Discovery of the First-in-Class Inhibitors of Hypoxia Up-Regulated Protein†1 (HYOU1) Suppressing Pathogenic Fibroblast Activation.

Fibroblasts are key regulators of inflammation, fibrosis, and cancer. Targeting their activation in these complex diseases has emerged as a novel strategy to restore tissue homeostasis. Here, we present a multidisciplinary lead discovery approach to identify and optimize small molecule inhibitors of pathogenic fibroblast activation. The study encompasses medicinal chemistry, molecular phenotyping assays, chemoproteomics, bulk RNA-sequencing analysis, target validation experiments, and chemical absorption, distribution, metabolism, excretion and toxicity (ADMET)/pharmacokinetic (PK)/in vivo evaluation. The parallel synthesis employed for the production of the new benzamide derivatives enabled us to a) pinpoint key structural elements of the scaffold that provide potent fibroblast-deactivating effects in cells, b) discriminate atoms or groups that favor or disfavor a desirable ADMET profile, and c) identify metabolic "hot spots". Furthermore, we report the discovery of the first-in-class inhibitor leads for hypoxia up-regulated protein 1 (HYOU1), a member of the heat shock protein 70 (HSP70) family often associated with cellular stress responses, particularly under hypoxic conditions. Targeting HYOU1 may therefore represent a potentially novel strategy to modulate fibroblast activation and treat chronic inflammatory and fibrotic disorders.

A Novel Human Interleukin-23A Overexpressing Mouse Model of Systemic Lupus Erythematosus.

OBJECTIVE: Interleukin-23 (IL-23) is a crucial cytokine implicated in chronic inflammation and autoimmunity, associated with various diseases such as psoriasis, psoriatic arthritis, and systemic lupus erythematosus (SLE). This study aimed to create and characterize a transgenic mouse model overexpressing human IL-23A (TghIL-23A), providing a valuable tool for investigating the pathogenic role of human IL-23A and evaluating the efficacy of anti-human IL-23A therapeutics. METHODS: TghIL-23A mice were generated via microinjection of CBA × C57BL/6 zygotes with a fragment of the human IL23A gene, flanked by its 5'-regulatory sequences and the 3' untranslated region of human ß-globin. The TghIL-23A pathology was assessed through hematologic and biochemic analyses, cytokine and antinuclear antibody detection, and histopathologic examination of skin and renal tissues. The response to the anti-human IL-23A therapeutic agent guselkumab was evaluated in groups of eight mixed-sex mice receiving subcutaneous treatment twice weekly for 10 weeks using clinical, biomarker, and histopathologic readouts. RESULTS: TghIL-23A mice exhibited interactions between human IL-23A and mouse IL-23/IL-12p40 and developed a chronic multiorgan autoimmune disease marked by proteinuria, anti-double-stranded DNA antibodies, severe inflammatory lesions in the skin, and milder phenotypes in the kidneys and lungs. The TghIL-23A pathologic features exhibited significant similarities to those observed in human patients with SLE, and they were reversed following guselkumab treatment. CONCLUSION: We have generated and characterized a novel genetic mouse model of SLE, providing proof-of-concept for the etiopathogenic role of human IL-23A. This new model has a normal life span and integrates several characteristics of the human disease's complexity and chronicity, making it an attractive preclinical tool for studying IL-23-dependent pathogenic mechanisms and assessing the efficacy of anti-human IL-23A or modeled disease-related therapeutics.

The long non-coding RNA HOTAIR contributes to joint-specific gene expression in rheumatoid arthritis.

