scholarly journals Screening for CEBPD-modulating compounds using a THP-1-derived reporter cell line in the context of rheumatoid arthritis

2021 ◽  
Author(s):  
◽  
Tatjana Ullmann
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
Drug Discovery ◽  
Mrna Expression ◽  
Hdac Inhibitor ◽  
Screening Assay ◽  
Base Pairs ◽  
Anti Inflammatory ◽  
Time Point ◽  
Inflammatory Effect

Development of treatment strategies of chronic inflammatory disorders relies on on-going progress in drug discovery approaches and related molecular biologics. This study presents a gene reporter-based approach of phenotypic screening for anti-inflammatory compounds in the context of rheumatoid arthritis (RA). CEBPD gene, used as the target gene for the screening readout, encodes CCAAT/enhancer binding protein delta (C/EBPδ) transcription factor (TF). Structural and regulatory characteristics of CEBPD gene as well as function of C/EBPδ TF in the context of inflammation satisfied assay requirements. C/EBPδ TF acts as a key regula-tor of inflammatory gene transcription in macrophages (Mϕ) and is observed to con-tribute to disease development in both a rodent model of RA and RA patient biopsies. Despite well-described pro-inflammatory effects of C/EBPδ TF, it functions as a cell context-specific signal integrator showing also an anti-inflammatory activity. Conse-quently, both activation and inhibition of CEBPD alike may display a desired anti-inflammatory effect. The aim of this study was to develop a high-throughput screening assay for CEBPD-modulating compounds and confirm hit compounds’ anti-inflammatory effects via gene expression analysis. Generation and characterization of a multi-gene-reporter cassette 1.0 encoding enzy-matic secreted alkaline phosphatase (SEAP) gene reporter was a priority during the assay development. Chemiluminescent SEAP assay demonstrating high assay sensitivi-ty, broad linear range, high reproducibility and repeatability was chosen to monitor activity of the defined CEBPD promoter (CEBPD::SEAP). PMA-differentiated and M1-polarized THP-1-derived Mϕ stably expressing multi-gene-reporter cassette 1.0 were used as the assay’s cellular system. mRNA expression of both reporter CEBPD::SEAP and endogenous CEBPD mirrored each other in response to a LPS and IFN-g-triggered inflammatory stimulus (M1 treatment), even though the defined CEBPD promoter re-gion, utilized in the assay, contained only the most proximal and known regulatory se-quences. SEAP chemiluminescence in the reporter cells´ supernatant reliably correlat-ed with the M1 treatment-induced CEBPD::SEAP gene expression. The final screening protocol was developed for semi-automatic screening in the 384-well format. In total, 2054 compounds from LOPAC®1280 and ENZO®774 libraries were screened twice using the enzymatic SEAP readout with subsequent analysis of 18 selected compounds: nine with the highest and nine with the lowest signals, further characterized by qPCR. Gene expression levels of endogenous CEBPD, CEBPD::SEAP reporter as well as, IL-6, IL-1β, and CCL2 as inflammatory markers were quantified. qPCR assays failed to corre-late to SEAP readout in 15 compounds within three standard deviations (SDs) from sol-vent control: nine low signal and six high signal compounds. Demonstrating both assay sensitivity and specificity, a correlation between qPCR gene expression and SEAP readout was observed for three hit compounds with signals above three SDs: BET inhib-itors (BETi) GSK 1210151A and Ro 11-1464 as well as an HDAC inhibitor (HDACi) vori-nostat. The control compound trichostatin A (TSA) that reproducibly upregulated SEAP readout is also an HDAC inhibitor with a similar structure to vorinostat and was there-fore included in the anti-inflammatory phenotype analysis. The observed suppression of IL-6, IL-1ß, and CCL2 gene expression by hit compounds suggested their anti-inflammatory effect in THP-1 reporter Mϕ. mRNA expression of IL-6 and CCL2 was suppressed by HDACi and BETi at both 4 and 24 hours, while BETi reduced IL-1β mRNA expression 24 hour time point. BETi significantly upregulated gene expression of both reporter CEBPD::SEAP and endogenous CEBPD, 4 hours after M1 treatment. At the same time point, HDACi completely abolished the mRNA expres-sion of the endogenous CEBPD, while simultaneously upregulating mRNA expression of the reporter CEBPD::SEAP. The use of the most proximal 300 base pairs region of en-dogenous CEBPD promoter, making the upstream regulatory elements unavailable in the assay, may account for differential expression levels of SEAP and C/EBPδ TF. This observation corroborated the need to include a longer and more extensive CEBPD´s gene regulatory area. Thus, an improved multi-gene-reporter cassette 2.0 was gener-ated to be used on the basis of a bacterial artificial chromosome (BAC) covering CE-BPD´s genomic area of about 200,000 base pairs. The generated screening assay is flexible, reliable, and sensitive displaying potential for drug discovery and drug repurposing. The pharmacological modulation of CEBPD gene expression, first reported for GSK 1210151A, Ro 11-1464, and vorinostat, contrib-utes to the understanding of inflammatory responses in Mϕ and may have RA thera-peutic applications.

