Articulo

Seminars in Immunology 26 (2014) 497511

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Seminars in Immunology

j ourna l ho me page: www.elsev ier .com/ locate /ysmim

Review

Cell death and autophagy in tuberculosis

Andrew H. Moraco, Hardy Kornfeld

Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA

a r t i c l e i n f o

Keywords:TuberculosisPhagocyteApoptosisNecrosisAutophagy

a b s t r a c t

Mycobacterium tuberculosis has succeeded evasion of innate and adaptive immunity. Tniche provided by mononuclear phagocytethe bacillus will or will not be delivered to infection or die, and whether the timing or the pathogen. Here we discuss cell deatbiology feature in all aspects of TB pathogeTB disease.

1. Introdu

Programcesses of celMycobacterincluding mremarkablecapacity to evade the innate antimicrobial effector mechanismsof mononuclear phagocytes (MPs) and leverage the intracellularenvironment as a replication niche. Infected MPs are faced with

Abbreviatioapoptotic protcFLIP, cellularprotein; COX,D; DAMP, daFas-associatedinterleukin; IMMAPK, mitogesition; Mtb, MNET, neutrophmembrane; PGPTP, permeabitein kinase; Smsuperoxide dismicroscopy; Ttor; TNFR, tumTUNEL, terminD receptor.

Corresponsachusetts MeTel.: +1 508 85

E-mail add

ogenlar e radmes

infeates rotec

variety of extracellular signals may activate the autophagic machin-ery of infected MP to drive Mtb into lethal autolysosomes asdescribed in Section 5. These responses set the stage for what arenow recognized as a very complex series of measures and coun-termeasures culminating in the survival or death of the infecting

http://dx.doi.o1044-5323/ ns: AIF, apoptosis-inducing factor; AMPK, AMP kinase; Apaf-1,ease activating factor; BMM, bone marrow-derived macrophages;

FLICE-like inhibitory protein; cIAP, cellular inhibitor of apoptosis cyclooxygenase; CTL, cytotoxic T lymphocytes; CYPD, cyclophilinmage-associated molecular pattern; DC, dendritic cell; FADD,

death domain; HrtA2/Ommi, high temperature requirement; IL,M, inner mitochondrial membrane; LT, leukotriene; LX, lipoxin;

n-activated protein kinase; MPT, mitochondrial permeability tran-ycobacterium tuberculosis; mTOR, mammalian target of rapamycin;il extracellular trap; NK, natural killer; OMM, outer mitochondrial, prostaglandin; PI, propidium iodide; PtdSer, phosphatidylserine;

lity transition pore; RIPK, receptor interacting serine/threonine pro-ac, second mitochondria-derived activator of caspases; SLR, SodA,

mutase; SLR, sequestasome-like receptor; TEM, transmission electronB, tuberculosis; TLR, Toll-like receptor; TNF, tumor necrosis fac-or necrosis factor receptor; TRADD, TNFR-associated death domain;al deoxynucleotidyl transferase dUTP nick end-labeling; VDR, vitamin

ding author at: Department of Medicine, LRB-303, University of Mas-dical School, 55 Lake Avenue North, Worcester, MA 01655, USA.6 2646.ress: [email protected] (H. Kornfeld).

pathogen or its host cell, the progression or resolution of immunepathology, and outcome of tuberculosis (TB) disease.

2. Overview of programmed cell death

A requirement for regulated cell death to support tissue devel-opment and homeostasis was conceived by Karl Vogt in 1842 butthe term apoptosis to describe a morphologically distinct form ofnon-traumatic cell death and the understanding of its biochem-ical mechanisms did not emerge until the late 20th century [2].Apoptosis is a tightly regulated process of cellular deconstruc-tion. It minimizes inammation and bystander injury by containingthe dismembered nuclear and cytoplasmic contents of dying cellswithin membrane-bound vesicles called apoptotic bodies that areengulfed by other phagocytes in a process called efferocytosis (Sec-tion 3.1.2). Binding of apoptotic bodies to specic receptors onMPs responding to nd me and eat me signals induces theexpression of anti-inammatory cytokines including transforminggrowth factor- and interleukin (IL)-10 to further insure the silent

rg/10.1016/j.smim.2014.10.0012014 Elsevier Ltd. All rights reserved.ction

med cell death and autophagy are fundamental pro-l biology intimately involved in the interaction betweenium tuberculosis (Mtb) and the phagocytes it infects,acrophages, dendritic cells (DC) and neutrophils. The

success of Mtb as a human pathogen results from its

a pathmolecuate frelysosofor theeliminhost-pin infecting one-third of the human race though inhibition orhe pathogen is a facultative intracellular parasite that uses thes for its advantage. Complex interactions determine whetheracidied lysosomes, whether the host phagocyte will surviveand mode of cell death works to the advantage of the hosth and autophagy in TB. These fundamental processes of cellnesis and may be exploited to the treatment or prevention of

2014 Elsevier Ltd. All rights reserved.

surviving in phagosomes that fail to incorporate themachinery needed to reduce vacuolar pH and gener-icals of oxygen or nitrogen, and that fail to fuse withto expose bacilli to damaging hydrolases [1]. Plan Bcted MP is to undergo programmed cell death, whichthe intracellular sanctuary and exerts other potentiallytive effects described in Section 3.1.2. Alternatively, a

498 A.H. Moraco, H. Kornfeld / Seminars in Immunology 26 (2014) 497511

elimination of cellular corpses [3,4]. The ultrastructural morphol-ogy of apoptosis is characterized by cell shrinkage and chromatincondensation (pyknosis), nuclear fragmentation (karyorrhexis),and blebbing of the outer cell membrane that culminates inapoptotic bnucleosomaon gel eleccomponentthe plasmaoutward-fathe cell surfclearance o

Necrosiscell membrcontents toto result onregulated m2.2) [5]. Thized by cytoand swellin[6]. These cplasma mement. Necrosiwhich disrumembrane molecular pmobility grmitochondr[7,8,812]. an innate insignals thatdiversity ofin immuneinvolving in

2.1. Apopto

Three mand perforianism withcytoskeletafamily of cytutively expthe rapid inindependenare activatecomplexes pases then of the execcaspases is ncumstances

2.1.1. ExtrinThe extr

necrosis facdomain in tcomplex foto TB are Ting trimerideath domaTNFR-assocptosis protecomplex I. lates pro-sufollowing dmic comple

domain (FADD) and caspase-8. Its formation is opposed by cellularFLICE-like inhibitory protein (cFLIP) that is induced by NFB [20].The TRADD-independent complex IIB (also called the ripoptosome)forms when TNFR1 is activated but cIAP1 is inhibited by mimetics

nd m of comas pro. Thi

ex bue-8 c

Intrin intrr strthat lizatiemb

activex carocamac he inreliemily

-2 fa,23].ase cers (eopto

activted cctiveto acovidic ap

Perfoird aof thes of5]. Pls thes th-3 anion i]. Theerfor

are aracnism

targe

gula

eldex sind on ecro

)-depcropronetextatedoptoody formation. Chromosomal DNA is cleaved at inter-l boundaries, demonstrated by laddering of DNA bands

trophoresis. Phosphatidylserine (PtdSer), a membrane that in viable cells is held facing the cytosolic side of

membrane by the enzyme ippase, translocates to thecing surface in apoptotic cells. Exposure of PtdSer onace plays an important role in membrane stability andf apoptotic bodies (Section 3.1.2).

is a much different death, dened by the loss of outerane integrity with release of cytoplasmic and nuclear

the extracellular space. Necrosis was originally thoughtly from accidental events (e.g. freezing or crushing) butechanisms of necrosis were later identied (Section

e ultrastructural morphology of necrosis is character-plasmic swelling (onicosis), cytoplasmic vacuolizationg of organelles including mitochondria and cell nucleihanges result from ATP depletion and the failure ofbrane ion pumps to maintain a stable osmotic gradi-

s can also result from direct plasma membrane damage,pts the cells without onicosis. Rupture of the plasmaprovokes inammation by releasing damage-associatedatterns (DAMPs) such as heat shock proteins, high-

oup box 1, S100 proteins, extracellular genomic andial DNA, ATP, monosodium urate, and heparin sulfateBinding of DAMPs to their cognate receptors activatesammatory response and sends endogenous adjuvant

can stimulate DC to promote T cell activation [9]. The protein and non-protein DAMPs ensures redundancy

stimulation but most converge on common pathwaysammasomes, IL-1 and leukotriene (LT)B4 [9,13].

sis signaling and execution

ajor pathways of apoptosis initiation (extrinsic, intrinsicn/granzyme) converge on a common execution mech-

degradation of chromosomal DNA and nuclear andl proteins. Both steps in this process involve caspases; asteine-dependent aspartate-directed proteases consti-ressed as zymogens. Caspases operate in a cascade forduction of apoptosis, which is energy-dependent butt of transcription [14]. Initiator caspases-8, -9, and -10d by dimerization following recruitment to signaling(Sections 2.1.1 and 2.1.2) [15]. Activated initiator cas-cleave and activate the pre-formed dimeric zymogensutioner caspases-3 and -7. Activation of executionerecessarily a tightly regulated event but can in some cir-

be mediated by proteases other than initiator caspases.

sic apoptosisinsic pathway begins with ligand binding to tumortor receptor (TNFR) family proteins containing a deathheir cytoplasmic tail which serves as the site for signalrmation [16]. The receptor/ligand pairs most relevantNF-/TNFR1 and Fas ligand/Fas [17,18]. TNF- bind-zes TNFR1 allowing recruitment of TNFR1-associatedin (TRADD), receptor interacting protein kinase (RIPK)1,iated factor (TRAF)2, TRAF5, cellular inhibitor of apo-in (cIAP) 1, and cIAP2 to form membrane-associatedSignals from complex I activate NFB that upregu-rvival genes [19]. Apoptosis is initiated from TNFR1issociation of complex I constituents to form cytoplas-xes. Complex IIA contains TRADD, Fas-associated death

of secomationcylindrrecruitactionscomplcaspas[19].

