BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The kinase inhibitor nintedanib specific for vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) significantly reduced the rate of decline of forced vital capacity versus placebo.AimTo determine the in vitro effect of nintedanib on primary human lung fibroblasts. Methods: Fibroblasts were isolated from lungs of IPF patients and from non-fibrotic controls. We assessed the effect of VEGF, PDGF-BB and basic FGF (bFGF) ± nintedanib on: (i) expression/activation of VEGFR, PDGFR, and FGFR, (ii) cell proliferation, secretion of (iii) matrix metalloproteinases (MMP), (iv) tissue inhibitor of metalloproteinase (TIMP), and (v) collagen.ResultsIPF fibroblasts expressed higher levels of PDGFR and FGFR than controls. PDGF-BB, bFGF, and VEGF caused a pro-proliferative effect which was prevented by nintedanib. Nintedanib enhanced the expression of pro-MMP-2, and inhibited the expression of TIMP-2. Transforming growth factor-beta-induced secretion of collagens was inhibited by nintedanib.ConclusionOur data demonstrate a significant anti-fibrotic effect of nintedanib in IPF fibroblasts. This effect consists of the drug’s anti-proliferative capacity, and on its effect on the extracellular matrix, the degradation of which seems to be enhanced.

Seidel P, Merfort I, Hughes JM, Oliver BG, Tamm M, Roth M. Dimethylfumarate inhibits NF-B function at multiple levels to limit airway smooth muscle cell cytokine secretion.

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BACKGROUND AND PURPOSEAirway remodelling is a consequence of long-term inflammation and MAPKs are key signalling molecules that drive pro-inflammatory pathways. The endogenous MAPK deactivator -MAPK phosphatase 1 (MKP-1) -is a critical negative regulator of the myriad pro-inflammatory pathways activated by MAPKs in the airway. EXPERIMENTAL APPROACHHerein we investigated the molecular mechanisms responsible for the upregulation of MKP-1 in airway smooth muscle (ASM) by the corticosteroid dexamethasone and the b2-agonist formoterol, added alone and in combination. KEY RESULTSMKP-1 is a corticosteroid-inducible gene whose expression is enhanced by long-acting b2-agonists in an additive manner. Formoterol induced MKP-1 expression via the b2-adrenoceptor and we provide the first direct evidence (utilizing overexpression of PKIa, a highly selective PKA inhibitor) to show that PKA mediates b2-agonist-induced MKP-1 upregulation. Dexamethasone activated MKP-1 transcription in ASM cells via a cis-acting corticosteroid-responsive region located between -1380 and -1266 bp of the MKP-1 promoter. While the 3′-untranslated region of MKP-1 contains adenylate + uridylate elements responsible for regulation at the post-transcriptional level, actinomycin D chase experiments revealed that there was no increase in MKP-1 mRNA stability in the presence of dexamethasone, formoterol, alone or in combination. Rather, there was an additive effect of the asthma therapeutics on MKP-1 transcription. CONCLUSIONS AND IMPLICATIONSTaken together, these studies allow us a greater understanding of the molecular basis of MKP-1 regulation by corticosteroids and b2-agonists and this new knowledge may lead to elucidation of optimized corticosteroid-sparing therapies in the future. AbbreviationsAd5, adenoviral serotype 5; ASM, airway smooth muscle; CREB, cyclic-AMP response element binding protein; GC, glucocorticoid; GRE, GC-responsive element; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; MKP-1, MAPK phosphatase 1; MOI, multiplicity of infection; 3'-UTR, 3′-untranslated region

Airway smooth muscle cells (ASMCs) secrete eotaxin and RANTES (regulated on activation, normal T-cell expressed and secreted) in response to tumour necrosis factor (TNF)-a, which is inhibited by the nuclear factor (NF)-kB inhibitor dimethylfumarate (DMF). NF-kB/IkB (inhibitor of NF-kB) glutathionylation and changes in chromatin remodelling can inhibit NF-kB activity. In this study, we determined whether NF-kB/IkB glutathionylation and reduced histone H3 phosphorylation might underlie the inhibitory effect of DMF on NF-kB activity, and eotaxin and RANTES secretion.Primary human ASMCs were treated with DMF, diamide and/or glutathione (GSH) ethylester (OEt) prior to TNF-a stimulation and were subsequently analysed by ELISA, electrophoretic mobility shift assay, immunofluorescence, co-immunoprecipitation or immunoblotting.DMF reduced intracellular GSH and induced IkBa glutathionylation (IkBa-SSG), which inhibited IkBa degradation, NF-kB p65 nuclear entry and NF-kB/DNA binding. In addition, DMF inhibited the phosphorylation of histone H3, which was possibly mediated by the inhibitory effect of DMF on mitogen-and stress-activated protein kinase (MSK)-1. However, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK and MAPK phosphatase-1, upstream of MSK-1, were not inhibited by DMF. Importantly, DMF-mediated effects on NF-kB, histone H3, eotaxin and RANTES were reversed by addition of GSH-OEt.Our data suggest that DMF inhibits NF-kB-dependent eotaxin and RANTES secretion by reduction of GSH with subsequent induction of IkBa-SSG and inhibition of histone H3 phosphorylation. Our findings offer new potential drug targets to reduce airway inflammation in asthma.