scholarly journals High-Throughput Screening for CEBPD-Modulating Compounds in THP-1-Derived Reporter Macrophages Identifies Anti-Inflammatory HDAC and BET Inhibitors

2021 ◽  
Vol 22 (6) ◽  
pp. 3022
Author(s):  
Tatjana Ullmann ◽  
Sonja Luckhardt ◽  
Markus Wolf ◽  
Michael J. Parnham ◽  
Eduard Resch
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
High Throughput ◽  
Transcription Initiation ◽  
High Throughput Screening ◽  
Inflammatory Responses ◽  
Hdac Inhibitors ◽  
Screening Assay ◽  
Bet Inhibitors ◽  
Anti Inflammatory

This study aimed to identify alternative anti-inflammatory compounds that modulate the activity of a relevant transcription factor, CCAAT/enhancer binding protein delta (C/EBPδ). C/EBPδ is a master regulator of inflammatory responses in macrophages (Mϕ) and is mainly regulated at the level of CEBPD gene transcription initiation. To screen for CEBPD-modulating compounds, we generated a THP-1-derived reporter cell line stably expressing secreted alkaline phosphatase (SEAP) under control of the defined CEBPD promoter (CEBPD::SEAP). A high-throughput screening of LOPAC®1280 and ENZO®774 libraries on LPS- and IFN-γ-activated THP-1 reporter Mϕ identified four epigenetically active hits: two bromodomain and extraterminal domain (BET) inhibitors, I-BET151 and Ro 11-1464, as well as two histone deacetylase (HDAC) inhibitors, SAHA and TSA. All four hits markedly and reproducibly upregulated SEAP secretion and CEBPD::SEAP mRNA expression, confirming screening assay reliability. Whereas BET inhibitors also upregulated the mRNA expression of the endogenous CEBPD, HDAC inhibitors completely abolished it. All hits displayed anti-inflammatory activity through the suppression of IL-6 and CCL2 gene expression. However, I-BET151 and HDAC inhibitors simultaneously upregulated the mRNA expression of pro-inflammatory IL-1ß. The modulation of CEBPD gene expression shown in this study contributes to our understanding of inflammatory responses in Mϕ and may offer an approach to therapy for inflammation-driven disorders.

Berberine Inhibits the gene Expression and Production of Proinflammatory Cytokines by Mononuclear cells in Rheumatoid Arthritis and Healthy Individuals

2020 ◽  
Vol 16 ◽  
Author(s):  
Niloofar Ghorbani ◽  
Maryam Sahebari ◽  
Mahmoud Mahmoudi ◽  
Maryam Rastin ◽  
Shahrzad Zamani ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
Mononuclear Cells ◽  
Inhibitory Effect ◽  
Healthy Individuals ◽  
High Concentration ◽  
Antiinflammatory Property ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Inflammatory Effect