2.1.2. The

cellulastress meabiinter-mtease complvates p[21]. Sfrom tsis by cIAP faby Bclsis [22to relemembpro-apgratedActivacally aof Bax Bid, printrins

2.1.3. A th

teases granulcells [2get celB cleavpases induct[26,27links pA and Cwell chmechaFas on

2.2. Re

Thecomplfocuseprise n(CYPD[5]. Nesis, pythe conis activanti-apitochondria-derived activator of caspases (Smac). For-omplex IIB also requires deubiquitination of RIPK1 bytosis. FasL binding to Fas recruits FADD which in turncaspase-8 and/or cFLIP via death-effector domain inter-s forms a membrane-associated death inducing signalt a secondary cytosolic complex of FADD, cFLIP, andan be released to further amplify apoptosis initiation

sic apoptosisinsic apoptosis pathway is induced by diverse intra-esses such as DNA damage, starvation, and oxidativelead to outer mitochondrial membrane (OMM) per-on. Cytochrome c released from the mitochondrialrane space binds the cytosolic protein apoptotic pro-

ating factor (Apaf-1) to form a multimeric signalinglled the apoptosome. The apoptosome recruits and acti-spase-9, which in turn activates executioner caspasesand the serine protease HtrA2/OMI are also releasedter-membrane space; they amplify intrinsic apopto-ving the constitutive caspase repression mediated by

members. Mitochondrial permeability is controlledmily proteins that either promote or inhibit apopto-

Pro-apoptotic Bax and Bak form pores in the OMMytochrome c. This is opposed by anti-apoptotic family.g. Bcl-2, Bclx-L, Mcl-1) but further promoted by othertic Bcl-2 proteins. Cell fate is determined by the inte-ities of pro- and anti-apoptotic Bcl-2 family proteins.aspase-9 cleaves pro-apoptotic Bid into an enzymati-

truncated form (tBid), which orchestrates the activitiescelerate cytochrome c release. Caspase-8 can also cleaveing a means for crosstalk between the extrinsic andoptosis pathways [24].

rin/granzyme mediated apoptosispoptosis induction pathway is mediated by serine pro-e granzyme family contained, along with perforin, in

cytotoxic T lymphocytes (CTL) and natural killer (NK)erforin creates pores in the plasma membrane of tar-rough which the granzymes are introduced. Granzymee initiator caspases -8 and -10 and the executioner cas-d -7, and it has other substrates relevant to apoptosisncluding inhibitor of caspase-activated DNase and Bid

former mediates DNA fragmentation while the latterin/granzyme to the mitochondrial pathway. Granzymesalso implicated in apoptosis although their roles are lessterized [28]. While perforin/granzymes is the primary

for killing, CTL can also cause apoptosis by engagingt cells with FasL to trigger the extrinsic pathway [29].

ted necrosis

of programmed cell death has become increasinglyce the discovery of apoptosis. Much recent interest haspathways of regulated necrosis, which currently com-ptosis, pyroptosis, pyronecrosis, ETosis, cyclophilin Dendent necrosis, parthanatos, and autophagic cell deathtosis is the best characterized pathway while pyropto-crosis and ETosis have been most closely identied in

of infection. Necroptosis occurs when TNFR1 signaling but caspase-8 is inhibited by drugs or virus-encodedtic proteins. This results in formation of a complex

A.H. Moraco, H. Kornfeld / Seminars in Immunology 26 (2014) 497511 499

called the necrosome, comprising RIPK1, RIPK3, FADD, and caspase-8 [30,31]. The necrosome recruits and activates mixed lineagekinase domain like which then translocates to the plasma mem-brane where it mediates TNF- and Ca2+-dependent necrotic celldeath (necring energy lysosomal mtion [5].

2.2.1. PyropPyroptos

text of intrListeria moninfectious tsubroutinesterminal plcommon tritor (NLR) a(PAMPs) frotics are the(human caand requirespeck-like ptease catheof lethality exneri at lopyroptosis but higher modes are 1 and IL-1multimers the extracemacrophag

2.2.2. CYPDMitocho

inner mem[39]. This in IMM calmitochondrmation althof cyclosposurvivable btion of pyrimatrix sweinter-memband apoptoway is activcrosstalk beindependenAIF also plamediated breviewed elresult of caCYPD-depeadenine nu

2.2.3. ETosiThis mo

trophils andspace, formptosis, chroPtdSer doesDNA presenfungi by eleing neutrop

kill bound pathogens [44]. Release of extracellular traps has beenidentied in eosinophils, mast cells, and macrophages, hence theterm ETosis [45,46].

Triggers for ETosis include lipopolysaccharide, interferon (IFN)-eria,st indephe Epletees N

targe seyeic

is furkabphil

tecti

ny asre use teium Ser on V amidal dL] asranscopyetwructu

depgateEM

rrogacriticant ent

ll De use y of ces anted. Tly dtine allens mat diffn a son exmbigtivatlk be

nececuttines

l dea

ptostracnd rs infoptosis) through several terminal mechanisms includ-depletion, reactive oxygen species (ROS) production,embrane permeabilization (LMP), and lipid peroxida-

tosis and pyronecrosisis and pyronecrosis were discovered in the con-acellular bacterial infection (e.g. Francisella tularensis,ocytogenes, and Shigella exneri [3234]) although non-riggers have also been identied [35]. These death

share dependence on inammasome constituents andasma membrane pore formation resulting onicosis. Agger for pyroptosis and pyronecrosis is NOD-like recep-ctivation by pathogen-associated molecular patternsm intracellular microbes. Distinguishing characteris-

dependence of pyroptosis on caspase-1 or caspase-11spase-4) while pyronecrosis is caspase-independents the inammasome component apoptosis-associatedrotein containing a CARD (ASC) and the lysosomal pro-

psin B. Pyroptosis and pyronecrosis share rapid kineticsand considerable overlap in triggers. As an example, S.w multiplicity of infection (MOI) induces macrophagedependent on the inammasome constituent NLRC4MOI triggers pyronecrosis via NLRP3 [36]. These deathhighly inammatory, with abundant production of IL-8 that is perpetuated by the release of aggregated ASC(ASC specks) which continue processing pro-IL-1 inllular space and even following phagocytosis by navees [37,38].

-dependent necrosisndrial permeability transition (MPT) occurs when thebrane (IMM) becomes permeable to solutes


wide spectrum of pathogenic microbes. Apoptosis offers severalpotential benets for the host including elimination of a replicationniche, exposure of pathogens to humoral immunity, and forcingintracellular pathogens to reestablish residence in nave host cells.Efferocytosof intracelluuration. It ato DC for e[54]. The rapnecrosis miment to concould be difacilitates pthrough eff

Macrophpathogenescannot gainary. Once Mnew hosts bcellular spaforms has bin vivo. A rea spectrumextrinsic, inseveral typemacrophagfates of infe3.3). While activation-iinduced dea

3.1. Apopto

Before tinfection, Mmonocytes while BCG necrosis. Inbeen mediation introdTB. Keane autonomouinfected wied by TEMTUNEL assaextrinsic pamanner ansignals sincThe attenuainducer thathat exogenmacrophagwas accombial effect omacrophag

A simpleTB emergedconrmed tviability [61associated tof apoptosof Fas on indownstreamtralized TNF

Recent macrophag

from the zebrash/M. marinum model show that apoptosis canfacilitate spread of infection to nave macrophages in vivo [57].Accelerated dissemination was also demonstrated in mice infectedwith a pro-apoptotic mutant of virulent Mtb (Section 3.1.1) in

mentroph

Mtb gajorf twacroacterptosediatrinson oe (So

demnd msult

SodAng insis ahe primtype

antin scred aM i

H de ther

secrh butose iuring37Rvs of

0.8 loial lodatasis ining tacte-typ

by d5]. T

andfecti

enhly dedent type ndenis noctivaor z-e autf mal chaurdemple

(IL-s. Desat thpoptis provides a means to defeat the virulence mechanismslar pathogens that inhibit vesicular trafcking and mat-lso delivers pathogens or their antigenic componentsfcient priming and cross presentation (Section 3.1.2)id, immunostimulatory demise of cells by programmedght benet the host by accelerating neutrophil recruit-trol fast-replicating bacteria [55]. Conversely, apoptosissease-promoting if it eliminates key host defense cells,enetration of epithelial barriers, or spreads infectionerocytosis [56,57].age-pathogen interactions play a central role in TBis. The bacillus is a facultative intracellular parasite that

a foothold in new hosts without this replication sanctu-tb has established infection in the lung, transmission toy infectious aerosols requires its transition to the extra-ce. It is therefore unsurprising that cell death in its manyeen identied in the context of Mtb infection in vitro andcent review on this topic cited 48 manuscripts reporting

of death modes and subroutines linked to TB includingtrinsic and perforin/granzyme-mediated apoptosis, and

of necrosis [58]. In this review we focus primarily one cell death resulting directly from Mtb infection but thected DC and neutrophils will also be discussed (Sectionbeyond the scope of this review, CTL-mediated death,nduced cell death, bystander death of T cells, and Mtb-th of epithelial cells all participate in TB pathogenesis.

