Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host–pathogen interaction

Structural features of PIMs, lipomannan and lipoarabinomannan

The majority of bacteria from suborder Corynebacterineae, including Corynebacterium diphtheriae, Corynebacterium glutamicum, pathogenic M. tuberculosis complex and nonpathogenic Mycobacterium smegmatis, possess the amphipathic lipoglycans, lipoarabinomannan and other related glycoconjugates, lipomannan and PIMs (Fig. 1). All Mycobacterium species possess two forms of acylated PIMs, tri- and tetra-acylated (Ac₁- and Ac₂-) phospho-myo-inositol-dimannoside (PIM₂) and tri- and tetra-acylated phospho-myo-inositol-hexamannoside (Ac₁/Ac₂PIM₆) (we have used Ac₁/Ac₂PIM_x for two different acylated versions of PIMs throughout the text, and PIM as a synonym where the acylation state is not clear), and different acylated versions of lipomannan and lipoarabinomannan (Khoo et al., 1995a), which are believed to be noncovalently attached to the cell membrane via a lipid anchor (Fig. 2) (Hunter & Brennan, 1990).

Biogenesis of PIMs, lipomannan and lipoarabinomannan

Accessibility of lipoglycans to the immune system: localization and trafficking

Lipoarabinomannan and related lipoglycans are not only essential for mycobacterial growth and cell viability (Haites et al., 2005; Kovacevic et al., 2006;), but are also thought to be important in the interactions between the mycobacteria and their host. The nature of these host–pathogen interactions is determined by the accessibility of the lipoglycans to the immune system, i.e. can cell wall-bound lipoglycans be recognized by the pattern-recognition receptors (PRRs) of the immune system and how do the lipoglycans traffic, once released from the mycobacterial cell wall?

The localization of PIMs and lipoarabinomannan in the mycobacterial cell envelope has been assessed in multiple ways, including biotin tagging of lipoarabinomannan and extraction of lipids from the cell wall with detergents or by mechanical treatment with glass beads. Because of the strong conditions needed to extract lipoarabinomannan from the cell wall, and the possibility to detect lipoarabinomannan with lipoarabinomannan-recognizing antibodies on whole cells, it was hypothesized that lipoarabinomannan is firmly attached via its MPI anchor to the surface of the cell (Chatterjee & Khoo, 1998). Biotin labeling, assumed to be restricted to the cell surface, showed two fractions of lipoarabinomannan: one anchored to the cytosolic membrane and one in the mycobacterial outer membrane (mycomembrane) (Hoffmann et al., 2008; Pitarque et al., 2008;). However, biotin is only a small molecule and may have easier access to lipoarabinomannan more buried in the cell wall as compared with the large PRRs, which may reduce the potential of lipoarabinomannan to be recognized by the immune system. Furthermore, native mycobacterial cells are surrounded by a capsule, which could cover lipoarabinomannan. The mycobacterial capsule mainly consists of polysaccharides and proteins (Daffé & Etienne, 1999). Electron microscopy (EM) with immunogold-labeled cells using ConA and anti-arabinan antibodies, combined with nuclear magnetic resonance studies, showed the presence of mannose-capped arabinomannan (Man-AM; i.e. without the lipid anchor present in Man-LAM) in the capsule (Ortalo-Magnéet al., 1995). The capsule has been reported to have only a very low lipid content, among which are small amounts of PIMs and virtually no lipoarabinomannan (Ortalo-Magnéet al., 1996a,b;). A recent study used immunogold-EM with monoclonal antibodies against PIM₆ (F183-24), and against the mannose cap (55.92.1A1) and the arabinan domain (F30-5) of Man-AM and Man-LAM to detect surface localization of these lipoglycans. Unperturbed mycobacterial cells bearing an intact capsule display good labeling with these antibodies (Sani et al., 2010), which confirms the presence of PIM₆ and Man-AM in the capsule. In contrast, mycobacteria without a surrounding capsule due to growth under perturbing conditions (in the presence of 0.05% Tween-80 and mechanical agitation) hardly become labeled with these antibodies. As lipoarabinomannan is localized in the cell wall and not in the capsule, this suggests limited surface exposure of lipoarabinomannan and related glycans buried in the mycomembrane, even if the capsule is not covering the cell wall. However, the amount of lipoarabinomannan or its accessibility in these cells grown under perturbing conditions has not been assessed further.

Although culture filtrate has been reported to contain only trace amounts of lipids (Lemassu & Daffé, 1994), studies with infected macrophages (Mϕ) showed intracellular trafficking of PIMs and lipoarabinomannan, suggesting that these glycolipids are substantially released from mycobacteria. Even release into noninfected bystander cells (Xu et al., 1994; Beatty et al., 2000; Rhoades et al., 2003;) and subsequent presentation through CD1 glycoproteins (Schaible et al., 2000) has been observed. Furthermore, isolated PIMs and lipoarabinomannan can be incorporated into the endomembranes and plasma membranes of different cell types, a process requiring the MPI anchor and the mannan core (Ilangumaran et al., 1995; Shabaana et al., 2005; Welin et al., 2008;). It can be hypothesized that the lipoglycans released are able to modulate the immune response, for example by interfering with phagosome maturation.

Phagosome maturation arrest

At least two strategies used by M. tuberculosis to survive in Mϕ have been described. One is delay of the phagosome maturation, i.e. prevention of fusion of the phagosome with late endosomal and lysosomal organelles, which normally leads to killing and digestion of a pathogen in an acid environment (Armstrong & Hart, 1971; Russell, 2001; Nguyen & Pieters, 2005;). The other strategy is based on escape from the phagosome to the cytosol (van der Wel et al., 2007). In the phagosome maturation arrest, a role for both Man-LAM and PIMs has been implicated (Vergne et al., 2003b; Welin et al., 2008;).

