Complex Negative Regulation of TLR9 by Multiple Proteolytic Cleavage Events

TLR9 is an innate immune receptor important for recognizing DNA of host and foreign origin. A mechanism proposed to prevent excessive response to host DNA is the requirement for proteolytic cleavage of TLR9 in endosomes to generate a mature form of the receptor (TLR9 471–1032 ). We previously described another cleavage event in the juxtamembrane region of the ectodomain that generated a dominant-negative form of TLR9. Thus, there are at least two independent cleavage events that regulate TLR9. In this study, we investigated whether an N-terminal fragment of TLR9 could be responsible for regulation of the mature or negative-regulatory form. We show that TLR9 471–1032 , corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by coexpression of the N-terminal fragment (TLR9 1–440 ), inclusion of the hinge region (TLR9 441–1032 ), or overexpression of UNC93B1, the last of which is critical for trafficking and cleavage of TLR9. TLR9 1–440 coimmunoprecipitates with full-length TLR9 and TLR9 471–1032 but does not rescue the native glycosylation pattern; thus, inappropriate trafficking likely explains why TLR9 471–1032 is nonfunctional. Lastly, we show that TLR9 471–1032 is also a dominant-negative regulator of TLR9 signaling. Together, these data provide a new perspective on the complexity of TLR9 regulation by proteolytic cleavage and offer potential ways to inhibit activity through this receptor, which may dampen autoimmune inflammation.