In the experiments described here, we have examined the role of TLRs on different immune cell types for the rapid production of inflammatory cytokines and for the activation of the adaptive immune response. Experiments employing a new conditional allele of the gene encoding the key TLR signaling adaptor molecule, MyD88, together with DC-specific expression of the Cre recombinase revealed a critical role for TLR signaling in DCs for many responses to TLR ligands. These experiments also revealed situations where other cell types can contribute importantly to the production of certain inflammatory cytokines and relieve the requirement for DC-intrinsic TLR signaling in order to stimulate a vigorous TH1 response.
These experiments provide evidence for the view that the direct recognition of microbial ligands by TLRs on DCs plays a prominent role for the initiation of the adaptive immune response and for directing polarization to a TH
1 response. When mice were immunized with ovalbumin and soluble CpG, TH
1 polarization of ovalbumin-specific CD4+ T cells was greatly diminished by deletion of the myd88
gene selectively in DCs. This result is consistent with a previous study using wild type and MyD88-deficient bone marrow chimeras (Sporri and Reis e Sousa, 2005
). However, in contrast to that study, we found that DCs lacking MyD88 had substantially compromised maturation in response to soluble CpG administrated i.v., as indicated by the reduced induction of co-stimulatory molecules. A major difference between those experiments and the ones described here is that in the bone-marrow chimeric mice, there was a mixture of MyD88-expressing DCs and MyD88-deficient DCs, whereas in the experiments described here, the vast majority of the DCs were MyD88-deficient and other cell types retained MyD88 expression. Therefore, the indirect maturation observed in the bone marrow chimeric mice probably reflects the action of cytokines produced primarily by neighboring DCs, acting in a paracrine manner. This interpretation is supported by observations reported here that DCs were the major cell types producing cytokines in response to soluble TLR ligands. Taken together, our results and those of Sporri and Reis e Sousa (2005)
demonstrate the importance of TLR signaling in DCs for CD4+
T cell responses, at least in the context of immunization with soluble TLR ligands
MyD88 function in DCs was found to be especially important for IL-12 production in response to TLR ligand stimulation. IL-12 is known to simulate innate immune cell types such as NK cells to express IFNγ, and to promote the TH
1 polarization of CD4+ T cells (Magram et al., 1996
), both of which can be enhanced by IL-18 (Takeda et al., 1998
). Indeed, we found that the early IFNγ production from NK cells and NKT cells in response to soluble CpG was totally dependent on IL-12. IFNγ from NK cells can initiate TH
1 polarization of antigen-stimulated CD4 T cells (Martin-Fontecha et al., 2004
) by inducing the fate-determining transcription factor T-bet (Afkarian et al., 2002
). In addition, IL-12 from DCs promotes fate stabilization of T cells polarizing to TH
1 cell by promoting their secretion of IFNγ. Given the critical roles of IL-12 and the fact that its induction by TLR ligands was highly dependent on direct TLR stimulation in DCs, it is very likely that IL-12 was an essential cytokine for promoting TH
1 polarization under these circumstances. Thus, TLR-MyD88 signaling in DCs likely contributes to adaptive TH
1 immune responses to immunization with soluble antigen and TLR ligands in two main ways: by contributing to DC maturation and by inducing IL-12 production.
We found that the physical form of the TLR ligand had a large effect on the ability of different cell types to contribute to the immune response in vivo
. CpG presented in an aggregated form by complexing it with the cationic lipid DOTAP induced a potent type 1 IFN response compared to CpG alone, as reported previously (Honda et al., 2005
). In contrast to the strong dependence of MyD88 function in DCs for the cytokine responses to soluble CpG, MyD88 function in both DCs and non-DC cell types made important contributions to this response. Indeed, whereas when soluble CpG was injected i.v., it was primarily CD8α+
DCs in the spleen that made cytokines initially, when CpG/DOTAP was injected i.v., both CD8α+
DC subsets responded rapidly, as did a F4/80+
cell type that may be the inflammatory monocyte. Interestingly, the uptake of CpG in these experiments was substantially enhanced when CpG was complexed with DOTAP. The altered spectrum of responding cell types may relate to changes in the mechanism of cell uptake, as it has been reported that CpG/DOTAP complexes enter cells through the endocytic pathway (Zabner et al., 1995
). Interestingly, interaction of self-DNA with a cationic amphipathic antimicrobial peptide LL37 has recently been implicated in the pathogenesis of psoriasis. This complex, which may be similar in its action to the CpG/DOTAP aggregate studied here, was found to greatly enhance activation of pDCs in the affected skin of patients with psoriasis (Lande et al., 2007
). It is likely that many microbial and endogenous TLR ligands exist in aggregated or particulate forms, so the ability of both DCs and other myeloid cell types to respond to TLR ligands presented in these complex forms may be relevant to many biological situations.
