- Email: [email protected]
Autophagy regulation in macrophages and neutrophils
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 8 7 –1 1 92
Available online at www.sciencedirect.com
www.elsevier.com/locate/yexcr
Review Article
Autophagy regulation in macrophages and neutrophils Cristina C. Mihalache, Hans-Uwe Simon⁎ Institute of Pharmacology, University of Bern, Bern, Switzerland
A R T I C L E I N F O R M A T I O N
A B S T R A C T
Article Chronology:
Autophagy is a conserved proteolytic mechanism that degrades cytoplasmic material including
Received 4 December 2011
cell organelles. Accumulating evidence exists that autophagy also plays a major role in
Accepted 26 December 2011
immunity and inflammation. Specifically, it appears that autophagy protects against infections
Available online 4 January 2012
and inflammation. Here, we review recent work performed in macrophages and neutrophils, which both represent critical phagocytes in mammalians. © 2012 Elsevier Inc. All rights reserved.
Keywords: Apoptosis Autophagy Cell death Innate immunity Macrophages Necrosis Neutrophils Reactive oxygen species
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autophagy induction by PAMP recognition receptors in macrophages Autophagy participates in macrophage death regulation . . . . . . Autophagy and neutrophil death regulation . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction Inflammation is a complex process generated in response to pathogens, damaged cells, tissue injury, allergens, toxic compounds, or irritant molecules [1]. The inflammatory process is largely initiat-
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ed by the increased production of cytokine and chemokines, whereby macrophages, besides other cells, play an important role. The cytokines and chemokines promote blood leukocyte recruitment, mainly neutrophils to the site of infection or injury [1]. Inflammation is associated with tissue damage and has been
⁎ Corresponding author at: Institute of Pharmacology, University of Bern, Friedbühlstrasse 40, CH-3010 Bern, Switzerland. Fax: + 41 31 632 4992. E-mail address: [email protected] (H.-U. Simon). 0014-4827/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.12.021
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linked to the development of autoimmune diseases and cancer [1]. Both macrophages and neutrophils are phagocytes able to uptake and kill pathogens intracellularly [2,3]. Neutrophils may additionally kill pathogens in the extracellular space by the generation of extracellular DNA traps [4]. Autophagy describes an evolutionary conserved catabolic process that enables cells to degrade and recycle cellular components. There are at least three forms of autophagy: Macro-, micro-, and chaperonmediated autophagy [5]. Since microautophagy has selectively been described in yeast and the role of chaperon-mediated autophagy has not been investigated in association with inflammation, this review deals with macroautophagy only. Please note, however, that, for simplicity, the term autophagy is used throughout the article. Autophagy is a process by which a portion of the cytosol is sequestered in characteristic double- or multi-membrane vesicles to form autophagosomes. The molecular mechanisms of autophagosome formation are conserved in evolution and involve several autophagyrelated genes. One of these genes is atg5, whose product, autophagyrelated gene 5 (ATG5) conjugates with ATG12, to generate an E3 ubiquitin ligase-like enzyme required for autophagy [6,7]. Mice deficient in the atg5 gene die on the first day after birth [8]. In addition to the ATG5–ATG12 conjugate, the ATG8 (LC3) conjugation system is also essential for autophagosome formation [6,7]. Once formed, autophagosomes then merge with lysosomes to form autolysosomes whose contents are degraded by lysosomal hydrolases [6,7]. For more details regarding the regulation of autophagy we refer to another recently published review article [5,7,9,10]. The physiological role of autophagy enables cellular homeostasis. Moreover, autophagy represents an adaptive response to cellular stress, such as nutrient deprivation or growth factor withdrawal [10], conditions, which also play a role in cells of the immune system. Other potential stress factors might be pathogens and other antigens, chemicals, radiation, hypoxia, reactive oxygen species (ROS), and others. This review focuses on autophagy regulation in macrophages and neutrophils that both represent key phagocytosing cells in innate immunity.
