autophagy as an innate defense against mycobacteria type vii secretion system. here, we review the...

MINIREVIEW

Autophagy as an innate defense against mycobacteriaEun-Kyeong Jo1,2

1 Department of Microbiology, Chungnam National University School of Medicine, Daejeon, Korea

2 Infection Signaling Network Research Center, Chungnam National University School of Medicine, Daejeon, Korea

A contemporary, comprehensive and timely review of autophagy in mycobacterial infections with particular relevance totuberculosis. A review of this proportion supported by excellent figures has long been needed.

Keywords

mycobacteria; autophagy; vitamin D; cyto-

kines; autophagy receptors; innate immunity.

Correspondence

Eun-Kyeong Jo, Department of Microbiology,

and Infection Signaling Network Research

Center, College of Medicine, Chungnam

National University, 6 Munhwa-dong, Jungku,

Daejeon 301-747, S. Korea.

Tel.: 82-42-580-8243

fax: 82-42-585-3686

e-mail: [email protected]

Received 29 September 2012; revised 12

January 2013; accepted 13 January 2013.

Final version published online 21 February

2013.

doi:10.1111/2049-632X.12023

Editor: Patrick Brennan

Abstract

Over the past several years, much has been revealed about the roles of autophagyand the mechanisms by which the autophagic pathway activates the host innateeffector response against Mycobacterium tuberculosis (Mtb) infection. In responseto invading mycobacteria, the host innate immune system not only recognizespathogen motifs through innate receptors, it also produces appropriate effectorproteins, including cytokines. These innate signals activate or regulate autophagicpathways during infection. It is now clear that vitamin D and functional vitamin Dreceptor signaling are critical in the activation of autophagic defenses against Mtbin human cells. Immunity-related GTPase family M proteins, including the cationicantimicrobial protein cathelicidin and autophagic receptor p62, participate inautophagic pathways that enhance antimicrobial activity against mycobacteria.Moreover, reactive oxygen species mediate antibacterial autophagy and success-ful antimicrobial responses during antibiotic chemotherapy. Recent work has alsoshown that pathogenic Mtb can be targeted by selective autophagy through anESX-1 type VII secretion system. Here, we review the triggers, host factors, andintracellular pathways that regulate host autophagy and its impact on antimicrobialhost defenses during mycobacterial infection.

Introduction

Tuberculosis (TB) is one of the most devastating infectiousdiseases globally. It is estimated that around one-third of thepopulation is latently infected. The WHO reported that therewere an estimated 8–9 million TB cases in 2010. TB is thesecond leading cause of death caused by an infectiousdisease, with a death rate of 1.2–1.5 million worldwide(including deaths from TB among HIV-positive individuals).Serious concerns have been raised regarding an increase inthe number of patients with MDR-TB (WHO, 2011). Auto-phagy is a ubiquitous intracellular process through whichdiverse cytoplasmic cargos are captured and destroyed toreplenish amino acids and energy sources during metabolicstress. During the host response to intracellular parasites,autophagy plays two roles: effector and regulatory roles.During mycobacterial infection, autophagy is essential formounting an effective host response (Gutierrez et al., 2004;Singh et al., 2006).If theMycobacterium tuberculosis (Mtb) phagosome is not

appropriately acidified through the selective exclusion of late

endosomal Rab7 (Via et al., 1997), it persists in immaturephagosomes within host macrophages. Autophagy acts asan immune effector, resulting in phagosomal maturation thatmediates mycobacteria clearance (Gutierrez et al., 2004).Interferon (IFN)-inducible effector proteins, including immu-nity-related GTPase family M (Irgm) proteins, play a hostprotective function against mycobacterial infection throughIFN-c-induced autophagy (Singh et al., 2006). Additionally,human genetic approaches have identified Irgm genevariants that protect against Mtb infection (Intemann et al.,2009).Recently, vitamin D receptor (VDR), a nuclear receptor

that mediates various biological functions of 1,25(OH)2D3

(1,25D3), has been found to play an essential role inantimycobacterial responses through the activation ofautophagy (Liu & Modlin, 2008; Fabri & Modlin, 2009; Jo,2010). Cargo receptors, including p62/SQSTM1, havebeen featured in selective autophagy through the connec-tion of ubiquitinated intracytoplasmic cargos into autopha-gic machinery (Kirkin et al., 2009). Moreover, the roles ofreactive oxygen species (ROS) in antibacterial autophagy

Pathogens and Disease (2013), 67, 108–118, © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved108

Pathogens and Disease ISSN 2049-632X

have been emphasized (Huang et al., 2009; Shin et al.,2010a). Antibiotic-mediated triggering of bacterial andcellular ROS generation is essential for controlling theactivation of autophagy, which is critical for a successfulantimicrobial response to mycobacterial infection (Kimet al., 2012a). Thus, autophagy and its interactions withantimicrobial pathways can restrict mycobacterial patho-gens in a coordinated and cooperative manner.Although numerous previous studies showed that exoge-

nous induction of autophagy can target mycobacteria, recentwork has also revealed that Mtb phagosomal permeabiliza-tion through ESX-1 secretion system induces the ubiquitin-mediated autophagy pathway during natural infection of Mtbthrough the selective autophagic receptors p62 and NDP52(Watson et al., 2012). In this review, we focus on autophagyas a host defensive strategy against mycobacterial infectionand provide a recent update on the pathways and mecha-nisms by which autophagy connects with innate immunity toenhance antimicrobial responses against Mtb. We alsoemphasize the regulatory roles of autophagy in the preven-tion of inflammation during mycobacterial infection.