Objective: Rheumatoid arthritis (RA) is the most prevalent autoimmune arthritis. Berberine is an alkaloid isolated from Berberis vulgaris and its anti-inflammatory effect has been identified. Method: Twenty newly diagnosed RA patients and 20 healthy controls participated. Peripheral mononuclear cells were prepared and stimulated with bacterial lipopolysachharide (LPS,1 µg/ml), exposed to different concentrations of berberine (10 and 50µM) and dexamethasone (10-7 M) as a reference. Toxicity of compounds was evaluated by WST-1 assay. Expression of TNF-α and IL-1β were determined by quantitative real-time PCR. Protein level of secreted TNF-α and IL1β were measured by using ELISA. Result: Berberine did not have any toxic effect on cells, whereas Lipopolysachharide (LPS) stimulation caused a noticeable rise in TNF-α and IL-1β production. Berberine markedly downregulated the expression of both TNF-α and IL1β and inhibits TNF-α and IL-1β secretion from LPS-stimulated PBMCs. Discussion: This study provided molecular basis for anti-inflammatory effect of berberine on human mononuclear cells through the suppression of TNF-a and IL-1secretion. Our findings highlighted the significant inhibitory effect of berberine on proinflammatory responses of mononuclear cells from rheumatoid arthritis individuals, which may be responsible for antiinflammatory property of Barberry. We observed that berberine at high concentration exhibited anti-inflammatory effect in PBMCs of both healthy and patient groups by suppression of TNF-a and IL-1cytokines at both mRNA and protein levels. Conclusions: Berberine may inhibit the gene expression and production of pro-inflammatory cytokines by mononuclear cells in rheumatoid arthritis and healthy individuals without affecting cells viability. Future studies with larger sample size is needed to prove the idea.

Evaluation of Toll-like Receptor 2 Gene Expression in Rheumatoid Arthritis and Correlation with the Disease Activity

2019 ◽  
Vol 13 (2) ◽  
pp. 140-148
Author(s):  
Mai Nasser ◽  
Noha M. Hazem ◽  
Amany Atwa ◽  
Amina Baiomy
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
Autoimmune Diseases ◽  
Disease Activity ◽  
Mrna Expression ◽  
Disease Activity Score ◽  
Activity Score ◽  
Tlr2 Expression ◽  
Positive Correlation ◽  
Tlr2 Mrna

Background: Rheumatoid Arthritis (RA) is an autoimmune, chronic, and systematic disease. It affects joints and bones. The exact etiology of RA is still unclear. Varied genetic and environmental factors have been associated with the increased risk for RA. Overactivation of Toll-Like Receptors (TLRs) could initiate the development of autoimmune diseases including RA. Objective: The aim of the study was to evaluate TLR2 gene expression in rheumatoid arthritis patients and investigate its correlation with the disease activity. Materials and Methods: This study included 60 patients and 20 healthy individuals. The patients were diagnosed with RA according to the 2010 American College of Rheumatology/ European League Against Rheumatism criteria (ACR/EULAR). All included subjects did not have any joint disorders and /or autoimmune diseases. RA disease activity was determined by the disease activity score of 28 joints. Whole blood was collected from all participants. Total RNA extraction was done. TLR2 mRNA expression was assessed by reverse transcription-PCR (RT-PCR). Results: TLR2 mRNA expression was found to be significantly higher in RA patients compared to healthy controls. Also, a strong positive correlation was found between TLR2 expression level and the disease activity score. A non significant positive correlation was found between TLR2 expression and serum Rheumatoid Factor (RF) level. Conclusion: TLR2 pathway may have an important role in RA pathogenesis and could be a new biomarker for diagnosis and monitoring disease activity.

scholarly journals The Anti-Rheumatoid Arthritis Property of the Folk Medicine Dianbaizhu (Gaultheria leucocarpa var. yunnanensis, Ericaceae)

2014 ◽  
Vol 9 (12) ◽  
pp. 1934578X1400901 ◽  
Author(s):  
Meng Xie ◽  
Yi Lu ◽  
Cheng Yan ◽  
Rui Jiang ◽  
Weirui Liu ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Tumor Necrosis ◽  
Folk Medicine ◽  
Plant Parts ◽  
Effective Components ◽  
Water Fractions ◽  
Anti Inflammatory ◽  
Preferred Species ◽  
Necrosis Factor ◽  
Inflammatory Effect

The Chinese folk medicine Dianbaizhu, consisting of Gaultheria species, is widely used for the treatment of rheumatoid arthritis by several minority nationalities. The species and plant parts of this genus used as Dianbaizhu in clinical application are confused. In order to elucidate the species and the medicinal parts, as well as to ascertain the effective components and the probable optimal source of Dianbaizhu, the different plant parts and polarity fractions of its mainstream species, G. leucocarpa var. yunnanensis were investigated. The inhibition of nitric oxide and tumor necrosis factor produced in macrophage J774 were used to assess the anti-inflammatory effect of those samples. G. leucocarpa var. yunnanensis may be the preferred species for anti-RA effect. The underground parts of this taxon showed the best anti-inflammatory and anti-RA activities; the n-butanol and water fractions of the underground parts may be the most anti-RA active.

