PU.1 controls fibroblast polarization and tissue fibrosis

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs

Oligomeric S100A4 Is Associated With Monocyte Innate Immune Memory and Bypass of Tolerance to Subsequent Stimulation With Lipopolysaccharides

Objectives: Most DAMPs in inflammatory diseases are TLR2- and TLR4-ligands and according to the current concept, repeated stimuli would result in tolerance. Aims of the study were to verify this assumption, to investigate whether epigenetic effectors are involved and to explore the situation in rheumatoid arthritis (RA).Methods: A trained immunity (TI) and tolerance protocol was established using peripheral blood monocytes from healthy donors, β-glucan and lipopolysaccharide (LPS). The training or tolerance capacities of RA-relevant DAMPs were tested.Results: β-Glucan-, oS100A4-, HMBG1-, and HSP90-pretreated monocytes showed increased IL-6 responses to LPS re-stimulation. β-Glucan, oS100A and tenascin C induced training of monocytes to release more TNFα. In comparison to β-glucan, most DAMPs tested induced less TI, with exception of oS100A4. Monocytes exposed to oS100A4 showed increased IL-1β, IL-6, and TNFα in response to LPS, in spite that both stimulate TLR4. RNASEq upon β-glucan or oS100A4 revealed similar changes in chemokines/cytokines and epigenetic effectors; 17 epigenetic effectors correlated with chemokine/cytokine gene expression; PRDM8 was associated with more chemokine and cytokine transcripts. Knockdown of PRDM8 abolished TI induced by oS100A4. In RA, plasma S100A4 correlated with increased CSF2, and increased PRDM8 transcription in RA monocytes was associated with increased plasma CCL5 and IL-6, as well as therapy-resistance.Conclusion: Bypass of tolerance by DAMPs might be a phenomenon as important as TI, since it could explain how chronic inflammation can be maintained in spite of an environment with multiple TLR2/TLR4-ligands. In RA monocytes, a PRDM8-dependent TI mechanism could be responsible for sustained chemokine/cytokines levels