Overview of autophagy and innate immunereceptors in mycobacterial infection

Autophagy is a process for maintaining intracellular qualitycontrol in the face of various stressors that mainly play ahousekeeping role. So far, three autophagic pathways havebeen identified: macroautophagy, microautophagy, andchaperone-mediated autophagy. Macroautophagy (referredto as autophagy throughout this review) initiates the forma-tion of a new vesicle, the phagophore, which is enlarged,elongated, and generated into a double-membraned orga-nelle, the autophagosome. The autophagosome fuses withlysosomes, maturing into an autolysosome. Here, cytoplas-mic cargos are degraded. This mechanism is recognized asan essential host defense mechanism able to eliminatebacteria in macrophages and nonphagocytic cells infectedwith numerous bacteria, including Streptococcus, Shigella,Legionella, and Salmonella typhimurium (Nakagawa et al.,2004; Amer et al., 2005; Ogawa et al., 2005; Birminghamet al., 2006). Various bacterial pathogens have evolvedstrategies to escape from or subvert host autophagy topersist in or invade host cells.Autophagy can also function as an innate effector and

regulate innate immunity to various viruses and bacteria,including Mtb (Levine & Deretic, 2007; Schmid & M€unz,2007; Yuk et al., 2012). Mtb is a successful intracellularpathogen that has evolved a successful strategy for evadinghost defenses by arresting phagosome maturation ininfected host cells (Fratti et al., 2001; Vergne et al., 2004).The activation of autophagy induces co-localization of themycobacterial phagosome with LC3 autophagosomesand delivers antimicrobial proteins to autolysosomal com-partments, achieving enhanced bactericidal activity inphagocytes (Gutierrez et al., 2004; Singh et al., 2006).Understanding autophagy’s role in regulating innate immu-nity and vice versa will help elucidate the molecular basis ofhost defenses against mycobacteria (Fig. 1).

During mycobacterial infection, toll-like receptor (TLR)signaling plays an important role in the recognition ofpathogens and elicits an inflammatory response, includingthe production of cytokines and antimicrobial effectors(Kleinnijenhuis et al., 2011; Yuk & Jo, 2011). Among theTLRs, TLR2, TLR9, and, possibly, TLR4 recognize Mtb andare involved in activation of the innate immune response(Kleinnijenhuis et al., 2011). Although there are controversialreports regarding the susceptibility of TLR2-deficient mice,in vivo studies using gene knockout mice of myeloid differ-entiation factor 88 (MyD88) and mice lacking both TLR2 andTLR9 revealed that these components are involved in hostdefensive pathways duringmycobacterial infection (reviewedin Kleinnijenhuis et al., 2011; Saiga et al., 2011; Yuk & Jo,2011). The protective role of a non-TLR, Nod-like receptor(NOD)2, was demonstrated in NOD2-deficient mice, whichshowed poor innate and adaptive immune responses andincreased bacterial loads (Divangahi et al., 2008). Moreover,NOD2 participates in nonredundant mechanisms and pro-motes the production of cytokines leading to TLR2 activation(Ferwerda et al., 2005). Additionally, NOD2 plays a crucialrole in the production of the inflammatory cytokines TNF-aand IL-1b in response to Mtb andMycobacterium bovis BCGin human macrophages (Brooks et al., 2011).Several atypical mycobacteria and their unknown compo-

nents induce Dectin-1 signaling (Yadav & Schorey, 2006;Shin et al., 2008; Lee et al., 2009). Further, trehalose-6,6′-dimycolate (TDM, also called cord factor), a mycobacterialcell wall glycolipid, induces macrophage activation and theinduction of Th17 immune responses through C-type lectinMincle receptors (Ishikawa et al., 2009; Schoenen et al.,2010). TDM-induced inflammatory signaling is mediatedthrough a class A scavenger receptor called macrophagereceptor with collagenous structure (MARCO) and TLR2/CD14 (Bowdish et al., 2009). These and future studies ofthe nature and shaping of innate immune responses throughthe recognition of various innate receptors by mycobacteriaand their components are important for understanding therole of autophagy and the molecular mechanisms by whichautophagy combats the pathogenic strategies of intracellularpathogens.Live Mtb infection and the Mtb protein ESAT-6 were

shown to induce IL-1b and NLRP3 transcription and toactivate the NLRP3/ASC inflammasome in human macro-phages (Mishra et al., 2010). This was not observed inmurine macrophages (Master et al., 2008). NLRP3 inflam-masome activation may have suppressive roles in thepromotion of autophagy and vice versa (Jo et al., 2012). It isbeyond the scope of this review to describe the relationshipbetween autophagy and the inflammasome. It is noteworthy,however, that specific mycobacterial components caninduce inflammasome activation, thus accelerating theescape from autophagic pathways.

