TLR4 Ligands Selectively Synergize to Induce Expression of IL-8

Abstract

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-a), 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-jB) 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. Keywords: fibronectin, TLR4, IL-8, inflammation, LPS INTRODUCTIONProlonged inflammation in response to injury is a contributory factor to the development of chronic wounds. Extracellular matrix (ECM)- fibronectin has also been shown to promote fibro-inflammatory responses in several mouse models. 9,17,18 The EDA domain of fibronectin can activate TLR4 signaling as either the individual domain, proteolytic fragment or in the context of the intact molecule. 19,20 Being responsible for matrix synthesis, assembly, and turnover, stromal fibroblasts are in constant dialogue with the fibronectin matrix in its polymerized and fragmented forms. Changes in tissue mechanical forces have been shown to caus...

Inflammatory gene expression during acute high‐altitude exposure

“…Activation of TLR4 might also contribute to the release of IL8, a chemotactic cytokine that recruits and mobilizes neutrophils to sites of infection. Previous studies have shown that damage to the extracellular matrix following cellular stress contributes to a positive feedback loop that drives a TLR‐dependent chronic inflammation, including chronic IL8 expression (Valenty et al., 2017). Our data revealed that IL8 mRNA expression is significantly upregulated on both the first and third days at high altitude (Fig.…”

Fibronectin Functions as a Selective Agonist for Distinct Toll-like Receptors in Triple-Negative Breast Cancer

“…In earlier studies we have shown that the addition of the fibronectin-derived DAMPS, FnEDA and FnIII-1c, to human embryonic skin fibroblasts, A1-F, stimulated the TLR4/NFκBdependent release of the proinflammatory cytokines, IL-8 and TNFα [25,27,39,40]. In the current study, we evaluated the effect of these fibronectin DAMPs on cytokine release in two subtypes of triple-negative breast cancer cells, MDA-MB-468 (basal-like1) and MDA-MB-231 (mesenchymal stem-like).…”

Recombinant Treponema pallidum protein Tp0136 promotes fibroblast migration by modulating MCP‐1/CCR2 through TLR4

“…The exogenous ligands of TLR4 are pathogen‐associated molecular patterns (PAMPs), and the most important exogenous ligand is LPS in gram‐negative bacteria . Endogenous ligands of TLR4 are damage‐associated molecular patterns (DAMPs), including fibronectin III1‐c and fibronectin III‐EDA, which are components of fibronectin in the extracellular matrix that are produced after enzymatic hydrolysis during injury . These DAMPs activate TLR4 and promote injury healing .…”

“…28 These DAMPs activate TLR4 and promote injury healing. 28 It is well known that T. pallidum lacks the most important virulence factor of gram-negative bacteria, LPS. 29 Interestingly, in this study, we demonstrated that Tp0136 could promote MCP-1 secretion through TLR4.…”

“…Endogenous ligands of TLR4 are damage‐associated molecular patterns (DAMPs), including fibronectin III1‐c and fibronectin III‐EDA, which are components of fibronectin in the extracellular matrix that are produced after enzymatic hydrolysis during injury . These DAMPs activate TLR4 and promote injury healing . It is well known that T. pallidum lacks the most important virulence factor of gram‐negative bacteria, LPS .…”