Immunol Res DOI 10.1007/s12026-015-8655-z

INTERPRETIVE SYNTHESIS REVIEW ARTICLE

Mcl-1 is vital for neutrophil survival Mark P. Murphy1,2 • Emma Caraher1,2

Ó Springer Science+Business Media New York 2015

Abstract Upon entry to the systemic circulation, neutrophils exhibit a short mean time to cell death. The viability of most cell types in a steady state is preserved by the interplay of the Bcl-2 family of proteins, wherein the anti-apoptotic members inhibit the action of their pro-apoptotic counterparts. Neutrophils, however, display absent or severely reduced expression of several anti-apoptotic Bcl-2 family proteins. Hence, they rely on the expression of Mcl-1, an anti-apoptotic member of the Bcl-2 family, for survival. This protein is uniquely short-lived relative to related proteins and its loss likely precipitates the induction of apoptosis in neutrophils. This review describes the role of Mcl-1 in the neutrophil in the context of apoptosis and highlights the proteins’ importance to the cell. We also address neutrophil apoptosis in the broader context of the cells’ response to pathogens, focussing particularly on the strategies used by pathogens to manipulate the apoptotic pathway to their own ends. Keywords Bcl-2 family proteins
Caspases
Host– pathogen interaction
Innate immunity
Neutrophil apoptosis

& Mark P. Murphy [email protected] Emma Caraher [email protected] 1

Centre for Microbial-Host Interactions, Institute of Technology Tallaght, Old Blessington Road, Tallaght, Dublin 24, Ireland

2

Centre of Applied Science for Health, Institute of Technology Tallaght, Dublin, Ireland

Introduction Inflammation is often the result of neutrophilia and has a number of undesirable sequelae. For this reason, targets to ameliorate the zeal of neutrophils can prove useful in controlling inflammation and improving health in some settings [1]. This review highlights neutrophil viability— and, specifically, the role of anti-apoptotic Mcl-1 in preserving same—as a potential focal point for modulating the cells’ activity. The resting neutrophil has a short survival time relative to other human cell types. Circulating neutrophils persist with a mean half-life generally accepted to be only 7 h in the absence of infection [2, 3]. This follows bone marrow residence of *7 days as a terminally differentiated cell [4]. This limited lifespan may be due to the unique expression profile of anti-apoptotic proteins which neutrophils exhibit. We detail in this review the cellular mechanisms of intrinsic and extrinsic cell death in neutrophils that follow from this expression, which renders the cells unusually reliant on the short-lived protein Mcl-1. Given the short lifespan of the neutrophil, the capacity of the cells to effect an anti-microbial response is enhanced by increases to their survival time. This is often mediated directly by cytokines being produced at the site of infection, such as granulocyte macrophage colony-stimulating factor (GM-CSF) [5–8]. Certain intracellular pathogens also actively enhance neutrophil viability in order to further their own survival. Conversely, many pathogens deliberately antagonise neutrophil viability for the obvious benefit of prevention of their destruction by the phagocytes. Examples of each of these strategies are discussed in this review.

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Regulatory mechanisms of neutrophil survival depend on Mcl-1 Cellular survival requires homoeostasis of a number of competing endogenous pro- and anti-apoptotic proteins. These include both pro- and anti-apoptotic members of the B cell lymphoma-2 (Bcl-2, Table 1) family of proteins, caspases, cyclin-dependent kinases (CDKs), inhibitors of apoptosis proteins (IAPs), as well as upstream effectors of cell death such as Fas, tumour necrosis factor receptor 1 (TNFR1) or TNF-related apoptosis-inducing ligand (TRAIL) receptor. The balance between these factors determines the cells’ fate. Circulating neutrophils exist in a state of constitutive apoptosis, possibly derived in part from the cells’ arrest in the G0 phase of the cell cycle, skewing the activity of CDKs [9]. They also lack expression of the anti-apoptotic protein, Bcl-2, rendering them largely reliant on the related protein, Mcl-1, for the prevention of apoptosis [10]. Indeed, it has been shown that the depletion of Mcl-1 alone is sufficient to induce apoptosis in U937 cells [11]. Mcl-1 is short-lived in the cell, with a turnover of approximately 3 h at both mRNA and protein levels [12]. This short half-life is the result of the presence of a number of motifs in Mcl-1, which target the protein for poly-ubiquitination and proteasomal degradation [13]. These motifs are absent from the other Bcl-2 family members expressed by neutrophils, giving them a far longer half-life and suggesting Mcl-1 as the key focal point of the cells’ survival. Indeed, complete inhibition of CDKs, by, for example, R-roscovitine, abrogates Mcl-1 expression and induces apoptosis [14–16]. While several studies have reported a loss of Mcl-1 mRNA expression resulting from CDK inhibition in neutrophils, the situation may not be so straightforward, as, if apoptosis is prevented following CDK inhibition—by addition of anti-apoptotic Bcl-2—the level of Mcl-1 mRNA does not reduce [17]. The authors of that study noted that proteasomal degradation of Mcl-1 was increased in that scenario. Thus, it can be inferred that CDKs function in part to mitigate degradation of Mcl-1. In the neutrophil, Mcl-1 acts most directly to bind certain pro-apoptotic Bcl-2 family members [18, 19]. In the viable cell, Mcl-1 sequesters Bak and Bax [18, 20] preventing their pro-apoptotic functions, thereby maintaining cell viability. Several overlapping anti-apoptotic Bcl-2 family members sequester their pro-apoptotic counterparts in the majority of cell types; however, many of these are absent in neutrophils. Therefore, neutrophils are more reliant on Mcl-1 than most cells. Countering Mcl-1, Noxa specifically binds and suppresses Mcl-1 unless its own expression is down-regulated [21]. In turn, the pro-apoptotic protein Bax can be