Autophagy induction by PAMP recognition receptors in macrophages There is evidence for a complex association between immunity, inflammation, and autophagy [11]. For instance, autophagy plays an important role in host defense against pathogens. Upon phagocytosis of bacteria by macrophages, autophagy induction was shown to be crucial in eliminating intracellular pathogens like Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica, Shigella flexneri, and Streptococcus pyogenes [12]. But how is autophagy induced under conditions of pathogen exposure? One possibility is that bacteria trigger autophagy via toll like receptors (TLRs), which recognize pathogen associated molecular patterns (PAMPs) and represent surface PAMP recognition receptors (PRRs) (Fig. 1). Indeed, activation of TLR4 and TLR7 by bacterial lipopolysaccharide (LPS) and single stranded DNA, respectively, induced autophagy in mouse macrophage RAW264.7 cells [13–15]. In these cells, TLR4 activation results in the recruitment of the adaptors MyD88 and Trif, which can bind Atg6/Beclin-1 to initiate autophagy [15]. Other PAMPs are able to trigger cytosolic PRRs and subsequent autophagy induction. For instance, both nucleotide-binding
Fig. 1 – Autophagy as a mechanism to prevent exaggerated endotoxin-induced IL-1β production in macrophages.
oligomerization domain (NOD)1 and NOD2 activation resulted in the up-regulation of autophagy by recruiting Atg16L1 to efficiently degrade bacteria [16]. In addition, although demonstrated in mouse embryonic fibroblasts only, double stranded RNA (dsRNA) of herpesvirus induces autophagy via IFN-inducible eIF2 kinase PKR [17]. Moreover, IFN-γ, which is produced following pathogen recognition, has also been reported to induce autophagy in macrophage cell lines, but less in primary mouse and human macrophages [18]. Taken together, these reports suggest that autophagy in macrophages can be triggered directly via at least some PRRs and indirectly via certain cytokines induced upon PRR activation. But why is autophagy induction in macrophages following pathogen exposure beneficial for the host? Clearly, autophagy can help to engulf and subsequently degrade intracellular pathogens [19]. However, autophagy plays additional roles in the innate immune response. For instance, autophagy also delivers ubiquitinated cytosolic proteins to autolysosomes, where they can be converted into bactericidal peptides [20,21]. In addition, autophagy appears to enhance the pathogen killing upon phagocytosis by promoting the fusion between phagosomes and lysosomes [22], although the exact molecular mechanisms remain to be determined. It is possible that autophagosomes are not involved in this process [22]. Interestingly, autophagy also
E XP ER I ME NT AL CE L L RE S E AR CH 3 18 (2 01 2 ) 11 8 7 –1 1 92
regulates phagocytosis of dead cells [23,24]. Moreover, it has been reported that autophagy-deficient mouse macrophages generate increased levels of IL-1β, causing additional immunopathology in an experimental colitis model [25]. Similarly, the accumulation of p62 due to deficient autophagy stimulates NF-κB, a major transcription factor for inflammatory cytokines [26] (Fig. 1). Autophagy has also been demonstrated to be important for MHC class II antigen presentation [27], although this mechanism might be more important in dendritic cells and less in macrophages. Taken together, autophagy in macrophages represents an important defense mechanism against pathogens and exerts additional anti-inflammatory effects during inflammatory responses (Table 1).
Autophagy participates in macrophage death regulation Macrophage activation by PAMPs or inflammatory stimuli was reported to activate both survival and death pathways in these cells. Single stimulating agents, such as LPS or TNF-α, do not induce macrophage death under in vitro conditions [28]. In fact, LPS-activated macrophages are more resistant towards death triggers [28–30]. Under these conditions, it is possible that the induction of autophagy plays a role for this protective effect [13]. Furthermore, IFN-γ was shown to induce autophagy, to inhibit ROS, to sustain Jak2-STAT1 activation, and to maintain cellular survival, at least in mouse embryonic fibroblasts [31]. On the other hand, LPS also activates caspases in macrophages [29,30]. Interestingly, pharmacological inactivation of caspases by benzyloxycarbonyl-Val-Ala-Asp (z-VAD) leads to strong macrophage death when concurrently activated by LPS [29,30]. In such cell death, the generation of ROS via p38 MAPK and STAT1 pathways seems to be important [29]. ROS may then induce autophagy in macrophages, perhaps due to overactivation of PARP [32]. Moreover, ROS has been shown to be able to activate the inflammasome resulting in IL-1β production [33]. Autophagy may here again be helpful not only to be cytoprotective, but also to limit ROS generation and subsequent IL-1β production and inflammation by removing defective mitochondria (Fig. 1).