sis of Mtb-infected macrophages

he discovery of an intrinsic apoptosis response to Mtbolloy et al. [59] reported that treating BCG-infected

with exogenous ATP induced apoptosis and killed bacilliviability was preserved after H2O2-induced monocyte

retrospect, the antimicrobial activity of ATP might haveted by autophagy (Section 5.2) [60] but this publica-uced the concept of apoptosis has host-protective inet al. [18] were the rst to describe apoptosis as ans response of primary human alveolar macrophagesth live but not heat-killed Mtb. Apoptosis was veri-, internucleosomal laddering of genomic DNA, and

y (the latter including human TB lung sections). Thethway was triggered TNF- in an autocrine/paracrined required infection-induced priming for TNFR deathe uninfected cells were resistant to exogenous TNF-.ted Mtb strain H37Ra was a much stronger apoptosisn virulent H37Rv. Oddo et al. [17] subsequently reportedous FasL or TNF- induced apoptosis of Mtb-infectedes (dened by annexin V/PI staining and by TUNEL)panied by reduced bacillary viability. No antimicro-ccurred with complement-induced necrosis of infectedes.

model of apoptosis as a host-protective response in from those early reports. Subsequent in vitro studieshe association of apoptotic cell death with reduced Mtb65]. The concept of apoptosis evasion as a virulence-rait of Mtb was substantiated and several mechanismsis evasion were described including downregulationfected macrophages, interference with death signals

of TNFR1, and shedding of soluble TNFR2 that neu-- [17,66,67].ndings have underscored the complexity of

e apoptosis in TB and its role in host defense. Data

experiof mac

3.1.1. A m

2007 osion mmycobsic apointermand inMutatimutasH37Rvcells aThis rewhereresultiapopto[73]. TT cell pheno

Thefunctioconferand BMI NADNOX2,TNF-in brothigh dlung dtype Hin termwith aBacterThese apoptofollowwhen bin wildportedmice [7ing TBpost-in

Therecentdepenphenoindepedeath pase ainhibitand thvival oaerosoterial bor a comationRveigest ththan as that also failed to support a direct antimicrobial effectage apoptosis in vivo [68].

enes linked to apoptosis evasion and induction advance in this eld was provided by the discovery ino Mtb genes (secA2 and nuoG) linked to the suppres-phage apoptosis, both acting reduce levels of ROS in theial vacuole [69,70]. This jibes with the models of extrin-is in Mtb-infected macrophages where ROS play anry role in TNF- signaling for apoptosis (and necrosis),ic apoptosis that can also be triggered by ROS [71,72].f secA2 in impairs secretion of bacterial superoxide dis-dA) and confers an apoptosis-inducing phenotype ononstrated by TUNEL and caspase activation in THP-1ouse bone marrow-derived macrophages (BMM] [70].was anticipated by an earlier study of H37Rv mutants

expression was knocked down with anti-sense RNA, vivo attenuation with less inammation and more MPfter high dose intravenous infection in C57BL/6 micesecA2 mutant strongly induced antigen-specic CD8+

ing in vivo, which was attributed to its pro-apoptotic(Section 3.1.2).-apoptotic activity of nuoG was revealed in a gain-of-reen in M. smegmatis. Deletion of nuoG in Mtb H37Rv

pro-apoptotic phenotype on infection of THP-1 cellsn vitro [69]. The nuoG gene encodes a subunit of a typehydrogenase that neutralizes ROS generated by hosteby inhibiting TNF--stimulated apoptosis as well asetion [74]. The mutant RvnuoG strain grows normally

it is attenuated in SCID and wild-type BALB/c mice afterntravenous challenge [69]. Growth of RvnuoG in the

the rst 3 weeks post-infection matched that of wild- and a complemented mutant strain; its attenuationbacterial burden was only evident at later times pointsg reduction compared to wild-type H37Rv at 20 weeks.ads in liver and spleen did not differ at any time point.

suggest that the host-protective role of macrophage TB may be restricted in time and tissue compartment,

he induction of adaptive immunity and in the periodrial burden in the lung is normally held at a plateau levele mice infected with wild-type Mtb. That notion is sup-ata from aerosol infection of protein kinase R (PKR)/

hese mice exhibit increased macrophage apoptosis dur- have lower lung CFU than wild-type mice at 70 dayson but not at 21 days.anced intracellular survival (eis) gene of Mtb is a morescribed pro-survival factor that acts through an ROS-pathway reminiscent of secA2 and nuoG [76]. Theof Rveis differs, however, by increasing a caspase-t cell death that is not clearly apoptotic in nature. Thist accompanied by a strong TUNEL signature or cas-tion and it is only partially blocked by the pan-caspaseVAD-fmk. The Eis protein also modulates autophagy,ophagy inhibitor 3-methyladeneine enhances the sur-crophages infected with Rveis. Following low dosellenge of wild-type C57BL/6 mice, lung and spleen bac-n was no different between Rveis, wild-type H37Rvmented mutant strain but PI-positive cells and inam-6 and TNF- levels) were higher in mice infected withpite the common theme of ROS reduction, the data sug-e Eis protein inhibits a predominantly necrotic ratherotic macrophage death mode. This may be explained


by its mechanism of action, based on acetylation of DUSP/MKP-7and inhibition of JNK-dependent autophagy and ROS generation(Section 5.4) [77].

An anti-apoptotic function was proposed for the Mtb proteinMPT64 actinThe Mtb Rvin suppresscaspase-8 w(Section 2.2suppress moxide stress

Far less ition. The Ma pore-formthe Esx-1 strate proteicandidate finfection-inESAT6 and Mtb-infectepase upregannexin-V been descriwith immoprimary maBCG, whichpotent indu

3.1.2. EfferoCells un

CXC3CL1, ATP, and Uapoptotic boperate in Trole for CXinteractionsfor the smaand so far threspond to of apoptotiapoptotic ccally inhibiapoptotic c

Caspase-plasma memultiple sigApoptotic cand/or phagrins, scavesome of whbospondin)activating tLPR1/MEGFprocess of receptor-macidied phexpression binds to SIR

The potecobacterial et al. [91] of M. aviumapoptotic contact-depmacrophagefferocytos

et al. [92]. Primary mouse macrophages infected with mCherry-expressing H37Rv in vitro were shown to undergo apoptosis viathe intrinsic pathway with subsequent delivery of apoptotic bodiesto nave macrophages. Transfer of Mtb from necrotic macrophages

serv aftephag

of Hith aorskooughinged ph+-Amononants. ing M, reciial lontroytos

cha at la

poteacterion w

CD8+

tb-inf nar resu, purgatotigen, wh

shoed bimulnd cahi efectemacr

cellsase-at thof ing of se in

cros

captive ironas de

mooundotheion oy sholy indnduc

andn suprect ain vitg in a pathway involving NF-B, miR21, and Bcl-2 [78].3654c and Rv3655c genes were shown to participateing extrinsic apoptosis by reducing the availability ofhich has the effect of promoting regulated necrosis) [79]. Finally, protein kinase E of Mtb was reported toacrophage apoptosis, specically in the context of nitric

[80].s known about Mtb genes required for apoptosis induc-tb 6 kDa early secretory antigenic target (ESAT6) ising, virulence-associated gene product exported by

ecretion system in a complex with 10-kDa culture l-n [81]. The pore-forming function of ESAT6 is a leadingor Mtb-mediated LMP that precedes several forms ofduced cell death. There is considerable data linkingthe Esx-1 secretion system to regulated necrosis ofd macrophages (Section 3.2) but ESAT6-mediated cas-ulation and induction of THP-1 apoptosis (dened bybinding and sensitivity to pan-caspase inhibition) hasbed [82]. It is uncertain whether these data obtainedrtalized monocytic cells reect death mechanisms incrophages infected Mtb. Others found that M. bovis

lacks ESAT6/CFP10 and the Esx-1 secretion system, is acer of apoptosis [61,83,84].

cytosis in TB defensedergoing apoptosis release nd me signals (e.g.lysophosphatidylcholine, sphingosine-1-phosphate,TP) that attract phagocytic cells for the clearance ofodies [85]. Which of these or other possible signalsB is presently conjectural but available data hint at a

3CL1 and its receptor CX3CR1 [86]. While ATP/P2X7R are implicated in TB defense [87], P2Y2 is the sensorll amounts of nucleotides released from apoptotic cellsis receptor has not been linked to TB. Neutrophils couldnd me signals but the non-inammatory clearancec corpses is maintained by lactoferrin released fromells. Lactoferrin serves as a stay away signal speci-ting neutrophil but not MP migration to the vicinity ofells [88].dependent exposure of PtdSer on the outer leaet ofmbranes is the best studied eat me signal althoughnals may be required to promote efferocytosis [85].

