The involvement of the endolysosomal compartment in proper functioning of the nucleic acid sensing TLRs (TLR3, 7, 9) is well-documented 11–16
, but the exact manner in which this environment contributes to the receipt and transmission of signals via these TLRs has not been established. We now demonstrate that TLR9 undergoes a proteolytic cleavage, executed by cysteine proteases, in the endolysosomal compartment. On a molar basis the C-terminal cleavage fragment of TLR9 binds CpG more strongly than full length TLR9, although full length TLR9 does bind CpG. The broadly specific inhibitor z-FA-FMK is most effective at blocking this cleavage, and accordingly abrogates production of TNF in cells exposed to the TLR9 agonist CpG. Our data are consistent with the involvement of more than one protease in this cleavage reaction, because we observe a blunted, rather than a completely blocked TNF response to CpG in Cathepsin L-deficient or Cathepsin S-deficient BMDCs. Furthermore, the combination of Cathepsin L - and Cathepsin S -selective inhibitors not only blocks TNF production in response to CpG, but also yields a cleavage intermediate, the pre-C-terminal fragment, the abundance of which is inversely correlated with TNF production. The expression of only the C-terminal fragment in Tlr9−/−
BMDCs restores their ability to produce TNF, as seen also for Cathepsin L-deficient BMDCs. We hypothesize that absence of cleavage in presence of chloroquine and bafilomycin A is best explained by elevated pH or reduced proteolytic activity.
The delivery of TLR9 to endolysosomal compartments requires its interaction with UNC93B1, mediated via the transmembrane segment of TLR922
. Eliminating this interaction, either by substituting the transmembrane segment of TLR9 or by deleting UNC93B1, causes TLR9 to traffic incorrectly to the cell surface16,23
. The capacity of a chimera consisting of the extracellular domain of TLR9 and the transmembrane and cytoplasmic domains of TLR4–which localizes to the cell surface–to induce signal transduction upon CpG binding might be attributed to the recruitment of adaptors other than Myd88 to the cytoplasmic tail of TLR416,32
We did not observe any inhibition of TNF production in Cathepsin K-deficient BMDCs or by imposition of a Cathepsin K blockade, in contrast to an earlier study that showed reduced IL12 production by Cathepsin K-deficient BMDCs in response to CpG21
. We further determined that responses requiring engagement of TLR7 were not affected by z-FA-FMK (), unlike the blockade in upregulation of CD86 and B cell proliferation reported earlier20
. IL12 production may require the involvement of Cathepsin K at steps downstream of TLR9 engagement, which could explain the observed discrepancy. Likewise, the surface expression of CD86 and steps that control B cell proliferation may require lysosomal protease involvement for reasons other than cleavage of the TLRs involved.
We have no evidence that TLR7 is cleaved in a z-FA-FMK sensitive manner, and we have been unable to detect cleavage fragments of TLR7. This raises the question of why signaling via TLR7 or via TLR3, for that matter, is nonetheless sensitive to the inclusion of lysosomotropic agents. Several of the TLRs are known to act in cooperation with partner proteins such as CD14 and MD2 for TLR433, 34
, or CD36 for TLRs 1, 2 and 635
, or Dectin-1 for TLR236
. We hypothesize that the activity of TLR3 and TLR7 may require cofactors whose involvement necessitates a proteolytic cleavage, or at least includes a pH sensitive step, affected by the inclusion of lysosomotropic agents.
The evolutionary significance of TLR9 cleavage is as yet unclear. Why should TLR9 be unique among lysosomal TLRs in requiring this mode of activation? The C-terminal cleavage fragment of TLR9 is capable of binding CpG DNA and signal transduction even when synthesized in the absence of the N-terminal portion of TLR9. A search for interaction partners of the N-terminal cleavage fragment might illuminate yet other aspects of TLR9 biology. Regardless of the identity of the TLR9 convertase(s) and the exact role of the cleavage products generated, full innate immunity requires lysosomal proteolysis no less than adaptive immunity.