Autophagy and neutrophil death regulation There is evidence that autophagy occurs in human and mouse neutrophils in both phagocytosis-independent and phagocytosisdependent manner [34,35], similar to what has been reported for macrophages [13–15,19]. However, the picture about inflammation-associated autophagy is less clear in neutrophils
Table 1 – Immune functions of macrophages regulated by autophagy. – – – – – – –
Phagosome maturation Antigen presentation Clearance of apoptotic cells/bodies Pathogen degradation (xenophagy) Release of antimicrobial peptides Regulation of cytokine production Cytoprotection and cell death regulation
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compared with macrophages, largely due to much less published reports. Since terminal differentiated neutrophils are metabolically relative inactive cells with a limited number of mitochondria only [36], autophagy may play a role during neutrophil differentiation as it has been described in the red cell lineage [37]. However, no published data are currently available that would support such an assumption. Most of the available data in mature neutrophils suggest that autophagy assists cell death and these studies are briefly reviewed here. Although vacuolized neutrophils have been noticed in the past, an autophagic-like morphology of neutrophils was just recently suggested [38]. It was demonstrated that upon GM-CSF priming and anti-Siglec-9 monoclonal antibody ligation, neutrophils undergo a non-apoptotic form of cell death characterized by cytoplasmic vacuolization, mitochondrial swelling, nuclear condensation and normal plasma membrane integrity [38]. Interestingly, preparations of human intravenous immunoglobulin (IVIg) containing natural anti-Siglec-9 autoantibodies were shown to bind Siglec-9 on neutrophils leading to autophagiclike cell death upon GM-CSF priming [39]. Furthermore, in agreement with these studies, we recently reported that neutrophils exposed to GM-CSF and other inflammatory cytokines followed by CD44 ligation undergo autophagy-associated caspase-independent death characterized by large cytoplasmic vacuoles, generated by fusion events between several organelles, including endosomes, autophagosomes and secondary granules [40]. These data point to the possibility that adhesion molecules have the capacity to trigger caspase-independent cell death associated with autophagy induction in neutrophils that is largely necrotic in its nature (Fig. 2). Vacuolized neutrophils were observed in septic shock, cystic fibrosis, rheumatoid arthritis and several skin diseases [40], suggesting that induction of autophagy in these cells is a general phenomenon of neutrophilic inflammatory responses independent of the trigger. In addition, NETosis another form of neutrophil death [4], characterized by the formation of neutrophil extracellular traps (NETs) [41], may require autophagy induction [42]. However, it should be noted that NETs can be generated in the absence of cell death [43]. Therefore, it is possible that NETs are generated before neutrophil death actually occurs. In addition, we observed a NETosis-like cell death in GM-CSF primed neutrophils following CD44 ligation in the absence of NETs formation [41]. Taken together, whether NETosis represents indeed a distinct form of cell death remains uncertain. It could be that NETosis is a necrotic form of cell death that may or may not require autophagy [44]. Interestingly, the non-apoptotic autophagic-like or necrotic types of neutrophil death depend on high intracellular ROS levels generated by the NADPH oxidase [38,40]. In contrast, lower levels of ROS may trigger spontaneous and TNF-induced apoptosis in these cells [45,46]. In many of these studies, the dependence on ROS was demonstrated by both genetic and pharmacological means [38,40,45,46]. These studies suggested that the type of death in singly neutrophils is likely regulated by intracellular ROS levels (Fig. 2). Therefore, when neutrophil populations are activated by certain death triggers in vitro, the actual type of death is not necessarily the same in each individual cell. Interestingly, the activation of the NADPH oxidase involves p38 MAPK and PI3K pathways, which are usually involved in survival signaling
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Fig. 2 – Autophagy in association with high ROS generation and its participation within necrotic cell death pathways in neutrophils. Please note that not necessarily all cells following death stimulation undergo the same type of death. If ROS levels are below a certain threshold, neutrophils undergo apoptosis, whereby mitochondria may or may not participate in the activation of the caspase cascade. The apoptotic cell death due to cytokine withdrawal was not discussed in this article. Original work demonstrating the roles of cathepsin D, caspase-8, and calpain is reported in Refs. [45] and [47].
