REVIEW

β-glucan signaling connects phagocytosis to autophagy

Jun Ma2 and David M Underhill1,,2,3

2Research Division of Immunology, Department of Biomedical Sciences,F. Widjaja Foundation Inflammatory Bowel and Immunobiology ResearchInstitute, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles,CA 90048, USA and 3Department of Pathology and Laboratory Medicine,David Geffen School of Medicine, University of California, Los Angeles,CA 90095, USA

Received on May 30, 2013; revised on June 3, 2013; accepted on June 4, 2013

A growing list of innate immune receptors is being definedthat recognize polysaccharides of microbial cell walls.Fungal β-glucan recognition by the receptor Dectin-1 trig-gers inflammatory immune responses in macrophages anddendritic cells that are appropriate for defense againstfungal pathogens. Among these responses is the specific re-cruitment of the autophagy-related protein light chain 3(LC3) to phagosomes containing fungi. Studies document-ing LC3’s recruitment to phagosomes containing β-glucanand other nonsugar particles suggest that LC3 plays a rolein regulating phagocytosis and its related immunologicalresponses.

Keywords: autophagy / C-type lectin / phagocytosis

Introduction

Innate immune receptors recognize conserved chemical motifstypically found on foreign cells, but not on self. Awell-studiedsuch “Pathogen-Associated Molecular Pattern” or “PAMP” isthe β-glucan polymer that comprises a large portion of the cellwalls of fungi. Fungal β-glucan is a glucose polymer that variesin length and typically consists of β-(1,3) linkages with occa-sional β-(1,6) branches (Figure 1A). Fungal β-glucan is recog-nized by innate immune cells such as macrophages, dendriticcells and neutrophils. The primary β-glucan receptor on thesecells has been identified as the C-type lectin pattern recognitionreceptor Dectin-1 that triggers phagocytosis, respiratory burstand inflammatory responses upon engaging fungi (Goodridgeet al. 2009). Dectin-1 is essential for effective mucosal immun-ity to fungi in mice and humans. Dectin-1 knockout mice aresubstantially more susceptible to infection with many differentfungi including Candida albicans, Pneumocystis carinii andAspergillus fumigatus (Saijo et al. 2007; Taylor et al. 2007;Ferwerda et al. 2009; Werner et al. 2009; Leal et al. 2010;

Marodi and Erdos 2010). Macrophages and dendritic cellsfrom mice deficient in Dectin-1 show a drastically reduced re-spiratory burst in response to fungi and generally produce lowerlevels of inflammatory cytokines. Importantly, humans withrare genetic deficiencies in Dectin-1 or CARD9, an adaptorprotein involved in Dectin-1 signaling, are specifically suscep-tible to fungal infections (Ferwerda et al. 2009; Glocker et al.2009; Marodi and Erdos 2010). Additional receptors havebeen implicated in detection of β-glucans including the comple-ment receptor 3 and lactosylceramide, although their specificcontributions to β-glucan-induced immune responses are lesswell-defined. This review will focus on how β-glucan recogni-tion by Dectin-1 leads to a phagocytic process tailored to opti-mize immune responses to fungi.

Dectin-1

Dectin-1 is a member of the C-type lectin family of pattern rec-ognition receptors. This is a growing family of receptors thatincludes, among others, Dectin-2, the mannose receptor,Mincle, DC-SIGN and CLEC5a (Vautier et al. 2010). Theserelated receptors all contain at least one extracellular C-typelectin domain and play roles in recognition of extracellularmicrobes. They differ in their ligand specificities and can differin their mechanisms of intracellular signaling.Dectin-1 is a type II transmembrane protein containing a

single extracellular C-type lectin domain that recognizesβ-glucan, and a short intracellular signaling tail (Brown andGordon 2001). The tail contains a signaling motif similar tothe immunoreceptor tyrosine-based activation motif (ITAM)found in T cell receptors, B cell receptors and Fc receptors, butwhile a true ITAM has two tyrosines that are essential for sig-naling, the Dectin-1 motif uses just one and is thus called a“hemITAM” (Rogers et al. 2005; Underhill et al. 2005). Thefunctional consequences of this difference are not yet clear.Like other ITAM-containing receptors, Dectin-1’s hemITAMbecomes phosphorylated by Src family kinases and recruits andactivates Syk. These kinases are responsible for triggeringphagocytosis and the recruitment and activation of the nicotina-mide adenine dinucleotide phosphate (NADPH) phagocyteoxidase leading to a release of antimicrobial reactive oxygeninto the phagosome (Gantner et al. 2003). In addition, Dectin-1activation triggers inflammatory transcriptional responsesthrough activation of nuclear factor of activated T cells (NFAT)and nuclear factor kappa-light-chain-enhancer of activated Bcells. A unique feature of Dectin-1 related to its role as aphagocytosis-inducing receptor is that while it binds bothsoluble and particulate forms of β-glucan, it is only activated by

1To whom correspondence should be addressed: Tel: +1-310-423-7654;e-mail: david.underhill@csmc.edu

Glycobiology vol. 23 no. 9 pp. 1047–1051, 2013doi:10.1093/glycob/cwt046Advance Access publication on June 6, 2013

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particulate glucans. This process is regulated by the presence ofCD45 and CD148 membrane phosphatases on phagocytic cells(Goodridge et al. 2011).