The phagosome–lysosome fusion process starts after cytosolic Ca²⁺ increase. The Ca²⁺/calmodulin-dependent PI3-kinase hVPS34 and its modulatory subunit p150 will then generate the membrane-trafficking lipid phosphatidylinositol 3-phosphate (PI3P) on the phagosomal membrane (Vergne et al., 2004a). Both PI3P and early endocytic small GTPase Rab5 mediate in the subsequent recruitment of membrane tethering protein early endosome autoantigen 1 (EEA1) to the phagosome (Vergne et al., 2003a) (Fig. 6). EEA1 plays an essential role in phagosome maturation by interacting directly with syntaxin-6, a soluble NSF attachment protein receptor (SNARE) protein involved in the delivery of cathepsins (lysosomal hydrolases) and V_oH⁺-ATPase from the trans-Golgi network to the phagosome (Simonsen et al., 1999). The normal cytosolic Ca²⁺ increase upon an infection is absent during mycobacterial uptake. This has been hypothesized to lead to a reduced activity of the PI3-kinase hVPS34 and an altered production of PI3P in the case of phagocytosis of mycobacteria (Malik et al., 2001; Chua & Deretic, 2004;) (Fig. 6).

This immune evasion strategy of blocking phagosome maturation can be mimicked by Man-LAM, which is also able to inhibit cytosolic Ca²⁺ increase (Fratti et al., 2003b; Vergne et al., 2003b;) (Fig. 6). In Mϕ infected with M. bovis BCG or Man-LAM-coated beads, EEA1 is excluded from the early endosome, thereby inhibiting phagosome maturation at the stage of recruitment of late endosomal and lysosomal constituents and, hence, preventing acidification (Fratti et al., 2001). The exact mechanism of [Ca²⁺]_cyt-modulation by Man-LAM is not known. For M. tuberculosis to infect Mϕ without the induction of a cytosolic Ca²⁺ increase, phagocytosis via the complement receptor is required (Malik et al., 2000), but the inhibition of phagosome maturation by Man-LAM appears to involve binding to the MMR instead (Kang et al., 2005). Furthermore, a role for macrophage phosphatase SHP-1 has been suggested, which is activated by Man-LAM and impairs Ca²⁺ signaling (Ono et al., 1997; Knutson et al., 1998; Vergne et al., 2004a;).

Another possibility of interference by Man-LAM in phagosome maturation, distinct from blocking the rise in [Ca²⁺]_cyt, considers a role for the activation of p38 mitogen-activated protein kinase (p38 MAPK). p38 MAPK activity may indirectly maintain Rab5 in an inactive GDP-bound form (Cavalli et al., 2001; Vergne et al., 2004a;). This is consistent with the report that the induction of p38 MAPK reduces the recruitment of Rab5-effector protein EEA1 to the early endosome (Fratti et al., 2003a). Moreover, the level of p38 MAPK activation is significantly increased upon infection with M. bovis BCG (Fratti et al., 2003a) and Man-LAM was hypothesized to be a triggering component (Vergne et al., 2004a). However, recently, it has been shown experimentally that p38 MAPK activation is neither induced nor influenced by isolated Man-LAM, and thus must be linked to other mycobacterial components (Welin et al., 2008).

Noteworthy, lipoarabinomannan is incorporated into membrane lipid rafts, a process that is also required for the phagosome maturation arrest (Welin et al., 2008). Lipid rafts are highly dynamic lipid domains, enriched in cholesterol and glycosphingolipids, and that have been associated with cell signaling (Simons & Toomre, 2000; Pike, 2009;). It has been suggested that the presence of lipoarabinomannan in the endomembrane causes drastic reorganization of the lipid domains and thereby fusion of the lipid vesicles (Hayakawa et al., 2007). However, PI-LAM from avirulent M. smegmatis is also incorporated into endomembranes, although to a lesser extent, but it cannot prevent phagosome–lysosome fusion nor inhibit cytosolic Ca²⁺ increase (Vergne et al., 2003b; Kang et al., 2005; Welin et al., 2008;). Given that the MMR only recognizes the mannose-capped Man-LAM and not Ara-LAM or PI-LAM (Schlesinger et al., 1994), this is further evidence that ligation of the Man-LAM to the MMR is required for the phagosome maturation block, which appears to be restricted to the more virulent Mycobacterium spp. A role for the MMR has been confirmed recently by showing that glycopeptidolipids from M. avium delay phagosome–lysosome fusion by interaction with the MMR and by an MMR siRNA knockdown in human monocyte-derived Mϕ, resulting in increased phagosome–lysosome fusion upon M. avium infection (Sweet et al., 2009).

