Toll-like receptor 9 is alternatively cleaved to generate a soluble form of the receptor that downregulates response to CpG-DNA

Proper regulation of nucleic acid sensing Toll-like receptors (TLRs), including TLR9, is critical to prevent autoimmune disease and promote host defense. The objective of our research is to elucidate the molecular mechanisms that control TLR9 signaling. Localization of TLR9 in endolysosomes is one regulatory mechanism that facilitates recognition of CpG-DNA and prevents response to self DNA. Restricted localization of TLR9 controls access to proteases, including cathepsins B and L and asparagine endopeptidase, which generate an active 80 kDa (p80) form of the receptor. Here we use mass spectrometry analysis to identify a 100 kDa soluble form of TLR9 (sTLR9, amino acids 1-740). The proteolytic cleavage of sTLR9 is distinct from that of p80 since it is not blocked by Z-FA-fmk or E-64 protease inhibitors, or by bafilomycin A1 or chloroquine. These data suggest that generation of sTLR9 does not occur in acidic endosomes. We show that the generation of sTLR9 requires cathepsin-S, a protease inhibitor active at neutral pH. Most importantly we show that the sTLR9 binds to CpG-DNA and down regulates CpG-DNA induced TLR9 signaling. Balance between proteolytic cleavage occurring at two distinct sites on TLR9 ectodomain is likely a major factor that modulates host innate immune responses to microbial infection and prevents development of autoimmune disease.