Research activities

Innate activation of the Synovial Fibroblast and early causalities driving chronic arthritis

Marietta Armaka, Vangelis Ntougkos, Christoforos Nikolaou

Rheumatoid arthritis (RA) is a most common chronic inflammatory joint disease debilitating 1-2% of the human population. It is also a relatively well studied disease both in humans and in animal models, offering exciting opportunities for the discovery of novel biological mechanisms relevant to a plethora of other chronic immunological disorders. While the exact mechanisms leading to RA pathogenesis remain elusive, recent experimental evidence indicates that chronic innate immune activation of the synovial fibroblast (SF) is an early event leading to synovial hyperplasia and inflammation. SFs are resident mesenchymal cells of the synovial membrane showing key disease promoting pro-inflammatory activities. While until recently such non-immune cells were regarded as merely targets of the inflammatory microenvironment, and thus secondary effectors, recent evidence suggests that SFs may be the inciting players in RA aetiopathogenesis.

Our current working hypothesis is that chronic activation signals targeting the synovial fibroblast lead to persistent alterations in chromatin structure, epigenetic and gene expression programs, transforming it into an autonomous and aggressive effector cell type inciting the whole spectrum of chronic pathogenic progression in arthritis. We are using a unique collection of mutant mouse models, genetic tools and functional genomic technologies, to identify mechanisms and hierarchies that result in the chronic, aggressive, immune-modulatory and invasive properties of SFs and to discover causalities in the epigenetic, biochemical, molecular and cellular changes that occur in this cell type during arthritis initiation and progression.

More specifically, in a functional genetics approach, we are investigating the SF-specific NF-κB and MAPK intracellular signals (mediated through IKKs, cyld, tpl2, tak1 etc) and evaluating their functional significance in the initiation and progression of the disease, in cellular assays and across different animal models of inflammatory arthritis. In the same context, we have recently identified actin cytoskeletal modifications in activated SFs as being of pathological and therapeutic significance in modelled disease, and are currently further exploiting this finding for mechanistic specificity.

Furthermore, based on the hypothesis that arthritogenic signals are sustained by multiple layers of epigenetic regulation, we are exploring the impact of epigenetic modifications on RA development in either a systemic or a SF-specific manner. Aiming at understanding the contribution of epigenetic phenomena, such as DNA methylation, as well as the role of micro RNAs, we wish to identify disease-associated epigenetic signatures by global, omics approaches, and in a more focused effort, to study the effect of particular epigenetic regulators and micro RNAs. To this end we work from both ends in a) the production of genome-scale transcriptomic, epigenetic and proteomic datasets and b) the development of novel computational tools for their analysis and interpretation.

Through the integration of multiple -omics analyses at systems level, we are interested in:

i) the way the onset and progression of the disease may be reflected on the structure of chromatin nucleus and the overall expression profile at both RNA and protein levels

ii) the genomic and epigenomic differences between the chronic and the acute states of the disease

iii) the way treatment with various therapeutic agents may affect the general state of the cells at various hierarchical levels (gene, pathways and systems)

Finally, our pursuit of better understanding of the biology of rheumatoid arthritis is complemented by more translational projects seeking novel small molecule-based therapies. Disease specific cellular and in vivo assays have been developed and are being used to screen compounds at a preclinical level aiming to provide successful candidate leads for clinical evaluation.

Selected Publications
Pandis I, Ospelt C, Karagianni N, Denis M, Reczko M, Camps C, Hatzigeorgiou A, Ragoussis J, Gay S and Kollias G. (2012) Identification of microRNA-221/222 and -323-3p Association with Rheumatoid Arthritis via Predictions using the Human TNF Transgenic Mouse Model. Ann Rheum Dis. 71:1716-23.

Douni E, Rinotas V, Makrinou E, Zwerina J, Penninger JM, Eliopoulos E, Schett G, Kollias G.  (2012) A RANKL G278R mutation causing osteopetrosis identifies a functional amino acid essential for trimer assembly in RANKL and TNF. Hum Mol Genet., Feb 15;21(4):784-98

Armaka M, Apostolaki M, Jacques P, Kontoyiannis DL, Elewaut D and Kollias G. (2008) Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases. J Exp Med 205:331-7.