As mammalian phosphoinositide PI3P plays an important role in phagosome maturation, next to lipoarabinomannan, other mycobacterial PI-analogs, PIMs and lipomannan, were investigated as well for their possible interference in phagosome maturation. While for lipomannan no role in phagosome maturation arrest could be detected (Kang et al., 2005), PIMs do have an effect, although in a way distinct from Man-LAM (Vergne et al., 2004b). Similar to lipoarabinomannan, PIMs can be incorporated into lipid rafts and, moreover, the addition of PIMs competitively inhibits lipoarabinomannan insertion (Ilangumaran et al., 1995; Welin et al., 2008;). Although PIMs seem to reverse the effect of lipoarabinomannan of preventing endosomal fusions (Welin et al., 2008), however, indications exist of a different role of PIMs in the phagosome maturation arrest. PIMs do prevent phagosome acidification, but not by reducing the recruitment of syntaxin-6 (Fratti et al., 2003b; Vergne et al., 2004b;). Instead, PIMs induce the acquisition of endosomal SNARE protein syntaxin-4 and the transferrin receptor (Fratti et al., 2003b; Vergne et al., 2004a,b;) (Fig. 6). Transferrin and its receptor are recycling endosomal markers involved in iron delivery (Clemens & Horwitz, 1996; Sturgill-Koszycki et al., 1996;). While Man-LAM arrests phagosome maturation by blocking the recruitment of late endosomal and lysosomal markers, PIMs appear to stimulate fusion with early endosomes and thereby retrieve nutrients necessary for mycobacteria residing in the phagosomal compartments (Kelley & Schorey, 2003; Vergne et al., 2004b;). This process is also Rab5-dependent (Gorvel et al., 1991), in particular when Rab5 activity is rate limiting, but whether PIMs affects Rab5 directly or indirectly is not yet known (Vergne et al., 2004b).

Interestingly, also in the effect that PIMs exert on the phagosome maturation, the MMR seems to play a role. While the lower-order PIMs (PIM₂) are not recognized by the MMR, the MMR has a high affinity for higher-order PIMs (PIM₅ and PIM₆) (Torrelles et al., 2006). This is consistent with the report that only in Mϕ stimulated with higher-order PIMs was a significant increase in phagosome-lysosome fusion seen upon MMR blockade (Torrelles et al., 2006). Thus, although PIMs and Man-LAM influence the phagosome maturation by distinct mechanisms, both may involve recognition by the MMR. This indicates a balance between Man-LAM preventing maturation into the phagolysosome on the one hand and PIMs stimulating early endosomal fusion to retrieve nutrients on the other (Vergne et al., 2004b).

Inhibition of phagosome maturation by pathogenic Mycobacterium spp. may be a critical first step for their intracellular survival. Mycobacteria probably display several mechanisms to prevent lysosomal transfer, of which one is interference of Man-LAM in the phagosome maturation process. A M. marinum mutant that only produces lipoarabinomannan devoid of mannose caps showed a significant increase in colocalization with phagolysosomes in murine Mϕ as compared with its parent strain. However, the absolute numbers remained low in this assay (7.4%, 12.0% and 5.0% for the wild-type, mutant and complemented strain, respectively), and importantly, no significant differences in bacterial survival were observed (Appelmelk et al., 2008). Other mechanisms of phagosome maturation blockade, independent of Man-LAM, have been reported, for example the secretion of SapM by M. tuberculosis, a lipid phosphatase that hydrolyzes the PI3P on the endomembranes (Vergne et al., 2005), and the secretion of a eukaryotic-like serine/threonine protein kinase G (PknG) (Walburger et al., 2004).

Clusters of differentiation (CD)1

CD-1 glycoproteins have been identified as important antigen-presenting molecules of the immune system, next to major histocompatibility complex (MHC) class I and II molecules. While MHC class I and II present peptide antigens, CD1 molecules present glycolipids, thereby covering the presentation of a large variety of both self as well as microbial antigens (Young & Moody, 2006; de Libero & Mori, 2009;). In mycobacterial infection, CD1 ensures the presentation of the glycolipids unique to the mycobacterial cell wall to activate CD1-restricted T cells and is thereby involved in shaping the immune response (Porcelli et al., 1998; Sieling et al., 1999; Barral & Brenner, 2007;).

Human CD1 molecules are expressed by a variety of antigen-presenting cells (APC) and can be divided into three groups: CD1a, CD1b and CD1c together form group 1, and CD1d and CD1e form group 2 and group 3, respectively. Murine homologs for group 1 CD1 molecules have not been identified, but mice do express CD1d. Group 1 CD1 molecules present lipids to a clonally diverse T-cell population in which the precursors have unique specificity for a single antigen (Barral & Brenner, 2007). The expression of group 1 CD1 on isolated human myeloid APC is hardly detectable, but it is upregulated to high levels within a couple of days after infection with M. tuberculosis or activation by mycobacterial lipids (Roura-Mir et al., 2005) (Felio et al., 2009). This demonstrates an apparent role of antigen presentation by the group 1 CD1 in the clonal expansion of T cells and, hence, the adaptive immune response against mycobacterial infection (Roura-Mir et al., 2005; Barral & Brenner, 2007;). CD1d presents lipids to CD1d-restricted natural killers T (NKT) cells including the subset of clonally less diverse invariant NKT cells that display a rapid innate-like response (Barral & Brenner, 2007). In contrast to group 1 CD1, CD1d molecules are constitutively expressed and are reported to be downregulated during mycobacterial infection, confirming their association with the innate immune response (Roura-Mir et al., 2005; Moody, 2006;). CD1e is only restricted to myeloid dendritic cells (DCs) and is not expressed at the cell surface and thus does not present antigens to TCRs. In this review, we focus on CD1b and CD1d, because these CD1 molecules bind and present PIMs and related lipoglycans. Group 2 CD1d has been reported to only bind lower-order PIMs, PIM₂ and PIM₄, but not lipomannan or Man-LAM (Fischer et al., 2004; Zajonc et al., 2006;). In contrast, group 1 CD1b binds several mycobacterial lipid including PIM₂ and Man-LAM (Sieling et al., 1995; Prigozy et al., 1997; Ernst et al., 1998;).

