TLR4 Ligands Selectively Synergize to Induce Expression of IL-8

Objective: Dysfunctional remodeling of the extracellular matrix contributes to the formation of TLR-dependent feed forward loops that drive chronic inflammation. We have previously shown that two Type III domains of Fibronectin, FnEDA and FnIII-1c, cooperate to induce the synergistic release of interleukin 8 (IL-8) from dermal fibroblasts. We now identify steps in the TLR4 pathway where synergy can be demonstrated as well as additional kinases functioning in fibronectin activation of TLR4 signaling. We also evaluate the ligand and cell-type specificity of this synergistic response. Approach: FnEDA, FnIII-1c, and lipopolysaccharide (LPS)-induced genes in fibroblasts were analyzed by a quantitative reverse transcription-polymerase chain reaction (qPCR) and protein was measured by an enzyme-linked immunosorbent assay (ELISA). Kinases functioning in gene expression were identified by using specific inhibitors. Activated TLR4-dependent effector molecules were identified by cell fractionation and Western blot and quantified by image analysis. Results: The addition of FnEDA and FnIII-1c to dermal fibroblasts resulted in a synergistic increase in the expression of IL-8, tumor necrosis factor alpha (TNF-α), and vascular cell adhesion molecule (VCAM-1). Synergy between these domains was detected at the level of nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) and inhibitor of kappa B kinase (IKK) activation. Induction of IL-8 by fibronectin ligands was partially attenuated in the presence of inhibitors to either epidermal growth factor receptor or Src kinases. FnIII-1c also synergized with LPS to induce IL-8 in dermal fibroblasts, whereas the combined effect of FnEDA and LPS on IL-8 synthesis was additive. In contrast, synergistic responses to these ligands were not observed in THP-1 monocytic cells. Innovation: The data suggest that chronic inflammation may be driven by matrix- and pathogen-derived TLR4 ligands that work in synergy to promote an exuberant innate response. Conclusion: The data suggest that the molecular mechanism underlying synergistic responses to TLR4 ligands lies upstream of IKK activation, likely in the molecular composition of the TLR4 receptor complex that assembles in response to each ligand. In addition, synergistic responses to TLR4 activation may be both cell-type and ligand specific.