orpses are recognized by a variety of tethering, signalinggocytic receptors on responding MPs (e.g. CD36, inte-nger receptors, TAM receptors, TIM4, lectins, and RAGE),ich require bridging molecules (e.g. MFG-E8 and throm-. These interactions initiate efferocytosis primarily byhe Rho family GTPase Rac via CrkII-Dock180-ELMO or10-GULP-ABCA1/ABCA7 pathways. The internalizationefferocytosis resembles macropinocytosis more thanediated phagocytosis and delivers apoptotic bodies toagolysosomes [89]. Specicity is further ensured by theof dont eat-me signals on viable sells (e.g. CD47 whichP to inhibit efferocytosis) [90].ntial for efferocytosis to enhance the innate antimy-properties of MPs was rst proposed by Fratazziin experiments using an apoptosis-inducing strain. Adding nave macrophages to cultures of infected,

macrophages reduced mycobacterial viability in aendent manner that was not seen if the infectedes were made necrotic. Denitive evidence foris-dependent killing of Mtb was provided by Martin

was obin vivomacrokillingway wwith fsis thrBacilli acidieuolar Hwas dearachidrecipiefollowmodelbactertype coefferocin vivomainly

Themycobactivattion ofwith Mtion oSimilasterileinvestiMtb anin vivofurthermediatalso stto DC aDivangMtb-inprone CD8+ Tof caspgest thdeath priminrespon

3.2. Ne

Thealternalar envMtb whumanThey fwhile inductquentlpotentPGE2 imationErdmaThe diunder ed in parallel and both phenomena were demonstratedr transfer of Rv/mCherry-infected DiO-labeled CD45.2+

es into CD45.1+ recipients. Efferocytosis-dependent37Rv was demonstrated in vitro by blocking this path-nti-TIM4 mAb or by pre-treating nave macrophageslin or prostaglandin (PG)E2 that inhibit efferocyto-

a mechanism involving increased intracellular cAMP.sted by efferocytosis were delivered to capacious,agolysosomes that co-localized with LAMP1 and vac-

TPase. An antimicrobial effect of efferocytosis in vivostrated by intraperitoneal transfer of H37Rv-infectedte 5-lipoxyengase (Alox5)/ macrophages into Rag/

Alox5/ macrophages are more prone to apoptosistb infection (Section 3.2). In this infection/transfer

pients treated with anti-TIM4 had roughly 2-fold higherad in spleen and lung than recipients treated with iso-l mAb. The data clearly demonstrate the potential foris-dependent killing of Mtb in TB although results fromllenge with RvnuoG [68] suggest that this operatester stages of TB disease (Section 3.1.1).ntial for efferocytosis to promote cross-presentation ofial antigens for MHC class-I- and CD1-dependent T cellas initially reported by Schaible et al. [54]. Restimula-T cells from donors with latent TB infection co-culturedfected macrophages required the intermediary func-

ve DCs that acquired antigen through efferocytosis.lts were achieved when nave DCs were pulsed with

ied vesicles derived from Mtb-infected MPs. Thesers subsequently demonstrated that immunization with-containing vesicles can cross-prime nave CD8+ T cellsich requires DC homing to lymph nodes [93]. Theywed that apoptotic bodies possess adjuvant activityy Toll-like receptor (TLR)-2, and that CD4+ T cells areated in vivo. Evidence of efferocytotic antigen transferross-priming occur during TB disease was reported byt al. [94] in experiments using intratracheal transfer ofd Alox5/ macrophages. Transfer of these apoptosis-ophages accelerated the activation of TB10.4-specic

and depended on apoptosis (suppressed by inhibition8 and caspase-9) as well as DC. Altogether, the data sug-e capacity of virulent Mtb to suppress the apoptoticfected macrophages could contribute to the delayedadaptive immunity that characterizes the early host

TB.

is of Mtb-infected macrophages

acity of virulent Mtb to suppress apoptosis implies anexit strategy for the bacillus to reach the extracellu-ment. The rst evidence of programmed necrosis inscribed by Duan et al. [95] using in vitro infection ofnocyte-derived macrophages with attenuated H37Ra.

that some infected macrophages died by apoptosisrs died by necrosis, which correlated with MPT. Thef MPT and CYPD-dependent necrosis by Mtb was subse-wn to be inhibited by cyclosporin A [96] and to be moreuced by H37Rv than H37Ra [97]. It was later shown thated by H37Ra protects against mitochondrial PTP for-

necrosis while lipoxin (LX)A4 induced by H37Rv andpresses PGE2 production and promotes necrosis [98].ntimicrobial effect of apoptosis against Mtb observedro culture conditions was not seen when infected cells


died by necrosis. These data supported the concept of programmednecrosis as means for replication-competent bacilli to exit hostmacrophages for spreading infection.

The apoptotic death of infected macrophages connes Mtb inmembrane-come is loscleared by determinanrst revealinhibits theserves to stfully condumacrophagdeterminanfate in this mmediated mvia the E2levels in mgenase (COeffects. InfeLXA4 resultprostaglandPGE2 and dsuggest thamon pathwplasma memnecrosis if LXA4 produacted by repof ESAT6 ismembrane uct of the Minduces necis not block3 mediatedblocked by ttosis) [5;10

The in vdefense is smodels, andtant to aeroTh1 biased ics of Rvnis only lowthat its protphase of TBmarinum mlase, which with this mover-produand multibLTA4H polymLTB4 [104,1and anti-ineffects of pagainst damin vivo pheand necrosieicosanoids

Inammpostulated ing intracepathogenic[55]. Slow so its replicgrammed n

gene product was reported to suppress caspase-1 and pyroptosisin macrophages [106].

Lytic viruses induce host cell necrosis after an optimal periodof replication to a burst size intracellular load. Similar dynam-

re sutine

bacif Ptd

housicleuiree neg, andenlospois [1ck oherel in cteriut itR mu

[108 theis if i

lim is nics oe tsdomricteamm

ls anted

ogra

crophta frophilshago

idenweeyteser th

and 14]. d by ed m

limf there oion aged se tonablthors

maceporo ned by

DNA of threqutrast,nd Hbound vesicles but this potentially host-protective out-t if the apoptotic bodies decompose before they areefferocytosis. Plasma membrane stability is anothert of the fate of Mtb-infected macrophages. This wased by experiments showing that virulent Mtb H37Rv

cross-linking of annexin-I bound to PtdSer, whichabilize apoptotic membranes [99]. In a series of care-cted, mechanistic studies the capacity of Mtb-infectedes to repair membrane damage was shown to be a majort of an apoptotic versus necrotic demise [94,100]. Cellodel hinges on eicosanoid regulation of the lysosome-embrane repair machinery, which is induced by PGE2

receptor. Virulent H37Rv promotes elevated LXA4acrophages, which in turn downregulates cyclooxy-X)2 mRNA thereby reducing PGE2 and its protectivection of Alox5/ macrophages that cannot synthesizes in increased apoptosis even with H37Rv. Conversely,in E synthase (Pges)/ macrophages cannot produceie by necrosis even with H37Ra infection. These datat virulent and attenuated Mtb strains trigger a com-ay that can terminate in apoptosis if mitochondrial andbrane integrity is protected by PGE2, or progresses to

LXA4 predominates. How virulent Mtb strains inducection and what causes the membrane injury counter-air remains to be determined. The pore forming activity

one suggested mechanism for LMP as well as plasmamicrodisruptions [101]. Another candidate is the prod-tb Rv3903c gene (CpnT) whose C-terminal fragment

rotic cell death with plasma membrane disruption thated by a pan-caspase inhibitor (therefore not caspase-

apoptosis or caspase-1 dependent pyroptosis) and nothe RIPK1 inhibitor necrostatin-1 (therefore not necrop-2].ivo relevance of excess lipoxins as detrimental to TBupported by genetic data from the mouse and zebrash

in human TB patients. Alox5/ mice are more resis-sol TB than wild-type controls and make a more robustadaptive immune response [103]. Reminiscent of kinet-uoG growth in vivo, lung bacterial load in Alox5/ miceer than wild-type 21 days post-infection, suggestingective effect is not manifest during the primarily innate

defense. A zebrash mutant hypersusceptible to M.apped to the lta4 h locus encoding leukotriene A4 hydro-catalyzes the last step in LTB4 synthesis [104]. Zebrashutation are decient in pro-inammatory LTB4 and

ce anti-inammatory lipoxins. Human resistance to TBacillary leprosy is associated with heterozygosity oforphisms that correlate with differential production of

05]. These data suggest that an optimal balance of pro-ammatory eicosanoids protects against the adverseoorly controlled bacillary replication on one hand andaging immune pathology on the other. Whether these

notypes reect differential regulation of MP apoptosiss, or some other effects on immunity of these pleiotropic

remains to be determinedasome activation and the induction of pyroptosis isas a defense mechanism that deprives rapidly grow-llular bacterial pathogens of a growth niche. Many

bacteria have mechanisms to subvert this responsegrowing Mtb requires the intracellular niche of MPs,ation would be restricted the rapid induction of pro-ecrosis of its host cells. In this regard, the Mtb Rv3364c

ics wesubrou2040tures owithintotic venot reqfrom thswellinindepeby cycnecrostic attacould tatypicaThe baned, bphoPMOI 25lackingnecrosof onlynotypedynamof micthe preunrestthe introphiassocia

