Poly(I:C) Drives Type I IFN- and TGFβ-Mediated Inflammation and Dermal Fibrosis Simulating Altered Gene Expression in Systemic Sclerosis

ObjectivesThere is considerable evidence that implicates dysregulation of type I interferon signalling (or type I IFN signature) in the pathogenesis of systemic sclerosis (SSc). Interferon regulatory factor 7 (IRF7) has been recognised as a master regulator of type I IFN signalling. The objective of this study was to elucidate the role of IRF7 in dermal fibrosis and SSc pathogenesis.MethodsSSc and healthy control skin biopsies were investigated to determine IRF7 expression and activation. The role of IRF7 in fibrosis was investigated using IRF7 knockout (KO) mice in the bleomycin-induced and TSK/+mouse models. In vitro experiments with dermal fibroblasts from patients with SSc and healthy controls were performed.ResultsIRF7 expression was significantly upregulated and activated in SSc skin tissue and explanted SSc dermal fibroblasts compared with unaffected, matched controls. Moreover, IRF7 expression was stimulated by IFN-α in dermal fibroblasts. Importantly, IRF7 co-immunoprecipitated with Smad3, a key mediator of transforming growth factor (TGF)-β signalling, and IRF7 knockdown reduced profibrotic factors in SSc fibroblasts. IRF7 KO mice demonstrated attenuated dermal fibrosis and inflammation compared with wild-type mice in response to bleomycin. Specifically, hydroxyproline content, dermal thickness as well as Col1a2, ACTA2 and interleukin-6 mRNA levels were significantly attenuated in IRF7 KO mice skin tissue. Furthermore, IRF7 KO in TSK/+mice attenuated hydroxyproline content, subcutaneous hypodermal thickness, Col1a2 mRNA as well as α-smooth muscle actin and fibronectin expression.ConclusionsIRF7 is upregulated in SSc skin, interacts with Smad3 and potentiates TGF-β-mediated fibrosis, and therefore may represent a promising therapeutic target in SSc.

Download Full-text

Histone modifications underlie monocyte dysregulation in patients with systemic sclerosis, underlining the treatment potential of epigenetic targeting

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2018-214295 ◽

2019 ◽

Vol 78 (4) ◽

pp. 529-538 ◽

Cited By ~ 10

Author(s):

Maarten van der Kroef ◽

Monica Castellucci ◽

Michal Mokry ◽

Marta Cossu ◽

Marianna Garonzi ◽

...

Keyword(s):

Gene Expression ◽

Systemic Sclerosis ◽

Chromatin Immunoprecipitation ◽

Transcription Start ◽

Histone Marks ◽

Transcription Start Sites ◽

Altered Gene Expression ◽

Bromodomain Proteins ◽

Altered Gene

Background and objectiveSystemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes.MethodsChromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFN-responsive gene expression.Results1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFNα induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression.ConclusionSSc monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc.

Download Full-text

UMP-CMP kinase 2 gene expression in macrophages is dependent on the IRF3-IFNAR signaling axis

PLoS ONE ◽

10.1371/journal.pone.0258989 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0258989

Author(s):

Hera Kim ◽

Yashwanth Subbannayya ◽

Fiachra Humphries ◽

Astrid Skejsol ◽

Sneha M. Pinto ◽

...

Keyword(s):

Gene Expression ◽

Type I Interferon ◽

Neutralizing Antibody ◽

Acid Synthesis ◽

Subcellular Fractionation ◽

Poly I:C ◽

Type I ◽

Type I Ifn ◽

Downstream Signaling ◽

Protein Levels

Toll-like receptors (TLRs) are highly-conserved pattern recognition receptors that mediate innate immune responses to invading pathogens and endogenous danger signals released from damaged and dying cells. Activation of TLRs trigger downstream signaling cascades, that culminate in the activation of interferon regulatory factors (IRFs), which subsequently leads to type I interferon (IFN) response. In the current study, we sought to expand the scope of gene expression changes in THP1-derived macrophages upon TLR4 activation and to identify interferon-stimulated genes. RNA-seq analysis led to the identification of several known and novel differentially expressed genes, including CMPK2, particularly in association with type I IFN signaling. We performed an in-depth characterization of CMPK2 expression, a nucleoside monophosphate kinase that supplies intracellular UTP/CTP for nucleic acid synthesis in response to type I IFN signaling in macrophages. CMPK2 was significantly induced at both RNA and protein levels upon stimulation with TLR4 ligand—LPS and TLR3 ligand—Poly (I:C). Confocal microscopy and subcellular fractionation indicated CMPK2 localization in both cytoplasm and mitochondria of THP-1 macrophages. Furthermore, neutralizing antibody-based inhibition of IFNAR receptor in THP-1 cells and BMDMs derived from IFNAR KO and IRF3 KO knockout mice further revealed that CMPK2 expression is dependent on LPS/Poly (I:C) mediated IRF3- type I interferon signaling. In summary, our findings suggest that CMPK2 is a potential interferon-stimulated gene in THP-1 macrophages and that CMPK2 may facilitate IRF3- type I IFN-dependent anti-bacterial and anti-viral roles.

Download Full-text

Molecular and Microscopic Analysis of Altered Gene Expression in Transgenic and Gene Targeted Mice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162521 ◽

1996 ◽

Vol 54 ◽

pp. 12-13

Author(s):

W. K. Jones ◽

J. Robbins

Keyword(s):

Gene Expression ◽

Pulmonary Veins ◽

Microscopic Analysis ◽

Mouse Tissue ◽

Adult Heart ◽

Heavy Chains ◽

Altered Gene Expression ◽

Altered Gene ◽

Pulmonary Myocardium

Two myosin heavy chains (MyHC) are expressed in the mammalian heart and are differentially regulated during development. In the mouse, the α-MyHC is expressed constitutively in the atrium. At birth, the β-MyHC is downregulated and replaced by the α-MyHC, which is the sole cardiac MyHC isoform in the adult heart. We have employed transgenic and gene-targeting methodologies to study the regulation of cardiac MyHC gene expression and the functional and developmental consequences of altered α-MyHC expression in the mouse.We previously characterized an α-MyHC promoter capable of driving tissue-specific and developmentally correct expression of a CAT (chloramphenicol acetyltransferase) marker in the mouse. Tissue surveys detected a small amount of CAT activity in the lung (Fig. 1a). The results of in situ hybridization analyses indicated that the pattern of CAT transcript in the adult heart (Fig. 1b, top panel) is the same as that of α-MyHC (Fig. 1b, lower panel). The α-MyHC gene is expressed in a layer of cardiac muscle (pulmonary myocardium) associated with the pulmonary veins (Fig. 1c). These studies extend our understanding of α-MyHC expression and delimit a third cardiac compartment.

Download Full-text