LC3-associated phagocytosis

Details of the signaling pathway activated by Dectin-1 uponbinding to β-glucan have been discussed in other recent

reviews (Kerrigan and Brown 2011; Goodridge et al. 2012;Sancho and Reis e Sousa 2012). We will focus our discussionhere on a relatively new addition to our understanding of thephagocytic response to β-glucans mediated by Dectin-1 called“LC3-associated phagocytosis”. Light chain 3 (LC3) is amicrotubule-associated protein highly conserved acrossspecies. While well-known for its role in autophagy, recentfindings have suggested that it may play a related but distinct

Fig. 1. Activation of LC3-associated phagocytosis by β-glucan. (A) Fungal cell wall β-glucan typically consists primarily of β(1,3)glucan with occasional β(1,6)branches. (B) Recognition of β-glucan-containing particles by Dectin-1 on myeloid phagocytes triggers phagocytosis, production of reactive oxygen species, andrecruitment of LC3. LC3 recruitment enhances phagosome maturation leading to more rapid accumulation of major histocompatibility complex class II and LAMP1.It also enhances the efficiency of presentation of antigens by MHCII.

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role in the process of traditional phagocytosis (Sanjuan et al.2007). Autophagy is a process whereby cells sequester cyto-solic debris, organelles and pathogens in double-membraneautophagosomes (Levine et al. 2011). These newly formedintracellular compartments fuse with lysosomes which degradeand recycle their contents. In a state of metabolic stress or cyto-solic infection, autophagy is activated and contributes to cellsurvival. Activation of autophagy and formation of autophago-somes is often measured by observing the accumulation of LC3puntae. The formation of these puntae is directed by an autop-hagy signaling pathway that involves ATG5, ATG7, ATG12,ATG4 and Beclin. Newly synthesized LC3 exists as LC3I in thecytosol, and signaling by this pathway causes LC3I to becomeconjugated to phosphatidylethanolamine. This lipidated LC3called LC3II becomes membrane-associated, localizing specif-ically to autophagosomes (Mizushima et al. 2008). Despite itswide use as a marker for autophagy, the exact role that LC3plays in the process is largely unknown.The appearance of LC3 as a highly specific marker of autop-

hagy has been recently challenged. In 2007, Green and cowor-kers found that phagosomes in which Toll-like receptor 2(TLR2) signaling was activated recruited GFP-tagged LC3expressed in macrophages (Sanjuan et al. 2007). Plastic beadscoated with a TLR2 ligand led to recruitment of LC3, whilebeads without a TLR ligand did not. By electron microscopy,the investigators confirmed that the organelle LC3 was recruitedto be a traditional phagosome enclosed in a single membrane,not the double-membrane compartment formed during autop-hagy. Even though LC3 is recruited to a compartment that is notan autophagosome, the recruitment nevertheless requires add-itional signaling components utilized in autophagy. The investi-gators noted that LC3 recruitment to these phagosomes requiredAtg5 and Atg7 using siRNA knockdown and genetically defi-cient macrophages. They suggested that the process was import-ant for phagosome maturation, since LAMP1 recruitment wasdelayed in cells lacking Atg5, and killing of live yeast wasimpaired in these cells. These findings suggest that we shouldbegin to think about LC3 (and perhaps some other componentsof the traditional autophagy pathway) as potential regulators ofimmunologically important processes beyond autophagy.We, therefore, investigated whether recognition of β-glucan

by Dectin-1 could trigger formation of phagosomes that specif-ically recruit LC3. We found that phagosomes containing yeastor β-glucan particles specifically and rapidly trigger conversionof LC3I into LC3II and accumulation of the protein on phago-somes (Ma et al. 2012) (Figure 1B). We further demonstratedby directly cross-linking the receptor that Dectin-1 signaling isentirely sufficient to trigger LC3-associated phagocytosis.In cells lacking Dectin-1, recruitment of LC3 to β-glucan-containing phagosomes was completely abrogated, suggestingthat Dectin-1 is the only receptor directing this response. Incontrast, we found that conversion of LC3I into LC3II was par-tially deficient in Dectin-1-deficient cells exposed to C. albi-cans yeast, suggesting that while Dectin-1 is a key receptor forthis response, others may also participate. As discussed above,TLRs are likely candidates.In 2009, Brummel and coworkers observed that Fcγ recep-

tors can trigger LC3 recruitment to phagosomes containingIgG-opsonized beads (Huang et al. 2009). Using inhibitors

