Complex Negative Regulation of TLR9 by Multiple Proteolytic Cleavage Events

Abstract

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 (TLR9471–1032). 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 TLR9471–1032, corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by coexpression of the N-terminal fragment (TLR91–440), inclusion of the hinge region (TLR9441–1032), or overexpression of UNC93B1, the last of which is critical for trafficking and cleavage of TLR9. TLR91–440 coimmunoprecipitates with full-length TLR9 and TLR9471–1032 but does not rescue the native glycosylation pattern; thus, inappropriate trafficking likely explains why TLR9471–1032 is nonfunctional. Lastly, we show that TLR9471–1032 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.