Crosstalk between autophagy and innateimmunity in the fight against mycobacteria

TLR signaling and autophagy are linked: TLR signalingrecognizes pathogen-associated molecular patterns through

Pathogens and Disease (2013), 67, 108–118, © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved 109

E.-K. Jo Autophagy in mycobacterial infection

pattern-recognition receptors (PRRs), and autophagic path-ways can eliminate harmful pathogens. As many Mtb mole-cules act as ligands for TLRs and non-TLRs (reviewed in Joet al., 2007; Kleinnijenhuis et al., 2011; Saiga et al., 2011),the recognition of mycobacterial molecules may initiate orregulate autophagy. Various TLR stimuli, in addition to Nod-like receptor-induced signals or CD40, can activate autopha-gic responses to induce host innate immunity and overcomeamycobacterial phagosome arrest (Sanjuan et al., 2007,2009; Xu et al., 2007; Delgado et al., 2008; Orvedahl &Levine, 2009). It is of interest that, regardless of the TLRligand stimulus, host autophagy activation can lead to theclearance of mycobacteria-containing phagosomes throughthe recruitment of autophagosomes and induction ofphagosomal maturation (Xu et al., 2007; Delgado et al.,2008). Further, several crucial components of TLR signalingsuch as MyD88, TIR-domain-containing adapter-inducinginterferon-b (TRIF), and mitogen-activated protein kinase(MAPK) are involved in the regulation of autophagy activation(Xu et al., 2007; Delgado et al., 2008; Shi & Kehrl, 2008).Indeed, several mycobacterial components per se can

activate or modulate host autophagy (Fig. 1). The well-known mycobacterial TLR2 ligand, 19-kDa lipoprotein,robustly activates autophagic responses in monocytes/

macrophages. This only occurs with sufficient amounts ofactive vitamin D metabolites or with Cyp27b1 gene activa-tion, followed by functional vitamin D signaling (Shin et al.,2010b). In contrast, Mtb eis downregulates autophagy,which is mediated by cellular- and NADPH oxidase-derivedROS generation (Shin et al., 2010a). Recent studies haverevealed that Mtb eis is an efficient N(e)-acetyltransferasethat acetylates Lys55 of dual-specificity protein phosphatase16 (DUSP16)/MAPK phosphatase-7 (MKP-7), a JNK-spe-cific phosphatase, thus inhibiting JNK phosphorylation andinflammatory cytokine generation (Kim et al., 2012b).In non-TLR-mediated autophagy, muramyl dipeptide

(MDP) leads to the formation of autophagosomes throughreceptor-interacting serine–threonine kinase-2 (RIPK-2),autophagy-related protein-5 (ATG5), ATG7, and ATG16L1(Cooney et al., 2010; Travassos et al., 2010). Both NOD1and NOD2 are involved in autophagy activation and bacte-ricidal responses through the recruitment of ATG16L1 to theplasma membrane and bacterial wrapping by autophago-somes (Travassos et al., 2010). Recent studies have foundthat NOD2 activation leads to expression of the autophagyproteins Irgm, LC3, and ATG16L1 in human alveolarmacrophages. These proteins contribute to improved intra-cellular control of virulent Mtb (Ju�arez et al., 2012). Addi-

TLR2/1TLR2/6

TLR4

TLR9

NOD2 endosome

Mtb phagosome

Dectin-1

MincleMyD88

Mtb phagosome

Antibacterial autophagy activation

NADPH oxidase

ROS

ROS

JNK

DUSP16/MKP-7

Eis

NLRP3inflammasome

ESAT6

zmp1

LpqH/19-kDaTLR2/1 ligand

VDR signaling

Autophagolysosome

Fusion with autophagosome

Cathelicidin

Fig. 1 Crosstalk between autophagy and innate immune signaling in mycobacterial infection. Mycobacteria trigger innate immune signaling through

pathways involving diverse innate receptors, including TLRs (TLR2, TLR4, and TLR9) and non-TLRs (Dectin-1, Mincle, and NOD2). Several

mycobacterial ligands activate innate immune pathways, thus affecting antibacterial autophagic responses in Mtb-infected macrophages. Mtb

eis inhibits autophagy through the modulation of ROS and acetylation of dual-specificity protein phosphatase 16 (DUSP16)/MAPK phosphatase-7

(MKP-7). ESAT-6 protein and Zmp1 may modulate autophagy through positive and negative regulation of the NLRP3 inflammasome, respectively.