and NADPH oxidase-deficient cells, they demonstrated that re-active oxygen production by the NADPH oxidase is requiredfor LC3’s recruitment to Fcγ receptor-triggered phagosomes.Since Dectin-1 activates the NADPH oxidase in a manner verysimilar to Fcγ receptors, we examined whether reactive oxygenproduction is similarly required for β-glucan-induced LC3 re-cruitment. β-glucan-induced LC3 recruitment was completelyblocked by inhibitors of the NADPH oxidase as well as in cellsgenetically deficient in the enzyme. Furthermore, we and othershave previously shown that Syk is required for Dectin-1 to acti-vate reactive oxygen production, so we examined whether thekinase is also required for Dectin-1-triggered LC3 recruitment.Inhibition or knockout of Syk blocked LC3 recruitment.While these data demonstrate that signaling through Sykand activation of reactive oxygen production are necessary forLC3-recruitment to Fcγ receptor and Dectin-1 phagosomes, theprecise mechanism by which reactive oxygen promotes LC3IIformation and recruitment has not yet been understood.Further, TLR signaling does not involve Syk and is generallynot associated with NADPH oxidase activation in most situa-tions, suggesting that the mechanism of TLR-induced LC3association might be different.In trying to understand the functional significance of LC3

recruitment to phagosomes, investigators have typically reliedon the use of inhibitors and genetic mutants affecting upstreamcomponents of the autophagy signaling pathway includingmammalian target of rapamycin, ATG5, ATG7 and ATG12(Sanjuan et al. 2007; Lee et al. 2010). Interpretation of suchstudies with respect to the specific role of LC3 recruitment tophagosomes can be complicated by the fact that these proteinshave been implicated in regulating diverse processes in additionto LC3II formation including roles in regulating apoptosis,metabolic regulation and nonfungal immunity (Levine et al.2011). We, therefore, made use of cells specifically deficient inLC3 in order to understand the significance of LC3 recruitmentto β-glucan-containing phagosomes.Two isoforms of LC3 have been identified in mice, LC3α

and LC3β. In macrophages and dendritic cells, LC3β appears tobe the only isoform expressed (Ma et al. 2012). Mice lackingthe gene for LC3β (Map1lc3b) have been made, and unlikemany other autophagy gene knockouts that are lethal, theanimals are healthy (Cann et al. 2008). Using fungi expressingthe model antigen ovalbumin, we showed that dendritic cellslacking LC3β are significantly less efficient at processing andpresenting fungal-derived antigens on major histocompatibilitycomplex class II (MHCII) compared with wild-type cells.Phagosomes in cells lacking LC3β demonstrated reducedability to accumulate MHCII molecules, suggesting that LC3might be important in directing phagosome association withMHCII-containing vacuoles (Figure 1B). LC3β did not appearto be important in regulating cross presentation on MHCI.These data are consistent with the above-discussed reports

suggesting that LC3 recruitment to phagosomes might regulatephagosome maturation. Other studies have also associated LC3recruitment with killing of ingested microbes and with clear-ance of apoptotic and necrotic cells, although these analyseshave been done using inhibition or genetic deletion of upstreamautophagy regulators which may participate in processes inde-pendent of LC3 (Sanjuan et al. 2007; Martinez et al. 2011).

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Further studies will be required to elucidate the specific role ofLC3 in these processes.

Future directions

In addition to triggering phagocytosis, respiratory burst anddirecting inflammatory responses that are essential for effectivehost defense against pathogenic fungi, recognition of β-glucan-containing particles by Dectin-1 directs LC3 recruitment to pha-gosomes. As discussed above, this process influences how fungiare handled by immune cells. We hypothesize that other innateimmune receptors mediating recognition of other microbialsugars (e.g., mannans or chitin) are similarly poised to activateLC3 recruitment to phagosomes. Dectin-2, a pattern recognitionreceptor implicated in sensing fungal mannans, is expressed at theplasma membrane associated with the Fc receptor common γchain and signals in a manner that is highly similar to Dectin-1(Sato et al. 2006). Thus, it would seem logical to anticipate thatthis receptor might also be able to regulate LC3 recruitment tophagosomes, even though unlike Dectin-1, Dectin-2 has not beendemonstrated to be phagocytic. As illustrated by data suggestingthat TLR signaling can trigger LC3 recruitment to phagosomes, itis not clear that a receptor must be “phagocytic” in order topromote LC3 association. Further studies will be required to de-termine which microbial sugars and which receptors are able tomodulate LC3 recruitment to phagosomes.Recent studies have started to elucidate the signaling

pathway which activates LC3 recruitment to phagosomes. Asnoted above, many upstream components of the autophagy sig-naling pathway including ATG5, ATG7 and ATG12 are alsorequired for LC3 recruitment during LC3-associated phagocyt-osis. However, a couple of recent studies have demonstratedthat components further upstream in the autophagy signalingpathway including ULK1, FIP200 and ATG13 are not required(Martinez et al. 2011; Henault et al. 2012). The data suggestthat there are specific signals upstream of ATG5, ATG7 andATG12 leading to LC3-associated phagocytosis that are distinctfrom the more widely studied pathway for autophagy. Futureinvestigations are necessary to fully elucidate how microbialsugar recognition by receptors such as Dectin-1 drivesLC3-associated phagocytosis and to define the role of thisprocess in orchestrating effective immunity.

Funding

This effort was supported in part by a grant from the NationalInstitutes of Health (R01 AI071116).

Conflict of interest

None declared.

Abbreviations

ITAM, immunoreceptor tyrosine-based activation motif; LC3,light chain 3; NFAT, nuclear factor of activated T cells.

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