Panagiotis Chouvardas

Inferring active regulatory networks from gene expression data using a combination of prior knowledge and enrichment analysis

BackgroundUnder both physiological and pathological conditions gene expression programs are shaped through the interplay of regulatory proteins and their gene targets, interactions between which form intricate gene regulatory networks (GRN). While the assessment of genome-wide expression for the complete set of genes at a given condition has become rather straight-forward and is performed routinely, we are still far from being able to infer the topology of gene regulation simply by analyzing its “descendant” expression profile. In this work we are trying to overcome the existing limitations for the inference and study of such regulatory networks. We are combining our approach with state-of-the-art gene set enrichment analyses in order to create a tool, called Regulatory Network Enrichment Analysis (RNEA) that will prioritize regulatory and functional characteristics of a genome-wide expression experiment.ResultsRNEA combines prior knowledge, originating from manual literature curation and small-scale experimental data, to construct a reference network of interactions and then uses enrichment analysis coupled with a two-level hierarchical parsing of the network, to infer the most relevant subnetwork for a given experimental setting. It is implemented as an R package, currently supporting human and mouse datasets and was herein tested on one test case for each of the two organisms. In both cases, RNEA’s gene set enrichment analysis was comparable to state-of-the-art methodologies. Moreover, through its distinguishing feature of regulatory subnetwork reconstruction, RNEA was able to define the key transcriptional regulators for the studied systems as supported from the literature.ConclusionsRNEA constitutes a novel computational approach to obtain regulatory interactions directly from a genome-wide expression profile. Its simple implementation, with minimal requirements from the user is coupled with easy-to-parse enrichment lists and a subnetwork file that may be readily visualized to reveal the most important components of the regulatory hierarchy. The combination of prior information and novel concept of a hierarchical reconstruction of regulatory interactions makes RNEA a very useful tool for a first-level interpretation of gene expression profiles.

Genomic Responses of Mouse Synovial Fibroblasts During Tumor Necrosis Factor-Driven Arthritogenesis Greatly Mimic Those in Human Rheumatoid Arthritis.

Aberrant activation of synovial fibroblasts is a key determinant in the pathogenesis of rheumatoid arthritis (RA). The aims of this study were to produce a map of gene expression and epigenetic changes occurring in this cell type during disease progression in the human tumor necrosis factor (TNF)–transgenic model of arthritis and to identify commonalities with human synovial fibroblasts.

Comorbid TNF-mediated heart valve disease and chronic polyarthritis share common mesenchymal cell-mediated aetiopathogenesis

Objectives Patients with rheumatoid arthritis and spondyloarthritisshow higher mortality rates, mainly caused by cardiac comorbidities. The TghuTNF (Tg197) arthritis model develops tumour necrosis factor (TNF)-driven and mesenchymalsynovial fibroblast (SF)-dependent polyarthritis. Here, we investigate whether this model develops, similarly to human patients, comorbid heart pathology and explore cellular and molecular mechanisms linking arthritis to cardiac comorbidities. Methods Histopathological analysis and echocardiographic evaluation of cardiac function were performed in the Tg197 model. Valve interstitial cells (VICs) were targeted by mice carrying the ColVI-Cretransgene. Tg197 ColVI-Cre Tnfr1 fl/fl and Tg197 ColVI-Cre Tnfr1 cneo/cneo mutant mice were used to explore the role of mesenchymal TNF signalling in the development of heart valve disease. Pathogenic VICs and SFs were further analysed by comparative RNA-sequencing analysis. Results Tg197 mice develop left-sided heart valve disease, characterised by valvular fibrosis with minimal signs of inflammation. Thickened valve areas consist almost entirely of hyperproliferative ColVI-expressing mesenchymal VICs. Development of pathology results in valve stenosis and left ventricular dysfunction, accompanied by arrhythmic episodes and, occasionally, valvular regurgitation. TNF dependency of the pathology was indicated by disease modulation following pharmacological inhibition or mesenchymal-specific genetic ablation or activation of TNF/TNFR1 signalling. Tg197-derived VICs exhibited an activated phenotype ex vivo, reminiscent of the activated pathogenic phenotype of Tg197-derived SFs. Significant functional similarities between SFs and VICs were revealed by RNA-seq analysis, demonstrating common cellular mechanisms underlying TNF-mediated arthritides and cardiac comorbidities. Conclusions Comorbidheart valve disease and chronic polyarthritis are efficiently modelled in the Tg197 arthritis model and share common TNF/TNFR1-mediated, mesenchymal cell-specific aetiopathogenic mechanisms.

