Until recently, it was assumed that Mu
was the only Mycobacterium
species producing mycolactone. In fact, other mycolactone-producing mycobacteria have been isolated from fish and frogs presenting ulcerative diseases (14
). All these species contain versions of the Mu
virulence plasmid, produce mycolactones, and show genetic relatedness to Mu
and M. marinum
, a Mycobacterium
species causing localized skin granulomas in humans and an acute lethal pathology in fish (18
). On the basis of a genetic analysis, Yip et al. (18
) suggested that mycolactone-producing mycobacteria may have evolved from a common M. marinum
progenitor to form two ecotypes causing disease in either ectotherms or endotherms, such as humans. The expression of mycolactone-like compounds nevertheless remains restricted to these particular species, and no such complex polyketides have thus far been isolated from other pathogenic mycobacteria.
Polyketides are a diverse class of secondary metabolites produced by polyketide synthases (PKSs). Mycobacteria express a remarkable arrays of PKSs, which generate multiple cell wall–associated lipids (19
). At the interface with the host immune system, some of these envelope components have been shown to be important virulence factors and immune modulators. For example, in M. tuberculosis
, glycosylation of the PKS products phenol phtiodiolones leads to the generation of phenolic glycolipids, which are potent inhibitors of proinflammatory cytokine release by monocyte-derived macrophages (20
). With respect to mycolactone, the genetic basis of its biosynthesis has been recently elucidated with the isolation of a 174-kb plasmid encoding three modular PKSs (21
). Interestingly, mycolactone PKSs present highly unusual features in terms of size and structure, and mycolactone is the first example of a complex polyketide isolated from pathogenic mycobacteria. In fact, the macrocyclic polyketide structure of mycolactone shares similarities with that of complex polyketides typically produced by filamentous soil actinomycetes such as Streptomyces
. Strikingly, analysis of the chromosomal sequence reveals that numerous PKS genes are inactivated in Mu
, suggesting that energy and PKS-specific substrates are preferentially allocated to mycolactone biosynthesis in this species (22
). In the present study, we demonstrate that this original polyketide has the unique capacity to modulate DC functions in a selective manner.
The ulcerative properties of Mu
have been attributed to the cytopathic action of mycolactone, which is able to cause growth arrest in cultured fibroblasts, as well as cell death via apoptosis in fibroblast and macrophage cell models after 3–5 d of exposure (23
). Cell susceptibility to mycolactone activity nevertheless varies with the cell type. For example, Mu
lipid extracts showed no cytostatic activity on Jurkat T cells, whereas they markedly suppressed the cell-cycle progression and viability of a mouse pre–B cell line (24
). In the present study, we found that the susceptibility of DCs to mycolactone cytotoxicity was higher during the immature state. Collectively, these observations suggest that the molecular target of mycolactone is ubiquitous in eukaryotic cells but may be differentially expressed, depending on the cell type or the cell activation state.