[40,46]. Due to these and other peculiarities of cell death pathways in neutrophils [44], the strategy of pharmacological inhibition of these pathways in inflammation may not necessarily be successful.
Concluding remarks Autophagy plays a role in the regulation of innate immunity. Enhancement of autophagy following PRR stimulation in macrophages contributes in pathogen defense mechanisms. On the other hand, dysregulated autophagy may allow pathogen survival and stimulates inflammatory pathways. The role of autophagy in neutrophil killing mechanisms against bacteria and/or fungi remains to be investigated. However, there is evidence that neutrophils increase autophagic activity under inflammatory conditions and that the autophagic pathways frequently participate in non-apoptotic cell death pathways. Taken together, the induction of autophagy in both macrophages and neutrophils seems to contribute to the prevention of chronic infectious and inflammatory diseases. Future studies are required to better understand the role of autophagy in the neutrophil defense against pathogens. Moreover, since the
majority of the studies have been performed under in vitro conditions, the in vivo role of autophagy in immunity needs to be clarified in experimental models and disease.
Acknowledgments Work in the laboratory of H.U.S. is supported by the Swiss National Science Foundation (grant no. 310030_129640), Stiftung zur Krebsbekämpfung, Zurich, and Allergie-Stiftung Ulrich MüllerGierok, Bern.
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Available online at www.sciencedirect.com
www.elsevier.com/locate/yexcr
Review Article
Autophagy regulation in macrophages and neutrophils Cristina C. Mihalache, Hans-Uwe Simon⁎ Institute of Pharmacology, University of Bern, Bern, Switzerland
A R T I C L E I N F O R M A T I O N
A B S T R A C T
Article Chronology:
Autophagy is a conserved proteolytic mechanism that degrades cytoplasmic material including
Received 4 December 2011
cell organelles. Accumulating evidence exists that autophagy also plays a major role in
Accepted 26 December 2011
immunity and inflammation. Specifically, it appears that autophagy protects against infections
Available online 4 January 2012
and inflammation. Here, we review recent work performed in macrophages and neutrophils, which both represent critical phagocytes in mammalians. © 2012 Elsevier Inc. All rights reserved.