Although patients with rheumatoid arthritis (RA) typically exhibit symmetrical joint involvement, some patients develop alternative disease patterns in response to treatment, suggesting that different molecular mechanism may underlie disease progression depending on joint location. Here, we identify joint-specific changes in RA synovium and synovial fibroblasts (SF) between knee and hand joints. We show that the long non-coding RNA HOTAIR, which is only expressed in knee SF, regulates more than 50% of this site-specific gene expression in SF. HOTAIR is downregulated after stimulation with pro-inflammatory cytokines and is expressed at lower levels in knee samples from patients with RA, compared with osteoarthritis. Knockdown of HOTAIR in knee SF increases PI-Akt signalling and IL-6 production, but reduces Wnt signalling. Silencing HOTAIR inhibits the migratory function of SF, decreases SF-mediated osteoclastogenesis, and increases the recruitment of B cells by SF. We propose that HOTAIR is an important epigenetic factor in joint-specific gene expression in RA.

SCALA: A complete solution for multimodal analysis of single-cell Next Generation Sequencing data.

Analysis and interpretation of high-throughput transcriptional and chromatin accessibility data at single-cell (sc) resolution are still open challenges in the biomedical field. The existence of countless bioinformatics tools, for the different analytical steps, increases the complexity of data interpretation and the difficulty to derive biological insights. In this article, we present SCALA, a bioinformatics tool for analysis and visualization of single-cell RNA sequencing (scRNA-seq) and Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq) datasets, enabling either independent or integrative analysis of the two modalities. SCALA combines standard types of analysis by integrating multiple software packages varying from quality control to the identification of distinct cell populations and cell states. Additional analysis options enable functional enrichment, cellular trajectory inference, ligand-receptor analysis, and regulatory network reconstruction. SCALA is fully parameterizable, presenting data in tabular format and producing publication-ready visualizations. The different available analysis modules can aid biomedical researchers in exploring, analyzing, and visualizing their data without any prior experience in coding. We demonstrate the functionality of SCALA through two use-cases related to TNF-driven arthritic mice, handling both scRNA-seq and scATAC-seq datasets. SCALA is developed in R, Shiny and JavaScript and is mainly available as a standalone version, while an online service of more limited capacity can be found at http://scala.pavlopouloslab.info or https://scala.fleming.gr.

Infection and inflammation stimulate expansion of a CD74+ Paneth cell subset to regulate disease progression.

Paneth cells (PCs), a specialized secretory cell type in the small intestine, are increasingly recognized as having an essential role in host responses to microbiome and environmental stresses. Whether and how commensal and pathogenic microbes modify PC composition to modulate inflammation remain unclear. Using newly developed PC-reporter mice under conventional and gnotobiotic conditions, we determined PC transcriptomic heterogeneity in response to commensal and invasive microbes at single cell level. Infection expands the pool of CD74+ PCs, whose number correlates with auto or allogeneic inflammatory disease progressions in mice. Similar correlation was found in human inflammatory disease tissues. Infection-stimulated cytokines increase production of reactive oxygen species (ROS) and expression of a PC-specific mucosal pentraxin (Mptx2) in activated PCs. A PC-specific ablation of MyD88 reduced CD74+ PC population, thus ameliorating pathogen-induced systemic disease. A similar phenotype was also observed in mice lacking Mptx2. Thus, infection stimulates expansion of a PC subset that influences disease progression.

Targeting senescence and inflammation in chronic destructive TNF-driven joint pathology.

We had shown that administration of the senolytic Dasatinib abolishes arthritis in the human TNF transgenic mouse model of chronic destructive arthritis when given in combination with a sub-therapeutic dose of the anti-TNF mAb Infliximab (1 mg/kg). Herein, we found that while the number of senescent chondrocytes (GL13+/Ki67-), assessed according to guideline algorithmic approaches, was not affected by either Dasatinib or sub-therapeutic Infliximab monotherapies, their combination reduced senescent chondrocytes by 50 %, which was comparable to levels observed with therapeutic Infliximab monotherapy (10 mg/kg). This combination therapy also reduced the expression of multiple factors of senescence-associated secretory phenotype in arthritic joints. Studies to elucidate the interplay of inflammation and senescence may help in optimizing treatment strategies also for age-related pathologies characterized by chronic low-grade joint inflammation.

Distinct innate and adaptive immunity phenotypic profile at the circulating single-cell level in Psoriatic Arthritis.