3.3. Pr

Mabut daneutroinant preadilying betmonocwhethnative[1121deneaccept

Themany oliteratuactivatchallenresponwere uThe auDC and[116] rundergrescuefeaturevationdeath In conmice aggested for Mtb, which triggers an atypical necrosisin macrophages at intracellular loads in the range oflli [106,108]. Dying cells exhibit early apoptotic fea-Ser externalization and nuclear pyknosis, but progressrs to necrosis without nuclear fragmentation or apop-

formation. This death is caspase-independent and does pro-apoptotic Bcl2 family proteins. This death differscrosis of bioenergetic collapse by the absence of osmoticnd it differs from pyroptosis and pyronecrosis, beingt of caspase-1 and cathepsin B [108]. It is not inhibitedrin A and is therefore distinct from CYDP-dependent07]. Death is initiated upon LMP, followed by lipoly-n mitochondrial, nuclear and outer cell membranes. Itfore be classied as a form of lysosomal cell death but isits dependence on lipase more than protease activities.al determinants of this death mechanism remain unde-

requires one or more genes of the PhoPR regulon since atant of H37Rv fails to cause LMP or kill macrophages at]. In contrast, BCG and a dened RvRD1espA mutant

ESX-1 secretion system are fully capable of provokingntroduced at high MOI. Both of those strains are capableited intracellular replication, so this pro-necrotic phe-ot evident when they are introduced at low MOI. Thef Mtb burden per cell in MPs after aerosol challenge

a burst size model of necrosis [109]. Neutrophils areinant phagocytes harboring Mtb cell during periods ofd bacillary replication [109,110]. This is consistent withatory nature of necrotic cell death which recruits neu-

d absence of the stay away signal lactoferrin that iswith apoptosis [13,88].

mmed death of other myeloid cells in TB

ages have been the focus of research on cell death in TBm the mouse model and human TB indicate that DC and

are important and in some circumstances the predom-cytes harboring Mtb in vivo [109111]. Neutrophils aretied by common laboratory methods but distinguish-

n tissue-resident macrophages, recruited macrophages,, and DC is more challenging and some have questionedese cell types represent distinct lineages or simply alter-plastic phenotypes a common monocytic progenitorRecognizing this controversy, we will refer to DC asthe authors of cited publications who used generallyethods to isolate and phenotype these cells.ited literature on the fate of Mtb-infected DC reects

same challenges to interpreting the often contradictoryn macrophage cell death. An early study compared thend survival of C56BL/6 mouse bone marrow derived DCwith Erdman at MOI 10 [115]. Immature DC matured in

infection and survived >48 h but (unlike macrophages)e to kill intracellular bacilli even with IFN- activation.

noted that high rates of cell death (trypan staining) inrophages at later time points. More recently, Ryan et al.ted that human peripheral blood monocyte-derived DCcrosis (PI staining) that was caspase-independent (not

Q-VD-Oph) and lacked nuclear fragmentation but did cleavage (nucleosomal particle ELISA) despite no acti-e executioner caspases 3 and 7. The induction of DC

ired live bacilli and was triggered by H37Rv and H37a. a study using bone marrow-derived DC from C57BL/637Rv (MOI 10) described activation of inammasome


and executioner caspases 3/7 with along with apoptosis (denedby TUNEL) but no necrosis (dened by adenylate kinase release)[117]. Based on these and other data the authors concluded thatMtb induces DC apoptosis dependent on Esx-1 but does not causepyroptosis infect and aensue depeand activatof DC coulddeath mighsince therecytes that cadaptive imhas a majorin mice [11

3.3.1. NeutrMuch re

play in TB [early eventerate immuand passingDC trafckiMtb strains priming wa[68]. In conhuman periand indepeaction, comone strain ththe other st

While nearly eventtissue injuras occurs w[65,124,125TB, which mtion and lun[126]. NETsis another fmacrophagpoorly contactivity agaMtb [127]. Rby IFN- [4mice with TTB feature ithese cells p

3.4. Transla

Manipulthe most prlation of krecombinanlisteriolysinoptimal intis more immand uniqueattributablein Section 3nology, VPM[130] and a

Opportuapoptosis a(e.g. T cells)

for secondary necrosis. Nonetheless, a more comprehensive under-standing of how different fates for MPs and neutrophils inuencethe effectiveness of host defense and the quantity and quality ofimmune pathology in TB is vital to understanding TB pathogenesis

w ta

mma

diveath i, myion sle ceressedomstudiutrophaghese te ditracen thnism

appase.f commenters lid LDat phat pnecrd byoughted a

rvie

opha cel

ing ote ceety osmi

autops. Mand wagicf cytagy ationophah mst m

othof th

incos. A

of cion.

Unc FIP2e reas weised or pyronecrosis. We are left to conclude that Mtb canctivate DC and that apoptotic or necrotic death mightnding on variables likely to include the origin, subtypeion state of the DCs prior to infection. Necrotic death

plausibly delay immune priming while an apoptotict facilitate priming. These are important considerations

is substantial evidence that DC are the major phago-onvey viable bacilli to lung-draining lymph nodes formune priming [110,118]. The kinetics of these events

impact on the subsequent outcome of disease at least9].

ophil cell death in TBcent interest has focused on the roles that neutrophils120]. A fascinating dynamic has been proposed for thes following inhalation of Mtb, where neutrophils accel-ne priming by undergoing infection-induced apoptosis

bacilli to migratory DC in a manner that facilitatesng to the lymph node [68,121]. Evidence that virulentinhibit neutrophil apoptosis and thereby delay immunes obtained using the pro-apoptotic Rv/nuoG mutanttrast, Mtb H37Rv was reported to trigger apoptosis ofpheral blood neutrophils that was dependent on TLR2ndent of TNF- [122]. Adding complexity to this inter-parison of two different clinical Mtb isolates identiedat strongly induced human neutrophil apoptosis while

rain did not induce apoptosis [123].eutrophils may exert a host protective function as an

in TB, they also appear to be important mediators ofy at later stages of disease if they are present in excessith poorly controlled Mtb infection in susceptible hosts]. Neutrophil lifespan is prolonged in I/St mice withay be a factor in the increased neutrophil accumula-g damage seen in that highly susceptible mouse strain

may contribute to lung tissue damage [47] and ETosisate that has been described for human neutrophils andes infected with Mtb in vitro [46,127], in the mice withrolled TB in vivo [109]. While NETs exert antimicrobialinst a range of bacteria, this activity does not extend toelease of traps by human macrophages is accelerated

6] but NETs were identied in lung lavage of IFN-/

B [109]. The diverse fates of Mtb-infected neutrophils inn the host-protective and damage-inducing roles thatlay in TB pathogenesis.

tion opportunities

ation of apoptosis in the context of vaccination holdsomise compared other therapeutic goals for early trans-nowledge about cell death in TB. The pro-apoptotict strain BCGureC::hly+ expresses the pore-forming

of L. monocytogenes and lacks urease C to ensure anraphagosomal pH for listeriolysin activity. This strainunogenic than the parental BCG for type 1 responses

ly induces type 17 as well [128,129]. These results are to exploitation of the efferocytotic pathway discussed.1.2. The human vaccine candidate based on this tech-1002, demonstrated safety in a phase I clinical trial

phase II trial is currently underway.nities for adjunctive TB therapies that directly enhancere less obvious and could risk killing necessary cells

and/or generating an excess of apoptotic bodies at risk

and ne

3.5. Su

Thecell destrainsactivatmultipcells stfate prmany and nemacroever, tpromolimit in

Givmechain vivoTB disetude oexperidisordoxidizeterol thIFN- terates inducecells thunrela

4. Ove

Autway inincludregulato varicytoplacalled cargoeto TB aautophtion oautophdegrad

Autily witfor yeaily andmany ers aresystemgroupsformatprisingAtg13,positivtively, comprrgets for treatment and diagnosis.

ry of cell death in TB

rse outcomes and conclusions of published studies onn TB (Table 1) reect differences in host species andcobacterial species and strains, cell types and theirtate, conditions of infection, and the likelihood thatll death programs can be simultaneously activated ined by Mtb infection. These variables determine whichinates in particular experimental systems. Results fromes support the concept that apoptosis of macrophagesphils contributes mainly host-protective effects, whilee necrosis is mainly linked to adverse outcomes. How-are clearly not absolute paradigms since apoptosis cansseminated infection and accelerated necrosis mightellular bacillary replication.e complex interplay of multiple host and bacterials to promote or prevent cell death, more research withroaches is needed to identify what truly matters in

A comprehensive model must also integrate a multi-mon factors that are often excluded in reductionist

al systems. Examples include the effects of metabolicke diabetes and hyperlipidemia where methylglyoxal,L cholesterol, or an excess of intracellular free choles-

romote apoptosis or necrosis [131,132]; cytokines, suchromotes survival of macrophages at low MOI but accel-osis with high bacillary loads [133,134]; and apoptosis

CTL. The best studies will conrm unequivocally thatt be to dying are indeed dying and ideally use multiplessays if making a case for one particular fate.

w of autophagy

gy (self-eating) is an evolutionarily conserved path-l biology that serves to control cytoplasmic contentrganelles, to recycle chemical resources in bulk, and tollular functions under basal conditions and in responsef stresses. Macroautophagy refers to the isolation of

c content by the formation of lipid bilayer vacuoleshagosomes which fuse with lysosomes to degrade theircroautophagy is the autophagic process most relevantill be simply called autophagy in this review. Other

processes such as microautophagy (direct sequestra-oplasm within lysosomes) and chaperone-mediatedthat targets proteins with a specic signal sequence for

are reviewed elsewhere [135].gy is controlled primarily by Atg proteins, a fam-ore than 30 members that were revealed in screensutants defective for autophagy [136]. The Atg fam-

er proteins operate in a complex conjugation cascade;e interactions and multiple functions of these play-mpletely understood, particularly in mammalian cell

simplied scheme for autophagy includes four sub-ore proteins involved in three major steps of vesicleAutophagy is initiated by the Atg1/ULK1 complex com--51 like autophagy activating kinases 1 and 2 (ULK1/2),000, and Atg101 [137]. Initiation is under negative andgulation by mTOR and AMP kinase (AMPK), respec-ll as mTOR-independent pathways. The Vps34 complexof the class III phosphatidylinositol-3 kinase Vps34,


Table 1Myeloid cell death and host defense in TB.