Keywords: Apoptosis Autophagy Cell death Innate immunity Macrophages Necrosis Neutrophils Reactive oxygen species
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autophagy induction by PAMP recognition receptors in macrophages Autophagy participates in macrophage death regulation . . . . . . Autophagy and neutrophil death regulation . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction Inflammation is a complex process generated in response to pathogens, damaged cells, tissue injury, allergens, toxic compounds, or irritant molecules [1]. The inflammatory process is largely initiat-
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ed by the increased production of cytokine and chemokines, whereby macrophages, besides other cells, play an important role. The cytokines and chemokines promote blood leukocyte recruitment, mainly neutrophils to the site of infection or injury [1]. Inflammation is associated with tissue damage and has been
⁎ Corresponding author at: Institute of Pharmacology, University of Bern, Friedbühlstrasse 40, CH-3010 Bern, Switzerland. Fax: + 41 31 632 4992. E-mail address: [email protected] (H.-U. Simon). 0014-4827/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.12.021
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linked to the development of autoimmune diseases and cancer [1]. Both macrophages and neutrophils are phagocytes able to uptake and kill pathogens intracellularly [2,3]. Neutrophils may additionally kill pathogens in the extracellular space by the generation of extracellular DNA traps [4]. Autophagy describes an evolutionary conserved catabolic process that enables cells to degrade and recycle cellular components. There are at least three forms of autophagy: Macro-, micro-, and chaperonmediated autophagy [5]. Since microautophagy has selectively been described in yeast and the role of chaperon-mediated autophagy has not been investigated in association with inflammation, this review deals with macroautophagy only. Please note, however, that, for simplicity, the term autophagy is used throughout the article. Autophagy is a process by which a portion of the cytosol is sequestered in characteristic double- or multi-membrane vesicles to form autophagosomes. The molecular mechanisms of autophagosome formation are conserved in evolution and involve several autophagyrelated genes. One of these genes is atg5, whose product, autophagyrelated gene 5 (ATG5) conjugates with ATG12, to generate an E3 ubiquitin ligase-like enzyme required for autophagy [6,7]. Mice deficient in the atg5 gene die on the first day after birth [8]. In addition to the ATG5–ATG12 conjugate, the ATG8 (LC3) conjugation system is also essential for autophagosome formation [6,7]. Once formed, autophagosomes then merge with lysosomes to form autolysosomes whose contents are degraded by lysosomal hydrolases [6,7]. For more details regarding the regulation of autophagy we refer to another recently published review article [5,7,9,10]. The physiological role of autophagy enables cellular homeostasis. Moreover, autophagy represents an adaptive response to cellular stress, such as nutrient deprivation or growth factor withdrawal [10], conditions, which also play a role in cells of the immune system. Other potential stress factors might be pathogens and other antigens, chemicals, radiation, hypoxia, reactive oxygen species (ROS), and others. This review focuses on autophagy regulation in macrophages and neutrophils that both represent key phagocytosing cells in innate immunity.
Autophagy induction by PAMP recognition receptors in macrophages There is evidence for a complex association between immunity, inflammation, and autophagy [11]. For instance, autophagy plays an important role in host defense against pathogens. Upon phagocytosis of bacteria by macrophages, autophagy induction was shown to be crucial in eliminating intracellular pathogens like Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica, Shigella flexneri, and Streptococcus pyogenes [12]. But how is autophagy induced under conditions of pathogen exposure? One possibility is that bacteria trigger autophagy via toll like receptors (TLRs), which recognize pathogen associated molecular patterns (PAMPs) and represent surface PAMP recognition receptors (PRRs) (Fig. 1). Indeed, activation of TLR4 and TLR7 by bacterial lipopolysaccharide (LPS) and single stranded DNA, respectively, induced autophagy in mouse macrophage RAW264.7 cells [13–15]. In these cells, TLR4 activation results in the recruitment of the adaptors MyD88 and Trif, which can bind Atg6/Beclin-1 to initiate autophagy [15]. Other PAMPs are able to trigger cytosolic PRRs and subsequent autophagy induction. For instance, both nucleotide-binding
Fig. 1 – Autophagy as a mechanism to prevent exaggerated endotoxin-induced IL-1β production in macrophages.