LpqH/19-kDa lipoprotein of mycobacteria promotes antibacterial autophagy through functional VDR signaling activation.


Autophagy in mycobacterial infection E.-K. Jo

tionally, mutant mice harboring variants of NOD2 (3020insC)showed elevated NF-KB activation in response to MDP andupregulated IL-1b secretion (Maeda et al., 2005). Moreover,mutant NOD2 (Crohn’s disease-associated NOD2 frame-shift mutation) failed to recruit ATG16L1 to the plasmamembrane and induce autophagy, suggesting that NOD2and autophagic pathways are functionally linked to increasebacterial killing and to maintain host immune homeostasis.The mycobacterial NOD2 ligand MDP, which is N-glycol-

ylated by N-acetyl muramic acid hydroxylase, shows morepotent NOD2-stimulating immune activation in mousemodels (Coulombe et al., 2008). Thus, mycobacterialN-glycolylated MDP may induce autophagy to a greaterextent than N-acetylated MDP, the most common form inbacteria. It remains unclear whether mycobacterial signalinginduced by the C-type lectin receptor Dectin-1 or Mincle,and by scavenger receptors, including MARCO, is involvedin the activation or regulation of the autophagic pathwayduring infection. An understanding of the receptor-specificregulation and induction of autophagy by individual myco-bacterial ligands or antigens may facilitate the developmentof effective therapeutic and vaccine strategies againsthuman TB.

Autophagy and cytokines in mycobacterialinfection

Expression of the Th1 cytokine IFN-c, mainly by CD4+ Tcells, is central to host defenses against TB (Harding &Boom, 2010). TNF-a plays a role in promoting hostprotective immunity during TB, as shown in cases of TB inpatients treated with anti-TNF-a agents in a range ofinflammatory/autoimmune diseases, including rheumatoidarthritis (Harris & Keane, 2010). Recent studies haveprovided insight into the roles of these protective cytokinesin the activation of autophagy, thus promoting antimyco-bacterial immune defenses. IFN-c enhances the autophagiccontrol of Mtb, whereas the Th2 cytokines IL-4 and IL-13inhibit those effects (Harris et al., 2007). IFN-c and itseffector mechanisms are mediated through multiple path-ways: the induction of antimicrobial effectors, includinginducible nitric oxide synthase 2, which may produce nitricoxide; the IFN-inducible GTPase family LRG47 (also knownas Irgm) in mice (Macmicking et al., 2003); and upregulatedexpression of MHC class II molecules, which play a centralrole in antigen processing and presentation (Harding &Boom, 2010). Autophagy activation promotes antigenprocessing and presentation by MHC class II moleculesand results in the bridging of innate and adaptive immuneresponses. In this aspect, IFN-c may facilitate host protec-tive immune responses through the connection of auto-phagy activation and innate immune activation forcontrolling Mtb infection. Mycobacteria also inhibit the Januskinase–signal transducer and activator of transcription (JAK-STAT) signaling pathway (Hussain et al., 1999), and STAT1is required for protective immunity against mycobacteria(Sugawara et al., 2004; Vairo et al., 2011). It is unknownwhether JAK-STAT signaling also promotes host autophagyactivation and antimicrobial clearance.

Proinflammatory TNF-a plays an important role in theactivation of phagosome maturation because TNF-a neu-tralization suppresses IFN-c-induced phagosome matura-tion in primary human peripheral blood monocyte-derivedmacrophages (Harris et al., 2008). These results indicatethat TNF-a may synergize IFN-c-induced antimicrobialresponses and autophagic responses against intracellularmycobacteria (Harris, 2011). Many other cytokines, includ-ing IL-2, CCL2, IL-6, and TGF-b, play key roles inpositively regulating autophagy in various cell types (e.g.IL-2 in CD4+ T cells, CCL2 and IL-6 in CD11b+ peripheralblood mononuclear cells, and TGF-b in hepatocarcinomacell lines) (reviewed in Harris, 2011). A variety of innatecytokines, including CCL2, IL-6, and TNF-a, are increasedduring the initiation of mycobacterial infection. Thesecytokines are involved in the development of granuloma,pathophysiology, and local immunity during TB (Cooperet al., 2011). Thus, it is important to determine theroles of each and/or combined cytokines in the controlof TB.Mtb-induced IL-12p40 and IL-23p19 expression is nega-

tively regulated in human macrophages by the mammaliantargets of rapamycin (mTOR)/70-kDa ribosomal S6 kinase 1(S6K1) pathway (Yang et al., 2006). These data suggestthat IL-12/IL-23 is likely induced during autophagic pathwayactivation in mycobacterial infection. Additionally, IL-23production by Mtb in dendritic cells is mediated by thecombined sensing of NOD2 and TLR2, whereas IL-12p70production requires IFN-c or the TLR7/8 ligand R848(Gerosa et al., 2008). All of these signals play a crucial rolein the activation of autophagy, suggesting a mechanisticlink between protective cytokines and the induction ofautophagy.Autophagy negatively regulates the activation of tran-