Epigenetic deregulation in rheumatoid arthritis synovial fibroblasts

Die Rheumatoid Arthritis (RA) ist eine chronische Autoimmunerkrankung mit fortschreitender Zerstörung der Gelenke. In der Pathogenese der Erkrankung spielen zwei zelluläre Kompartimente eine wesentliche Rolle. Das eine beinhaltet aktivierte Immunzellen wie T-Zellen, B-Zellen und Makrophagen. Kennzeichnend für diese Gruppe ist das Freisetzen von entzündlichen Botenstoffen (Zytokine) wie Interleukin (IL)- 1 beta, IL-6 und „tumor-necrosis-factor“ (TNF) alpha sowie Autoantikörpern von Plasmazellen. Das andere umfasst die aktivierten synovialen Fibroblasten (RASF), Osteoklasten und Chondrozyten, alles Effektorzellen, die durch Aktivierung zur Zerstörung der Gelenke beitragen. Bis heute konnte kein genetischer Polymorphismus aufgezeigt werden, der den aktivierten Phänotyp dieser synovialer Fibroblasten ausreichend erklärt. Unsere Hypothese ist, dass epigenetische Modifikationen, insbesondere veränderte DNA Methylierung zur Aktivierung dieser Zellen beitragen und so die Zerstörung von Knorpel- und Knochensubstanz herbeiführen. Wir zeigen hier erstmalig eine Untersuchung des Methylierungsmusters synovialer Fibroblasten von Patienten mit RA. In in vivo Untersuchungen wiesen synoviale Gewebe von Patienten mit RA eine signifikante Hypomethylierung in den untersuchten Zellkernen auf im Vergleich zu Geweben von Patienten mit Osteoarthritis (OA). Und das betrifft insbesondere die synovialen Fibroblasten wie bei in vitro Untersuchungen isolierter Zellkerne gezeigt werden konnte (Chapter 2, Figure 1). Inflammatorische Zytokine wie TNF alpha und IL-1 beta hatten einen positiven Einfluss auf den Zellzyklus und gleichzeitig führten sie zu einer reduzierter Menge an 5-Methylcytosin in den Zellkernen der untersuchten Zellen. Wir zeigen hier eine reduzierte Expression von DNA-Methyltransferase 1 (DNMT1) in RASF, die möglicherweise zur progressiven Hypomethylierung dieser Zellen beiträgt. In diesem Zusammenhang unterstützen proinflammatorische Zytokine den Prozess der DNA Hypomethylierung durch Aktivierung der Zellproliferation, sie sind aber nicht die Ursache des niedrigen basalen Levels an DNMT1 in RASF. Darüber hinaus konnten wir aufzeigen, dass repetitive Sequenzen wie LINE-1 in RASF demethyliert sind, was die Hypothese einer globalen Hypomethylierung in RASF unterstützt. Um die für den aktivierten Phänotyp synovialer RA Fibroblasten verantwortlichen Gene zu identifizieren, wurden synoviele Fibroblasten von gesunden Probanden über einen Zeitraum von 2 Monaten mit nicht toxischen Mengen des DNMT1-Inhibitors 5-azacytidine (5-azaC) behandelt, und anschießend eine Genexpressionsanalyse durchgeführt. Wir konnten zeigen, dass mehr als die Hälfte der so überexprimierten Gene mit denen übereinstimmen, die mit der Pathogenese der RA assoziiert wurden. Dies sind Gene, die zur Gelenkzerstörung beitragen wie Matrix-Metalloproteinasen (MMPs), Integrine und viele weitere, die im Kapitel 2 aufgeführt sind (Suppl. Tables 1,2). In Kapitel 3 zeigen wir die Untersuchung eines spezifischen Gens mit verändertem Methylierungsmuster in RASF. Aktivierte RASF exprimieren unterschiedliche Chemokine, die so weitere Immunzellen in die betroffenen Gelenke locken. CXCL12 (SDF-1α) ist eines dieser Chemokine, welches in RASF überexprimiert wird. Wir konnten nachweisen, dass im Vergleich zu OASF, in RASF der Promotor von CXCL12 weniger methyliert ist. Weiterhin korrelierte die Expression von CXCL12 auf mRNA Level in diesen Zellen signifikant mit der Hypomethylierung im CXCL12 Promotor. Die durch Hypomethylierung verstärkte Expression von CXCL12 in RASF induzierte dann in den Zellen eine verstärkte Expression von MMPs durch die Bindung an den dazugehörigen Rezeptor CXCR7. Wir beschreiben hier einen endogenen Aktivierungsmechanismus in RASF, der zur progressiven Zerstörung der Gelenke beiträgt. Basierend auf unserer Daten verursacht eine veränderte globale und genspezifische DNA Methylierung den aktivierten invasiven Phänotyp der RASF. Summary Rheumatoid arthritis is a chronic autoimmune disease involving destruction of affected joints. Two cellular compartments are involved in the pathogenesis of RA. The first one involves activated T cells, B cells and macrophages. They secrete a variety of pro-inflammatory cytokines such as interleukin- 1beta, IL-6 and tumor necrosis factor alpha (TNFalpha) as well as a variety of autoantibodies. The second compartment involves activated rheumatoid arthritis synovial fibroblasts (RASF), osteoclasts and chondrocytes that are the effector cells of joint destruction. The thesis focuses on the epigenetic mechanisms leading to the activated phenotype of RASF. Since to date no genetic polymorphism can explain the hyperactive phenotype of synovial fibroblasts, we hypothesised that epigenetic modifications, particularly impaired DNA methylation, can cause the activation of synovial fibroblasts and lead to joint destruction. For the first time, the methylation status of cells was analysed in the synovium of patients with rheumatoid arthritis (RA). RA synovial tissues were found to have hypomethylated nuclei (Chapter 2, Figure 1). Especially RASF had low amounts of 5-methylcytosine. Pro-inflammatory cytokines such as TNF alpha and IL-1 beta induced cell cycle progression and reduced further the amount of 5-methylcytosine in the nuclei. We reported a deficiency of DNA methyltransferase 1 (DNMT1) in RASF that can be an important factor involved in the progressive demethylation. In this context, pro-inflammatory cytokines favor DNA hypomethylation by increasing the rate of cell proliferation; however, they were not responsible for low basal levels of DNMT1. Furthermore, repetitive sequences such as LINE-1 were demethylated in RASF, supporting the hypothesis of an active global hypomethylation in these cells. To mimic the chronic hypomethylation state of synovial fibroblasts and identify gene targets, normal synovial fibroblasts were treated over a long period of time (2 months) with a non-toxic dose of the DNMT1 inhibitor 5- azacytidine (5-azaC) and a gene expression analysis was performed. More than half of the genes that were found to be overexpressed by this treatment were previously associated with the pathogenesis of RA. These included genes associated with joint destruction such as matrix metalloproteinases (MMPs), integrins and others summarized in Chapter 2 (Suppl. Tables 1,2). Finally, a specific gene target that may have an impaired DNA methylation in RASF was analysed in Chapter 3. Activated RASF secrete chemokines that attract a variety of inflammatory cells into the joint. CXCL12 (SDF-1α) is a chemokine overexpressed and secreted by RASF. We reported that the promoter of CXCL12 is less methylated in RASF than in osteoarthritis synovial fibroblasts (OASF). The mRNA expression of CXCL12 significantly correlated with the CXCL12 promoter methylation. The upregulation of CXCL12 could stimulate RASF to produce more MMPs via the receptor CXCR7. Thereby, we describe an endogenously activated pathway in RASF which promotes joint destruction. In conclusion, this study confirms the hypothesis that global and gene specific DNA methylation alterations are responsible for the activated phenotype of RASF

