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Effects of Toll-Like Receptor 4 Inhibition on Transforming Growth Factor-β2 Signaling in the Human Trabecular Meshwork.

ABSTRACT: Purpose: Transforming growth factor-β2 (TGFβ2) and Toll-like receptor 4 (TLR4) crosstalk have been implicated in extracellular matrix regulation in the trabecular meshwork (TM) and ocular hypertension in mice. We investigated TLR4 expression in normal and glaucomatous human trabecular meshwork (HTM) sections and utilized a human perfusion organ culture model to determine TGFβ2-TLR4 signaling crosstalk in glaucoma. Methods: Expression of TLR4 was determined in TM of normal and glaucomatous human eyes. Anterior segments of paired human eyes were perfused at a constant flow rate (2.5 μL/min) for 4 days to acquire stable baseline intraocular pressures (IOPs). We treated paired eyes with two different treatment paradigms: (1) TGFβ2 in one eye and vehicle control in the paired eye, (2) TGFβ2 in one eye and TGFβ2 + TLR4 inhibitor TAK-242 in the paired eye. Perfusate and TM tissue were collected and analyzed for fibronectin (FN) and collagen IV (COLIV) expression. Results: We observed increased TLR4 expression in glaucomatous HTM sections compared to normal (age-matched) (P < 0.05). Significant elevation of IOP was detected in 47% of TGFβ2-treated anterior segments (P < 0.01) compared to control, and in TGFβ2 treated compared with co-treatment with TGFβ2 + TLR4 inhibitor (P < 0.0001). An increase in FN and COLIV expression was observed after TGFβ2 treatment, and inhibition of TLR4 signaling decreased TGFβ2-induced FN and COLIV expression in perfusate (P < 0.05). Conclusions: These studies identify TGFβ2-TLR4 crosstalk as a novel pathway in glaucoma. They provide a potential new target to lower IOP and explore glaucoma pathogenesis.

SUBMITTER: Sharma TP

PROVIDER: S-EPMC7175615 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundElevated transforming growth factor (TGF)-β2 in aqueous humor (AH) has been suggested to contribute to trabecular meshwork (TM) fibrosis and intraocular pressure (IOP) regulation in primary open-angle glaucoma (POAG), but TGF-β2 is downregulated in secondary open-angle glaucoma (SOAG). Because autotaxin (ATX) is upregulated in SOAG, we investigated the relationships and trans-signaling interactions of these mediators.MethodsThe level of ATX in AH was determined using a two-site immunoenzymetric assay, and TGF-β levels were measured using the Bio-Plex Pro TGF-β Assay. RNA scope was used to assess the expression of ATX and TGF-β2 in human's eye specimen. And in vitro studies were performed using hTM cells to explore if trans-signaling of TGF-β2 regulates ATX expressions.ResultsTGF-β2/ATX ratio was significantly high in AH of control or POAG compared with SOAG, and negatively correlated with IOP. RNA scope revelated positive expressions of both TGF-β2 and ATX in ciliary body (CB) and TM in control, but ATX expressions was significantly enhanced in SOAG. In hTM cells, ATX expressions were regulated by TGF-β2 with concentration-dependent manner. In counter, ATX also induced TGF-β1, TGF-β2 and TGFBI upregulations and activation of the Smad-sensitive promoter, as well as upregulation of fibrotic markers, and these upregulation was significantly suppressed by both TGF-β and ATX inhibition.ConclusionsTrans-signaling of TGF-β2 regulates ATX expressions and thereby induced upregulations of TGF-βs or fibrosis of hTM. TGF-β2 trans-signaling potently regulate ATX transcription and signaling in hTM cells, which may reflect different profile of these mediators in glaucoma subtypes. Trial Registration This prospective observational study was approved by the Institutional Review Board of the University of Tokyo and was registered with the University Hospital Medical Information Network Clinical Trials Registry of Japan (ID: UMIN000027137). All study procedures conformed to the Declaration of Helsinki. Written informed consent was obtained from each patient.

Project description:Variations in the Toll-interacting protein (TOLLIP) gene have been identified in genome-wide association studies to correlate with risk of disease, mortality, and response to N-acetylcysteine therapy in idiopathic pulmonary fibrosis. Although TOLLIP is known to modulate innate immune responses, its relevance in organ fibrogenesis remains unknown. Prior work in the literature suggests TOLLIP dampens transforming growth factor beta (TGFβ) signaling in human cell lines. In this study, we examined the role of TOLLIP in mouse lung fibroblast (MLF) responses to TGFβ and in the bleomycin model of experimental lung fibrosis using Tollip-/- mice. We hypothesize that if TOLLIP negatively regulates TGFβ signaling, then Tollip-/- mouse lung fibroblasts (MLFs) would have enhanced response to TGFβ treatment, and Tollip-/- mice would develop increased fibrosis following bleomycin challenge. Primary MLFs were stimulated with TGFβ (1 ng/mL) for 24 h. RNA was obtained to assess global transcriptional responses by RNA-seq and markers of myofibroblast transition by qPCR. Functional assessment of TGFβ-stimulated MLFs included cell migration by scratch assay, cell proliferation, and matrix invasion through Matrigel. In the in vivo model of lung fibrosis, Tollip-/- mice and wild-type (WT) littermates were administered bleomycin intratracheally and assessed for fibrosis. We further examined TGFβ signaling in vivo after bleomycin injury by SMAD2, ERK1/2, and TGFβR1 Western blot. In response to TGFβ treatment, both WT and Tollip-/- MLFs exhibited global transcriptional changes consistent with myofibroblast differentiation. However, Tollip-/- MLFs showed greater number of differentially expressed genes compared to WT MLFs and greater upregulation of Acta2 by qPCR. Functionally, Tollip-/- MLFs also exhibited increased migration and Matrigel invasiveness compared to WT. We found evidence of enhanced TGFβ signaling in Tollip-/- through SMAD2 in vitro and in vivo. Tollip-/- mice experienced lower survival using a standard weight-adjusted dosing without evidence of differences in fibrosis at Day 21. With adjustment of dosing for sex, no differences were observed in fibrosis at Day 21. However, Tollip-/- mice had greater weight loss and increased bronchoalveolar lavage fluid total protein during early resolution at Day 14 compared to WT without evidence of differences in acute lung injury at Day 7, suggesting impaired resolution of lung injury.

Project description:ObjectiveAlthough 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.MethodsExpression 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.ResultsSkin 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.ConclusionIn 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.

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Effects of Toll-Like Receptor 4 Inhibition on Transforming Growth Factor-β2 Signaling in the Human Trabecular Meshwork.

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