Cigarette Smoke–Induced Emphysema Exhausts Early Cytotoxic CD8+ T Cell Responses against Nascent Lung Cancer Cells

Chronic obstructive pulmonary disease is a chronic inflammatory disorder with an increased incidence of lung cancer. The emphysema component of chronic obstructive pulmonary disease confers the greatest proportion to lung cancer risk. Although tumors create inflammatory conditions to escape immunity, the immunological responses that control growth of nascent cancer cells in pre-established inflammatory microenvironments are unknown. In this study, we addressed this issue by implanting OVA-expressing cancer cells in the lungs of mice with cigarette smoke–induced emphysema. Emphysema augmented the growth of cancer cells, an effect that was dependent on T cytotoxic cells. OVA-specific OTI T cells showed early signs of exhaustion upon transfer in emphysema tumor hosts that was largely irreversible because sorting, expansion, and adoptive transfer failed to restore their antitumor activity. Increased numbers of PD-L1– and IDO-positive CD11c+ myeloid dendritic cells (DCs) infiltrated emphysema tumors, whereas sorted emphysema tumor DCs poorly stimulated OTI T cells. Upon adoptive transfer in immunocompetent hosts, T cells primed by emphysema tumor DCs were unable to halt tumor growth. DCs exposed to the emphysema tumor microenvironment downregulated MHC class II and costimulatory molecules, whereas they upregulated PD-L1/IDO via oxidative stress–dependent mechanisms. T cell activation increased upon PD-L1 blockade in emphysema DC–T cell cocultures and in emphysema tumor hosts in vivo. Analysis of the transcriptome of primary human lung tumors showed a strong association between computed tomography–based emphysema scoring and downregulation of immunogenic processes. Thus, suppression of adaptive immunity against lung cancer cells links a chronic inflammatory disorder, emphysema, to cancer, with clinical implications for emphysema patients to be considered optimal candidates for cancer immunotherapies.

Mesenchymal TNFR2 promotes the development of polyarthritis and comorbid heart valve stenosis

Mesenchymal TNF signaling is etiopathogenic for inflammatory diseases such as rheumatoid arthritis and spondyloarthritis (SpA). The role of Tnfr1 in arthritis has been documented; however, Tnfr2 functions are unknown. Here, we investigate the mesenchymal-specific role of Tnfr2 in the TnfΔARE mouse model of SpA in arthritis and heart valve stenosis comorbidity by cell-specific, Col6a1-cre-driven gene targeting. We find that TNF/Tnfr2 signaling in resident synovial fibroblasts (SFs) and valvular interstitial cells (VICs) is detrimental for both pathologies, pointing to common cellular mechanisms. In contrast, systemic Tnfr2 provides protective signaling, since its complete deletion leads to severe deterioration of both pathologies. SFs and VICs lacking Tnfr2 fail to acquire pathogenic activated phenotypes and display increased expression of antiinflammatory cytokines associated with decreased Akt signaling. Comparative RNA sequencing experiments showed that the majority of the deregulated pathways in TnfΔARE mesenchymal-origin SFs and VICs, including proliferation, inflammation, migration, and disease-specific genes, are regulated by Tnfr2; thus, in its absence, they are maintained in a quiescent nonpathogenic state. Our data indicate a pleiotropy of Tnfr2 functions, with mesenchymal Tnfr2 driving cell activation and arthritis/valve stenosis pathogenesis only in the presence of systemic Tnfr2, whereas nonmesenchymal Tnfr2 overcomes this function, providing protective signals and, thus, containing both pathologies.

04.14 Aortic valve disease co-develops with polyarthritis in the tnf-driven models and shares a common mesenchymal cell-mediated causality

Introduction and objectives Rheumatoid arthritis (RA) is a chronic condition characterised by inflammation of the joints as well as destruction of bone and cartilage. Studies on TNF transgenic models of polyarthritis established Tumour Necrosis Factor (TNF) targeting the mesenchymal-origin Synovial Fibroblasts (SFs) as a key event instigating the pathology. RA patients often show higher mortality rates, mainly due to the development of extraarticular disease including cardiovascular, gut and skin manifestations. The Tg197 and TnfΔARE/+ mouse models overexpress TNF and develop spontaneous chronic polyarthritis, fully mimicking human RA pathology. Here, we investigate whether these models develop, similarly to human patients, co-morbid heart pathology. Materials and methods We used the Tg197 and TnfΔARE/+ mouse models to evaluate possible arthritis-related cardiovascular disease. For further ex vivo analysis, we isolated Valve Interstitial Cells (VICs), which are the mesenchymal-origin fibroblasts constituting the aortic valve. We used a GFP reporter mouse labelling specifically mesenchymal-origin cells (ColVICre;mTm/mGFP) to target VICs. Similarities of pathogenic VICs and SFs were analysed by comparing their expression profiling with RNA sequencing analysis. Results Both arthritis models examined develop TNF-dependent aortic valve thickening, as indicated by the amelioration of the pathology following treatment with anti-TNF biologics. Aortic valves exhibited significant fibrosis with minimal signs of inflammatory cell infiltration and thickened areas, consisting almost entirely of VICs, indicating proliferation of this cell type as a hallmark of the observed phenotype. Isolated VICs from mutant mice exhibited a more proliferative and migratory phenotype and expressed high levels of TNF. Interestingly, VIC activation resembles the activated phenotype of pathogenic SFs isolated from the same mice. A significant functional correlation between these two pathogenic cells of mesenchymal origin was also supported by RNA-seq analysis, suggesting common cellular mechanisms operating in RA and RA-related heart pathology. Conclusion TNF-driven arthritis models, apart from their arthritic symptoms, develop co-morbid heart valve disease, similarly to reported comorbidities in RA patients. These two co-morbid diseases are shown here to share common mesenchymal-cell driven aetiopathogenesis.