Epigenetic changes: the missing link

The association of rheumatoid arthritis (RA) with a number of genetic risk loci is well established; however, only part of the risk to develop the disease is based on genetics. Environmental factors significantly contribute to the pathogenesis. A gene-environment interaction for smoking and certain major histocompatibility complex (MHC) class II alleles has been shown to promote anti-citrullinated protein antibody (ACPA)-positive RA; however, the molecular mechanisms of interaction remain unclear. In contrast to the genetic background, epigenetic factors are responsive to external stimuli and can modulate gene expression. Therefore, epigenetic mechanisms may function as intermediaries between genetic risk alleles and environmental factors. In this review, epigenetic mechanisms are explained and the evidence for epigenetic changes relevant for the pathogenesis of RA and potential therapeutic applications are discussed

Inhibition of spermidine/spermine n1-acetyltransferase activity: a new therapeutic concept in rheumatoid arthritis

OBJECTIVE Changes in polyamine-modulated factor 1 (PMF-1) promoter methylation might favor the expression of spermidine/spermine N1-acetyltransferase 1 (SSAT-1), causing excessive consumption of S-adenosyl methionine (SAM). This study was undertaken to evaluate the effect of SSAT-1 activity inhibition, either alone or in combination with SAM. METHODS Synovial fibroblasts were isolated from patients with rheumatoid arthritis (RA) or osteoarthritis (OA). PMF-1 promoter methylation was determined by pyrosequencing. Small interfering RNAs (siRNAs) against SSAT-1 were transfected weekly in RA synovial fibroblasts (RASFs). In addition, synovial fibroblasts were treated with diminazene aceturate (DA), an inhibitor of SSAT-1. SSAT-1, 5-methylcytosine (5-MeC), adenosyl methionine decarboxylase (AMD), PMF-1, DNA methyltransferase 1 (DNMT-1), CXCL12, β1 integrin, and CD44 levels were measured by flow cytometry. Putrescine levels were determined by colorimetry. Levels of matrix metalloproteinases were measured by enzyme-linked immunosorbent assay. Cell adhesion was tested. The SCID mouse model of RA was used to monitor the invasiveness of RASFs. RESULTS RASFs showed elevated SSAT-1, AMD, and PMF-1 levels. However, PMF-1 promoter methylation was unchanged. Transfection of siRNA targeting SSAT-1 increased 5-MeC levels within 21 days. Similarly, DA increased 5-MeC levels in RASFs. In addition, DA increased the levels of DNMT-1, decreased the levels of AMD, putrescine, activation markers, and MMP-1, and altered the adhesion of RASFs. DA was more efficient in RASFs with higher levels of SSAT-1. Most interestingly, the combination of DA and SAM reduced the invasiveness of RASFs by 70%. CONCLUSION The use of DA alone or in combination with SAM/l-methionine might introduce a new therapeutic concept in RA. This is the first therapy that would directly target RASFs and thereby inhibit ongoing joint destruction