The structure of CD1 molecules has similarities to the MHC class I molecules, but shows some important differences in its binding groove, which is deeper and facilitates the binding of two acyl chains as present in the MPI anchor of PIMs and Man-LAM (Zeng et al., 1997; Porcelli et al., 1998; Fischer et al., 2004;). While the lipid anchoring in the hydrophobic CD1 groove is relatively nonspecific, the TCR recognizes the hydrophilic carbohydrate head group of the antigens with high specificity (Moody et al., 1997). As compared with CD1b, additional interactions between the center of the binding groove of CD1d and the polar head group of the PIM₂ are of additive importance for the formation of a stable glycolipid complex and subsequent T cell recognition (Zajonc et al., 2006). In the presentation of PIM₄ by CD1d, the two additional α(1→6)-linked Manp residues are probably orientated away from the binding groove (Zajonc et al., 2006). Considering the low abundance of PIM₄ in the mycobacterial cell wall in contrast to (diacylated) PIM₂ (Gilleron et al., 2001), presentation of PIM₄ by CD1d may not be of high biological significance. For group 1 CD1b, mycobacterial antigens with head groups much larger than present in PIM₂ have been described, which raises questions regarding how these large carbohydrates fit in the narrow space between the TCR and CD1 (Young & Moody, 2006). Higher-order PIM₆ needs processing into the smaller PIM₂ before being able to stimulate CD1b-restricted T cells. A role in this antigen processing has been implicated for CD1e, because the presence of CD1e is required for the activation of CD1b-restricted T cells by PIM₆, and not by PIM₂ (de la Salle et al., 2005). As mentioned above, CD1e does not present lipid antigens at the cell surface, but probably aids in endosomal/lysosomal α-mannosidase activity to produce PIM₂ by binding PIM₆ similar to the other antigen-presenting CD1 molecules (de la Salle et al., 2005). Secondly, CD1e may facilitate the loading of other CD1 molecules (de Libero & Mori, 2009). How Man-LAM is presented in the interaction between the TCR and the CD1b-Man-LAM complex has not yet been resolved. Man-LAM may be partly digested similar to PIM₆ (Ernst et al., 1998), which is most likely, as already PIM₆ with its short carbohydrate head group requires processing. Of note, CD1b and Man-LAM do colocalize in the cell (Prigozy et al., 1997) and CD1b is able to bind Man-LAM (Ernst et al., 1998). Two other options have been suggested by Young & Moody (2006). One possibility is flattening of the glycan part of Man-LAM between the TCR and CD1, so that only one or two carbohydrate units are positioned directly between the TCR and CD1. Multiple TCR-docking orientations may play a role in this as well. In the second option, Man-LAM is not presented by CD1b, but stimulates the process of CD1-dependent T cell activation indirectly via the mechanisms discussed below (Young & Moody, 2006).

CD1b is the predominant group 1 CD1 molecule present in the late endosomes/lysosomes and MHC class II compartments (Prigozy et al., 1997; Ernst et al., 1998; Schaible et al., 2000;) and shares with MHC class II molecules the requirement for acidification in order to function (Benaroch et al., 1995; Sugita et al., 1999;). PIMs and Man-LAM have been observed to be released in the phagosomes of infected cells and transported into the same intracellular compartments (Xu et al., 1994; Prigozy et al., 1997; Schaible et al., 2000;). The low pH in these compartments causes conformational changes in the structure of CD1b including relaxation of certain parts of its binding groove to facilitate subsequent antigen loading (Ernst et al., 1998; Sugita et al., 1999; Kronenberg & Sullivan, 2008; Relloso et al., 2008; de Libero & Mori, 2009;). As described above, PIMs and Man-LAM interfere in phagosome maturation and in particular Man-LAM has been shown to prevent endosomal acidification (Fratti et al., 2001). Hence, PIMs and Man-LAM likely impede their own presentation by CD1b. On the other hand, mycobacterial lipids have been shown to induce the transcription and expression of group 1 CD1 glycoproteins at the surface of the APC by signaling through TLR-2 (Roura-Mir et al., 2005; Moody, 2006;). Possible lipids involved were reported to be PIM₂ and Ara-LAM extracted from the mycobacterial cell wall (Roura-Mir et al., 2005). However, PIM₂ and Ara-LAM are poor TLR2 ligands as discussed in the next section (Nigou et al., 2008). Copurified lipopeptides, which are more potent inducers of TLR2 signaling, may also have induced CD1 expression in this assay (Nigou et al., 2008; Zahringer et al., 2008;).

Mycobacteria are able to interfere with the immune response against mycobacterial infection including the modulation of peptide antigen presentation by MHC class I and II molecules (Kaufmann & Schaible, 2005). Therefore, the lipid antigen presentation via four different CD1 glycoproteins forms an important alternative mechanism to induce an effective immune response (Sugita et al., 1999). Although the function and expression of CD1 molecules can be impaired by mycobacteria or mycobacterial components such as capsular α-glucan (Gagliardi et al., 2007, 2009; Balboa et al., 2010), the many distinct pathways for antigen sampling from various intracellular localizations and their subsequent presentation circumvents the immune evasion strategies exploited by mycobacteria (Sugita et al., 1999; Kaufmann & Schaible, 2005; de Libero & Mori, 2009;). Furthermore, both group 1 and group 2 CD1 presentation of lipid antigensseem to play a potential role in the protection against tuberculosis by vaccination with BCG (Watanabe et al., 2006; Venkataswamy et al., 2009;).