Although the mechanism by which mycolactone acts as a cytotoxic agent is intriguing, we have focused on the immunosuppressive effects of nontoxic doses. We found that mycolactone strongly affects the maturation of both mouse and human DCs. In particular, we show a failure to up-regulate the phenotypic markers of the mature DCs CD83 and CD25, and to a lower extent CD80 and CD40. This maturation defect was not reversed by removal of mycolactone from cell-culture medium, suggesting that mycolactone durably affects DC maturation. In addition, we demonstrate that mycolactone-treated DCs show a reduced allostimulatory capacity and that they are inhibited in their ability to cross-present antigens to T cells (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20070234/DC1
). That said, other features of maturing DCs remained intact. In particular, phagocytic activity of DCs, as measured by the quantity of intracellular fluorescent beads after 4 h, was not reduced by coincubation of the cells with 10–100 ng/ml mycolactone (unpublished data). The fact that mycolactone limits the migratory properties of DCs, as well as their ability to mature and activate antigen-specific T cells in vivo (Fig. S3), thus further suggests that mycolactone suppresses the capacity of DCs to prime cellular immune responses. These findings may help explain the global defect in IFN-γ production by T cells in BU patients and the fact that once BU lesions are excised, there is a restoration of Th1 cellular responses (12
We also explored the effect of mycolactone on the ability of DCs to secrete proinflammatory molecules. Surprisingly, we observed a selective effect on inducible chemokines. In particular, mycolactone blocked the production of the monocyte and lymphocyte chemoattractants MIP-1α, MIP-1β, IP-10, RANTES, and MCP-1. MIP-1α, MIP-1β, and RANTES are ligands for CC chemokine receptor 5, which is specifically expressed by iDCs and Th1 T cells (25
). Defective production of these inflammatory chemokines is therefore likely to interfere with both the innate and adaptive immune responses to Mu
infection. Specifically, decreased β-chemokine production by mycolactone-exposed DCs may limit the recruitment and activation of iDC precursors to the inflammation site. In addition, defective production of these CC chemokine receptor 5 ligands, as well as the CXC chemokine receptor 3 ligand IP-10, is likely to interfere with the homing capacity of IFN-γ–secreting T cells to infected tissues. This hypothesis is strongly supported by the observation that, in contrast to DCs infected with mup045
, DCs infected with the WT strain failed to produce the chemokines MIP-1α, MIP-1β, and RANTES. In accordance with the histopathological features of BUs, bacterial production of mycolactone may therefore be responsible for suppressing the innate immune responses of iDCs, thus preventing the trafficking of inflammatory cells to the ulcerative lesion.
The exogenous addition of mycolactone to mup045-infected DCs resulted in a strong reduction of their β-chemokine response to infection, further demonstrating the critical importance of the toxin in this process. Interestingly, the inhibition of β-chemokine production by exogenous mycolactone in mup045-infected DCs was incomplete, particularly in the case of MIP-1β, suggesting that mycolactone may regulate the expression of other immunosuppressive agents.
Suppression of these inflammatory chemokines was nearly complete with minimal doses of mycolactone, whereas the expression of the inflammatory cytokines TNF-α, IL-12, and IL-6 was only marginally modified. Accordingly, IL-12, IL-1β, and TNF-α mRNA were detected at considerable levels in lesions from BU patients and were not substantially modulated between the nodular and ulcerative stages of the disease (6
), suggesting that mycolactone has little impact on the level of expression of these cytokines in vivo. Th 1 cellular responses, as measured by IFN-γ production, are vigorous during the nodular stage of BU and become defective at later stages of the disease. From our results, we can propose that, in contrast to inflammatory cytokines, defective inflammatory chemokines may constitute a hallmark of early Mu
infection, as well as a sensitive indicator of BU progression from the nodular to the ulcerative stage.
Given the structural similarity between mycolactone and the immunosuppressive drugs FK506 and rapamycin (Fig. S1), it is interesting to examine their respective activities. FK506 and rapamycin are structural analogues binding the same intracellular receptor, FKBP12, even though the resulting complex targets a different molecule. Although FK506 and rapamycin are used clinically to suppress activated T cells, recent data suggest that they also affect DC differentiation and function (27
). Interestingly, FK506 has been shown to modulate the production of β-chemokines (29
). However, FK506 blocks the production of inflammatory cytokines such as IL-12 and TNF-α (27
), as well as IL-8 (31
), all molecules that are not altered in mycolactone-treated DCs. Our results therefore suggest that mycolactone binds a different molecular target than FK506 or rapamycin. Further work will be required to map the precise mechanism underlying mycolactone immunosuppressive activity. In conclusion, we demonstrate in this study that mycolactone interferes with DC biology in a unique manner, which helps account for the pathologic features of BU. Moreover, we show that mycolactone inhibits DC maturation with a selective effect on the chemotactic signals critical for the initiation of an inflammatory response. This specificity of action defines a novel class of potentially useful immunosuppressive agents.