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inactivated by being bound by Mcl-1 once Bax has been phosphorylated by Akt or ERK [22, 23]. Moreover, degradation of Mcl-1 is ordinarily expedited by its phosphorylation by glycogen synthase kinase (GSK)-3b. Prolongation of neutrophil survival in response to certain endogenous cytokines, such as IL-32c, involves maintenance of Mcl-1 protein levels through inactivation of GSK3b [24]. Antagonism of Mcl-1 by GSK-3b can be inhibited by its phosphorylation, transduced by activated Toll-like receptors (TLRs) or the formyl peptide receptor (FPR) [25, 26], thus emphasising a role for Mcl-1 in prolonging the neutrophil response to bacterial challenge. Work by Franc¸ois et al. [27], which focussed on the outcome of agonism of neutrophil TLRs by their respective, canonical ligands with respect to the survival of the cells, described that the activation of TLRs 2, 4, 7/8 and 9—by peptidoglycan, lipopolysaccharide (LPS), R-484 and CpG-DNA, respectively—prolonged the survival of human neutrophils in vitro. Moreover, this extension of neutrophil lifespan coincided with up-regulation of the anti-apoptotic Mcl-1 and A1 proteins. Each of the effective pro-survival TLRs acted independently through both nuclear factor jB (NF-jB) and phosphatidylinositol-3 kinase (PI3K), and it was subsequently corroborated that TLR9 agonism by CpG-DNA activates NF-jB, resulting in up-regulation of Mcl-1 [28]. The observation that agonism of the remaining neutrophil-associated TLRs, 1, 5 and 6, failed to improve survival was linked to the ability of the former set of TLRs to mediate this activity through PI3K, a functionality which the latter group lack [29]. TLR4-mediated extension of neutrophil lifespan is a short-term phenomenon (B4 h) where neutrophils are stimulated as a homogeneous cell population. However, the presence of a minor cohort of monocytes enables the prevention of apoptosis in neutrophils for greater than 20 h [30]. This coincides with an enhanced secretion of GMCSF, interleukin-8 (IL-8) and IL-1b by the monocytes [31]. Helicobacter pylori bacteria secrete a neutrophil-activating protein (HP-NAP) during colonisation of the gastric mucosa which prolongs the survival of neutrophils, hence potentiating inflammation [32]. HP-NAP achieves this indirectly, by stimulating the secretion of IL-1b from monocytes. PI3K signalling is also integral to the survival extension conferred by GM-CSF, as is Mcl-1 [33, 34]; in fact, GMCSF-stimulated PI3K and protein kinase B (Akt) signalling enhance Mcl-1 expression [10, 20, 35]. Similarly, TNFa, when present in low concentrations, can stabilise Mcl-1 by inducing extracellular signal-related kinase (ERK)-1/2mediated phosphorylation of the protein [36–38]. Hence, it is apparent that the actions of many pro-survival effectors converge through the kinases PI3K and Akt, not only

Immunol Res Table 1 Members of the Bcl-2 family of apoptosis inducing and/or regulating proteins which are expressed by human neutrophils Function

Protein

BH domains

Anti-apoptotic

A1

BH1–BH4

Bcl-XL Mcl-1 (Myeloid cell leukaemia-1) Pro-apoptotic effectors

Bak (Bcl-2 homologous antagonist/killer)

