autophagy in human monocytes/macrophages (95). Of note, it is probable that autophagy firstly responds to neutrophil secretions before bacteria endocytosis, suggesting that induction of this autophagy in these cells is a general phenomenon independent of the main trigger. Autophagy and phagocytosis

Phagocytosis constitutes an essential antimicrobial process whereby sequestered bacteria are targeted to highly organized endocytic compartments, the phagosomes, and delivered to lysosomes for degradation (96). Neutrophils and macrophages constitute the professional phagocytic cells, and are able, with a unique capacity, to swallow and thereby eliminate microbes and cell debris (97). Phagocytic vesicles are equipped with specialized receptors to recognize their targets (98) and thus mediate internalization and initiation of a variety of destructive mechanisms that end in killing and disposal of the sequestered particles. Moreover, autophagy, an ancient process of lysosomal self-digestion of

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organelles, protein aggregates and cytosolic pathogens, has only recently become appreciated for its dynamic relationship with phagocytosis (99). Previously, several studies have highlighted the link between autophagy and phagocytosis in host defense (100–102). This connection has been defined by a ‘hybrid’ process (autophagic-phagocytosis) such as microtubule-associated protein 1 LC3associated phagocytosis (103). Given the commonalities between phagocytosis and the autophagic process, neutrophils use both mechanisms to kill pathogenic bacteria. However, during phagocytosis and in response to invasive bacteria, which can escape phagosomes, autophagy becomes the only neutrophil weapons to clean intracellular pathogens. In addition, neutrophils activate autophagy, independently of phagocytosis, in response to rapamycin (29) and LPS treatment (data not shown). Recently, Lu et al. showed that LPS triggered lysosomal destabilization in neutrophils which was phagocytosis independent, because treatment of neutrophils with the phagocytosis inhibitor cytochalasin D did not affect LPS triggered lysosomal destabilization or capase-1 activation (104). Here, we note that neutrophils as well as several other cell types activate their autophagy in response to intra and extracellular stimuli. Particularly, autophagy is activated both in presence and also in the absence of phagocytosis in infected neutrophils. However, some pathogenic bacteria block autophagy in neutrophils (13). Affection of the phagocytic process in this case remains unknown.

AUTOPHAGY CONTROLS INFLAMMATION AND CELL DEATH Autophagy is a fundamental, homeostatic process by which cells renew their own compounds. The primordial function of this degradation pathway is the adaptation to nutrient deficiency. However, in complex multicellular organisms, the basic autophagy machinery orchestrates different aspects of cell and tissue responses to other dangerous stimuli such as infectious agents (21, 22, 90). Recent advances have revealed a crucial role of the autophagic process in inflammation and cell death. Neutrophils are an important cell population in acute bacterial infections and inflammation (86) and autophagy possesses a regulatory and effector role influencing neutrophils response against intracellular pathogens in many different pathways (13). There is evidence that autophagy occurs in human and mouse neutrophils in both phagocytosis-independent and phagocytosis-dependent manners (29, 105, 106). This established role of autophagy as a

