CpG-containing ODNs activate TLR9 in endosomes and trigger the secretion of inflammatory cytokines by a mechanism that is dependent on the adaptor protein MyD88 (Krieg, 2002b
; Takeda et al., 2003
). We demonstrate that cellular activation by immunostimulatory ODNs is more complex than previously described and requires a TLR9-independent SFK-driven pathway. This SFK-dependent signaling cascade is not parallel but upstream of the chloroquine-sensitive TLR9–MyD88 pathway.
We first provide evidence that stimulation with CpG, but not the control GpC, induces a complex pattern of tyrosine phosphorylation in monocytes. While characterizing several of these phosphorylated proteins, we identified two SFKs expressed in myeloid cells, Hck and Lyn. Treatment of monocytes with the SFK inhibitor PP2 inhibited the CpG-induced tyrosine phosphorylation cascade. Interestingly, treatment with chloroquine and quinacrine, two known inhibitors of TLR9 signaling, failed to block tyrosine phosphorylation.
In addition to the kinases Hck and Lyn, we found that CpG also induced the phosphorylation of Pyk2, Cbl, and the previously described CpG-activated Vav (Stovall et al., 2004
). These proteins are required for adhesion and migration responses that involve rearrangement of the actin cytoskeleton (Suzuki et al., 2000
; Latour and Veillette, 2001
). For example, the phosphorylation of Pyk2 by SFKs, along with the participation of other focal adhesion proteins, promotes actin cytoskeleton rearrangements that induce cell adhesion, spreading, and lamellipodia formation (Suen et al., 1999
). Cbl, which interacts with Pyk2, Vav, and SFKs, has been associated with increased motility of macrophages (Caveggion et al., 2003
). Vav, a multidomain signal integrator, transduces signals to cytoskeleton-dependent pathways, which include the PI3K pathway and the activation of extracellular signal–regulated kinase and NF-κB (Tybulewicz et al., 2003
). The phosphorylation of Pyk2, Cbl, and Vav upon CpG stimulation suggested that actin reorganization plays an important role upon CpG stimulation. Indeed, our experiments confirmed that CpG triggered the reorganization of the actin cytoskeleton, promoting cell adhesion and migration. We found that CpG treatment induced a transient, SFK-dependent cell adhesion in human monocytes but not when cells were stimulated with either control GpC-ODN or a mouse-selective CpG-ODN. Again, the TLR9 inhibitor chloroquine was unable to block CpG-induced adhesion and migration of THP-1 monocytes and adhesion on human monocyte–derived DCs; however, those cellular responses were completely blocked with an SFK inhibitor.
CpG-induced SFK signaling was upstream and required for NF-κB activation and subsequent cytokine secretion. SFK inhibitors blocked CpG-induced secretion of IL-6 in splenocytes, IFN-α production in human pDCs, and the up-regulation of activation markers on murine B cells. Furthermore, we found that SFK activation was intact in MyD88−/− mice, indicating that this pathway is both upstream and independent of MyD88.
The rapid kinetics of tyrosine phosphorylation suggested that this event was initiated at the cell surface and independent of the endosomal localized TLR9. The use of CpG-coated beads, too large to be internalized, revealed that CpG induced robust, rapid actin reorganization at the bead–cell contact area, further suggesting that these events did not require internalization of CpG. The use of TLR9−/−
monocytes indicated that these cellular events were independent of TLR9 and MyD88 in addition to being chloroquine insensitive. Together, these studies indicated that CpG-induced SFK activation at the plasma membrane drives cytoskeletal reorganization upstream and independently of the TLR9–MyD88 pathway that could result in cell adhesion and migration. In support of our findings, it was recently reported that, upon DNA viral infection of mice, leukocyte recruitment to the liver is not altered in TLR9−/−
and MyD88 −/−
mice, whereas the secretion of cytokines in vivo was dramatically reduced (Delale et al., 2005
Our findings indicate that the recognition of CpG-ODN is more complex than previously described, but as reported (Krieg et al., 1995
; Manzel and Macfarlane, 1999
), this TLR9-independent pathway cannot induce cytokine secretion without ODN internalization. Although plate-bound CpG induced the formation of large lamellipodia in macrophages from wild-type, TLR9−/−
, and MyD88−/−
mice, it was unable to induce IL-6 secretion.