TLRs

Three TLRs have been implicated to play a role in the mycobacterial infection: TLR2, TLR4 and TLR9 (Ozinsky et al., 2000; Quesniaux et al., 2004a; Jo, 2008;). PIMs, lipomannan and lipoarabinomannan have all been examined for signaling via TLR2 and via TLR4, of which an overview is given here.

Lipoproteins are the major ligands for TLR2 (Brightbill et al., 1999), but MPI-anchored mannosylated lipoglycans can signal via TLR2 as well, depending on their degree of acylation and mannosylation (Gilleron et al., 2006; Doz et al., 2007; Nigou et al., 2008;). TLR2 dimerizes with either TLR1 or TLR6 in order to function (Ozinsky et al., 2000). TLR1/TLR2 heterodimers mainly recognize triacylated lipoproteins, while the diacylated forms bind TLR2/TLR6 (Takeuchi et al., 2002; Akira & Takeda, 2004;). Lipoglycan-induced signaling occurs via the TLR1/TLR2 complex (Elass et al., 2005; Gilleron et al., 2006; Nigou et al., 2008;). A positive relation exists between the length of the mannan chain and the ability of the lipoglycan to activate TLR2 (Nigou et al., 2008). The lipoglycan bearing the largest accessible mannan chain (i.e. not substituted with an arabinan domain) – lipomannan – showed to be a potent inducer of TLR2-signaling (Quesniaux et al., 2004b), although this activity is restricted to the tri- and tetra-acylated forms (Ac₁/Ac₂LM) (Gilleron et al., 2006; Doz et al., 2007;). Next to the induction of cytokines, Mϕ stimulated with lipomannan displayed increased production of matrix metalloproteinase (MMP)-9 (Elass et al., 2005). This was due to the downregulation of the transcription of the MMP-9 inhibitor, tissue inhibitor of metalloproteinases-1, and dependent on TLR2. This implies a role for lipomannan in tissue destruction by MMP-9 during mycobacterial infection via interaction with TLR2 (Elass et al., 2005). Furthermore, lipomannan induces granuloma macrophage fusion in an in vitro granuloma model in a TLR2-dependent way (Puissegur et al., 2007).

In the group of PIMs, both Ac₁/Ac₂PIM₂ and Ac₁/Ac₂PIM₆ have been reported to signal via TLR2, irrespective of their acylation pattern (Jones et al., 2001; Gilleron et al., 2003;). Further, in two studies, cellular activation via TLR2 by non-mannose-capped lipoarabinomannan (PI-LAM/Ara-LAM) from rapidly growing species has been observed (Means et al., 1999; Underhill et al., 1999;), but not for M. tuberculosis or BCG-derived Man-LAM. In addition, an inflammatory response induced by PI-LAM from M. smegmatis in mice appeared to be TLR2 dependent (Wieland et al., 2004). However, in a comparative study of all lipoglycans, Ac₁/Ac₂PIM₂, PI-LAM and Ara-LAM were shown to be poor inducers of TLR2 signaling as compared with lipomannan and Ac₁/Ac₂PIM₆ (Nigou et al., 2008). This is consistent with an earlier study showing that in contrast to lipomannan, neither Ara-LAM from M. chelonae nor Man-LAM and Ac₁/Ac₂PIM₂ from Mycobacterium kansasii mediate TLR2-dependent activation (Vignal et al., 2003). Moreover, chemical degradation of the arabinan domain of Man-LAM from M. kansasii restored its ability to induce cytokine secretion via TLR2, which suggests that the arabinan domain prevents the proper interaction of Man-LAM with TLR2 (Vignal et al., 2003). This was confirmed by a recent study by Birch et al. (2010) in which lipoarabinomannan containing a truncated arabinan domain from an M. smegmatis AftC knockout mutant showed enhanced TLR2 signaling as compared with wild-type lipoarabinomannan. The positive effects of lipoarabinomannan in the earlier reports could be due to contamination of the lipoarabinomannan extract with lipopeptides (Nigou et al., 2008; Zahringer et al., 2008; Geurtsen et al., 2009; Birch et al., 2010;). Overall, the data indicate that lipomannan, and in a minor respect PIM₆, are the only significant TLR2 ligands from this group of mycobacterial lipoglycans.

For TLR4, only a few mycobacterial lipoglycans have been reported as ligands. Using nonactivated Mϕ, only tri- and tetra-acylated lipomannan from M. tuberculosis and M. bovis BCG, respectively, show signaling via TLR4 (Gilleron et al., 2006; Doz et al., 2007;). In contrast to TLR2, which can only signal by the recruitment of adaptor proteins, Myeloid differentiation primary response gene (MyD)-88 and Toll-interleukin 1 receptor (TIR) domain-containing adapter protein (TIRAP), TLR4 can also signal via TIR-domain-containing adapter-inducing interferon-β and translocating chain-associating membrane protein (Akira & Takeda, 2004; Jo, 2008;). The secretion of proinflammatory cytokines, by Mϕ stimulated with lipomannan, is strongly dependent on the MyD88/TIRAP pathway (Doz et al., 2007). Most probably, cell wall-associated or soluble factors other than lipoglycans [e.g. heat shock proteins (Bulut et al., 2005)] stimulate TLR4 signaling in M. tuberculosis infection; while both live and ‘heat-killed’M. tuberculosis activate cells via TLR2, only live M. tuberculosis induces TLR4-dependent activation excluding a major role for the heat-stable glycolipids (Means et al., 1999). In Table 2, an overview is given of all TLR signaling observed for lipomannan using TLR1/2/4/6 knockout cells or antibodies against specific TLRs.