Laser-assisted microdissection and real-time PCR detect anti-inflammatory effect of perfluorocarbon

2003 ◽  
Vol 285 (1) ◽  
pp. L55-L62 ◽  
Author(s):  
Katharina von der Hardt ◽  
Michael Andreas Kandler ◽  
Ludger Fink ◽  
Ellen Schoof ◽  
Jörg Dötsch ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Mrna Expression ◽  
Vascular Smooth Muscle Cells ◽  
Alveolar Macrophages ◽  
Real Time Pcr ◽  
Muscle Cells ◽  
Cell Types ◽  
Anti Inflammatory ◽  
Inflammatory Effect

The aim of this study was to identify cell types involved in the anti-inflammatory effect of ventilation with perfluorocarbon in vivo. Fifteen anesthetized, surfactant-depleted piglets received either aerosolized perfluorocarbon (Aerosol-PFC), partial liquid ventilation (rLV) at functional residual capacity (FRC) volume (FRC-PLV), or intermittent mandatory ventilation (control). After laser-assisted microdissection of different lung cell types, mRNA expression of IL-8 and ICAM-1 was determined using TaqMan real-time PCR normalized to hypoxanthine phosphoribosyltransferase (HPRT). IL-8 mRNA expression (means ± SE; control vs. Aerosol-PFC) was 356 ± 142 copies IL-8 mRNA/copy HPRT mRNA vs. 3.5 ± 1.8 in alveolar macrophages ( P <0.01); 208 ± 108 vs. 2.7 ± 0.8 in bronchiolar epithelial cells ( P <0.05); 26 ± 11 vs. 0.7 ± 0.2 in alveolar septum cells ( P <0.01); 2.8 ± 1.0 vs. 0.8 ± 0.4 in bronchiolar smooth muscle cells ( P <0.05); and 1.1 ± 0.4 vs. 0.2 ± 0.05 in vascular smooth muscle cells ( P <0.05). With FRC-PLV, IL-8/HPRT mRNA expression was significantly lower in macrophages, bronchiolar epithelial, and vascular smooth muscle cells. ICAM-1 mRNA expression in vascular endothelial cells remained unchanged. Predominantly, alveolar macrophages and bronchiolar epithelial cells were involved in the inflammatory pulmonary process. The anti-inflammatory effect of Aerosol-PFC was most pronounced.

scholarly journals Different DOACs Control Inflammation in Cardiac Ischemia-Reperfusion Differently

2020 ◽  
Author(s):  
Ihsan Gadi ◽  
Sameen Fatima ◽  
Ahmed Elwakiel ◽  
Sumra Nazir ◽  
Mohd Mohanad Al-Dabet ◽  
...  
Keyword(s):  
Gene Expression ◽  
Infarct Size ◽  
Ischemia Reperfusion ◽  
Sterile Inflammation ◽  
Inflammasome Activation ◽  
Differential Effects ◽  
Anti Inflammatory ◽  
Anticoagulant Function ◽  
Inflammatory Effect

Rationale: While thrombin is the key protease in thrombus formation, other coagulation proteases, such as fXa or activated protein C (aPC), independently modulate intracellular signaling via partially distinct receptors. Objective: To study the differential effects of fXa or fIIa inhibition on gene expression and inflammation in myocardial ischemia-reperfusion injury (IRI). Methods and Results: Mice were treated with a direct fIIa inhibitor (fIIai) or direct fXa inhibitor (fXai) at doses that induced comparable anticoagulant effects ex vivo and in vivo (tail bleeding assay and FeCl3-induced thrombosis). Myocardial IRI was induced via LAD ligation. We determined infarct size and in vivo aPC generation, analyzed gene expression by RNAseq, and performed immunoblotting and ELISA. The signaling-only 3K3A-aPC variant and inhibitory antibodies that blocked all or only the anticoagulant function of aPC were used to determine the role of aPC. Doses of fIIai and fXai that induced comparable anticoagulant effects resulted in a comparable reduction in infarct size. However, unbiased gene expression analyses revealed marked differences, including pathways related to sterile inflammation and inflammasome regulation. fXai but not fIIai inhibited sterile inflammation by reducing the expression of proinflammatory cytokines (IL-1beta, IL-6, and TNFalpha) as well as NF-κB and inflammasome activation. This anti-inflammatory effect was associated with reduced myocardial fibrosis 28 days post myocardial IRI. Mechanistically, in vivo aPC generation was higher with fXai than with fIIai. Inhibition of the anticoagulant and signaling properties of aPC abolished the anti-inflammatory effect associated with fXai, while inhibiting only the anticoagulant function of aPC had no effect. Combining 3K3A-aPC with fIIai reduced the inflammatory response, mimicking the fXai-associated effect. Conclusions: We showed that specific inhibition of coagulation via DOACs had differential effects on gene expression and inflammation, despite comparable anticoagulant effects and infarct sizes. Targeting individual coagulation proteases induces specific cellular responses unrelated to their anticoagulant effect.