scription, processing, and secretion of several proinflam-matory cytokines, including IL-1a, IL-1b, and IL-18 (Saitohet al., 2008; Cris�an et al., 2011; Harris et al., 2011;Nakahira et al., 2011; Zhou et al., 2011). It is beyond thescope of this review to comprehensively discuss the role ofautophagy in the inhibition of inflammasome activation.However, the autophagic pathway negatively controls IL-1bproduction by at least two mechanisms (i.e. by targetingpro-IL-1b for degradation in lysosomal compartments andby inhibiting NLRP3 inflammasome activation) (Harriset al., 2011; Jo et al., 2012). Conversely, it was alsoreported that autophagy may play an important role in theinduction of TNF-a transcription and secretion (Cris�anet al., 2011). Another recent paper showed an unappreci-ated contribution of autophagy to the synthesis andsecretion of the proinflammatory cytokine IL-1b (Dupontet al., 2011). Multiple cytokines and mediators participatein the regulation of autophagic pathways to promote orinhibit host defenses (Fig. 2). The crosstalk betweenautophagic pathways and various activating/inhibiting cyto-kine stimuli may be influenced by the microenvironment,which affects the behavior of immune cells, or by functionalproperties of the innate receptors or signaling moleculesthat contribute to cytokine generation and the autophagicprocess.



Vitamin D and autophagy duringmycobacterial infection

Global vitamin D insufficiency and proper VDR signaling areimportant in a variety of human diseases, including TB(Bouillon et al., 2008; Hewison, 2011a, 2012a). Recentstudies have emphasized a role for the coordinated functionof VDR signaling in the regulation of innate and adaptiveimmunity (Baeke et al., 2010; Hewison, 2011a, b, 2012b).Through the autocrine or paracrine synthesis of 1,25D3 fromthe precursor 25-hydroxyvitamin D(3), vitamin D plays animportant role in antibacterial activities through the inductionof antibacterial proteins in various cell types, includingphagocytes (Adams et al., 2007; Liu & Modlin, 2008; Jo,2010). Several signals, including TLR2/1 stimulation,activate innate immune cells to express the enzyme25-hydroxyvitamin D-1a-hydroxylase (Cyp27b1), which iscritical for the intracrine conversion of circulating 25-hydrox-yvitamin D to active 1,25D3 (Liu et al., 2006; Krutzik et al.,2008; Liu & Modlin, 2008; Jo, 2010; Fabri et al., 2011a, b).Vitamin D-induced human antimicrobial activity against

Mtb is mediated through the cationic antibacterial proteincathelicidin LL-37 (Liu et al., 2006, 2007; Yuk et al., 2009;Shin et al., 2010b). Cathelicidins and defensins are part ofinnate immune antibiotics found in many species. Theseantimicrobial peptides function not only in the innate hostdefense against pathogens, especially in the airway, butalso in the regulation of a variety of physiological functions,including immune activation, wound healing, angiogenesis,and migration (Zanetti, 2004; Bowdish et al., 2006; Tecleet al., 2010; Shin & Jo, 2011). Additionally, human cathelic-

idin LL-37/hCAP-18 mediates the induction of vitaminD-dependent autophagy and TLR-mediated vitaminD-dependent autophagy through the transcriptional regula-tion of the essential autophagy-related genes Beclin-1 andAtg5 and is recruited into autophagosomes after 1,25D3 orTLR2/1 stimulation (Yuk et al., 2009; Shin et al., 2010b).Further, the Th1 cytokine IFN-c, but not the Th2 cytokineIL-4, upregulates cathelicidin and b-defensin 2 in humanmonocyte-derived macrophages cultured in media contain-ing 25-D3 (Fabri et al., 2011a, b). In the same study, it wasdemonstrated that IFN-c-induced autophagy and phago-some–lysosome fusion require vitamin D sufficiency and theactivation of functional VDR signaling (Fabri et al., 2011a,b). These data suggest that vitamin D-dependent autophagyactivation and antimicrobial responses are important forinnate (TLR) and acquired (IFN-c-dependent) mechanisms(Fig. 3), although these two immune arms use distinctreceptors and signaling pathways (i.e. MyD88 and STAT1,respectively) (Yuk et al., 2009; Shin et al., 2010b; Fabriet al., 2011a, b).Co-infection with Mtb and HIV potentiates the deteriora-

tion of immune functions and leads to premature death(Pawlowski et al., 2012). Additionally, both infections arehighly associated with vitamin D insufficiency (Campbell &Spector, 2011; Luong & Nguyen, 2011; Khoo et al., 2012).In human macrophages, vitamin D induces the activation ofautophagy, which is dependent on pathways involvingphosphatidylinositol 3-kinase, Atg5, and Beclin-1. Autopha-gic pathways are required for the suppression of HIV-1replication in vitro (Campbell & Spector, 2011). BlackAfricans in Cape Town are highly susceptible to active TB

Antibacterialautophagy

Interferon-γInterferon-inducible GTPasesCathelicidin/β-defensinJAK-STAT?