Victoratos P, Kollias G. (2009) Induction of autoantibody-mediated spontaneous arthritis critically depends on follicular dendritic cells. Immunity, 30(1):130-42

Aidinis V., Carninci P., Armaka M., Witke W., Harokopos V., Pavelka N., Koczan D., Argyropoulos C., Thwin MM., Möller S., Kazunori W., Gopalakrishnakone P., Ricciardi-Castagnoli P., Thiesen HJ., Hayashizaki Y., Kollias G. (2005) Cytoskeletal rearrangements in synovial fibroblasts as a novel pathophysiological determinant of modeled rheumatoid arthritis. PLoS Genetics 1(4):455-466.

Kontoyiannis D., Pasparakis M., Pizzaro Th., Cominelli F. and Kollias G. (1999) Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies. Immunity 10:387-398.

Plows D., Kontogeorgos G. and Kollias G. (1999) Mice lacking mature T and B lymphocytes develop arthritic lesions after immunization with Type II collagen. J. Immunol. 162:1018-1023.

Alexopoulou L., Pasparakis M. and Kollias G. (1997) A murine transmembrane Tumour Necrosis Factor (TNF) transgene induces arthritis by cooperative p55/p75 tumour necrosis factor receptor signalling. Eur. J. Immunol. 27:2588-2592

Probert L., Plows D., Kontogeorgos G. and Kollias G. (1995) The type I IL-1 receptor acts in series with TNF to induce arthritis in TNF transgenic mice. Eur. J. Immunol., Vol. 25, pp.1794-1797.

Probert L., Keffer J., Corbella P., Cazlaris H., Patsavoudi E., Stephens S., Kaslaris E., Kioussis D. and Kollias G. (1993). Wasting, ischaemia and lymphoid abnormalities in mice expressing T cell targeted human tumour necrosis factor transgenes. J. Immunol., 151, 1894-1906.

Keffer J., Probert L., Cazlaris H., Georgopoulos S., Kaslaris E., Kioussis D., Kollias G. (1991) Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J. 10, 4025-4031


Mesenchymal-specific pathways in intestinal inflammation and carcinogenesis

Vasiliki Koliaraki, Manolis Roulis

 Intestinal structure and function during development and in adulthood and the maintenance of intestinal homeostasis following pathogenic challenges is shaped by complex and dynamic interactions between the intestinal epithelium, the underlying mesenchymal cell (MC) compartment and the various lymphoid tissues and immune cell types. These interactions are necessary both for the beneficial cooperation of hosts with commensal microbiota and for the development of defence strategies against pathogens.
Intestinal mesenchymal cells (MCs) comprise a group of cells in the lamina propria, which are involved in a variety of normal or pathological processes, including morphogenesis, inflammation and carcinogenesis, although their exact mechanisms of action are not yet completely understood.

We have recently demonstrated that intestinal myofibroblast-specific signals play an important physiological role in chronic intestinal inflammation and inflammation-induced cancer. A growing list of mediators released by MCs, such as growth and proangiogenic factors, reactive oxygen species, chemokines and cytokines and proinvasive matrix-degrading enzymes could serve as effectors during these processes. Especially in cancer, MCs appear to be crucial components of the tumor microenvironment and to actively participate in complex interactive networks with the pre-neoplastic or cancer cells to establish a pro- or anti-tumorigenic milieu.

Our hypothesis is that MCs have innate characteristics and are thus capable of directly sensing and metabolizing innate signals in order to regulate intestinal homeostasis during acute or chronic inflammatory and tumorigenic conditions.

To verify this hypothesis in the context of intestinal carcinogenesis we are using recently developed conditional knockout mice carrying relevant mutant alleles, such as Tpl2, IKK2, CYLD and MK2 and the mesenchymal-specific Cre mouse line TgColVI-Cre, previously developed in our lab to specifically ablate these genes in MCs. We then explore the effect of MC-specific gene ablation in in vivo models of intestinal cancer and inflammation-induced cancer, such as the APC1638N and the AOM/DSS model.
Extensive phenotypic analysis along with molecular and biochemical characterization of deregulated pathways in isolated MCs is further performed in order to elucidate the detailed regulatory signalling pathways that control the functions of MCs in intestinal tumorigenesis.