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defense mechanism against intracellular bacteria and protozoa has been demonstrated by studies using Atg5. In mice, knockout of Atg5 in macrophages and neutrophils increases susceptibility to infection with L. monocytogenes and the protozoan Toxoplasma gondii (107). The best studied and well characterized process is xenophagy, where microbes undergo direct enzymatic degradation by autolysosomes. In contrast to non selective autophagy induced by nutrient deprivation or rapamycin, xenophagy involves autophagic receptors for selective degradation of strange invaders (108). This process is triggered by innate immunity receptors or pattern recognition receptors, such as TLRs and nucleotide-binding oligomerization-domain-like receptors, following the detection of various ligation of pathogen-associated molecular patterns on the cellular surface or the cytosol (29, 94, 109). Almost all members of the TLR family are thought to be directly or indirectly involved in the initiation and regulation of the autophagic machinery against intracellular pathogens (110). In most of these studies, the model of mycobacterial infection has been used. For instance, TLR4 stimulation by LPS induces autophagy in macrophages enhancing mycobacterium colocalization with the autophagosomes (93). It seems that TLR4 signalling mediates the recruitment of Beclin-1 through dissociation of Bcl-2 inhibitor, promoting autophagy (111). In addition, Mihalache’s group found that neutrophils demonstrating vacuolization undergo rapid cell death. Their study showed that neutrophils death depends on receptor-interacting protein 1 kinase activity and papain family protease, but not caspases (112). Vacuolated neutrophils are present in infectious and autoimmune diseases under in vivo conditions (113). Besides, isolated neutrophils from such patients are very sensitive toward ROS production, CD44-mediated phosphatidylinositol 3kinase (PI3K) activation, and cell death, suggesting that the autophagic form of programmed neutrophil death plays an important role in inflammatory responses (112). Recently, we showed that autophagy plays a similar role in AIEC-infected neutrophil-like cells (13). AIEC infection of PLB 985 cells significantly induced the expression of inflammatory cytokine IL-8. Blocking autophagy with Atg5 shRNA markedly amplified IL-8 production, while enhancing the autophagic process by starvation or rapamycin treatment, had the contrary effect. Modulation of IL-8 production by autophagy was induced at the level of gene transcription and correlated with the intracellular bacterial load. Indeed, the non-pathogenic E. coli K12 strain, which does not survive within differentiated PLB-985 cells, was a weak inducer of IL-8 mRNA expression (13).

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Therefore, in neutrophils, autophagy controls the production of inflammatory cytokines by bacterial sequestration and/or elimination. Neutrophils are the most common type of white blood cells and apoptosis is the major mechanism of cell death. Since a long time ago, autophagy has been identified as a modulator of cell survival. However several studies showed that autophagy has emerged as another mechanism involved in cell death independently of apoptosis and necrosis (114–116). The concept of autophagic cell death was first established based on observation of increased autophagic markers in dying cells. Furthermore, different studies showed the induction of autophagy in response to bacterial infection (Table 1). Neutrophils and macrophages have been

Table 1. Autophagy responses to bacterial infection Cellular types Bacteria Adherent invasive Neurtophils Escherichia Differentiated coli (AIEC) PLB-985 Human neutrophils

Escherichia coli

Mice monocytes and neutrophils Human breast carcinoma epithelial cells Human epithelial HeLa cell line HL-60 cells

Listeria monocytogenes

Macrophages and granulocytes mice cells (Atg5) WT and Atf6 MEFs cells HeLa cells

/

Salmonella typhimurium SL1344 Anaplasma phagocytophilum Mycobacterium tuberculosis Salmonella typhimurium Group A streptococcus

Macrophages

AIEC

Macrophage Mice cell

Listeria monocytogenes Mycobacterium tuberculosis H37Rv

Human primary monocytes, MDMs, THP-1 cells Gastric epithelial cells

HeLa human epithelial cells Peripheral blood mononuclear cell

Helicobacter pylori

Salmonella typhimurium SL1344 Mycobacterium tuberculosis

© 2014 APMIS. Published by John Wiley & Sons Ltd

particularly used to establish the interplay between pathogenic agents and the autophagic process. However, the image about autophagy induction and cell death is less clear in neutrophils compared with macrophages. Neutrophil death is a key event in fighting infection. Indeed, it is well established that blood neutrophils are short-lived cells, programmed to die by apoptotic cell death (117). However, several lines of evidence challenge this dogma. At an inflammatory site, a number of cytokines and bacterial products block neutrophils apoptosis to fight bacterial infection. In such an inflammatory environment, Simon’s team recently reported nonapoptotic neutrophil death that was related to massive PI3K-dependent autophagic vacuolization (112, 118).

