Integration of signals derived from Dectin-1 and TLR2 directs the coordinated response of myeloid phagocytes to fungal particles. Previous studies indicated that the transcriptional induction of pro-inflammatory mediators is controlled by TLR2-MyD88-induced NF-κB activation, a signal that can be amplified by Dectin-1, and Dectin-1-mediated ITAM-like signaling which activates NFAT transcription factors(8
). A recent report also indicated that Dectin-1 signals can directly activate NF-κB in DC via the adaptor protein CARD9(12
). In our current study we have revealed an additional layer of regulation of Dectin-1/TLR2-induced pro-inflammatory cytokine production.
Our data show that Dectin-1 signaling via CARD9 is differentially regulated in myeloid cells, resulting in the ability of only certain macrophage/DC populations to produce TNF-α upon Dectin-1 ligation alone. bmM, thioglycollate-elicited peritoneal macrophages (TEPM), and Flt3L-derived DC failed to produce TNF-α in response to Dectin-1 ligation, whereas bmDC, GM-CSF/IFN-γ-primed bmM, resident peritoneal cells and alveolar macrophages all displayed robust TNF-α production following Dectin-1 ligation ( and ). Expression of Dectin-1, CARD9 and its interacting partner Bcl10 did not correlate with the ability of the cells to produce TNF-α in response to Dectin-1 ligation (). However, we demonstrated that overexpression of Dectin-1 in bmM is sufficient to permit CARD9-dependent TNF-α induction (), indicating that these cells possess an inhibitor of Dectin-1-CARD9 signaling.
Meyer-Wentrup et al. recently demonstrated that the tetraspanin CD37 associates with Dectin-1 and limits pro-inflammatory cytokine induction(26
). Induction of IL-6 by zymosan was 10-fold higher in TEPM from CD37 knockout mice compared to wild type TEPM. Furthermore, while wild type TEPM failed to produce IL-6 in response to the β-glucan curdlan, IL-6 was induced by curdlan treatment of CD37-deficient TEPM. Our data suggest that CD37 may block Dectin-1-CARD9-mediated cytokine induction in macrophage/DC populations unresponsive to depleted zymosan. Consistent with this, we observed higher CD37 expression by bmM than bmDC, and a reduction in CD37 expression following treatment of bmM with GM-CSF and IFN-γ (data not shown). Meyer-Wentrup et al. also observed a reduction in CD37 expression upon differentiation of DC from human monocytes(26
Tetraspanins are small transmembrane scaffold proteins that anchor proteins in membrane microdomains(27
). Hence CD37 may sequester Dectin-1 into a microdomain that selectively modulates the recruitment of key signaling molecules downstream of CARD9. Lower CD37 expression in bmDC/primed bmM, coupled with higher Dectin-1 expression in these cells, would be predicted to result in a larger pool of Dectin-1 that is not associated with and limited by CD37. Further studies are required to determine whether a CD37-based mechanism is responsible and sufficient for the differential CARD9 signaling we have observed in this study.
Dectin-1 has also been reported to associate with another tetraspanin, CD63, in human monocyte-derived DC, although the function of this interaction is unknown(28
). Hence the relative levels of different tetraspanins may also determine the type of signal generated. Detailed investigation of the regulation of Dectin-1 signaling by CD37 and CD63 is required to further assess the ability of tetraspanins to differentially regulate Dectin-1 signals.
Gross et al. had demonstrated that Dectin-1-CARD9 signaling results in NF-κB activation in bmDC(12
), which contrasted with our previous failure to observe NF-κB activation in RAW264.7 macrophages and bmM(8). We have now shown that Dectin-1 can indeed activate NF-κB directly in certain myeloid cells, but that this signaling connection is not always made. This is in contrast to other Dectin-1 signals, including Src-Syk activation(6
), p38 phosphorylation (), and NFAT activation(9
), which are triggered robustly in bmM (and RAW264.7 macrophages) as well as bmDC. Whether other signals downstream of CARD9 are also absent in the “unresponsive” macrophages, contributing to the inability of Dectin-1 signals to directly induce TNF-α, remains to be established.
The cell type-specific variability of CARD9-mediated NF-κB activation downstream of Dectin-1 ligation stands in sharp contrast to MyD88-mediated NF-κB activation downstream of TLRs. As far as we are aware TLR signaling via MyD88 always results in NF-κB activation, regardless of cell type. In contrast, we have shown that Dectin-1 signaling via CARD9 does not always result in NF-κB activation. Variability in Dectin-1 signaling in different cell types represents a novel mechanism by which myeloid cells can be fine-tuned to regulate anti-fungal immunity, with different myeloid populations having distinct inflammatory and anti-microbial properties. Whereas alveolar macrophages are inherently ready to release cytokines and initiate an inflammatory response to β-glucan particles, other macrophages may require information about an ongoing infection, such as IFN-γ production, to become primed for this response. Furthermore, as we have observed fundamentally different pro-inflammatory signaling in GM-CSF- and Flt3L-derived bmDC, there may be significant further heterogeneity in how different DC subsets prime adaptive immunity. Future studies will define the consequences of this variability on cell function and the contribution of differentially responsive cells to anti-fungal immunity.
Despite the apparent restricted ability of CARD9 to activate NF-κB in bmM, CARD9 is not completely silent in these cells, as indicated by the defect in Dectin-1-induced p38 phosphorylation in CARD9-deficient bmM (). Furthermore, CARD9 appears to be responsible for the collaboration of Dectin-1 signals with TLR2 signals, as evidenced by the defect in TNF-α production by zymosan-stimulated CARD9-deficient bmM (). These data are consistent with our observations of CARD9 recruitment to Dectin-1 phagosomes even in macrophages where Dectin-1 signaling is insufficient to drive TNF-α production ().
Hara et al. recently showed that CARD9 mediates signaling downstream of the FcγR, which, like Dectin-1, is an ITAM-containing phagocytic receptor(29
). Hence we predict that CARD9 is similarly recruited to FcγR phagosomes. That the CARD domain of CARD9 is enough to drive the protein to the Dectin-1 phagosome suggests that phagosomes might be an important scaffold for coordinating CARD-CARD signaling interactions. Future studies will have to define how the presence or absence of additional signaling molecules on the phagosomes of different cell types regulates inflammation and immunity.