The ability of CpG/DOTAP to induce large amounts of type 1 IFNs from pDCs, as well as from other non-DC cell type is likely to explain its ability to induce strong IFNγ production from NK cells, robust DC maturation, and a vigorous TH
1 response in the absence of MyD88 expression in DCs. Other have reported that type 1 IFNs can synergize with IL-18 to induce IFNγ even in IL-12-deficient splenocyte cultures (Freudenberg et al., 2002
), and we found that the NK cell IFNγ response to CpG was largely dependent on the expression of type 1 IFN receptors. In addition, type 1 IFNs are known to be able to induce maturation of DCs (Hoebe and Beutler, 2004
). Indeed, it has been shown that co-administration of IFNα with antigen induces delayed-type hypersensitivity (Gallucci et al., 1999
), as well as IgG2a antibody production (Le Bon et al., 2001
), both of which are typical TH
1 responses. Thus, the robust production of type 1 IFNs by cell types other than DCs is likely to explain why MyD88 function in DCs was not necessary for DC maturation or for a vigorous TH
1 response in DC-MyD88 KO mice immunized with OVA + CpG/DOTAP.
In our experiments, LPS had a behavior that was very similar to that of CpG/DOTAP. LPS is an amphipathic component of gram-negative bacterial cell walls that forms large aggregates in solution, so it is not surprising that is has the ability to induce robust cytokine responses from DC subsets and also from additional myeloid cell types similarly to CpG/DOTAP. However, it is worth noting that LPS, like CpG/DOTAP, neither induced IFNγ from NK cells and NKT cells (Supplemental Figure S3
) nor promoted TH
1 polarization in MyD88 KO mice (Schnare et al., 2001
). Although TLR9 and TLR4 both signal via MyD88, TLR4 also signals via the TRIF adaptor and this pathway induces abundant type 1 IFNs. Indeed, previous work showed that LPS stimulation of MyD88 KO mice failed to promote TH
1 responses despite a vigorous type I IFN response and clearly evident DC maturation (Pasare and Medzhitov, 2004
). Thus, type I IFN production alone is insufficient to drive TH
1 responses. These results suggest that one or more MyD88-dependent cytokines in addition to type 1 IFNs coming from cells other than DCs are also required for the innate IFNγ and TH
1 response seen in DC-MyD88 KO mice immunized with CpG/DOTAP or LPS. IL-18 is known to synergize with type 1 IFNs or IL-12 to induce IFNγ (Freudenberg et al., 2002
; Nakanishi et al., 2001
) and therefore is a strong candidate for the additional MyD88-dependent cytokine required for these responses.
In any case, our results clearly show that the functional fate of a T cell is not only affected by the DC that the T cell is interacting with, but also by surrounding cells responding via TLRs and MyD88. These surrounding cells include other DCs and non-DC cell types in the infected tissues. This trans
effect may allow cooperation between cell types expressing different sets of TLRs. For example, CD8α+
DCs, which do not express TLR7 (Edwards et al., 2003
), can receive important cytokine signals from pDCs, which secrete type 1 IFNs after TLR7 stimulation, allowing them to activate T cells. Other recent studies have also provided evidence for regulatory effects of macrophages on DCs for polarizing T cell differentiation. For example, lamina propria macrophages from the gut were found to express anti-inflammatory cytokines even after TLR stimulation in vitro
and to promote development of FoxP3+
regulatory T cells, which restrain immune responses to commensal microbes and dietary antigens, whereas DCs from the same location responded to the same stimulus by producing proinflammatory cytokines and promoting IL-17 producing T cell responses (Denning et al., 2007
). Clearly, much remain to be learned about how different types of innate immune cells communicate with one another and combine to direct the nature of the adaptive immune responses.
In summary, our results indicate that MyD88-dependent signaling in both DCs and non-DC cell types can support TH1 differentiation depending on the type of TLR stimulation. Whereas direct TLR stimulation is likely to be the most efficient way for activating DCs and for activating adaptive responses, we have found that other cell types stimulated with TLR ligands in complex forms secrete substantial amount of cytokines that can make important contributions to both innate and adaptive immune responses. It should be very interesting to use the mice described here to dissect further the role of TLR signaling in different cell types for activation of adaptive immune responses in more complicated and biologically important situations, such as infections with pathogens and autoimmune diseases.