Mass cytometry was employed to investigate 47 circulating leukocyte subsets in patients with active psoriatic arthritis (PsA, n = 16) compared to healthy controls (n = 13), seropositive (RF and/or anti-CCP, n = 12) and seronegative (n = 9) RA patients. Comparing PsA to controls, different cell frequencies were found in both innate and adaptive immunity cell subsets, as well as in cells bridging innate and adaptive immunity. In some T cell subsets increased costimulatory molecules' expression in PsA, was also noted.No changes were observed in patients who remained disease-active after 3 months of treatment, in contrast to those who achieved remission/low-disease activity. Comparing PsA to seropositive RA, elevated frequencies of naïve and activated CD8+ T cells, B cells, MAIT/iNKT and ILCs were found, while the opposite was the case for terminal effector, senescent, and Th2-like cells. Strikingly, the composition of the leukocyte pool in PsA was comparable to seronegative RA, providing evidence for the pathogenetic similarities between these two entities.

Repurposing the antipsychotic drug amisulpride for targeting synovial fibroblast activation in arthritis.

Synovial fibroblasts (SFs) are key pathogenic drivers in rheumatoid arthritis (RA). Their in vivo activation by TNF is sufficient to orchestrate full arthritic pathogenesis in animal models, and TNF blockade proved efficacious for a high percentage of patients with RA albeit coinducing rare but serious side effects. Aiming to find new potent therapeutics, we applied the L1000CDS2 search engine, to repurpose drugs that could reverse the pathogenic expression signature of arthritogenic human TNF-transgenic (hTNFtg) SFs. We identified a neuroleptic drug, namely amisulpride, which reduced SFs' inflammatory potential while decreasing the clinical score of hTNFtg polyarthritis. Notably, we found that amisulpride function was neither through its known targets dopamine receptors D2 and D3 and serotonin receptor 7 nor through TNF-TNF receptor I binding inhibition. Through a click chemistry approach, potentially novel targets of amisulpride were identified, which were further validated to repress hTNFtg SFs' inflammatory potential ex vivo (Ascc3 and Sec62), while phosphoproteomics analysis revealed that treatment altered important fibroblast activation pathways, such as adhesion. Thus, amisulpride could prove beneficial to patients experiencing RA and the often-accompanying comorbid dysthymia, reducing SF pathogenicity along with its antidepressive activity, serving further as a "lead" compound for the development of novel therapeutics against fibroblast activation.

Dosimetric accuracy of the Convolution algorithm for Leksell Gamma Plan radiosurgery treatment planning: Evaluation in the presence of clinically relevant inhomogeneities.

PURPOSE: The Leksell Gamma Plan Convolution algorithm (LGP-Convolution) has not been widely adopted. This mainly stems from the higher calculated beam-on times relative to the standard ray tracing-based LGP-TMR10 dose calculation algorithm. This study aims to evaluate the accuracy of the LGP-Convolution in scenarios where the treated lesions are in the vicinity of or encompassed by bone and/or air inhomogeneities. METHODS: The solid water dosimetry phantom provided by the vendor was modified to include bone and air inhomogeneities. Two treatment planning scenarios were investigated involving a single shot and multiple shots, respectively. Treatment planning and dose prescription were performed using the LGP-Convolution algorithm. Triple channel film dosimetry was performed using GafChromic EBT3 films calibrated in terms of absorbed dose to water in a 60 Co beam. Monte Carlo (MC) simulation dosimetry was also performed in the inhomogeneous experimental geometry using the EGSnrc MC platform and a previously validated sector-based phase-space source model. MC simulations were also employed to determine correction factors required for converting EBT3 measurements at points within the bone and air inhomogeneities from dose-to-water values to the corresponding dose to medium values. RESULTS AND CONCLUSIONS: EBT3 dose to medium correction factors ranged with field size (4, 8, or 16 mm) within 0.941-0.946 for bone and 0.745-0.749 for air inhomogeneities. An excellent agreement was found between the LGP-Convolution calculations with corresponding EBT3 and MC dose to medium results at all measurement points, except those located inside the air inhomogeneity. The latter is of no clinical importance and excluding them yielded gamma index passing rates of nearly 100% for 3% local dose difference and 1 mm distance-to-agreement criteria. The excellent agreement observed between LGP-Convolution calculations and film as well as MC results of dose to medium indicates that the latter is the quantity reported by the LGP-Convolution.