Cell death type Effects on host-pathogeninteraction

Consequences for hostdefense

References

Extrinsic and

totic

CYPD-depen

M necrosis sM necrosisCpnT-mediaM ETosis DC apoptosi

DC necrosis Neutrophil a

NETosis

M, macroph

Beclin 1 (hAtg14, alsotwo compleomegasomreticulum (been descrtion cascadER, buildinglike base of ubiquitinatinvolves cowhich thenlows cleava(LC3), yieldthrough conII to the isodened, theisolation meclosure of thsolic constitautophagoswhere dige[138].

The ratioopposed tomicroscopyThey are uautophagicated autophthe relatedtosis (LAP),phagosomebeen implicamong othe

Autophastasis of un

tors raturfectiolipin initiangendenutopies er ad intrinsic M apoptosis Eliminates replication niche Induces anti-inammatorycytokinesLactoferrin inhibits neutrophilrecruitmentPackages Mtb and antigens in apopvesicles

dent necrosis of M Releases bacteria to extracellularspace

due to inhibition of membrane repair Induces pro-inammatory cytokine with high Mtb burden ted M necrosis

Unknown s Packages Mtb and antigens in

apoptotic vesiclesUnknown

poptosis Antigen transfer to DC

Binds Mtb to extruded DNA but noantimicrobial effect in vitro

age.

omolog of yeast Atg6), p150 (Vps15 in yeast) and participates in autophagy induction. Together, thesexes promote vesicle nucleation with formation of thee, a cup-shaped protrusion from the endoplasmicER). Although other vesicle nucleation sources haveibed, ER is the best understood. An ensuing conjuga-e directs formation of a phagophore from repurposed

a double-layered isolation membrane on the ring-omegasome. Elongation of this membrane requires twoion-like reactions and two protein complexes. The rst

of factempeand incardio[144]) wide rindepetions, astrategto theinjugation of the ubiquitin-like protein Atg12 to Atg5, form a complex with Atg16L. The second reaction fol-ge of microtubule associated protein-1 light chain-3ing cytosolic LC3-I (Atg8) that is converted to LC3-IIjugation to phosphatidylethanolamine, tethering LC3-lation membrane. Through mechanisms not yet fullyse complexes facilitate expansion and bending of thembrane. Autophagosome formation is completed upone isolation membrane, sequestering the targeted cyto-uents. In the nal step, mediated by SNARE proteins, theome fuses with a lysosome to form an autolysosomestion of the sequestered vacuolar contents proceeds

of LC3-II to LC3-I on immunoblots and a punctate as cytosolic distribution of LC3 identied by uorescence

are commonly used as assays to quantify autophagy.seful but can be misleading; alternative measures of

ux may be required to distinguish between acceler-agosome biogenesis versus reduced turnover [139]. In

but distinct pathway called LC3-associated phagocy- LC3-II is recruited to conventional, single-membranes and promotes their fusion with lysosomes. LAP hasated in the clearance of apoptotic and necrotic corpsesr settings [140].gy proceeds at basal levels to maintain the homeo-stressed cells and is further activated by a variety

intracellulato host deA virus), bacus, and Lprotozoa (ecellular patXenophagyto infection

5. Autoph

As notesome biogemost otheranism. Desbacilli are dated by maautophagy rez et al. inin Mtb-infethe mTOR iand increasalso found or transfecreport, a surole that auLower bacillary load [17,18,59,6165]Less immune pathology [3,4]

Less immune pathology [88]

Efferocytosis by DCpromotes immune primingand cross presentation

[54,93,94]

Efferocytosis by M killsMtb

[91,92]

Efferocytosis promotesspreading infection

[57,68]

Promotes spreadinginfection and transmission

[95,96]

More immune pathology [94,100][107109][102]

Unknown [46]Unknown [117]

Unknown [116]Accelerated immunepriming

[68]

Unknown-restricted spread of Mtb?-increased tissue injury?

[109,127]

including starvation, hypoxia, extremes of pH ore, growth factor withdrawal, oxidative stress, ER stress,n [141143]. Targeting signals (e.g. externalization ofto the limiting membrane of damaged mitochondriaate autophagosome formation, which is subject to a

of regulatory inuences, some dependent and somet of mTOR and AMPK [145]. Among its many func-hagy provides a mechanism to counteract the variousmployed by intracellular pathogens to use host cellsvantage. The detection and autophagic destruction of

r pathogens, also called xenophagy, has been linkedfense against certain viruses (e.g. HIV and inuenzacteria (e.g. Shigella, Salmonella, Group A Streptococ-isteria), fungi (e.g. Candida and Cryptococcus), and.g. Toxoplasma) [146148]. Conversely, certain intra-hogens subvert autophagy to promote infection [149].

plays an integral role in innate and adaptive immunity with Mtb that is the focus of this review.

agy in TB

d in Section 1, the capacity of Mtb to inhibit phago-nesis and survive inside macrophages that eliminate

phagocytosed bacteria is an essential virulence mech-pite this countermeasure, a proportion of internalizedirected to acidied compartments and this is acceler-crophage activating factors such as IFN-. The role ofin this successful outcome was rst identied by Gutier-

2004 [150]. They reported that induction of autophagycted macrophages by starvation or by treatment withnhibitor rapamycin delivered bacilli to phagolysosomesed co-localization of Mtb with LC3 and Beclin 1. Theythat autophagy was induced by treatment with IFN-tion with the IFN- effector LRG-47. Since that initialbstantial body of evidence has established the majortophagy plays in TB defense.


5.1. Targeting Mtb for autophagic elimination

Following receptor-mediated phagocytosis, most Mtb bacillireside in early endosome-like vacuoles that resist acidication orfusion withment of mvesicular co[151153]. nutrients annerability tin part by innate cytodependent and their dthe ubiquitine/threonimodel of MRD1 and STfurther ideDRAM1 in TB defense ceptibility oin myeloid

Redundatargeting ouitin ligaseof dysfuncta critical rmacrophagxenophagy chondria evubiquitin, b(p62/SQSTMassociated adaptor brtophagosomubiquitinatgets for ubimediators f[159].

Innate cfor intracellbacterial pein macrophNOD2 in Mtof NF-, related genhuman cathcytokines IL

The captwith lysosoreplete witnitrogen. Mrecently shery of ribosthe cytosolneo-antimsor proteinsor if killingvesicles wimined.

5.2. Regula

Followinden rises i

replication is curtailed by adaptive immunity and particularlyIFN- produced mainly by Th1 cells [163]. Inducible nitric oxidesynthase was considered the major host-protective macrophageresponse to IFN- activation but it is now clear that acceler-

utoprotecf thep47 ave eviouIFN-s JA) [16s at phag

autot autors lr low

a vi asso

acti[168mince bbsen

by tyte cdiol

wheh PLC

androxycallyg to press71]. phagimuumaagy VDR t in n 5.6umbphaetweagy,

and [173d Mrophown

MO promion aen lithat sis oautops howayill bs of

irect ent ( lysosomes. This niche is permissive for some move-acromolecules to and from the cytoplasm, to othermpartments, and even the extracellular environmentSuch bidirectional transfer is required for Mtb to acquired manipulate host cell functions but also confers vul-

o host defense [154,155]. The LMP generated at leastESAT6 (Section 3.1.1) enables detection of PAMPs bysolic sensors. Genomic DNA of Mtb activates the STING-cytosolic pathway, leading to ubiquitination of bacillielivery to autophagosomes in a process dependent onin-autophagy receptors p62 and NDP52, and the ser-ne protein kinase TBK1 [154]. Data from the zebrash. marinum infection corroborated the involvement ofING in autophagic defense against mycobacteria, andntied the participation of the autophagy modulatorthis response [156]. The contribution of autophagy toin mammals was demonstrated by the increased sus-f Lyz-Cre-Atg5/ mice with targeted deletion of Atg5

cells [154,157].nt pathways for innate recognition and autophagicf intracellular Mtb have been identied. The ubiq-