Observations LC3 expression IL8 production Vaculezed neutrophils Autophagic death Phagocytosis ? TLR activation ? ROS Autophagy induction ROS ? phagosomes PH augmented Antimicrobial autophagy induction Bacteria within vesicles Amino acid starvation ? mTor inhibition ? autophagy Bacteria escape from autophagic vesicle Secreting Ats-1 ? Beclin 1-Atg14L Autophagy induction Defective autophagy ?ROS ? Cytokine Autophagy controls cytokines level Autophagy protects Mice from M. tuberculosis IFN (cytokines) ? ATF6 ? Autophagy induction Rab9A and Rab23 GTPases ? Forming autophagosomes Autophagy process Intracellular bacteria ? autophagy Induction of autophagy decreased AIEC and cytokine release TLR2 and RIP2 ? ERK ? LC3-II increases Autophagy activation Lipoprotein LpqH ? TLR2/1/CD14 and VitD ? autophagy activation Autophagy a host defence Vacuolating cytotoxin (VacA) ? sialic acid and catechins Autophagy represent a host mechanism to limit toxin-induced cellular damage Rab1 ? LC3-bacteria ? autophagosome formation Autophagy process Bacteria ? autophagy process Autophagy controls cytokine production

References (13)

(29) (81) (119)

(120) (121)

(122) (123) (124) (125) (126) (127)

(128) (129)

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Fig. 2. Autophagy blocking gives vacuolated neutrophil and cell death. Pathogenic bacteria stimulate autophagy via different pathways, use the autophagic vesicles to multiplication and escape from them. Accumulation of autophagic vesicles such as autphagosome and/or autolysosome accumulation give vacuolated neutrophil and cell death.

In our recent study we provided evidence of caspase-independent and PI3K-dependent cell death in pathogenic bacteria-infected neutrophils. At the ultrastructural level, the hallmark of AIEC-infected cells was a massive autophagic vacuolization, both in freshly isolated neutrophils and in the neutrophil-like PLB-985 cells (13). In contrast, dying neutrophils were not characterized by apoptotic features such as nuclei condensation/fragmentation and cell shrinkage. Likewise, the plasma membrane of AIEC-infected neutrophils was intact, excluding classical necrotic cell death. Of interest, this cell death was associated with autophagic markers but not apoptotic markers. Corresponding to these observations, AIEC-infected cells were rescued by a pharmacological inhibitor of autophagy but not of apoptosis (13). These findings suggested that pathogenic bacteria, such as AIEC, underwent autophagic neutrophils death. However, we cannot explain how the survival process kills infected-neutrophils. But, it is becoming clear that autophagic activity and autophagic level are not regularly identical, and the blockade of the autophagic process may be confused with excessive autophagy due to the possible accumulation of autophagosomes which give vacuolated neutrophils and cell death (Fig. 2). On the other hand, the role of autophagy in mature neutrophils, after non-pathogenic bacteria clearance, remains unknown.

CONCLUSION Autophagy is generally considered as a homeostasis and survival mechanism with a protective function against intracellular pathogens. However, 8

when infection severity or duration increases, it may promote cell death. In neutrophils, short-lived cells, autophagy has been considered as an essential process by which neutrophils degrade foreign invaders. Altogether, the report shows that autophagy is implicated in different levels of neutrophil defense against pathogens. Indeed, the link between NETs formation, antimicrobial secretion and phagocytosis explains well the role of autophagy in innate immunity. It is also reported that autophagy controls inflammation by the regulation of cytokine production in infected neutrophils, though subversion of the autophagic process under pathogenic bacteria induces neutrophils death. So, more investigations are being done and will be necessary to reveal the relationship between the regulation of autophagy and pathogenic infection which will lead to better understanding of how infectious diseases develop cancer, especially lung and intestinal cancers.

ABBREVIATIONS AIEC: adherent invasive Escherichia coli; mTOR: mammalian target of rapamycin; Atg: autophagy genes; ULK: UNC-51-like kinase; ROS: reactive oxygen species; MPO: myeloperoxidase; LC3: light chain 3; NETs: neutrophil extracellular traps; LPS: lipopolysaccharides; IL-8: interleukin 8; TNF: tumor necrosis factor; TLR(s): toll-like receptor(s); PI3K: phosphatidylinositol 3-kinase; PRRs: pattern recognition receptors; NOD: nucleotide-binding oligomerization-domain; NLRs: NOD-like receptors; PAMPs: pathogen-associated molecular patterns.

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We thank Pr Lassaad El Aouni for its assistance in English language and Dr Mograbi Baharia for its assistance in illustration construction. Research work in the authors’ laboratory was supported by grants from University of Tunis Elmanar and ‘Infectiop^ ole Sud PACA’.

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