Activation and Functions of Col6a1+ Fibroblasts in Colitis-Associated Cancer.

Cancer-associated fibroblasts (CAFs) comprise a group of heterogeneous subpopulations with distinct identities indicative of their diverse origins, activation patterns, and pro-tumorigenic functions. CAFs originate mainly from resident fibroblasts, which are activated upon different stimuli, including growth factors and inflammatory mediators, but the extent to which they also maintain some of their homeostatic properties, at least at the earlier stages of carcinogenesis, is not clear. In response to cytokines, such as interleukin 1 (IL-1) and tumor necrosis factor (TNF), as well as microbial products, CAFs acquire an immunoregulatory phenotype, but its specificity and pathophysiological significance in individual CAF subsets is yet to be determined. In this study, we analyzed the properties of Col6a1-positive fibroblasts in colitis-associated cancer. We found that Col6a1+ cells partly maintain their homeostatic features during adenoma development, while their activation is characterized by the acquisition of a distinct proangiogenic signature associated with their initial perivascular location. In vitro and in vivo experiments showed that Col6a1+ cells respond to innate immune stimuli and exert pro-tumorigenic functions. However, Col6a1+-specific inhibition of TNF receptor 1 (TNFR1) or IL-1 receptor (IL-1R) signaling does not significantly affect tumorigenesis, suggesting that activation of other subsets acts in a compensatory way or that multiple immune stimuli are necessary to drive the proinflammatory activation of this subset. In conclusion, our results show that adenoma-associated CAF subsets can partly maintain the properties of homeostatic fibroblasts while they become activated to support tumor growth through distinct and compensatory mechanisms.

ETS1 governs pathological tissue-remodeling programs in disease-associated fibroblasts.

Fibroblasts, the most abundant structural cells, exert homeostatic functions but also drive disease pathogenesis. Single-cell technologies have illuminated the shared characteristics of pathogenic fibroblasts in multiple diseases including autoimmune arthritis, cancer and inflammatory colitis. However, the molecular mechanisms underlying the disease-associated fibroblast phenotypes remain largely unclear. Here, we identify ETS1 as the key transcription factor governing the pathological tissue-remodeling programs in fibroblasts. In arthritis, ETS1 drives polarization toward tissue-destructive fibroblasts by orchestrating hitherto undescribed regulatory elements of the osteoclast differentiation factor receptor activator of nuclear factor-κB ligand (RANKL) as well as matrix metalloproteinases. Fibroblast-specific ETS1 deletion resulted in ameliorated bone and cartilage damage under arthritic conditions without affecting the inflammation level. Cross-tissue fibroblast single-cell data analyses and genetic loss-of-function experiments lent support to the notion that ETS1 defines the perturbation-specific fibroblasts shared among various disease settings. These findings provide a mechanistic basis for pathogenic fibroblast polarization and have important therapeutic implications.

Single-cell multimodal analysis identifies common regulatory programs in synovial fibroblasts of rheumatoid arthritis patients and modeled TNF-driven arthritis.