oligomerization domain (NOD)1 and NOD2 activation resulted in the up-regulation of autophagy by recruiting Atg16L1 to efficiently degrade bacteria [16]. In addition, although demonstrated in mouse embryonic fibroblasts only, double stranded RNA (dsRNA) of herpesvirus induces autophagy via IFN-inducible eIF2 kinase PKR [17]. Moreover, IFN-γ, which is produced following pathogen recognition, has also been reported to induce autophagy in macrophage cell lines, but less in primary mouse and human macrophages [18]. Taken together, these reports suggest that autophagy in macrophages can be triggered directly via at least some PRRs and indirectly via certain cytokines induced upon PRR activation. But why is autophagy induction in macrophages following pathogen exposure beneficial for the host? Clearly, autophagy can help to engulf and subsequently degrade intracellular pathogens [19]. However, autophagy plays additional roles in the innate immune response. For instance, autophagy also delivers ubiquitinated cytosolic proteins to autolysosomes, where they can be converted into bactericidal peptides [20,21]. In addition, autophagy appears to enhance the pathogen killing upon phagocytosis by promoting the fusion between phagosomes and lysosomes [22], although the exact molecular mechanisms remain to be determined. It is possible that autophagosomes are not involved in this process [22]. Interestingly, autophagy also
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regulates phagocytosis of dead cells [23,24]. Moreover, it has been reported that autophagy-deficient mouse macrophages generate increased levels of IL-1β, causing additional immunopathology in an experimental colitis model [25]. Similarly, the accumulation of p62 due to deficient autophagy stimulates NF-κB, a major transcription factor for inflammatory cytokines [26] (Fig. 1). Autophagy has also been demonstrated to be important for MHC class II antigen presentation [27], although this mechanism might be more important in dendritic cells and less in macrophages. Taken together, autophagy in macrophages represents an important defense mechanism against pathogens and exerts additional anti-inflammatory effects during inflammatory responses (Table 1).
Autophagy participates in macrophage death regulation Macrophage activation by PAMPs or inflammatory stimuli was reported to activate both survival and death pathways in these cells. Single stimulating agents, such as LPS or TNF-α, do not induce macrophage death under in vitro conditions [28]. In fact, LPS-activated macrophages are more resistant towards death triggers [28–30]. Under these conditions, it is possible that the induction of autophagy plays a role for this protective effect [13]. Furthermore, IFN-γ was shown to induce autophagy, to inhibit ROS, to sustain Jak2-STAT1 activation, and to maintain cellular survival, at least in mouse embryonic fibroblasts [31]. On the other hand, LPS also activates caspases in macrophages [29,30]. Interestingly, pharmacological inactivation of caspases by benzyloxycarbonyl-Val-Ala-Asp (z-VAD) leads to strong macrophage death when concurrently activated by LPS [29,30]. In such cell death, the generation of ROS via p38 MAPK and STAT1 pathways seems to be important [29]. ROS may then induce autophagy in macrophages, perhaps due to overactivation of PARP [32]. Moreover, ROS has been shown to be able to activate the inflammasome resulting in IL-1β production [33]. Autophagy may here again be helpful not only to be cytoprotective, but also to limit ROS generation and subsequent IL-1β production and inflammation by removing defective mitochondria (Fig. 1).
Autophagy and neutrophil death regulation There is evidence that autophagy occurs in human and mouse neutrophils in both phagocytosis-independent and phagocytosisdependent manner [34,35], similar to what has been reported for macrophages [13–15,19]. However, the picture about inflammation-associated autophagy is less clear in neutrophils
Table 1 – Immune functions of macrophages regulated by autophagy. – – – – – – –
Phagosome maturation Antigen presentation Clearance of apoptotic cells/bodies Pathogen degradation (xenophagy) Release of antimicrobial peptides Regulation of cytokine production Cytoprotection and cell death regulation
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compared with macrophages, largely due to much less published reports. Since terminal differentiated neutrophils are metabolically relative inactive cells with a limited number of mitochondria only [36], autophagy may play a role during neutrophil differentiation as it has been described in the red cell lineage [37]. However, no published data are currently available that would support such an assumption. Most of the available data in mature neutrophils suggest that autophagy assists cell death and these studies are briefly reviewed here. Although vacuolized neutrophils have been noticed in the past, an autophagic-like morphology of neutrophils was just recently suggested [38]. It was demonstrated that upon GM-CSF