BH1–BH4

Bax (Bcl-2-associated 9 protein) Pro-apoptotic direct activators

Bid (Bcl-2 interacting domain death agonist)

BH3 only

Bim (Bcl-2 interacting mediator of cell death) Pro-apoptotic derepressors

Bim

BH3 only

Noxa

through their direct modification of pro-apoptotic Bcl-2 members, but also by engaging NF-jB to up-regulate expression of Mcl-1 [28]. Neutrophils express a subset of the Bcl-2 family members thus far discovered. Those discussed in this review are categorised above based on their known functions in the cell. Also indicated are those Bcl-2 homology (BH) domains—which enable protein–protein interactions with other family members—which are possessed by each group [39].

Neutrophil cell death is a requirement for resolution of infection and itself requires Mcl-1 activity to be overcome That neutrophils undergo apoptosis is an apparent prerequisite for the resolution of the inflammation which accompanies infection. During their anti-microbial response, neutrophils potentiate inflammation by paracrine signalling and by release of tissue-destructive reactive oxygen species (ROS) and proteases. Such distressed tissue produces leukotriene B4 and other chemoattractants, the outcome of which is an accumulation of neutrophils and other innate and adaptive leucocytes. Once a critical cell quantity is reached, the intravascular association between platelets and the cohort of neutrophils awaiting extravasation prompts release of platelet-derived lipoxin A4 (LXA4) [40]. This, along with other pro-resolving mediators, such as resolvins and protectins, functions to restrict neutrophil recruitment while promoting the phagocytic activity of macrophages [41–45]. Thus, despite the extended survival the cells experience, the neutrophil must engage in anti-microbial activity within a short time period before it undergoes activation-mediated cell death. During infection, cytokines derived from the epithelium, along with microbially associated molecular patterns (MAMPs), encourage neutrophil survival in order

to complete the destruction of challenging micro-organisms. This can have tissue-damaging consequences however, and as such, having phagocytosed some quantity of microbes, the neutrophil will undergo phagocytosis-induced cell death (PICD). Phagocytosis induces the activation of the apoptotic effector, caspase-3, suggesting PICD as a deliberate apoptotic mechanism [46, 47]. PICD is preceded by generation of reactive oxygen species (ROS) by the neutrophil via the assembled NADPH oxidase complex, blockade of which prevents caspase activation. This is corroborated by the fundamental observation that phagocytosis of opsonised (IgG-coated) zymosan particles by neutrophils induces apoptosis in the cells, which is preventable with inhibitors of ROS generation [48]. The authors of that study supported their finding using neutrophils derived from people with chronic granulomatous disease (CGD), who lack functional NADPH oxidase. The CGD neutrophils phagocytosed the opsonised particles successfully, but did not undergo apoptosis. Hence, it can be inferred that PICD is an NADPH oxidase-dependent form of apoptosis. Furthermore, the phenotypic traits observed during PICD appear to be mediated by ROS generated cytoplasmically rather than intraphagosomally [49], such as that occurs following engagement of complement-opsonised bacteria by the b2 integrin, Mac-1 (CD11b) [50]. PICD prompts macrophages to ingest and clear the dying cells, in a process termed efferocytosis. Macrophages which ingest sufficient neutrophils become quiescent, secreting mediators to resolve inflammation, but prior to this, they may assist the recruited neutrophil population to die; they can secrete cell death-inducing ligands such as FasL or TNFa, which induce extrinsically mediated apoptosis in neutrophils through binding to their respective receptors, Fas (CD95) and TNFR1 [51]. Recent in vivo studies have suggested that Fas is a dominant route through which neutrophil numbers are deliberately controlled during infection [52].

