Partial role of TLR4 as a receptor responding to damage-associated molecular pattern

Part of pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) activate antigen-presenting cells through Toll-like receptors (TLRs) to initiate immune responses. However, controversy remains if TLR4 mediates DAMP signaling due to the confounding effects of potential LPS contamination. To test if TLR4 functions as a true receptor for DAMP, we compared TLR4pos- and TLR4neg-responders in vitro and in vivo after stimulation with whole necrotic cell (NC) lysates. Using CHO reporter cells transfected with anti-TLR4-siRNAs, TLR4 was found to partially mediate NF-κB activation in response to NC lysates. TLR4neg DCs exhibited less I-Ab expression and nitric oxide secretion than TLR4pos DCs upon NC stimulation and this defect was well correlated with diminished presentation of H-Y antigen by TLR4neg DCs to I-Ab-restricted CD4pos Marilyn T cells in vitro. Similarly, TLR4neg DCs showed significantly less expression of I-Ab, CD80, CD86, and CD40 than TLR4pos DCs when NC lysates were injected into peritoneal cavity. Finally, delayed type hypersensitivity response to OVA was significantly decreased in TLR4neg mice when NCs were used as an adjuvant. Taken together, our data support the idea that part of the endogenous ligands presented by NCs could activate APCs thru TLR4 and contribute to the development of antigen-specific adaptive immunity. Therefore, endogenous DAMP ligands themselves, not contaminated LPS, activate TLR4 signaling leading to activation of professional antigen-presenting cells.