Lipopolysaccharide responsiveness in vocal fold fibroblasts

Abstract

BackgroundVocal fold fibroblast’s (VFF) strategic location in the lamina propria and their ability to respond to external stimuli by producing inflammatory molecules suggest their possible direct involvement in innate immunity. Toll-like receptors (TLRs) are an essential signaling component to this response, as they allow for recognition of various microorganisms, leading to subsequent induction of pro-inflammatory genes. The objective of this study was to elucidate the role of VFF in the host immune response and subsequent influence on inflammatory cytokine secretion.MethodsVFF derived from polyp, scar, and normal tissue were treated with 5 μg/ml lipopolysaccharide (LPS). TLR1 through 9, CD14, and MD-2 were measured during stable conditions by polymerase chain reaction (PCR). Expression of TLR4 and IL-1R type-1 genes were quantified after 24 hrs LPS stimulation by reverse transcription-PCR. LPS responsiveness was determined by NF-κB nuclear translocation as measured by subunit p65 expression in nucleus with immunocytochemistry. Downstream effects were confirmed with immunoassay measuring IL-8 concentrations in supernatant after 8 hrs.ResultsAll VFFs constitutively expressed TLR1 to 6, TLR9, CD14, and MD-2 mRNA. Polyp VFF exhibited significantly higher TLR4 transcript levels (p < 0.001) in comparison to scar and normal VFF. LPS stimulated scar and polyp VFF exhibited increased levels of p65 in the nucleus (p < 0.01) and secreted greater IL-8 protein (p < 0.0001) compared to normal VFF.ConclusionVFF constitutively express genes for the receptors essential to the host immune response. Scar and polyp VFF produced greater LPS responsiveness resulting in over-activated inflammatory patterns. These findings support VFF role in the pathogenesis of inflammatory vocal fold disorders and suggests their presence in the wound bed could lead to chronic inflammation.

Characterization of the Leukocyte Response in Acute Vocal Fold Injury

“…Vocal fold macrophages response to LPS may be reflective of differences across organs. Fibroblasts are the most abundant cell type in the vocal fold [ 1 ] and have been shown in vitro to activate TLR4 ligation in response to LPS, although they do not appear to produce measureable levels of TNFα, IL–12 or IL–10 cytokines in response [ 22 ]. Further work, has demonstrated that vocal fold fibroblasts can influence the function of activated macrophages when co-cultured together [ 32 ].…”

“…Targets were amplified using GoTaq Hot Start Polymerase (Promega Corporation, Madison, WI) with the PTC- 200 Peltier Thermal Cycler according to the manufacturer’s protocol. Amplifications were optimized for each primer and carried out for 40 cycles as previously described [ 22 ]. Yields underwent electrophoresis on 2% agarose gel using ethidium bromide staining for visualization.…”

Cellular source and proinflammatory roles of high-mobility group box 1 in surgically injured rat vocal folds

“…Extracellular HMGB1 interacts with multiple cell surface receptors such as the receptor for advanced glycation end products, Toll‐like receptor (TLR) 2, and TLR 4, which in turn activates the nuclear factor‐κB (NF‐κB) signal pathway. NF‐κB is a transcription factor found in almost all mammalian cell types, including human vocal fold fibroblasts . The NF‐κB family of transcription factors plays a key role in the immune response by regulating a wide range of gene expression, including interleukins (ILs), tumor necrosis factors (TNFs), chemokines, growth factors, adhesion molecules, and inhibitors of apoptosis.…”

Liquid-type non-thermal atmospheric plasma ameliorates vocal fold scarring by modulating vocal fold fibroblast

“…To mimic the inflammatory conditions of VF injury, the following experiments were designed to treat with LTP after LPS treatment. 14 First, 30 or 60 s of LTP with/without LPS showed no toxic effects on the viability of human VF-MSCs. No significant cytotoxicity was observed in LPS-only-treated human VF-MSCs.…”