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FasL recognition causes formation of a death-inducing signalling complex (DISC) comprising Fas, Fas-associated protein with death domain (FADD) and procaspase 8 [53]. The formation of DISC, in turn, leads to the cleavage of procaspase-8 into active caspase-8 [54]. Activated caspase8 then causes the effector caspase, caspase-3, to become activated, which conducts the onset of many apoptosisrelated morphological changes. Caspase-8 conducts the intrinsic apoptotic pathway to activate caspase-3, but is also capable of activating caspase-3 directly, albeit inducing apoptosis at a slower rate [55]. Intriguingly, the ligand for TNFR1, TNFa, can extend the neutrophils’ lifespan at low concentrations, instigating DISC-mediated apoptosis only at higher concentrations [38]. This dichotomy may reflect the progress of a response to a nascent infection, whereby accumulation of TNFa over time prompts the death of neutrophils, ultimately enabling the resolution of inflammation. In a study of the ‘dose response’ of neutrophils to increasing concentrations of Escherichia coli bacteria, Matsuda et al. [56] observed that neutrophil apoptosis was inhibited when faced with low concentrations of bacteria—likely the result of agonism by MAMPs and endogenous cytokines—but that at high relative concentrations of bacteria, neutrophil apoptosis was elevated. The latter observation was likely due to phagocytosis-induced ROS. Hence, it is apparent that neutrophil survival is prolonged long enough to engage and ingest pathogens, but then deliberately ceases as a direct outcome of that activity. ROS exert their pro-apoptotic effect by causing lysosomal membrane permeabilisation (LMP) resulting in the release of multiple proteases. The lysosomal protease, cathepsin D, is among the primary drivers of apoptosis [57]. It achieves this by discrete activation of Bid and caspase-8 proteins [57, 58]. Caspase-8 then further aids in activation of Bid [59]. The resultant truncated Bid (tBid) propagates the process of apoptosis firstly by contributing to LMP [60, 61] and, secondly, by interacting with Bax to form pores in mitochondrial outer membranes [62, 63]. Concurrently, Bax is de-phosphorylated by the action of calpain-1—itself no longer inhibited due to the caspase-8mediated inactivation of the calpain-1 inhibitor, calpastatin [64]. Bax is then able to disengage Mcl-1—which otherwise sequesters Bak and Bax—allowing their translocation to mitochondria [62, 65–67]. Bak and Bax form homooligomeric pores in the mitochondrial outer membrane, conformations which are directly facilitated by tBid and Bim, respectively (though with functional crosstalk) [68]. Mitochondrial outer membrane permeabilisation (MOMP), in turn, allows the release of cytochrome C, Smac and Diablo proteins (among others), which act to produce active caspase-3 from its pro-caspase form [64, 69]. Caspase-3 degrades inhibitor of caspase-activated DNase (ICAD), thereby releasing caspase-activated DNase

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(CAD), which digests chromosomal DNA to yield the DNA fragmentation pattern characteristic of apoptosis [70]. Caspase-3 also accelerates the foregoing process by degradation of Mcl-1 [66]. Given that caspase-8-activated tBid can induce MOMP, LMP is not essential for MOMP and hence, apoptosis, but can greatly enhance it by cathepsin-mediated activation of caspase-8 and Bid. Lysosomes (as do other neutrophil granules) harbour the serine protease, proteinase 3 (PR3). PR3 is capable of directly activating caspase-3, without the requirement of mitochondrially derived effectors [71]. LMP is observed in ageing neutrophils, and PR3 has recently been shown to contribute to the spontaneous apoptosis experienced by aged neutrophils [72]. This direct intervention of caspase-8 and tBid in enabling Bak and Bax to induce MOMP is contingent on the failure of Mcl-1 (and related proteins) to sequester Bak and Bax. As such, these pro-apoptotic factors are aided in induction of apoptosis by a group of Bcl-2 family proteins, which themselves sequester their anti-apoptotic relatives. In neutrophils, the protein Noxa is perhaps most acutely relevant in this regard as it specifically binds Mcl-1 [73]. Bim, too, moonlights from its role as an activator of Bak oligomerisation in order to sequester Mcl-1 [74]. The view may also be taken that Mcl-1 sequesters Bim, to prevent the activation of Bak. Hence, the balance of respective concentrations of a cells’ pro- and anti-apoptotic proteins determines its viability. In support of this notion, the expression of Noxa in particular is modulated by certain death signals, such as ROS or TNFa, via the tumour-suppressing transcription factor, p53 [75, 76]. In p53-/- mice, neutrophils experience prolonged life, exhibit greater responsiveness to TLR agonism and demonstrate heightened phagocytosis [77]. These features enabled enhanced clearance of infections by the mice, implying a role for p53 in moderating the neutrophil anti-microbial response. Thusly, a signal calling for apoptosis is propagated through multiple, closely interconnected pathways, converging on caspase-3, whose activation is largely reliant on the proteasomal degradation or sequestration of antiapoptotic Mcl-1. The interactions between the actors discussed in this review are illustrated in Fig. 1.