A few studies report on an immunosuppressive effect of lipomannan, Ac₁LM and PIMs on lipopolysaccharide-activated Mϕ (Quesniaux et al., 2004b; Doz et al., 2007, 2009). These inhibitory effects were independent of their signaling via TLR2, which partially compensated the decrease of proinflammatory cytokine secretion (Quesniaux et al., 2004b; Doz et al., 2007;). Which receptor-lipoglycan ligation is then responsible for the immunosupression is not yet known, but it is unlikely to be the MMR (Doz et al., 2007): in contrast to the higher-order PIMs, the MMR recognizes lipomannan and Ac₁/Ac₂PIM₂ only with low affinity (Torrelles et al., 2006), while they show similar inhibitory effects on lipopolysaccharide-activated Mϕ (Doz et al., 2009). Importantly, neither lyso-PIM (monoacylated PIM) nor PI were inhibitory in this assay, indicating the requirement of mannosyl moiety and a diacylated MPI anchor (Doz et al., 2009). Of note, in all these experiments, lipopolysaccharide was used, which is a major ligand for TLR4. PIMs might interfere in the interaction between lipopolysaccharide and TLR4, thereby causing its inhibitory effect, but this seems unlikely (Doz et al., 2009). However, lipopolysaccharide is a nonmycobacterial ligand, which makes this assay system rather artificial for the comprehension of TLR-dependent immunomodulation by PIMs and related lipoglycans during mycobacterial infection. Finally, the immunomodulatory properties of Man-LAM on both lipopolysaccharide-activated Mϕ and DCs have been described as well (Geijtenbeek et al., 2003; Pathak et al., 2005;). These effects of Man-LAM can be attributed to its binding by the MMR and DC-SIGN, respectively, which will be discussed in the next sections.

In vivo studies using TLR knockout mice are not fully in agreement on whether TLRs are crucial for mycobacterial clearance and host defense (Drennan et al., 2004; Ferwerda et al., 2005;) or not (Sugawara et al., 2003a,b; Nicolle et al., 2004; Holscher et al., 2008;). Importantly, TLR signaling via the MyD88 pathway has been shown in multiple studies to be dispensable for the induction of an efficient adaptive immune response (Fremond et al., 2004; Nicolle et al., 2004; Ryffel et al., 2005; Holscher et al., 2008;). A critical role for TLRs then has been hypothesized in the early recognition and killing of mycobacteria (Quesniaux et al., 2004a; Ryffel et al., 2005; Jo, 2008;). However, this innate immune response mainly depends on MyD88 and may also involve a MyD88-mediated pathway different from via TLR (Feng et al., 2003; Holscher et al., 2008;), which is the type I interleukin-1 receptor (IL-1R1)-mediated signaling (Fremond et al., 2007; Reiling et al., 2008;). Alternatively, TLR2 activation may have a second function as a regulator by preventing an exaggerated inflammatory immune response in a later stage of mycobacterial infection (Drennan et al., 2004; Sutmuller et al., 2006; Jo, 2008; Harding & Boom, 2010;). Pattern recognition by multiple TLRs does contribute to the control of mycobacterial infection (Bafica et al., 2005; Ryffel et al., 2005; Jo et al., 2007;), but may play a less significant role than originally assigned (Reiling et al., 2008).

DC-SIGN

In contrast to the interaction between mycobacteria and Mϕ, which can be mediated by several different receptors (e.g. complement receptors, CD14, MMR, scavenger receptor) (Ernst, 1998), binding of mycobacteria to DCs is for the largest part mediated by DC-SIGN (CD209) (Tailleux et al., 2003). DC-SIGN is a type II transmembrane tetrameric C-type lectin containing one carbohydrate recognition domain per monomer. It was originally hypothesized that via interaction with DC-SIGN, mycobacteria modulate the immune response and thereby escape immune surveillance (Geijtenbeek et al., 2003; van Kooyk & Geijtenbeek, 2003;). However, the exact role of DC-SIGN in tuberculosis infection has not yet been clarified and is still under debate (Neyrolles et al., 2006; Ehlers, 2009; Tanne & Neyrolles, 2010;). Studies on the relation between the level of expression of DC-SIGN in human populations and susceptibility to tuberculosis are contradicting each other, varying from a protective effect to increased susceptibility to tuberculosis by high DC-SIGN expression or no correlation at all (Barreiro et al., 2006; Gomez et al., 2006; Ben-Ali et al., 2007; Vannberg et al., 2008;).

In a binding study using a DC-SIGN-expressing cell line, the lectin showed a high preference for the Mycobacterium spp. from the tuberculosis complex, while it bound little or not to strains from outside the complex (Pitarque et al., 2005). On the basis of differences in the mannose-capping degree of lipoarabinomannan between these species and the fact that DC-SIGN only recognizes lipoarabinomannan if it is mannose-capped, it was expected that Man-LAM would establish the interaction between mycobacteria and DC-SIGN as present on DCs (Geijtenbeek et al., 2003; Maeda et al., 2003;). Furthermore, DC-SIGN has a high-affinity α(1→2)-linked Manp residue, which increases with chain length (Koppel et al., 2004). The predominant mannose cap on Man-LAM of M. tuberculosis and M. bovis BCG consists of α(1→2)-linked dimannosides, and α(1→2)-linked trimannosides are also present (Pitarque et al., 2005). Surprisingly, an M. bovis BCG mutant, which only produces lipoarabinomannan without mannose cap, did not bind less to DC-SIGN and DCs as compared with wild-type BCG (Appelmelk et al., 2008); thus, ligands for DC-SIGN other than Man-LAM must be present in the cell wall.

