Niki Karagianni

Identification of microRNA-221/222 and microRNA-323-3p association with rheumatoid arthritis via predictions using the human tumour necrosis factor transgenic mouse model

Objective To identify novel microRNA (miR) associations in synovial fibroblasts (SF), by performing miR expression profiling on cells isolated from the human tumour necrosis factor (TNF) transgenic mouse model (TghuTNF, Tg197) and patients biopsies. Methods miR expression in SF from TghuTNF and wild-type (WT) control mice were determined by miR deep sequencing (miR-seq) and the arthritic profile was established by pairwise comparisons. Quantitative PCR analysis was utilised for profile validation, miR and gene quantitation in patient SF. Dysregulated miR target genes and pathways were predicted via bioinformatic algorithms and validated using gain-of-function coupled with reporter assay experiments. Results miR-seq demonstrated that TghuTNF-SF exhibit a distinct pathogenic profile with 22 significantly upregulated and 30 significantly downregulated miR. Validation assays confirmed the dysregulation of miR-223, miR-146a and miR-155 previously associated with human rheumatoid arthritis (RA) pathology, as well as that of miR-221/222 and miR-323-3p. Notably, the latter were also found significantly upregulated in patient RA SF, suggesting for the first time their association with RA pathology. Bioinformatic analysis suggested Wnt/cadherin signalling as a putative pathway target. miR-323-3p overexpression was shown to enhance Wnt pathway activation and decrease the levels of its predicted target β-transducin repeat containing, an inhibitor of β-catenin. Conclusions Using miR-seq-based profiling in SF from the TghuTNF mouse model and validations in RA patient biopsies, the authors identified miR-221/222 and miR-323-3p as novel dysregulated miR in RA SF. Furthermore, the authors show that miR-323-3p is a positive regulator of WNT/cadherin signalling in RA SF suggesting its potential pathogenic involvement and future use as a therapeutic target in RA.

Safe TNF-based antitumor therapy following p55TNFR reduction in intestinal epithelium

TNF has remarkable antitumor activities; however, therapeutic applications have not been possible because of the systemic and lethal proinflammatory effects induced by TNF. Both the antitumor and inflammatory effects of TNF are mediated by the TNF receptor p55 (p55TNFR) (encoded by the Tnfrsf1a gene). The antitumor effect stems from an induction of cell death in tumor endothelium, but the cell type that initiates the lethal inflammatory cascade has been unclear. Using conditional Tnfrsf1a knockout or reactivation mice, we found that the expression level of p55TNFR in intestinal epithelial cells (IECs) is a crucial determinant in TNF-induced lethal inflammation. Remarkably, tumor endothelium and IECs exhibited differential sensitivities to TNF when p55TNFR levels were reduced. Tumor-bearing Tnfrsf1a⁺⁺/⁻ or IEC-specific p55TNFR-deficient mice showed resistance to TNF-induced lethality, while the tumor endothelium remained fully responsive to TNF-induced apoptosis and tumors regressed. We demonstrate proof of principle for clinical application of this approach using neutralizing anti-human p55TNFR antibodies in human TNFRSF1A knockin mice. Our results uncover an important cellular basis of TNF toxicity and reveal that IEC-specific or systemic reduction of p55TNFR mitigates TNF toxicity without loss of antitumor efficacy.

Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis.

Significance Tumor progression locus-2 (Tpl2) is a proinflammatory gene genetically associated with inflammatory bowel diseases. This study provides a mechanistic interpretation for this association showing a dominant Tpl2-mediated homeostatic mechanism protecting mice from epithelial injury-induced colitis. This function of Tpl2 is mediated specifically by subepithelial intestinal myofibroblasts, a cell type supporting crypt stem cells. Tpl2 in myofibroblasts is essential for the compensatory proliferative response of the epithelium by promoting arachidonic acid metabolism and cyclooxygenase-2 (Cox-2)/prostaglandin E2 activation. Notably, in Crohn’s Disease patients, Tpl2 is downregulated in myofibroblasts isolated from the inflamed ileum. These results challenge current concepts on a solely proinflammatory function of Tpl2 and highlight the dominant role of subepithelial myofibroblasts in sensing inflammation and tissue damage and promoting intestinal homeostasis through Tpl2-Cox-2-prostaglandin E2. Tumor progression locus-2 (Tpl2) kinase is a major inflammatory mediator in immune cell types recently found to be genetically associated with inflammatory bowel diseases (IBDs). Here we show that Tpl2 may exert a dominant homeostatic rather than inflammatory function in the intestine mediated specifically by subepithelial intestinal myofibroblasts (IMFs). Mice with complete or IMF-specific Tpl2 ablation are highly susceptible to epithelial injury-induced colitis showing impaired compensatory proliferation in crypts and extensive ulcerations without significant changes in inflammatory responses. Following epithelial injury, IMFs sense innate or inflammatory signals and activate, via Tpl2, the cyclooxygenase-2 (Cox-2)-prostaglandin E2 (PGE2) pathway, which we show here to be essential for the epithelial homeostatic response. Exogenous PGE2 administration rescues mice with complete or IMF-specific Tpl2 ablation from defects in crypt function and susceptibility to colitis. We also show that Tpl2 expression is decreased in IMFs isolated from the inflamed ileum of IBD patients indicating that Tpl2 function in IMFs may be highly relevant to human disease. The IMF-mediated mechanism we propose also involves the IBD-associated genes IL1R1, MAPK1, and the PGE2 receptor-encoding PTGER4. Our results establish a previously unidentified myofibroblast-specific innate pathway that regulates intestinal homeostasis and may underlie IBD susceptibility in humans.