BACKGROUND: Synovial fibroblasts (SFs) are specialized cells of the synovium that provide nutrients and lubricants for the proper function of diarthrodial joints. Recent evidence appreciates the contribution of SF heterogeneity in arthritic pathologies. However, the normal SF profiles and the molecular networks that govern the transition from homeostatic to arthritic SF heterogeneity remain poorly defined. METHODS: We applied a combined analysis of single-cell (sc) transcriptomes and epigenomes (scRNA-seq and scATAC-seq) to SFs derived from naïve and hTNFtg mice (mice that overexpress human TNF, a murine model for rheumatoid arthritis), by employing the Seurat and ArchR packages. To identify the cellular differentiation lineages, we conducted velocity and trajectory analysis by combining state-of-the-art algorithms including scVelo, Slingshot, and PAGA. We integrated the transcriptomic and epigenomic data to infer gene regulatory networks using ArchR and custom-implemented algorithms. We performed a canonical correlation analysis-based integration of murine data with publicly available datasets from SFs of rheumatoid arthritis patients and sought to identify conserved gene regulatory networks by utilizing the SCENIC algorithm in the human arthritic scRNA-seq atlas. RESULTS: By comparing SFs from healthy and hTNFtg mice, we revealed seven homeostatic and two disease-specific subsets of SFs. In healthy synovium, SFs function towards chondro- and osteogenesis, tissue repair, and immune surveillance. The development of arthritis leads to shrinkage of homeostatic SFs and favors the emergence of SF profiles marked by Dkk3 and Lrrc15 expression, functioning towards enhanced inflammatory responses and matrix catabolic processes. Lineage inference analysis indicated that specific Thy1+ SFs at the root of trajectories lead to the intermediate Thy1+/Dkk3+/Lrrc15+ SF states and culminate in a destructive and inflammatory Thy1- SF identity. We further uncovered epigenetically primed gene programs driving the expansion of these arthritic SFs, regulated by NFkB and new candidates, such as Runx1. Cross-species analysis of human/mouse arthritic SF data determined conserved regulatory and transcriptional networks. CONCLUSIONS: We revealed a dynamic SF landscape from health to arthritis providing a functional genomic blueprint to understand the joint pathophysiology and highlight the fibroblast-oriented therapeutic targets for combating chronic inflammatory and destructive arthritic disease.

The second decade of anti-TNF-a therapy in clinical practice: new lessons and future directions in the COVID-19 era.

Since the late 1990s, tumor necrosis factor alpha (TNF-α) inhibitors (anti-TNFs) have revolutionized the therapy of immune-mediated inflammatory diseases (IMIDs) affecting the gut, joints, skin and eyes. Although the therapeutic armamentarium in IMIDs is being constantly expanded, anti-TNFs remain the cornerstone of their treatment. During the second decade of their application in clinical practice, a large body of additional knowledge has accumulated regarding various aspects of anti-TNF-α therapy, whereas new indications have been added. Recent experimental studies have shown that anti-TNFs exert their beneficial effects not only by restoring aberrant TNF-mediated immune mechanisms, but also by de-activating pathogenic fibroblast-like mesenchymal cells. Real-world data on millions of patients further confirmed the remarkable efficacy of anti-TNFs. It is now clear that anti-TNFs alter the physical course of inflammatory arthritis and inflammatory bowel disease, leading to inhibition of local and systemic bone loss and to a decline in the number of surgeries for disease-related complications, while anti-TNFs improve morbidity and mortality, acting beneficially also on cardiovascular comorbidities. On the other hand, no new safety signals emerged, whereas anti-TNF-α safety in pregnancy and amid the COVID-19 pandemic was confirmed. The use of biosimilars was associated with cost reductions making anti-TNFs more widely available. Moreover, the current implementation of the "treat-to-target" approach and treatment de-escalation strategies of IMIDs were based on anti-TNFs. An intensive search to discover biomarkers to optimize response to anti-TNF-α treatment is currently ongoing. Finally, selective targeting of TNF-α receptors, new forms of anti-TNFs and combinations with other agents, are being tested in clinical trials and will probably expand the spectrum of TNF-α inhibition as a therapeutic strategy for IMIDs.

Harnessing murine models of Crohn's disease ileitis to advance concepts of pathophysiology and treatment.