Parkin, which mediates the autophagic eliminationional mitochondria (mitophagy), was shown to playole in the colocalization of ubiquitin and Mtb ines [158]. The dual role of Parkin in mitophagy andfor intracellular bacteria is unsurprising since mito-olved from intracellular prokaryotes. Once tagged withacteria may be targeted by cytoplasmic sequestasome1)-like receptors (SLRs). By virtue of its ubiquitin-

domain and LC3 interaction region, p62 serves as anidging ubiquitinated bacilli and LC3 on the preau-e [146]. A role of p62 in recruiting an E3 ligase to

e Mtb has also been proposed but the precise tar-quitination on bacterial cells and the full spectrum ofor this conjugation reactions are incompletely dened

ytosolic receptors bind PAMPS, providING surveillanceular infection. The NLR subfamily receptor NOD2 bindsptidoglycans like muramyl dipeptide and is activatedages infected with Mtb [160]. Signaling downstream ofb-infected macrophages induces nuclear translocationresulting in increased expression of the autophagy-es IRGM, LC3, and ATG16L1, the antimicrobial peptideelicidin (hCAP-18/LL-37), and the pro-inammatory-1, IL-6, and TNF- [161].ure of Mtb in autophagosomes that subsequently fusemes delivers bacilli to a hostile, acidied compartmenth hydrolytic enzymes and free radicals of oxygen andicrobicidal activity against Mtb in autolysomes wasown involve another role for p62, namely the deliv-omal precursor and other ubiquitinated proteins from

to autolysosomes where they are cleaved to generateicrobial peptides [162]. Whether the cytosolic precur-

are delivered directly to autolysosomes containing Mtb depends on subsequent fusion of peptide-containingth Mtb-containing phagosomes remains to be deter-

tion of autophagy in TB

g inhalation of Mtb by nave mice, lung bacterial bur-n a log linear manner for 3 weeks until bacterial

ated aHost-pbers ocalled IFN- hIRG prnative require(MAPKthere imacroinhibitagainsactivathigh omay betion isinnatemTOR

Vitaclearanbut is aTLR2/1monoccalcifemodelthrougylation1A hydbiologibindinthe ex[170,1macroVDR-st in hautophof the interes(Sectio

A nto autolinks bautophMyd88of Mtbinfecteof Macalso shat lowtion 2)inducthas bedence apoptoerates the Raof pathMPs win termhelp dtreatmhagic ux is another important effect of IFN- [150].tive effects of IFN- are mediated in part by mem-

immunity-related GTPase family (IRG proteins, alsoGTPases). Two pathways for autophagy induction bybeen reported. One involves Irgm1, a STAT1-dependentsly called Lrg47 (IRGM in humans) [164]. The alter- autophagy pathway is independent of STAT1 butK 1/2, PI3K, and p38 mitogen-activated protein kinase5]. The Th1 cytokine TNF- also induces autophagy andleast indirect evidence for this effect in Mtb-infectedes [166]. In contrast, the Th2 cytokines IL-4 and IL-13phagy at least in mice [167]. There is evidence for andophagy induction by Mtb in the absence of exogenousike IFN-, and different mycobacterial strains may be

inducers [168]. Indeed, the suppression of autophagyrulence mechanism of the bacillus (Section 5.4). Infec-ciated with increased mTOR activity, indicating that

vation autophagy by mycobacteria is independent of].

D receptor (VDR) signaling complements IFN- for Mtby a pathway that may be highly signicant in humanst in mice. Shin et al. [169] reported that stimulation ofhe Mtb lipoprotein LpqH induces autophagy in humanultures supplemented with the pro-vitamin D hormone(25-dihydroxyvitamin D3) [169]. Their data support are signaling by TLR2/1 and CD14 induces Ca2+ inux- activation. Calcium ux results in AMPK phosphor-

activation of p38 MAPK that in turn upregulates VD3lase, which catalyzes the hydroxylation of calcifediol to

active calcitriol (1,25-dihydroxyvitamin D3). CalcitriolVDR induces expression of cathelicidin that increasesion Beclin-1 and Atg5 and promotes autophagic uxThe antimicrobial activity of IFN- activated humanes requires a sufcient level of calcifediol, linking thelated autophagic pathway the protective effect of IFN-n TB [172]. The VDR pathway is not required forinduction by rapamycin or starvation [172]. Discoverypathway and its role in TB defense stimulated renewedthe therapeutic potential of vitamin D in human TB).er of other mediators and pathways have been linkedgy induction in Mtb-infected macrophages. Given theen intracellular and cell surface innate receptors and

it is unsurprising that the TLR4 ligand LPS and IL-1 (via TBK1) have been linked to the autophagic elimination,174]. The growing list autophagy activators in Mtb-

Ps includes the scavenger protein Apoptosis Inhibitorages (a target of liver X receptor activation) that was

to inhibit apoptosis of THP1 cells challenged with MtbI [175]. Extracellular ATP (a DAMP discussed in Sec-otes autophagy via P2X7 receptor, linking autophagy

nd the host response to necrosis [60]. MicroRNA-155nked to TB defense in several reports including evi-its induction is dependent on ESAT6, that it regulatesf M. bovis BCG-infected macrophages, and that it accel-hagy and the killing of intracellular Mtb by suppressing

molog Rheb [176178]. It is likely that a wider arrays and mediators regulating xenophagy in Mtb-infectede identied in the future. Prioritizing these pathways

their protective effects against TB disease in vivo willtranslational research on harnessing autophagy for TBSection 5.6).


5.3. Autophagy-mediated regulation of immunity in TB

Autophagy participates in TB defense beyond its effector func-tion to kill intracellular bacilli. Since Mtb inhibits conventionalphagosomesomes provonto MHC cSeto et al. [1infected wicapture of ulysosomes. This is consfrom BCG) thowever, inand enhanc[179].

Autophatargeted depreventing vate the NLeffect of thiby the CastAtg5/ LysMH37Rv featucontrols busue necrosiIL-17, and Ceffects of atory responincreased inAtg7/ LysBCG. Cytokisusceptibiliincreased sMARCO andbound KEAPing in incrscavenger rdifference ithe Esx-1 se

5.4. Evasion

Evasion phagocytosvirulence afamily GTPmeasures tectopic expmation in wexpression et al. [187] cDC infectedautophagosThe capaciplementatiocomplementhat the Esxbe overcom

The Mtmacrophagand downreinhibits autRveis wasship betweautophagy

stressed cells that may succumb to one or another subroutine ofprogrammed death [51]. The Mtb cell wall component lipoarabi-nomannan is another virulence factor associated with autophagyinhibition. Phagocytosis of lipoarabinomannan-coated latex meads

264eadgmateveadom

netic

ody oologargend I

57,18s infent clvolved essMtb ted eral agy-

exityestigas n

courains7876iptioe G

tion tudie

with, Ching efn difagy iseasgnicThesc bacl inM ge

erap

goa wit

encethe pnouercue amate b

in Trazin-infepha

d hyd mode ant in and i biogenesis, its capture and degradation in autolyso-ides an opportunity to generate peptides for loadinglass II molecules. This outcome was demonstrated by59] in murine bone marrow derived DC and DC2.4 cellsth Mtb Erdman. The recruitment of MHC II followed thebiquitinated bacilli in autophagosomes and fusion withIn contrast to Erdman, BCG was not ubiquitinated in DC.istent with a requirement for ESAT6 (which is deletedo cause LMP. Treating BCG-infected DC with rapamycin,creases colocalization of bacilli with autophagosomeses the immunogenicity and protective efcacy of BCG

gy also functions to modulate inammation throughgradation of cytokines such and TNF- and IL-1, and bythe accumulation of damaged mitochondria that acti-RP3 inammasome via ROS and mtDNA [180184]. Thes regulation to limit inammation in TB was reportedillo et al. [157]. Pulmonary TB in autophagy-decient-Cre+ mice challenged with a low aerosol dose of Mtbred increased bacterial burden compared to wild-typet also increased neutrophilic inammation with tis-s, Th17 skewing, and elevated levels of IL-1, IL-12,XCL1. The authors concluded that the host-protectiveutophagy in TB include modulation of the inamma-se to infection. Bonilla et al. [185] similarly reportedammation and bacillary load in autophagy-decient

M-Cre+ mice challenged with a high intranasal dose ofne levels were not reported and the authors attributedty to accelerated phagocytosis of mycobacteria due tourface expression of the scavenger (and Mtb) receptors

MSR1. An excess of p62 in the Atg7/ macrophages1, a suppressor of the transcription factor Nrf2, result-

eased expression of Nrf2 regulated genes includingeceptors. Parenthetically, these authors observed non autophagy induction between BCG (lacking ESAT6 andcretion system) and Mtb H37Rv.

of autophagy by Mtb

of the vesicular trafcking systems that deliver mosted bacilli to acidied phagolysosomes is a key to Mtbnd is mediated through inhibition of Ca2+ ux and Rabases [1]. Similarly, the bacillus has evolved counter-o evade xenophagy. Zhang et al. [186] reported thatression of ESAT6/CFP10 inhibited autophagosome for-ith H37Rv-infected RAW264.7 cells, along with reducedof Atg8 and other autophagy-related genes. Romangoliompared autophagic ux in human monocyte-derived

with H37Rv, H37Ra or BGG, nding inhibition ofome-lysosome fusion only in DC infected with H37Rv.ty to inhibit autophagic ux was restored by com-n of BCG with the Esx-1 region from Mtb and bytation of H37Ra with the PhoP gene. They also showed-1 dependent autophagic block exerted by H37Rv coulde with rapamycin.b Eis protein, which inhibits infection-inducede cell death through acetylation of DUSP16/MKP-7gulation of JNK-induced ROS generation (Section 3.1.1)ophagy as well [76]. Macrophage cytolysis triggered by

attributed to autophagic cell death but the relation-en autophagy and cytotoxicity is controversial sinceis commonly activated as a prosurvival response in

by RAWwhile bM. smelikely rthe pre

5.5. Ge

A bas a biwith tAtg7, a[154,11) celldiffereally indeneferent associa

Sevautophcomplies invIRGM wAfricanMtb st(rs963transcrthe samprotecother sallelesIranianopposiallele iautophof TB dtical si[195]. genetimentathe IRG

