Toll‐like Receptor 9 Signaling Is Augmented in Systemic Sclerosis and Elicits Transforming Growth Factor β–Dependent Fibroblast Activation

Abstract

Objective Although transforming growth factor β (TGFβ) is recognized as being a key trigger of fibroblast activation in systemic sclerosis (SSc), prominent innate immunity suggests that additional pathways contribute to disease persistence. Toll‐like receptor 9 (TLR9) is implicated in autoimmunity and fibrosis; however, the expression, mechanism of action, and pathogenic role of TLR9 signaling in SSc remain uncharacterized. The aim of this study was to explore the expression, activity, and potential pathogenic role of TLR9 in the context of skin fibrosis in SSc and in mouse models of experimental fibrosis. Methods Expression and localization of TLR9 were evaluated in SSc skin biopsy specimens and explanted skin fibroblasts. Fibrotic responses elicited by type A CpG oligonucleotide and mitochondrial DNA (mtDNA) were examined in human skin fibroblasts by a combination of real‐time quantitative polymerase chain reaction, Western blot analysis, transient transfection, immunofluorescence microscopy, and functional assays. Expression of TLR9 was examined in 2 distinct mouse models of experimental fibrosis. Results Skin biopsy specimens obtained from 2 independent cohorts of SSc patients showed up‐regulation of TLR9, and myofibroblasts were the major cellular source. Moreover, SSc skin biopsy specimens showed evidence of TLR9 pathway activation. CpG induced robust TLR9‐dependent fibrotic responses in explanted normal fibroblasts that could be blocked by bortezomib and were mediated through the action of endogenous TGFβ. Mice with experimental fibrosis showed a time‐dependent increase in TLR9 localized primarily to myofibroblasts in the dermis. Conclusion In isolated fibroblasts, TLR9 elicits fibrotic responses mediated via endogenous TGFβ. In patients with SSc, mtDNA and other damage‐associated TLR9 ligands in the skin might trigger localized activation of TLR9 signaling, TGFβ production, and consequent fibroblast activation. Disrupting this fibrotic process with inhibitors targeting TLR9 or its downstream signaling pathways might therefore represent a novel approach to SSc therapy.

Role of Toll‐like receptor 9 signaling on activation of nasal polyp–derived fibroblasts and its association with nasal polypogenesis

“…TGF‐β is an important profibrotic molecule that mediates fibroblast activation, proliferation, and myofibroblastic differentiation . Previous studies have demonstrated that induction of TLR9 protein expression in lung and skin fibroblasts from idiopathic pulmonary fibrosis (IPF) and systemic sclerosis patients can be mediated by profibrotic cytokines when stimulated with TGF‐β . In the present study, TGF‐β stimulation induced a significant increase in TLR9 mRNA compared to untreated NPDFs, and low levels of TGF‐β mRNA were observed by CpG A stimulation.…”

“…showed that IL‐6 and IFN‐α were increased in lung fibroblasts stimulated with CpG DNA, but in contrast, TNF‐α expression did not become elevated. Fang et al . stimulated skin fibroblasts in patients with systemic sclerosis with CpG DNA, which led to detectable TLR9‐mediated responses.…”

TLR4-dependent fibroblast activation drives persistent organ fibrosis in skin and lung

“…Hierarchical clustering stratified the biopsies into the previously described diffuse-proliferative and inflammatory gene expression subsets (31,32). Inflammatory intrinsic subset biopsies ( Figure 9C, lower panel), accounting for ~34% of all SSc biopsies in the present study, showed significant enrichment with the TLR4-responsive gene signature (Pearson correlation = 0.2) compared with other biopsy subsets (2 samples, 2-tailed t test, P < 0.001) (33). Analysis of a replication microarray dataset comprising 28 skin biopsies from an independent cohort of 15 dcSSc patients (GSE32413) (32) confirmed significantly higher TLR4-responsive gene signatures in biopsies mapping to the inflammatory intrinsic subset (comprising 50% of all SSc biopsies; average Pearson correlation = 0.1, t test P < 0.05) (Supplemental Figure 9).…”

“…Nuclei were identified using DAPI. Subcellular distribution of immunofluorescence was evaluated under an immunofluorescence microscope or Zeiss UV Meta 510 confocal microscope or a Nikon C2 or A1Si confocal microscope and quantitated using ImageJ (12,33).…”

Evaluation of the antifibrotic potency by knocking down SPARC, CCR2 and SMAD3

“…Mmp9, Ccr5, Mgst1), the SUMO modification (e.g. Sumo1, Ildr2), NOS/hypoxia signaling (e.g.Itga2, Fn1, Ndufa6, Cox6a2), and Toll-like receptor (TLR) pathways (e.g.Tlr9, Oas3) [3,28,[32][33][34][35]. Some of the genes including Notch3, Mmp9, Ildr2, Sumo1, Rpl39, and Ndufa6 also altered in human lung biopsy samples of pulmonary fibrosis with the similar tendency to that in our mouse models, data from GEO database (Suppl.…”