Crohn's disease (CD) and ulcerative colitis (UC) are both characterized by chronic inflammation and severe dysfunction of the gastrointestinal tract. These two forms of inflammatory bowel disease (IBD) represent distinct clinical disorders with diverse driving mechanisms; however, this divergence is not reflected in currently approved therapeutics that commonly target general proinflammatory pathways. A compelling need therefore remains to understand factors that differentiate the topology and the distinct clinical manifestations of CD versus UC, in order to develop more effective and specialized therapies. Animal models provide valuable platforms for studying IBD heterogeneity and deciphering disease-specific mechanisms. Both the established and the newly developed ileitis mouse models are characterized by various disease initiating mechanisms and diverse phenotypic outcomes that reflect the complexity of human CD-ileitis. Microbial dysbiosis, destruction of epithelial barrier integrity, immune cell deregulation, as well as the recently described genome instability and stromal cell activation have all been proposed as the triggering factors for the development of ileitis-associated pathology. In this review, we aim to critically evaluate the mechanistic underpinnings of murine models of CD-ileitis, discuss their phenotypic similarities to human disease, and envisage their further exploitation for the development of novel targeted and personalized therapeutics.

INFRAFRONTIER quality principles in systemic phenotyping.

Improving reproducibility and replicability in preclinical research is a widely discussed and pertinent topic, especially regarding ethical responsibility in animal research. INFRAFRONTIER, the European Research Infrastructure for the generation, phenotyping, archiving, and distribution of model mammalian genomes, is addressing this issue by developing internal quality principles for its different service areas, that provides a quality framework for its operational activities. This article introduces the INFRAFRONTIER Quality Principles in Systemic Phenotyping of genetically altered mouse models. A total of 11 key principles are included, ranging from general requirements for compliance with guidelines on animal testing, to the need for well-trained personnel and more specific standards such as the exchange of reference lines. Recently established requirements such as the provision of FAIR (Findable, Accessible, Interoperable, Reusable) data are also addressed. For each quality principle, we have outlined the specific context, requirements, further recommendations, and key references.

TNFα induces endothelial dysfunction in rheumatoid arthritis via LOX-1 and arginase 2: reversal by monoclonal TNFα antibodies.

AIMS: Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting joints and blood vessels. Despite low levels of low-density lipoprotein cholesterol (LDL-C), RA patients exhibit endothelial dysfunction and are at increased risk of death from cardiovascular complications, but the molecular mechanism of action is unknown. We aimed in the present study to identify the molecular mechanism of endothelial dysfunction in a mouse model of RA and in patients with RA. METHODS AND RESULTS: Endothelium-dependent relaxations to acetylcholine were reduced in aortae of two tumour necrosis factor alpha (TNFα) transgenic mouse lines with either mild (Tg3647) or severe (Tg197) forms of RA in a time- and severity-dependent fashion as assessed by organ chamber myograph. In Tg197, TNFα plasma levels were associated with severe endothelial dysfunction. LOX-1 receptor was markedly up-regulated leading to increased vascular oxLDL uptake and NFκB-mediated enhanced Arg2 expression via direct binding to its promoter resulting in reduced NO bioavailability and vascular cGMP levels as shown by ELISA and chromatin immunoprecipitation. Anti-TNFα treatment with infliximab normalized endothelial function together with LOX-1 and Arg2 serum levels in mice. In RA patients, soluble LOX-1 serum levels were also markedly increased and closely related to serum levels of C-reactive protein. Similarly, ARG2 serum levels were increased. Similarly, anti-TNFα treatment restored LOX-1 and ARG2 serum levels in RA patients. CONCLUSIONS: Increased TNFα levels not only contribute to RA, but also to endothelial dysfunction by increasing vascular oxLDL content and activation of the LOX-1/NFκB/Arg2 pathway leading to reduced NO bioavailability and decreased cGMP levels. Anti-TNFα treatment improved both articular symptoms and endothelial function by reducing LOX-1, vascular oxLDL, and Arg2 levels.

Col6a1+/CD201+ mesenchymal cells regulate intestinal morphogenesis and homeostasis.