IL-12/IL-23

Protective cytokines against Mtbpromote antibacterial autophagy

Th2 cytokines

IL-4 IL-13

Inhibitory cytokines against Mtbsuppress antibacterial autophagy

Tumor necrosis factor-αSynergize interferon-γ?

CCL2

IL-6

Proinflammatory cytokines may synergize/promote autophagy

Autophagy crosstalks with inflamma-some/inflammatory cytokines

Autophagy positively or negatively regulates

TNF-α IL-1αIL-1β IL-1β

IL-18

Fig. 2 Autophagy and the cytokine network in mycobacterial infection. Mycobacteria and their antigens induce numerous cytokines. Autophagy is

induced by various cytokines and crosstalk with this cytokine network. Protective cytokines (IFN-c and IFN-12) involved in host defense during

mycobacterial infection promote antibacterial autophagy, whereas inhibitory cytokines (IL-4 and IL-13) may suppress autophagy. Additionally,

mycobacteria are effective at inducing host proinflammatory responses. Inflammatory cytokines and chemokines may synergize or promote

autophagic responses. However, TNF-a seems to synergize IFN-c-mediated antimicrobial responses and phagosomal maturation in Mtb-infected

cells. Defective autophagy can lead to increased inflammasome activation, thus upregulating the production and maturation of IL-1b and IL-18.

Conversely, autophagy is necessary for the biosynthesis and secretion of TNF-a and IL-1b.



in the presence and absence of HIV infection, and they havea reciprocal relationship between seasonal variations inserum 25D3 status and the incidence of TB (Martineauet al., 2011). A meta-analysis demonstrated that certainVDR gene polymorphisms are significantly linked with therisk of TB (Gao et al., 2010). Moreover, multiple genes,including IFNG, NRAMP1, and NOS2A, have been sug-gested as candidate genes in association studies (M€oller &Hoal, 2010). Depending on the vitamin D concentration, itwould be interesting to examine whether certain VDRpolymorphisms and the activation of autophagy are stronglyassociated with TB incidence and/or susceptibility. A com-bination of genetic and physiological knowledge is essentialto understand the key factors driving pathogenesis and

protective immunity against TB. This knowledge will improvethe therapeutic development of customized patient profiles.

Immunity-related GTPases and autophagy inmycobacterial infection

Immunity-related GTPases (IRG proteins) are an importantinnate resistance system against a variety of intracellularpathogens, including Mtb and Toxoplasma gondii (Macm-icking et al., 2003; Bekpen et al., 2010; Hunn et al., 2011).The IRG protein family consists of IFN-inducible GTPasesbelonging to two subfamilies (i.e. GMS and GKS) (Boehmet al., 1998; Bekpen et al., 2010). Among them, the GMSsubfamily (Irgm1, Irgm2, and Irgm3) has a methionine (M)instead of a lysine (K) in the G1 motif, and it plays anessential function in the regulation of the GTPase cycle ofGKS proteins and their normal accumulation at the parasi-tophorous vacuolar membrane after intracellular parasiticinfection (Hunn et al., 2008; Bekpen et al., 2010). The bestcharacterized IRG protein, IRGM1, plays an essential role inIFN-c-dependent intracellular bacterial elimination throughthe activation of autophagy (Gutierrez et al., 2004). Addi-tionally, it has various roles in immune regulation such ascontrolling macrophage motility by positioning of the GKSIRG protein (Irgb6) to the advancing lamellipodia (Henryet al., 2010) and in the protection of mature effector CD4+ Tlymphocytes from IFN-c-induced autophagic cell death(Feng et al., 2008). The GKS protein Irga6 also plays arole in the IFN-c-mediated fusion of intracellular pathogenswith autophagosomes and bacterial elimination (Al-Zeeret al., 2009).Human Irgm, the sole human IRG, plays a key function in