Many micro-organisms influence neutrophil survival through modulation of apoptosis The picture of neutrophil–pathogen interactions, with respect to neutrophil survival, is complex and species-dependent, with differing outcomes. Several pathogens are known to attack neutrophils through secreted toxins, for example. The prominent opportunistic bacterium,

Immunol Res

Fig. 1 Overview of the relationships between several pro- and anti-apoptotic Bcl-2 family member proteins and other extrinsic and intrinsic effectors of neutrophil survival or cell death. CtD cathepsin D, Cyt C cytochrome C, PYO pyocyanin

Pseudomonas aeruginosa, can secrete the leukotoxic pigment pyocyanin, which accelerates neutrophil cell death by inducing apoptotic pathways mediated through loss of Mcl1 and induction of caspase-3 [78, 79]. Pyocyanin induces apoptosis by permeabilising lysosomal membranes, resulting in cathepsin-mediated cell death [79]. Furthermore, pyocyanin also interferes with the ability of macrophages to ingest apoptotic neutrophils in these settings [78]. Besides the secretion of pyocyanin and other phenazine derivatives, P. aeruginosa also secrete rhamnolipids with proven anti-neutrophil efficacy [80]. P. aeruginosa

rhamnolipid was shown to be capable of inducing necrosis in neutrophils within 5 min of exposure. Moreover, rhamnolipid-deficient mutant strains have been shown to be unable to prevent their phagocytosis by neutrophils [81, 82]. Staphylococcus aureus can express its own leukotoxic compound, Panton-Valentine leukocidin (PVL). Importantly, PVL not only induces apoptosis, but dose-dependently shifts neutrophils into necrosis [83]. Streptococcus pneumoniae is similarly destructive, possessing a pneumolysin which contributes to necrosis in neutrophils [84].

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The facultative intracellular pathogen M. tuberculosis also manipulates neutrophil survival, doing so to enable its dissemination. The bacteria enhance neutrophil apoptosis following their TLR2-mediated recognition and subsequent phagocytosis [85]. The balance of the pro- and anti-apoptotic Bcl-2 family concentrations is altered in neutrophils harbouring M. tuberculosis [46]. This reflects an induction of apoptosis, which is ROS-driven, given the observation that M. tuberculosis do not effectively induce apoptosis in neutrophils derived from CGD patients [86]. The involvement of ROS has been postulated to result from permeabilisation of the phagosome from within by-products secreted by the bacteria [87]. Recently, it has been shown that a prominent strain of community-associated, methicillin-resistant S. aureus alters the normal course of PICD onset following ingestion by neutrophils [88]. Neutrophils, having internalised the MRSA, express CD47, a signal which discourages macrophages from ingesting the cell. These neutrophils then undergo lysis within 6 h through an apparent necroptotic mechanism (programmed necrosis) [89]. In creating this circumstance, the pathogen is freed to continue proliferation. Extension of neutrophil lifespan is also a survival strategy employed by certain micro-organisms. The obligate intracellular parasite Toxoplasma gondii can increase the expression of Mcl-1 to preserve its host cell [90]. This is accompanied by concomitant or consequent inhibition of caspase-3 activity. Similarly, the obligate intracellular bacterium Anaplasma phagocytophilum, for which the neutrophil is the preferred host, manipulates the cells’ survival to its advantage. Once internalised, the bacteria cause the maintenance of Mcl-1 expression and inhibition of caspase-3 activation [91, 92]. Neisseria gonorrhoeae achieves a similar extension of neutrophil survival from an intracellular vantage, provoking NF-jB signalling [93]. Neutrophils harbouring N. gonorrhoeae experience sustained expression of apoptosis inhibiting X-IAP and cIAP-2 proteins [94]. Additionally, they may suppress the neutrophils’ oxidative burst, an outcome dependent on the expression by the bacteria of cell surface opacity-associated proteins as well as the multiplicity of infection [95, 96]. Francisella tularensis bacteria also interfere with NADPH oxidase activity as part of their self-preservation [97]. Intriguingly, these bacteria appear to induce inactivating cross-linking of Fas in order to further ensure the survival of their host cell [98].

Concluding remarks Mcl-1 is an important anti-apoptotic protein in neutrophils, which serves to block the action of related pro-apoptotic members of the Bcl-2 family of proteins and neutrophil survival is predicated upon the sustained expression of

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Mcl-1. Many pathogens strategically modulate neutrophil survival to facilitate their own persistence in the host, exploiting the intrinsic apoptosis pathway to do so. Therefore, this pathway, and particularly Mcl-1, represents an effective therapeutic target. Preservation of Mcl-1 and, hence, neutrophil viability can improve neutrophil anti-microbial efficacy. Conversely, promoting down-regulation or degradation of Mcl-1 may temper the cascade of mutual activation which contributes to inflammation. Acknowledgments This work was supported in part from a grant provided by Science Foundation Ireland (No. RFP2816). Conflict of interest of interest.

The authors declare that they have no conflict

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