Intestinal mesenchymal cells encompass multiple subsets, whose origins, functions, and pathophysiological importance are still not clear. Here, we used the Col6a1Cre mouse, which targets distinct fibroblast subsets and perivascular cells that can be further distinguished by the combination of the CD201, PDGFRα and αSMA markers. Developmental studies revealed that the Col6a1Cre mouse also targets mesenchymal aggregates that are crucial for intestinal morphogenesis and patterning, suggesting an ontogenic relationship between them and homeostatic PDGFRαhi telocytes. Cell depletion experiments in adulthood showed that Col6a1+/CD201+ mesenchymal cells regulate homeostatic enteroendocrine cell differentiation and epithelial proliferation. During acute colitis, they expressed an inflammatory and extracellular matrix remodelling gene signature, but they also retained their properties and topology. Notably, both in homeostasis and tissue regeneration, they were dispensable for normal organ architecture, while CD34+ mesenchymal cells expanded, localised at the top of the crypts, and showed increased expression of villous-associated morphogenetic factors, providing thus evidence for the plasticity potential of intestinal mesenchymal cells. Our results provide a comprehensive analysis of the identities, origin, and functional significance of distinct mesenchymal populations in the intestine.

Changes in Thyroid Hormone Signaling Mediate Cardiac Dysfunction in the Tg197 Mouse Model of Arthritis: Potential Therapeutic Implications.

Background Rheumatoid Arthritis (RA) patients show a higher risk of heart failure. The present study investigated possible causes of cardiac dysfunction related to thyroid hormone (TH) signaling in a RA mouse model. Methods A TNF-driven mouse model of RA[TghuTNF (Tg197)] was used. Cardiac function was evaluated by echocardiography. SERCA2a and phospholamban protein levels in left ventricle (LV) tissue, thyroid hormone levels in serum, TH receptors in LV and TH-related kinase signaling pathways were measured. T3 hormone was administered in female Tg197 mice. Results We show LV and atrial dilatation with systolic dysfunction in Tg197 animals, accompanied by downregulated SERCA2a. We suggest an interaction of pro-inflammatory and thyroid hormone signaling indicated by increased p38 MAPK and downregulation of TRß1 receptor in Tg197 hearts. Interestingly, female Tg197 mice showed a worse cardiac phenotype related to reduced T3 levels and Akt activation. T3 supplementation increased Akt activation, restored SERCA2a expression and improved cardiac function in female Tg197 mice. Conclusions TNF overexpression of Tg197 mice results in cardiac dysfunction via p38 MAPK activation and downregulation of TRß1. Gender-specific reduction in T3 levels could cause the worse cardiac phenotype observed in female mice, while T3 administration improves cardiac function and calcium handling via modified Akt activation.

In Silico Identification and Evaluation of Natural Products as Potential Tumor Necrosis Factor Function Inhibitors Using Advanced Enalos Asclepios KNIME Nodes.

Tumor necrosis factor (TNF) is a regulator of several chronic inflammatory diseases, such as rheumatoid arthritis. Although anti-TNF biologics have been used in clinic, they render several drawbacks, such as patients' progressive immunodeficiency and loss of response, high cost, and intravenous administration. In order to find new potential anti-TNF small molecule inhibitors, we employed an in silico approach, aiming to find natural products, analogs of Ampelopsin H, a compound that blocks the formation of TNF active trimer. Two out of nine commercially available compounds tested, Nepalensinol B and Miyabenol A, efficiently reduced TNF-induced cytotoxicity in L929 cells and production of chemokines in mice joints' synovial fibroblasts, while Nepalensinol B also abolished TNF-TNFR1 binding in non-toxic concentrations. The binding mode of the compounds was further investigated by molecular dynamics and free energy calculation studies, using and advancing the Enalos Asclepios pipeline. Conclusively, we propose that Nepalensinol B, characterized by the lowest free energy of binding and by a higher number of hydrogen bonds with TNF, qualifies as a potential lead compound for TNF inhibitors' drug development. Finally, the upgraded Enalos Asclepios pipeline can be used for improved identification of new therapeutics against TNF-mediated chronic inflammatory diseases, providing state-of-the-art insight on their binding mode.