Myeloid Takl Acts as a Negative Regulator of the LPS Response and Mediates Resistance to Endotoxemia

TGFβ-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, is considered a key intermediate in a multitude of innate immune signaling pathways. Yet, the specific role of TAK1 in the myeloid compartment during inflammatory challenges has not been revealed. To address this question, we generated myeloid-specific kinase-dead TAK1 mutant mice. TAK1 deficiency in macrophages results in impaired NF-κB and JNK activation upon stimulation with lipopolysaccharide (LPS). Moreover, TAK1-deficient macrophages and neutrophils show an enhanced inflammatory cytokine profile in response to LPS stimulation. Myeloid-specific TAK1 deficiency in mice leads to increased levels of circulating IL-1β, TNF and reduced IL-10 after LPS challenge and sensitizes them to LPS-induced endotoxemia. These results highlight an antiinflammatory role for myeloid TAK1, which is essential for balanced innate immune responses and host survival during endotoxemia.

Improved topical delivery of tacrolimus: A novel composite hydrogel formulation for the treatment of psoriasis

We have developed a composite hydrogel for improved topical delivery of the poorly soluble drug Tacrolimus (TAC) to psoriasis lesions. TAC is efficiently solubilized in methoxy poly- (ethylene glycol) hexyl substituted poly-(lactic acid) (mPEGhexPLA) based nanocarriers. For convenient and patient-friendly topical administration, TAC loaded polymeric nanocarriers were incorporated in a Carbopol® based hydrogel, to yield a composite hydrogel formulation (TAC composite hydrogel). TAC composite hydrogel was designed to have superior pharmaceutical formulation properties, delivery efficiency and local bioavailability, compared to currently available paraffin-based TAC ointments. Composite hydrogel formulations had good local tolerance and showed no signs of immediate toxicity after repeated topical administration in healthy mice. Skin delivery of TAC composite hydrogel in an imiquimod-induced psoriasis mouse model was found to be twice as high as for the commercial formulation Protopic™, used as benchmark. TAC composite hydrogel showed significant improvement in the in vivo and histopathological features of the imiquimod-induced psoriasis model.

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.

Extensive phenotypic characterization of a new transgenic mouse reveals pleiotropic perturbations in physiology due to mesenchymal hGH minigene expression

The human growth hormone (hGH) minigene used for transgene stabilization in mice has been recently identified to be locally expressed in the tissues where transgenes are active and associated with phenotypic alterations. Here we extend these findings by analyzing the effect of the hGH minigene in TgC6hp55 transgenic mice which express the human TNFR1 under the control of the mesenchymal cell-specific CollagenVI promoter. These mice displayed a fully penetrant phenotype characterized by growth enhancement accompanied by perturbations in metabolic, skeletal, histological and other physiological parameters. Notably, this phenotype was independent of TNF-TNFR1 signaling since the genetic ablation of either Tnf or Tradd did not rescue the phenotype. Further analyses showed that the hGH minigene was expressed in several tissues, also leading to increased hGH protein levels in the serum. Pharmacological blockade of GH signaling prevented the development of the phenotype. Our results indicate that the unplanned expression of the hGH minigene in CollagenVI expressing mesenchymal cells can lead through local and/or systemic mechanisms to enhanced somatic growth followed by a plethora of primary and/or secondary effects such as hyperphagia, hypermetabolism, disturbed glucose homeostasis, altered hematological parameters, increased bone formation and lipid accumulation in metabolically critical tissues.

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.