the induction of autophagy (Fig. 3). Several pathwaysinvolving rapamycin (mTOR inhibition) and IFN-c lead tothe activation of Irgm-mediated autophagy (Singh et al.,2006, 2010). Irgm contributes to the activation of autophagyby translocating to mitochondria, and it is required formitochondrial fission, which is crucial for the autophagiccontrol of intracellular mycobacteria (Singh et al., 2010).Irgm also affects the localization of a second family of IFN-induced GTP-binding proteins, guanylate-binding proteins(GBPs). This leads to the accumulation of Gbp2 in LC3/p62-expressing intracellular compartments (Traver et al., 2011).Several GBP families, including Gbp1, Gbp2, and Gbp7,also play a key role in antimicrobial immunity againstmycobacterial infection within phagocytes and in micethrough coordination with multiple defense systems, includ-ing phagocyte oxidase, antimicrobial peptides, and auto-phagy effector activation (Kim et al., 2011). GBPs promoteautophagy via an interaction with specific autophagy pro-teins; Gbp1 interacts with the SLR p62, while Gbp7 interactswith Atg4 (Kim et al., 2011). IRG proteins may contribute tohost antibacterial defenses via the enforcement of anautophagy activation flux, the terminal stage of autophagicdegradation, through mediating vesicular trafficking, fusionbetween organelles, and lysosomal function to clear intra-cellular cargos (Howard, 2008).Human genetic studies have indicated the potential role of

Irgm in the pathogenesis of TB and Crohn’s disease.

Mtb phagosome

TLR2/1TLR2/6

Cyp27b1

1,25D3 25D3

CAP18/LL-37

β-Defensin

Interferon-γ

IRGM

Bactericidalpeptidegeneration

Mitochondrial fission

Autophagy

Inhibition of mTORAutophagosome

UbiquitinP62

VDR

VDRE

Fig. 3 Innate and adaptive immune pathways converge at antibacterial

autophagy activation to eliminate intracellular mycobacteria. TLR

(innate)-mediated intracellular signaling cascades induce Cyp27b1

expression in macrophages under vitamin D-sufficient conditions. This

results in the expression of cathelicidin and b-defensin, which translo-

cate to autolysosomal compartments to eliminate bacteria. IFN-c

(adaptive) not only induces autophagy through IRGM-dependent mito-

chondrial fission and antibacterial defenses, but also activates cathelic-

idin in human macrophages through functional VDR signaling activation.

Moreover, the autophagic receptor p62 selectively targets ubiquitinated

cytosolic proteins (for the generation of bactericidal neopeptides) or

even mycobacteria (for degradation) into LC3 autophagosomes. Thus,

both innate (TLR) and acquired (IFN-b-dependent) pathways converge

at antibacterial autophagy activation through functional VDR signaling

activation in Mtb-infected macrophages. The activation of IRGM-

dependent antibacterial defenses, mitochondrial function, antibacterial

protein generation, and autophagic receptors (e.g. p62) may coopera-

tively act in antimicrobial killing against mycobacteria.



Genetic analysis has shown that an Irgm1 allele with twosingle-nucleotide polymorphisms (rs13361189 T/C andrs10065172 C/T) and a 20-kb deletion upstream of thecoding region are associated with Crohn’s disease (Parkeset al., 2007). The allele with the 20-kb deletion is alsoassociated with decreased Irgm levels (Mccarroll et al.,2008). These data suggest that defective Irgm expressionaffects the autophagic process against intracellular bacteria,resulting in the pathogenesis of Crohn’s disease (Mccarrollet al., 2008). Moreover, the Crohn’s disease-related T alleleof rs10065172 is associated with TB susceptibility amongAfrican-Americans (King et al., 2011). The Irgm variant-261TT genotype is also significantly related to protectionagainst human pulmonary TB caused by Mtb, but notM. africanum strains (Intemann et al., 2009). This polymor-phism Irgm -261T is associated with enhanced expressionof mature Irgm, suggesting that increased Irgm promotesthe autophagic degradation of intracellular bacteria (Inte-mann et al., 2009).Although these studies suggest the pivotal roles of IRG

and GBP proteins in host innate immunity, the detailedmechanisms by which IRG proteins influence immuneresistance against infectious and inflammatory diseasesare unknown. In addition, it is interesting how the sameprotein is involved in host susceptibility to both TB andCrohn’s disease. A better understanding of the roles of Irgmwill be gained by comprehensive clinical genetic studies.

Autophagy receptors during mycobacterialinfection

Recent studies have suggested the roles of autophagyreceptors, including p62, NBR1 and NDP52, in selectiveantibacterial autophagy (von Muhlinen et al., 2010; Most-owy et al., 2011; Randow, 2011; Deretic, 2012; Kuballaet al., 2012). Ubiquitin-decorated bacteria are recognized bythe autophagic adapters p62 and NBR1, which act ascytosolic bridges for pathogens/proteins to autolysosomes(Johansen & Lamark, 2011; Randow, 2011). p62 and NBR1have domains that recognize cargo and participate ininflammatory responses. These proteins can also be selec-tively degraded by autophagy [reviewed in (Deretic, 2012)].During mycobacterial infection, the autophagy receptor p62eliminates Mtb through processing and the generation ofantimicrobial peptides from cytosolic components (Ponpuaket al., 2010). In addition, p62 participates in the activation ofthe M. abscessus-induced NLRP3 inflammasome in humanmacrophages (Lee et al., 2011), suggesting a role for p62in the induction of inflammatory processes. Althoughnumerous studies have elucidated the roles of optineurin,NDP52, or NBR1 in bacterial targeting to autophagosomes(reviewed in Kuballa et al., 2012), much less is known aboutthe roles of these autophagic adaptors during mycobacterialinfection. Recent report has revealed that selective auto-phagy pathway targets phagosomal Mtb via recognition ofMtb DNA, which is translocated into cytosol through MtbESX-1 secretion system (Watson et al., 2012). Interestingly,cytosolic access of Mtb DNA leads to ubiquitination ofbacteria that can be targeted by autophagy system via