Epigenome analysis reveals TBX5 as a novel transcription factor involved in the activation of rheumatoid arthritis synovial fibroblasts

In this study, we analyzed the methylation status of human promoters in rheumatoid arthritis synovial fibroblasts (RASF). Differentially methylated genes between RASF and osteoarthritis synovial fibroblasts (OASF) were identified by methylated DNA immunoprecipitation and hybridization to human promoter tiling arrays. The methylation status was confirmed by pyrosequencing. Gene and protein expression of differentially methylated genes was evaluated with real-time PCR, Western blot, and immunohistochemistry. Chromatin immunoprecipitation was used to measure the gene promoter-associated acetylation and methylation of histones. Transcription factor-specific targets were identified with microarray and luciferase assays. We found that the transcription factor T-box transcription factor 5 (TBX5) was less methylated in rheumatoid arthritis (RA) synovium and RASF than in osteoarthritis (OA) samples. Demethylation of the TBX5 promoter in RASF and RA synovium was accompanied by higher TBX5 expression than in OASF and OA synovium. In RA synovium, TBX5 expression was primarily localized to the synovial lining. In addition, the TBX5 locus was enriched in activating chromatin marks, such as histone 4 lysine 4 trimethylation and histone acetylation, in RASF. In our functional studies, we observed that 790 genes were differentially expressed by 2-6-fold after overexpression of TBX5 in OASF. Bioinformatic analysis of these genes revealed that the chemokines IL-8, CXCL12, and CCL20 were common targets of TBX5 in OASF. Taken together, our data show that TBX5 is a novel inducer of important chemokines in RASF. Thus, we conclude that RASF contribute to the inflammatory processes operating in the pathogenesis of RA via epigenetic control of TBX5

TET1 is an important transcriptional activator of TNFα expression in macrophages

Activation of macrophages and overexpression of TNFα is associated with the pathogenesis of chronic inflammatory diseases. However, the mechanisms leading to TNFα overexpression are still unknown. 5-methylocytosine (5-mC) is an epigenetic modification that is associated with silenced genes. Recent studies showed that it is converted to 5-hydroxylmethylocytosine (5-hmC) and reactivates gene expression through the action of the family of Ten-Eleven-Translocation (TET1-3) enzymes. In this study, we show that 5-hmC levels are increased globally and specifically in the TNFα promoter during the differentiation of monocytes to macrophages. In addition, the levels of 5-hmC are increased upon LPS stimulation of macrophages. Furthermore, CRIPSR stable knockout of TET1 decreases the expression of TNFα and other pro-inflammatory cytokines. In conclusion, we showed that TET1 contributes to the activation of macrophages possibly through regulation of 5-hydroxymethylation in the promoter of pro-inflammatory cytokine genes. The TET1 enzyme could be a promising therapeutic target to inhibit the persistent inflammation caused by macrophages in chronic inflammatory diseases

Epigenetic control in rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis synovial fibroblasts (RASFs) are the effector cells of cartilage and bone destruction. These cells show an 'intrinsically' activated and aggressive phenotype that results in the increased production of matrix-degrading enzymes and adhesion molecules, and is conserved over long-term passage in vitro. The three main mechanisms of epigenetic control -- DNA methylation, histone modifications and microRNA activity -- interact in the development of the RASF phenotype. The extent of global DNA methylation is reduced in synoviocytes in situ and RASFs in vitro. In addition, histone hyperacetylation occurs and specific microRNAs are expressed in RASFs. Normal synovial fibroblasts cultured in a hypomethylating milieu acquire an activated phenotype similar to that of RASFs. These findings suggest that epigenetic control, in particular the control of DNA methylation, is deficient in RASFs. Genome-wide analyses of the epigenome will enable the detection of additional genes involved in the pathogenesis of rheumatoid arthritis, the identification of epigenetic biomarkers, and potentially the development of a therapeutic regimen that targets activated RASFs

Oligomeric S100A4 Is Associated With Monocyte Innate Immune Memory and Bypass of Tolerance to Subsequent Stimulation With Lipopolysaccharides

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