scholarly journals Increased FURIN expression in rheumatoid arthritis patients and its anti‐inflammatory effect

2020 ◽  
Vol 34 (12) ◽  
Author(s):  
Rong Cao ◽  
Ying Zhang ◽  
Juping Du ◽  
Shuaishuai Chen ◽  
Na Wang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Anti Inflammatory ◽  
Inflammatory Effect

scholarly journals The Role of Simvastatin in the Therapeutic Approach of Rheumatoid Arthritis

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Lucia Cojocaru ◽  
Andrei Constantin Rusali ◽  
Cristina Şuţa ◽  
Anca Mihaela Rădulescu ◽  
Maria Şuţa ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cardiovascular Disease ◽  
Active Rheumatoid Arthritis ◽  
Therapeutic Potential ◽  
Efficacy And Safety ◽  
Good Safety Profile ◽  
Study Support ◽  
Anti Inflammatory ◽  
Inflammatory Effect

The pleiotropic effects of statins, especially the anti-inflammatory and immunomodulatory ones, indicate that their therapeutic potential might extend beyond cholesterol lowering and cardiovascular disease to other inflammatory disorders such as rheumatoid arthritis. Therefore, we undertook a prospective cohort study to evaluate the efficacy and safety of simvastatin used for inflammation control in patients with rheumatoid arthritis. One hundred patients with active rheumatoid arthritis divided into two equal groups (the study one who received 20 mg/day of simvastatin in addition to prior DMARDs and the control one) were followed up over six months during three study visits. The results of the study support the fact that simvastatin at a dose of 20 mg/day has a low anti-inflammatory effect in patients with rheumatoid arthritis with a good safety profile.

The Situation of Chemokine Ligands and Receptors Gene Expression, Following the Oral Administration of Drug Mannuronic Acid in Rheumatoid Arthritis Patients

2020 ◽  
Vol 14 (1) ◽  
pp. 69-77
Author(s):  
Mona Aslani ◽  
Arman Ahmadzadeh ◽  
Zahra Rezaieyazdi ◽  
Seyed S. Mortazavi-Jahromi ◽  
Anis Barati ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
Clinical Trial ◽  
Oral Administration ◽  
Mrna Expression ◽  
Phase Iii ◽  
Inadequate Response ◽  
Mannuronic Acid ◽  
Target Molecules ◽  
Chemokine Ligands

Background: Regarding the leukocytes infiltration into the synovium of Rheumatoid Arthritis (RA) patients is mostly mediated by chemokine ligands and receptors, and following the efficient and motivating results of international Phase III clinical trial of β-D-Mannuronic acid (M2000) patented EP067919 (2017), as a novel anti-inflammatory drug, in patients with RA, the present research was designed. Objectives: This study aimed to assess the oral administration effects of this new drug on gene expression of some chemokine receptors and ligands, including CXCR4, CXCR3, CCR2, CCR5 and CCL2/MCP-1 in PBMCs of patients with active form of RA. Methods: Twelve patients suffering from RA, with inadequate response to conventional drugs were selected (Clinical trial identifier IRCT2017100213739N10) and 1000mg/day of M2000 was orally administrated to them for 12 weeks. The mRNA expression of target molecules was then evaluated in PBMCs of the patients before and after treatment with M2000 using real-time PCR and was compared to healthy controls. Patents related to this study were also reviewed. Results: The results showed that M2000 was able to significantly down-regulate the mRNA expression of CXCR4, CCR2 and CCL2/MCP-1 in the PBMCs of the RA patients. It should be noted that the gene expression situation of the target molecules was in coordinate with the clinical and paraclinical assessments in the patients. Conclusion: Taken together, the results of this investigation revealed the part of molecular and immunological mechanisms of drug Mannuronic acid (M2000) in the treatment of RA, based on chemokine ligands and receptors mediated processes.

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