Next to Man-LAM, PIMs have been examined for their binding to DC-SIGN as well (Torrelles et al., 2006; Driessen et al., 2009;). In particular, the higher-order PIMs (PIM₅ and PIM₆), with terminal α(1→2)-linked Manp residues reminiscent of the mannose cap of Man-LAM, were of interest. As expected, DC-SIGN recognizes the higher-order PIMs with high affinity as compared with the lower-order PIMs (Boonyarattanakalin et al., 2008; Driessen et al., 2009;). Besides PIMs, other potential ligands in the mycobacterial cell wall for DC-SIGN have been identified: lipomannan, Man-AM and mannosylated lipoproteins 19 and 45 kDa (Pitarque et al., 2005), and more recently, capsular α-glucan (Geurtsen et al., 2009) (a list is provided in Table 3). All these compounds have been shown to bind DC-SIGN and/or inhibit binding of M. tuberculosis to DC-SIGN. This suggests that binding of mycobacteria to DC-SIGN and DCs is not mediated by one dominant ligand, but that there is a redundancy in ligands for DC-SIGN present on the cell surface (Pitarque et al., 2005). This was supported by the observation that even a double knockout M. bovis BCG strain that neither produces the mannose cap on lipoarabinomannan nor higher-order PIMs did not show any reduction in binding to DC-SIGN and DCs as compared with its parent strain (Driessen et al., 2009). Moreover, this mutant strain did not induce an altered cytokine profile in DCs (Driessen et al., 2009). All of the reported ligands for DC-SIGN can be found in DC-SIGN nonbinding Mycobacterium spp. as well. Hence, other factors probably determine the binding to DC-SIGN instead of only the presence or the absence of one or a few ligands. The affinity of DC-SIGN for specific Mycobacterium spp. may be related to differences in the mannosylation pattern of potential ligands, the relative abundance of various compounds at the cell surface (in particular, α(1→2)-linked Manp-containing compounds), cell wall structure and/or capsule composition. Finally, not all potential ligands, when cell wall bound, may be accessible to DC-SIGN and thus of equal importance for the binding of mycobacteria to DC-SIGN.

To investigate the DC-SIGN-dependent modulation of the immune response by mycobacteria or their mannosylated lipoglycans, several in vitro and in vivo studies have been performed. Purified mycobacterial Man-LAM, not Ara-LAM, inhibits both lipopolysaccharide- as well as BCG-induced maturation of DCs by reducing the expression of MHC class II and costimulatory molecules (CD80, CD83 and CD86) as compared with untreated lipopolysaccharide- or BCG-activated cells (Geijtenbeek et al., 2003). Furthermore, Man-LAM has been shown to induce the secretion of anti-inflammatory IL-10 in lipopolysaccharide-activated DCs (Geijtenbeek et al., 2003). Ligation of Man-LAM to DC-SIGN appears to interfere with lipopolysaccharide signaling via TLR4 by the activation of serine-threonine kinase Raf-1, which subsequently leads to acetylation of NF-κB subunit p65. Acylation of p65 prolongs and enhances the transcription of IL10, thereby increasing IL-10 production (Gringhuis et al., 2007). Together with a study on mice lacking SIGN receptor (SIGNR)-1, a murine homolog for DC-SIGN, which displayed a stronger T helper 1 response upon M. tuberculosis infection and a reduced level of IL-10 production (Wieland et al., 2007), this led to the initial thought that the interaction with DC-SIGN is beneficial for mycobacteria.

Recently, it has been shown that Man-LAM alters the cytokine profile in lipopolysaccharide-activated DCs by increasing the secretion of not only IL-10, but also of proinflammatory IL-12 and IL-6 (Gringhuis et al., 2009). Binding of Man-LAM or mannose-containing pathogens like M. tuberculosis induces the recruitment of effector proteins to a DC-SIGN signalosome, which is required for the activation of Raf-1, followed by enhancement of the transcription of the genes encoding IL-12 and IL-6 in a similar manner as for IL-10 (Gringhuis et al., 2009). Noteworthy, lipopolysaccharide is a nonmycobacterial ligand and both Man-LAM (Geijtenbeek et al., 2003) as well as intact mycobacteria (Driessen et al., 2009) do not induce IL-10 secretion in nonactivated DCs. In the context of mycobacterial infection, further research on interference of DC-SIGN signaling induced by Man-LAM with signaling via TLR2 by for example BCG-Ac₁LM or the 19 kDa antigen would be of interest.

A study using mouse DCs showed a higher induction of suppressor of cytokine signaling-1 expression by SIGNR1 stimulation as compared with signaling via TLR2 (Srivastava et al., 2009). It is important to mention that seven murine homologs for human DC-SIGN are known. Each homolog differs in certain properties from the human DC-SIGN, among which are the specific mannose- and fucose-structures it recognizes, and this may alter their functions significantly (Powlesland et al., 2006; Tanne et al., 2009;). Strikingly, human DC-SIGN transgenic mice exhibited decreased pathology and prolonged survival during mycobacterial infection (Schaefer et al., 2008). A clear protective role has recently also been shown for the murine ortholog SIGNR3 (Tanne et al., 2009). Resistance to M. tuberculosis infection was impaired in SIGNR3-deficient mice, but not in mice lacking SIGNR1 and SIGNR5 (Tanne et al., 2009).