5.6. Th

Theplianceemergest in endogeantitubmay bcandideffectsand pyin Mtbof autoderivethe ythat that leasanide .7 cells inhibited the accumulation of autophagosomess coated with E. coli LPS or phophatidyl-myo-inositol ofis had no suppressive effect [188]. Further research willl more host and pathogen countermeasures regulatinginantly host-protective effects of autophagy in TB.

associations of autophagy and TB

f genetic evidence supports the relevance of autophagyically signicant host defense mechanism in TB. Miceted mutations in the autophagy-related genes Atg5,rgm1 all demonstrate increased susceptibility to Mtb5,189]. In a genome-wide siRNA screen in human (THP-cted with Mtb H37Rv or a several clinical isolates of

ades, Kumar et al. [190] identied 275 genes function-d in control of infection. Seventy-four of these genesential components active against the spectrum of dif-isolates and within that subset, more than half werewith the regulation of autophagy.studies identied association of polymorphic alleles inrelated genes with TB susceptibility or resistance. The

of these interactions is highlighted by several stud-ating polymorphisms in IRGM. The -261TT allele ofegatively associated with TB in a cohort from the westntry of Ghana but this applied only to disease caused by

of the Euro-American lineage [191]. The -261TT allele) is predicted to eliminate binding sites for inhibitoryn factors and therefore increase IGRM expression. Inhanaian population, this allele was not associated withfrom TB caused by M. africanum or M. bovis. Severals variously identied associations of polymorphic IRGM

protection or susceptibility to TB in African-American,nese and Korean populations [191194]. In some casesfects on TB susceptibility were associated with the sameferent populations. Analysis of 22 polymorphisms in 14genes in an Indonesian population found an associatione with polymorphisms in LAMP1 and MTOR but statis-ance was lost after correction for multiple comparisonse divergent associations may reect differences in hostkground, locally prevalent Mtb strains and/or environ-uences but regardless of the effect the data clearly linkne to TB defense.

eutic opportunities

l of TB elimination is hampered by challenges of com-h prolonged multi-drug antibiotic regimens and the

of drug-resistant Mtb strains. This has stimulated inter-otential for host-directed therapies (HDTs) to amplifys effector mechanisms and accelerate the response tolous chemotherapy [196]. Among the pathways thatenable to this approach, autophagy is an attractiveased on its anti-mycobacterial and anti-inammatoryB. Interestingly, the rst line anti-TB drugs isoniazidamide were reported to induce autophagy selectivelycted but not uninfected macrophages [196]. Activationgy was dependent on ROS and attributed to bacteria-roxyl radicals produced in response to these drugs. Inel of M. marinum infection, these authors showed thatti-mycobacterial activity of the antibiotics dependedpart on autophagy. The anti-protozoal drug nitazox-ts metabolite tizoxanide inhibit mammalian target of


rapamycin complex 1 (mTORC1) signaling and stimulate autophagyby a mechanism attributed to suppression of the quinone oxi-doreductase NQO1 [197]. This drug also kills replicating andnon-replicating Mtb in broth culture but its antimicrobial activ-ity was grepotent thanstimulatingtreatment.

The besttext of TB isare associatment outcoof these stufrom a case-els below aodds for TBtation in TBplacebo-conpatients staerol on enroD treatmenoutcome) osmear-posiwho had corandomizator placebo ging TB treatcalcifediol cTB treatmenculture conSputum culD supplemegenotype ofre-analyzedfullled pervitamin D sand resolutpro-inammMtb antigen

Some drstimulate autive theraporgan transcoronary sttion of mTOunacceptabdelivery of rwith less trohave activitthroughputmacrophagwhich also sin mice andBALB/c mic

Many otalthough ththese, the ocandidate. Mwell as cell interest in stimulate ato increasedmTOR [213]lators of autThree of thapproved a

approved for cardiovascular indications (niguldipine, nicardipine,amiodarone), and one (loperamide) is approved for treatment ofdiarrhea. None of these compounds modulated mTOR phosphor-ylation, indicating that they induce autophagy through pathways

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roteinMS Imatest in Mtb-infected macrophages and it was more rapamycin. Drugs with dual antibiotic and autophagy-

effects may particularly attractive candidates for TB

studied non-antibiotic effector of autophagy in the con- vitamin D (Section 5.2). Low serum levels of calcifedioled with increased TB susceptibility and adverse treat-mes in different geographic locations [198203]. Nonedies established causation, and a cautionary note comescontrol study in Greenland where serum calcifediol lev-nd above 75140 nmol/l was associated with increased

[202]. Two trials have tested vitamin D supplemen-. The rst of these was a randomized, double-blind,trolled trial conducted in Guinea-Bissau [204]. Adultrting TB treatment received 100,000 IU of cholecalcif-llment and after 5 and 8 months of inclusion. Vitamint did not reduce the clinical TB severity score (primaryr 12-month mortality. The second study enrolled, newtive adult pulmonary TB patients in the United Kingdomrrected serum calcium concentration >2.65 mmol/l forion to receive four oral doses of 2.5 mg cholecalciferoliven at baseline and then 14, 28 and 42 days after start-ment. Supplementation was shown to increase serumoncentrations in the patients receiving intensive-phaset but did not signicantly inuence the time to sputumversion (primary endpoint) across the whole cohort.ture conversion was, however, accelerated by vitaminntation in the subgroup of participants having the tt

the TaqI VDR polymorphism. Data from that trial were a separate publication that included only patients who-protocol analysis criteria [205]. In this patient group,upplementation accelerated sputum smear conversionion of lymphopenia and monocytosis, and it suppressedatory cytokines in the circulation and in cultures of-stimulated whole blood.ugs already in clinical use for other indications cantophagy and therefore might be considered for adjunc-

y in TB. Rapamycin is used for immunosuppression inplantation and to inhibit endothelial proliferation onents [206,207]. It potently suppresses mTOR via inhibi-RC1 [208]. Systemic immunosuppression is clearly anle effect for TB patients but this might be mitigated byapamycin in inhalable particles [209]. Alternative drugsubling side effects include statins that where shown toy against Mtb in vitro and in vivo [210,211]. A high-

screen for small molecules that restrict Mtb growth ines identied activity for getinib and uoxetine, both oftimulate autophagy [166]. Of these, getinib was tested

restricted Mtb growth following aerosol infection ofe.her approved drugs are known to induce autophagyeir application to TB has not yet been reported. Amongral anti-diabetic biguanide metformin is an attractiveetformin activates AMPK and stimulates autophagy as

death in malignant cells and has attracted considerablefor cancer treatment [212]. Metformin also appears toutophagy by an AMPK-independent mechanism linked

expression of REDD1, which is a negative regulator of. Zhang et al. [214] identied eight small molecule regu-ophagy using an image-based high-throughput screen.e compounds are U.S. Food and Drug Administration-nti-psychotic drugs, three are Ca2+ current inhibitors

distincin anotviouslyautophinteresadvantand inence thmechaprovidBurkhothat glindepeof inalater rtic comsulfonycytokinwith B

6. Con

Autkey viresis. Itbacilli,moduland theral cyfor dessolic anstimulregulapendenof thesbe detmal deThe exalso bethat haautoly

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[2] El20

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[7] PepFEm rapamycin. Minoxidil and clonidine were identiedcreen for autophagy enhancers restricted to drugs pre-d in humans without major side effects [215]. As morepromoting drugs are evaluated against Mtb it will beto learn if particular pathways offer better efcacy

as HDTs for TB. At the same time it will be necessaryative to test whether candidate HDTs adversely inu-lance of protective versus damaging immunity throughs like mTOR inhibition. A cautionary, if unrelated, note is

the experience with glyburide in patients infected witha pseudomallei (melioidosis). Koh et al. [216] reportedide improved survival in diabetic melioidosis patientst of glycemic control; an effect attributed to inhibitionasome assembly. A contrasting effect of glyburide wased by Liu et al. [217] who found higher rates of sep-tions in melioidosis patients treated with (unspecied)as and suppressive effects of glyburide on inammatoryoduction by peripheral blood mononuclear cells treateddomallei antigen.

ions

gy provides a means for infected MPs to overcome thece mechanism of Mtb; inhibition of phagosome biogen-tributions to TB defense include killing of intracellularoting MHC class II restricted antigen presentation, and

the inammatory responses that cause tissue damageical manifestations of TB disease. Phagocytes use sev-

lic sensors recognize intracellular bacilli and tag themion. Further research will likely identify additional cyto-ll surface receptors involved in the detection of Mtb and

of autophagic ux. Autophagy is managed by a complexnetwork with evidence of mTOR-dependent and inde-thways linked to TB defense. The relative contributionferent pathways to positive outcomes in TB remains toned. Such knowledge might be leveraged for the opti-

of host-directed therapies that stimulate autophagy.g translational opportunities for autophagy in TB willanced by better understanding of the countermeasuresvolved in the bacillus to escape detection and inhibite biogenesis.

gement

ed in part by NIH grant HL081149 (to H.K.).

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