priming and anti-Siglec-9 monoclonal antibody ligation, neutrophils undergo a non-apoptotic form of cell death characterized by cytoplasmic vacuolization, mitochondrial swelling, nuclear condensation and normal plasma membrane integrity [38]. Interestingly, preparations of human intravenous immunoglobulin (IVIg) containing natural anti-Siglec-9 autoantibodies were shown to bind Siglec-9 on neutrophils leading to autophagiclike cell death upon GM-CSF priming [39]. Furthermore, in agreement with these studies, we recently reported that neutrophils exposed to GM-CSF and other inflammatory cytokines followed by CD44 ligation undergo autophagy-associated caspase-independent death characterized by large cytoplasmic vacuoles, generated by fusion events between several organelles, including endosomes, autophagosomes and secondary granules [40]. These data point to the possibility that adhesion molecules have the capacity to trigger caspase-independent cell death associated with autophagy induction in neutrophils that is largely necrotic in its nature (Fig. 2). Vacuolized neutrophils were observed in septic shock, cystic fibrosis, rheumatoid arthritis and several skin diseases [40], suggesting that induction of autophagy in these cells is a general phenomenon of neutrophilic inflammatory responses independent of the trigger. In addition, NETosis another form of neutrophil death [4], characterized by the formation of neutrophil extracellular traps (NETs) [41], may require autophagy induction [42]. However, it should be noted that NETs can be generated in the absence of cell death [43]. Therefore, it is possible that NETs are generated before neutrophil death actually occurs. In addition, we observed a NETosis-like cell death in GM-CSF primed neutrophils following CD44 ligation in the absence of NETs formation [41]. Taken together, whether NETosis represents indeed a distinct form of cell death remains uncertain. It could be that NETosis is a necrotic form of cell death that may or may not require autophagy [44]. Interestingly, the non-apoptotic autophagic-like or necrotic types of neutrophil death depend on high intracellular ROS levels generated by the NADPH oxidase [38,40]. In contrast, lower levels of ROS may trigger spontaneous and TNF-induced apoptosis in these cells [45,46]. In many of these studies, the dependence on ROS was demonstrated by both genetic and pharmacological means [38,40,45,46]. These studies suggested that the type of death in singly neutrophils is likely regulated by intracellular ROS levels (Fig. 2). Therefore, when neutrophil populations are activated by certain death triggers in vitro, the actual type of death is not necessarily the same in each individual cell. Interestingly, the activation of the NADPH oxidase involves p38 MAPK and PI3K pathways, which are usually involved in survival signaling
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Fig. 2 – Autophagy in association with high ROS generation and its participation within necrotic cell death pathways in neutrophils. Please note that not necessarily all cells following death stimulation undergo the same type of death. If ROS levels are below a certain threshold, neutrophils undergo apoptosis, whereby mitochondria may or may not participate in the activation of the caspase cascade. The apoptotic cell death due to cytokine withdrawal was not discussed in this article. Original work demonstrating the roles of cathepsin D, caspase-8, and calpain is reported in Refs. [45] and [47].
[40,46]. Due to these and other peculiarities of cell death pathways in neutrophils [44], the strategy of pharmacological inhibition of these pathways in inflammation may not necessarily be successful.
Concluding remarks Autophagy plays a role in the regulation of innate immunity. Enhancement of autophagy following PRR stimulation in macrophages contributes in pathogen defense mechanisms. On the other hand, dysregulated autophagy may allow pathogen survival and stimulates inflammatory pathways. The role of autophagy in neutrophil killing mechanisms against bacteria and/or fungi remains to be investigated. However, there is evidence that neutrophils increase autophagic activity under inflammatory conditions and that the autophagic pathways frequently participate in non-apoptotic cell death pathways. Taken together, the induction of autophagy in both macrophages and neutrophils seems to contribute to the prevention of chronic infectious and inflammatory diseases. Future studies are required to better understand the role of autophagy in the neutrophil defense against pathogens. Moreover, since the
majority of the studies have been performed under in vitro conditions, the in vivo role of autophagy in immunity needs to be clarified in experimental models and disease.
Acknowledgments Work in the laboratory of H.U.S. is supported by the Swiss National Science Foundation (grant no. 310030_129640), Stiftung zur Krebsbekämpfung, Zurich, and Allergie-Stiftung Ulrich MüllerGierok, Bern.
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