DC-SIGN specifically recognizes the pathogenic Mycobacterium spp. from the tuberculosis complex (Pitarque et al., 2005). By expressing many ligands for DC-SIGN, DC-SIGN is the most important uptake receptor for mycobacteria to infect DCs. However, concluding from these last studies, DC-SIGN-binding appeared not to be mainly beneficial for the pathogens, and DC-SIGN may function primarily in the protection of the host. How DC-SIGN signaling exactly optimizes the immune response against mycobacteria, i.e. by strengthening the proinflammatory response or by preventing excessive inflammation, remains to be established (Ehlers, 2009) (Fig. 7).

Mannose receptor

The MMR (CD206) is a type I transmembrane monomeric C-type lectin with eight carbohydrate recognition domains. In addition to the complement receptors, the MMR mediates for a large part in the phagocytic uptake of mycobacteria by Mϕ (Schlesinger, 1993). The MMR recognizes virulent M. tuberculosis strains Erdman and H37Rv, but not avirulent H37Ra, which has been suggested to be caused by subtle structural differences in Man-LAM (Schlesinger, 1993; Schlesinger et al., 1996;). Furthermore, the MMR also shows a low affinity for nontuberculosis mycobacteria M. smegmatis, M. phlei and M. kansassi, of which the first two species only bear lipoarabinomannan without a mannose cap (Astarie-Dequeker et al., 1999). Although the presence of a mannose cap on Man-LAM is essential for the recognition of lipoarabinomannan by the MMR (Schlesinger et al., 1994), it does not completely explain the differences in MMR-mediated uptake by Mϕ between the Mycobacterium spp. (Schlesinger et al., 1996; Astarie-Dequeker et al., 1999; Appelmelk et al., 2008;). Next to the recognition of the mannose cap on Man-LAM, the MMR furthers binds higher-order PIM₅ and PIM₆ with a preference of the triacylated species above the tetra-acylated ones, and glycopeptidolipids, but not to lower-order PIM₂, lipomannan and Ara-LAM or PI-LAM (Schlesinger et al., 1994; Villeneuve et al., 2005; Torrelles et al., 2006;). Because of their structural similarities, mannosylated proteins and (phosphorylated) arabinomannans and mannan may be involved as well (Ortalo-Magnéet al., 1995; Dobos et al., 1996; Maes et al., 2007; Torrelles et al., 2008a;). Man-LAM and related lipoglycans all contribute to the MMR-mediated phagocytosis of mycobacteria (Villeneuve et al., 2005; Torrelles et al., 2008b;). Overexpression of Mt-ManB in M. smegmatis results in the overproduction of PIMs, lipomannan and lipoarabinomannan. Subsequent increased association with Mϕ is probably due to the higher amount of higher-order PIMs, as lipoarabinomannan from M. smegmatis does not have a mannose cap (McCarthy et al., 2005).

A role for the MMR has been implicated in many of the immunomodulatory effects by Man-LAM and the related glycans described above. Next to uptake of mycobacteria by Mϕ via the MMR, the MMR is also involved in the transport of glycolipids to uninfected bystanders cells and intracellular trafficking to the late endosomal compartments (Prigozy et al., 1997). However, in DCs, uptake of both mycobacteria as well as of the glycolipids is performed mainly by DC-SIGN, leaving a minor role for the MMR (Schaible et al., 2000; Geijtenbeek et al., 2003; Driessen et al., 2009;). Binding of Man-LAM to the MMR has been further linked to the delay of phagosome maturation (described in more detail in the corresponding section above) (Kang et al., 2005), the induction of MMP-9 by M. tuberculosis and Man-LAM similar to that observed for TLR2 activation by lipomannan (Rivera-Marrero et al., 2002) and the expansion of regulatory T cells (Tregs) (Garg et al., 2008). Furthermore, DCs from MMR knockout mice secrete more IL-12p40 upon infection with M. tuberculosis as compared with DCs from wild-type mice (Ehlers, 2009). Thus, the MMR seems to have an immunosuppressive function on DCs, which was earlier shown by cross-linking the MMR on DCs with anti-MMR antibodies and several natural ligands (Chieppa et al., 2003). Finally, in Mϕ, Man-LAM suppresses lipopolysaccharide-induced IL-12p40 secretion via the induction of IL-1 receptor-associated kinase (IRAK)-M expression (Pathak et al., 2005). IRAK-M negatively regulates TLR signaling (Kobayashi et al., 2002; Jo et al., 2007;) and its induction is probably triggered by binding of Man-LAM to the mannose receptor (Pathak et al., 2005).

Concluding, the MMR signaling may induce an anti-inflammatory response. A study by Torrelles et al. (2008b) reported, for clinical isolates of M. tuberculosis (HN885 and HN1554) with both truncated Man-LAM and a reduced amount of higher-order PIMs, a significant decrease in association with the MMR as compared with the Erdman strain, while uptake via the CR was not altered. Phagocytosis via the MMR may shape the nature of the immune response, and it was suggested that due to its immunosuppressive activity, uptake via the MMR may lead to a latent infection instead of active disease (Torrelles et al., 2008b). Related to this, an M. tuberculosis pimB-mutant strain (Rv0557/mgtA), which expresses less lipomannan and Man-LAM in its cell wall, showed an increased rate of macrophage death upon infection as compared with its parent strain (Torrelles et al., 2009). This does not necessarily mean increased virulence, but may even be advantageous for the host as the more virulent strains have been reported to inhibit apoptosis (Torrelles et al., 2009). Although the pimB-mutant phenotype could not be linked to reduced association with the MMR, the presence of high amounts of PIMs, lipomannan and Man-LAM results in a less inflammatory immune response and indicates adaptation of the mycobacteria to their host (Torrelles et al., 2009; Torrelles & Schlesinger, 2010;) (Fig. 7).