Our current studies provide compelling evidence that TLR9 transits through the Golgi complex and that Golgi export is critical for optimal response to CpG DNA. Synthesis of transmembrane and secreted proteins begins on rough ER where the nascent protein is concurrently translated and transferred through the ER membrane. For proteins with N-linked glycosylations, a preformed oligosaccharide precursor containing several mannose residues is then transferred to an asparagine (in the sequence Asn-X-Ser/Thr) on the nascent protein (high mannose Glycoform). When the protein is ready for export, it transits through the Golgi complex where modification of the oligosaccharide is completed. These modifications include trimming of mannose residues (hybrid Glycoform) and the incorporation of N-acetylglucosamine, galactose and N-acetylneuraminic acid (complex Glycoform). Here we show that TLR9 is glycosylated in the ER, since it binds GN, a lectin specific for high mannose and hybrid glycoforms (). TLR9 also binds to DS, a lectin with high affinity for hybrid and complex glycoforms, demonstrating it contains Golgi-dependent glycan modifications. We further show, using a newly developed furin cleavage assay, that TLR9 is exposed to the Golgi-resident protease furin during intracellular movement (). These data are in contrast to the current model, which proposes that TLR9 translocates directly from the ER to endosomes by a mechanism similar to ER-mediated phagocytosis. This model was based on the observation that glycans on TLR9 remained sensitive to digestion with EndoH despite TLR9’s movement to endosomes. We now clearly show that the glycans on TLR9 remain sensitive to EndoH digestion upon treatment with BFA despite a change in EndoH sensitivity of the fusion chimera TLR4-9. Furthermore, optimal TLR9 signaling is blocked by pre-treatment of cells with BFA demonstrating that TLR9 not only passes through the Golgi complex, but that Golgi export is critical for TLR9 signaling (). These results are important for defining the intracellular trafficking pathway of TLR9, a pathway that may be amenable to interruptive drug treatment to prevent TLR9 signaling in diseases such as systemic lupus erythematosus (SLE).
Another important finding of our studies is that TLR9 is not fully retained in the ER, but constitutively moves through the cell and reaches the lysosomal compartment (). These data are consistent with previous studies, which have suggested that under certain conditions that some TLR9 can be detected in an endosomal compartment6, 32
. There are two possible explanations as to why TLR9 is detected in the lysosomal compartment of untreated cells. It could be a mechanism for continuous turnover of TLR9, or it could provide a pool of TLR9 immediately ready to respond to endocytosed CpG DNA. High levels of TLRs in general lead to constitutive activation and constant turnover in the lysosome may help to regulate these levels. However, we provide data demonstrating that the pool of TLR9 that has exited the Golgi is constitutively associated with MyD88 and thus likely contributes to signaling (). Therefore, lysosomal TLR9 is poised to rapidly respond to an influx of CpG DNA. Early signaling events such as the phosphorylation of p38, although delayed, still occur in BFA treated cells. Thus, we concluded that the extra-Golgi TLR9 can respond to CpG DNA. Since our organelle separation studies demonstrate that the TLR9 that exits the Golgi complex moves to lysosomes, it is likely that signaling can be initiated from this compartment. What role this initial signaling plays in the optimal TLR9 signaling, which requires additional TLR9 translocation from the ER to endolysosomes, is not clear. TLR9 translocation to endolysosomes in response to CpG DNA still occurs in MyD88 deficient dendritic cells, so it is unlikely that signaling initiated by the small pool of TLR9 in endolysosomes is responsible for inducing the additional movement of TLR97
. Further studies are necessary to decipher the contribution of lysosomal TLR9 in signaling.
Recently, a partially cleaved form of TLR9 that contains approximately half of the ecto-domain and all of the transmembrane domain and cytoplasmic tail has been described31
. This form is enriched in phagosomes and may be the functional receptor. We have detected a lower molecular weight form of TLR9 in some experiments using either transient or stable cell lines expressing tagged or untagged TLR9. Surprisingly, we detect this lower molecular weight form with an antibody that recognizes amino acids 273–288, an epitope that would be lost based on the predicted cleavage site31
. However, we have failed to consistently detect this form in the lysosomal fractions from either HEK293 cells expressing HA-TLR9 (detected with antibody to TLR9) or BJAB cells expressing endogenous TLR9 (Chockalingam A, 2008 unpubl. data). It is possible that cell lineage, growth conditions or endocytosis/phagocytosis influence the amount of cleaved TLR9 detected in unstimulated cells.
A remaining question is what regulates TLR9 translocation from the ER through the Golgi complex to endolysosomes. Several proteins have been described to control response of TLR9 to CpG DNA including, Hsp90, cathepsins HMGB1, and UNC93B117, 30, 33–36
. A macrophage specific Hsp90 deficiency in mouse resulted in loss of response to all TLRs, suggesting that this chaperone is a master regulator of TLR signaling35
. Other studies have suggested that the function of Hsp90 is to control intracellular TLR trafficking37
. Cathepsins have also been implicated in regulating TLR9 response to CpG DNA. Given the cleavage product of TLR9 described above31
, it is interesting to note that dendritic cells treated with cathepsin K inhibitors or deficient in cathepsin K failed to respond to stimulation with CpG DNA36
. However, the role of cathepsin K in regulating TLR9 signaling is unclear since several recent papers provide conflicting data on the role of different cathepsins in regulating TLR9 signaling31, 36, 38
. HMGB1 also regulates TLR9 signaling and can directly bind to both TLR9 and immune complexes containing DNA that are found in SLE serum30
. TLR9 directly associates with the HMGB1 receptor, RAGE and this association regulates TLR9 response. An Fc-RAGE fusion blocks TLR9 response to DNA; however, we do not know if this regulation is at the level of receptor translocation. UNC93B1 is an ER resident protein that interacts with nucleic acid recognizing TLRs via their transmembrane domains and thus regulates their intracellular trafficking17, 33
. UNC93B1 deficient mice fail to respond to CpG DNA, as well as TLR7 and TLR3 ligands17, 33, 34
. UNC93B1 is likely required for TLR9 to exit the ER, but it is unclear if it plays any additional role in regulating TLR9 movement. Here we have established that TLR9 traffics through Golgi complex and that Golgi export is required for optimal TLR9 signaling. It will be interesting to determine how these TLR-interacting proteins regulate TLR9 movement from the ER to endolysosomes via the Golgi complex.
In summary, we have demonstrated that TLR9 does not bypass the Golgi complex during translocation to endolysosomes, and that Golgi export is required for optimal signaling. A small pool of TLR9 is constitutively localized in endolysosomes and this pool is bound to MyD88 and likely initiates signaling. Elucidating the mechanisms regulating TLR9 movement within cells is critical to understand how TLR9 normally remains quiescent when cells are exposed to self DNA. A better understanding of the molecular mechanisms controlling these events will allow in identification and testing of inhibitors that target the regulatory pathways of TLR9 trafficking and prevent recognition of self DNA in autoimmune diseases such as SLE. It is intriguing to think that some of these pathways may be shared with other TLRs, such as TLR7, that also contribute to SLE. Therefore, manipulating the translocation of nucleic acid recognizing TLRs could be a novel approach to treat debilitating autoimmune inflammation.