autophagic receptors p62 and NDP51, as well as TBK1(Watson et al., 2012). Additional studies are needed toreveal the exact functions and mechanisms of severalautophagic receptors and the crosstalk between innateimmune signaling during mycobacterial infection.

Role of ROS in the activation of hostautophagy by antibiotic chemotherapyagainst mycobacteria

An essential role for host autophagy activation has beenreported in successful antimicrobial responses during anti-biotic chemotherapy (Kim et al., 2012a). Isoniazid or pyraz-inamide treatment significantly induces the autophagic fluxin macrophages infected with Mtb. Antibiotics initiate thefusion of mycobacterial phagosomes with autophagosomes(Kim et al., 2012a), suggesting that antibiotic treatmentleads to phagosomal maturation in Mtb-infected cells. Asantimicrobial killing effects have been observed at latertimes (e.g. 3 days after), the activation of autophagy byantibiotic treatment plays an important role in intracellularkilling in murine macrophages in vitro and in M. marinum-infected flies in vivo (Kim et al., 2012a). Previous studieshave shown that bactericidal drug action is potentiated viahydroxyl radicals and oxidative damage of bacterial deathpathways, regardless of the drug targets (Kohanski et al.,2007, 2008). Importantly, ROS derived from antibiotic-killedbacteria triggered the release of cellular ROS originatingfrom NOX2 and mitochondria, which activated host cellautophagy (Kim et al., 2012a).These findings provide new insight into the initial mech-

anism of pyrazinamide inside phagocytes and in vivo.Pyrazinamide does not have sterilizing activity againstMtb, unless it is activated in an acidic environment (pH5.0–5.5) in vitro (Mcdermott & Tompsett, 1954). However,it is a paradox how pyrazinamide can be activated insidecells and in vivo. Pyrazinamide action may occur as drug-triggered hydroxyl radicals and host autophagy lead to thetargeting of bacteria to more acidic compartments, whichconstitute a more appropriate environment for pyrazinamideaction as well as autolysosomal degradation (Fig. 4). Thesedata indicate that antituberculosis drugs promote hostautophagy through ROS generation in infected cells andthat the activation of autophagy is required for efficientbacterial clearance during chemotherapy against mycobac-terial infection.

Concluding remarks

Studies of antimycobacterial autophagy have made consid-erable progress. Studies of innate immunity during myco-bacterial infection have unveiled the crosstalk between PRRsignaling and autophagic pathways. Essential roles forimmune regulators such as cytokines in the modulation ofautophagy and a new role for antimicrobial proteins incontrolling autophagic pathways are gaining support. Theactivation of functional VDR signaling in macrophagesrepresent a mechanism for inducing autophagy and antimi-crobial responses against mycobacterial infection. Addition-



ally, IRG proteins play a crucial role in the activation ofautophagy, and the actions of these proteins have clearclinical relevance to humans. Newly described cargoreceptors such as p62 are being recognized to mediatexenophagy against mycobacteria through the selectiveclearance of ubiquitinated target bacteria or proteins. Pro-gress in understanding the roles of ROS and antibacterialautophagy may help answer key questions such as how todevelop new therapeutics targeting host autophagy againstmycobacterial infection. Antibacterial autophagy either leadsto the resolution of intracellular bacterial invasion or regu-lates excessive host inflammatory responses during myco-bacterial infection. Future efforts will be directed not onlytoward further elucidation of the basic mechanisms of theautophagic pathways in innate immunity to mycobacteria,but also toward achieving protective and therapeutic bene-fits in human TB.

Acknowledgements

I thank Dr R. L. Modlin (University of California, CA, USA) forinvaluable discussion and critical reading of the manuscript;D. M. Shin and J. J. Kim for kind support in figurepreparation; H. M. Lee and J. M. Yuk for critical reading ofthe manuscript. I am indebted to current and past membersof our laboratory for discussions and investigations thatcontributed to this article. This work was supported by theNational Research Foundation of Korea (NRF) grant fundedby the Korea government (MEST) (2012-0005763) throughthe Infection Signaling Network Research Center at Chung-nam National University. I apologize to colleagues whosework and publications could not be referenced owing tospace constraints. The authors have no financial conflict ofinterests.

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autophagy as an innate defense against mycobacteria type vii secretion system. here, we review the...

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