DCs are the major antigen-presenting cells of the mammalian immune system. As fungal cell walls are rich in β-glucans, it is likely that the nature of the immune response will be influenced by the interaction between DCs and β-glucans. The data presented herein establish that while DCs recognize β-glucans, the cytokine response is selective in the sense that only some cytokines are stimulated. Most notably, while β-glucans stimulated strong TNF-α production, levels of IL-12p70 were undetectable. Both particulate and soluble β-glucans stimulated similar cytokine responses. This is important because cduring the course of a fungal infection, both in situ and shed β-glucans may be available to interact with Dectin-1 (41
Studies comparing responses in wild-type and Dectin-1−/−
mice established that Dectin-1 is the major β-glucan receptor on BMDCs. First, phagocytosis of YGPs by Dectin-1−/−
BMDCs was markedly reduced. Second, Dectin-1−/−
BMDCs secreted nearly undetectable amounts of TNF-α following stimulation with YGPs or SCG. Nevertheless, because Dectin-1−/−
BMDCs exhibited some laminarin-inhibitable phagocytosis and TNF-α release, the possibility that other β-glucan receptors may be involved cannot be excluded. As noted above, in addition to Dectin-1, β-glucans reportedly are recognized by CR3, lactosylceramide, scavenger receptors, and CD5 (12
). Moreover, stimulation of human CD5 with β-glucans was recently shown to induce mitogen-activated protein kinase activation and cytokine release (49
The fungal cell wall is complex, and there is much interspecies and intraspecies variation, including differences in the amounts of β-glucans that are surface exposed and thus available to interact with Dectin-1 (43
). Studies using whole fungi as stimulants, while biologically relevant, are nevertheless difficult to interpret because the contributions of the individual cellular components are not easily dissected. The widely used cell wall derivative of S. cerevisiae
, zymosan, contains multiple PAMPs, including β-glucans, mannans, and ligands for TLR2 and TLR4 (40
). This complexity greatly hinders the interpretation of experimental results using zymosan as a model β-glucan. In contrast, YGPs, while also derived from S. cerevisiae
, are considerably purer than zymosan. By biochemical analysis, YGPs are composed of >85% glucan polymers, ~2% chitin, and <1% lipids and protein, with the remainder primarily ash and moisture (23
). Stimulation of BMDCs with YGPs was dependent on Dectin-1 but independent of MyD88, TLR2 (data not shown), and the mannose receptor (data not shown).
It has been proposed that signals from TLR agonists are necessary for efficient initiation of a CD4+
T-cell response by DCs (4
). Our data suggest that the situation may be more complicated for fungi because β-glucans modulate the cytokine response to TLR agonists. Remarkably, depending upon the cytokine studied, additive, antagonistic, and indifferent effects were seen when the combinations of β-glucans and PAMPs were compared with the responses stimulated by individual PAMPs.
IL-12p70 is a heterodimer composed of p40 and p35 subunits. Both soluble and particulate sources of β-glucans (SCG and YGPs) downregulated BMDC-mediated secretion of IL-12p70 stimulated by the TLR agonists, Pam3
, LPS, imiquimod, and CpG. However, the β-glucans did not affect secretion of the IL-12p40 subunit. The possibility of the formation of inhibitory p40 homodimers cannot be excluded (20
). However, we speculate that the downregulatory effect of β-glucans on IL-12p70 secretion stimulated by TLR agonists is most likely due to reduced IL-12p35 production. While regulation of the IL-12p35 subunit is not well defined, it is secreted only as part of a heterodimer with IL-12p40 (47
). Recently, it was demonstrated that β-glucans downregulated IL-12p35 mRNA levels in DCs treated with Pam3
but not LPS (10
It is tempting to speculate though that the mechanisms responsible for downregulation of IL-12p70 following ligation of Dectin-1 may be similar to that seen following Fcγ receptor ligation (15
). Of particular note is that Dectin-1 and Fcγ receptors have ITAM-like motifs in their cytoplasmic tails. In addition, Ca2+
fluxes are observed following ligation of both of these receptors (15
). Regardless of the mechanism, β-glucan-induced downregulation of IL-12p70 was observed in both human and murine DCs and was not overcome by IFN-γ priming. Recently, it was demonstrated that in the presence of antigen-stimulated CD8 T cells, the β-glucan curdlan stimulated BMDCs to produce IL-12p70 (29
). Thus, there appear to be feedback mechanisms whereby T cells can, under certain conditions, prime DCs for a IL-12p70 response to β-glucans.
IL-12p70 is a key cytokine involved in initiating T helper 1 (Th1)-type CD4+
T-cell responses (32
). Therefore, the inhibitory effects of β-glucans on BMDC IL-12p70 production could have consequences vis-à-vis Th skewing during the course of a fungal infection. Indeed, fungal infections are often associated with Th17-type responses (24
). Another member of the IL-12 superfamily, IL-23, is important for propagating Th17 responses (27
). IL-23 is a heterodimer that shares the p40 subunit with IL-12p70 but has a unique p19 subunit. Following stimulation of BMDCs with YGPs and/or TLR agonists, we could not detect significant levels of IL-23 secretion by ELISA (detection limit of 31 pg/ml; data not shown). However, Dennehy et al. did detect low levels of IL-23 following stimulation of BMDCs with β-glucans (10
). Moreover, they found that while β-glucans did not stimulate p19 mRNA, the combination of β-glucans plus Pam3
stimulated higher levels of p19 mRNA than Pam3
alone did. Nevertheless, because the levels of secreted IL-23 are low to undetectable, even in the presence of TLR agonists, these results raise the possibility that other stimuli besides β-glucans contribute to the Th17 skewing seen during fungal infections.
As noted above, fungi have multiple PAMPs that are recognized by host PRRs (34
). Other examples of cross talk among fungal PAMPs have been demonstrated. Cryptococcus neoformans
mannoproteins act synergistically with CpG to enhance TNF-α and IL-12p70 release in BMDCs (8
). Moreover, zymosan cooperates with the TLR2 agonist Pam3
to increase TNF-α secretion and upregulate IL-12p40 mRNA (14
). Furthermore, curdlan synergizes with TLR2 and TLR4 agonists to boost TNF-α and IL-10 production in human monocytes and macrophages (13
). We also observed synergistic increases in IL-10 production when BMDCs were stimulated with β-glucans and TLR2 and TLR9 agonists. As IL-10 is generally anti-inflammatory, this may serve as a mechanism for the host to minimize damage from an overly exuberant immune response following fungal invasion.
In addition to their potential to affect the outcome of fungal infections, the propensity of fungal PAMPs to stimulate cross talk has implications for the design of vaccines and immunomodulators containing β-glucans. β-Glucans have been proposed as antigen delivery systems (3
) and (despite a lack of definitive data) are touted as supplements to boost immune responses (36
). Our data emphasize that the DC cytokine response following β-glucan stimulation is quite intricate, particularly when the β-glucans are combined with other PAMPs. In vivo responses are likely to be even more complex, as opsonization with complement and anti-β-glucan antibodies contribute additional ligands (7
). Further understanding of the combined responses to β-glucans and PAMPS is critical if β-glucans are to be safely and effectively used as components of vaccine adjuvants and immunomodulators.