The TLR9 receptor responds differently to the three identified ODN classes (A, B, and C). The mechanism by which these diversified responses are generated remains unresolved (Vollmer et al., 2004
). It has been hypothesized that TLR9 requires additional coreceptors/cofactors, as is the case for TLR4 (Verthelyi and Zeuner, 2003
), for the recognition of CpG–A class ODNs (Gursel et al., 2002
). Our findings support this hypothesis because SFK activation and downstream cellular events are most strongly triggered by CpG–A class ODN when compared with B class ODN. Based on our preliminary characterization of this pathway, we hypothesize that a CpG-sensing coreceptor/cofactor is localized at the plasma membrane, which could also potentially be internalized with CpG-ODN, resulting in endosomal TLR9 activation (). Until this pathway is further characterized, it remains possible that this coreceptor/cofactor is also localized in the endosome in resting cells.
Figure 9. A model for the two-step activation by CpG-DNA. We propose that the recognition of A class CpG-DNA is a two-step process that can begin at the plasma membrane (PM) by a TLR9-independent mechanism. We hypothesize the existence of a receptor, localized (more ...)
The role of PI3K family on TLR signaling is poorly understood. PI3K interacts with the cytosolic Toll/IL-1 receptor domain of TLR2 (Arbibe et al., 2000
) and has been found activated downstream of IRAK1 in response to IL-1β (Neumann et al., 2002
). Recently, Akt, a downstream effector of the SFK–PI3K pathway (Franke et al., 1997
), was shown to be phosphorylated in response to CpG by a TLR9-independent mechanism that involves DNA-dependent protein kinase (Dragoi et al., 2005
). In our experimental system, Akt phosphorylation was intact in chloroquine-treated cells, indicating that the MyD88 cascade is not involved in Akt activation. However, Akt phosphorylation was completely blocked by both PP2 and the PI3K inhibitor wortmannin. These data indicate that the TLR9-independent tyrosine phosphorylation cascade that we describe is upstream of Akt activation.
Finally, we provide evidence that the SFK pathway does intersect with the TLR9–MyD88 pathway. TLR9 is tyrosine phosphorylated upon CpG stimulation, and this event is independent of chloroquine but blocked by PP2. Furthermore, Syk coimmunoprecipitates with TLR9 upon CpG stimulation, and this interaction can be blocked by PP2. Because Syk is normally recruited to ITAM-containing receptors, which is not the case of TLR9, it is likely that the TLR9–Syk association is indirect and that other proteins participate in the formation of a complex. Two potential ITAM chains, the γ chain of the Fc receptor and DAP12 (DNAX-activating protein of 12 kD) were examined, but no phosphorylation was detected upon CpG stimulation (unpublished data). Although our experiments do not reveal whether Syk associates directly with TLR9, coimmunoprecipitation after CpG stimulation is not affected by chloroquine, indicating that this event is driven by the initial SFK pathway independently of TLR9. This link between tyrosine kinase signaling and TLRs is further supported by a recent study involving TLR3, a receptor for double-stranded RNA. Like TLR9, TLR3 is tyrosine phosphorylated, and this event is linked to PI3K-mediated enhancement of the interferon regulatory factor 3 pathway (Sarkar et al., 2004
). Unlike our findings with TLR9, TLR3 phosphorylation does not regulate NF-κB activation.
In conclusion, we describe a TLR9-independent, SFK-driven signaling cascade induced by CpG. This cascade is upstream of the MyD88-dependent endosomal pathway and is initiated at the plasma membrane. This chloroquine-insensitive pathway initiates complex cytoskeletal rearrangements necessary for cell biological events such as adhesion and migration. Moreover, these two pathways interact, as inhibition of the SFK pathway blocks activation events downstream of endosomal TLR9/MyD88 such as NF-κB activation and cytokine secretion. Our findings suggest that a potential CpG-sensing receptor is localized at the plasma membrane and might interact with CpG before the activation of endosomal TLR9. Future studies are required to identify candidate proteins that would perform this function. The identification of a TLR9 coreceptor would potentially provide a new target for pharmaceutical intervention that could result in new treatments for autoimmune diseases.