Selected Publications
Koliaraki V, Roulis M, Kollias G. (2012) Tpl2 regulates intestinal myofibroblast HGF release to suppress colitis-associated tumorigenesis. J. Clin. Invest. 122(11):4231–4242.

Roulis M, Armaka M, Manoloukos M, Apostolakis M and Kollias G. (2011) Intestinal epithelial cells as producers but not targets of chronic TNF suffice to cause murine Crohn-like pathology. Proc. Natl. Acad. Sci. USA 29, 108:5396-401.

Kontoyiannis D, Boulougouris G, Manoloukos M, Armaka M, Apostolaki M, Pizzaro T, Kotlyarov A, Forster I, Flavell R, Gaestel M, Tsichlis P, Cominelli F. and Kollias G. (2002) Genetic dissection of the cellular pathways and signaling mechanisms in modeled tumor necrosis factor-induced Crohn’s-like inflammatory bowel disease. J. Exp. Med. 196:1563-74.

Apostolaki M, Manoloukos M, Roulis M, Wurbel MA, Müller W, Papadakis KA, Kontoyiannis DL, Malissen B,Kollias G. (2008) Role of beta7 integrin and the chemokine/chemokine receptor pair CCL25/CCR9 in modeled TNF-dependent Crohn's disease. Gastroenterology134(7):2025-35

Victoratos P, Lagnel J, Tzima S, Alimzhanov MB, Rajewsky K, Pasparakis M, Kollias G. (2006) FDC-specific functions of p55TNFR and IKK2 in the development of FDC networks and of antibody responses. Immunity 24(1):65-77

Kontoyiannis D., Kotlyarov S., Carballo E., Alexopoulou L., Blackshear PJ., Gaestel M., Davis R., Flavell R. and Kollias G. (2001) Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology. EMBO J. 20:3760-70

Neurath M., Fuss I., Pasparakis M., Alexopoulou L., Haralambous S., Meyer zum Buschendelde K.-H., Strober W. and Kollias G. (1997) Predominant pathogenic role of Tumor Necrosis Factor in experimental colitis in mice. Eur. J. Immunol. 27:1743-1750.

Pro- and Anti-inflammatory mechanisms in Experimental Autoimmune Encephalomyelitis (EAE), the animal model of Multiple Sclerosis (MS)

Niki Tsakiri

MS is the most common neuroimmunological disease, characterized by disseminated foci of inflammatory demyelination in the brain and spinal cord that lead to significant neurological disability, especially affecting young women between the age of 20 and 40. Typically MS has a relapsing and remitting course followed by the development of irreversible neurological disability due to persistent axonal dysfunction and neuronal loss. CNS demyelination, the pathological hallmark of MS is brought about by autoreactive autoimmune infiltrations. As MS is the prototypical autoimmune CNS disease, it is associated with dysfunctional immune suppressive/ tolerogenic mechanisms, which regulate aggressive autoimmune behaviour. Chronicity and relapses in autoimmune diseases including MS seem to involve defective integration of inflammatory and anti-inflammatory cues. Numerous studies have dissected the multiple and opposing roles of cytokine-producing cells in the autoimmune pathogenesis of MS and EAE.

To date the exact mechanisms driving chronicity and relapses in MS remain unknown and they are most commonly modelled in murine Experimental Autoimmune Encephalomyelitis (EAE). EAE is induced by immunization with myelin proteins and it produces T cell-driven demyelination with components from cellular and humoral immunity leading to axonal injury associated with disability ranging from mild weakness to severe paralysis.

Our goal is to utilize genetically modified animals to better understand the role of molecules TNF, TNFR2 and Tpl2, which are critically involved in inflammatory processes in EAE. These molecules can produce a complex array of responses both pro-inflammatory and protective, and be involved in tissue damage repair and remyelination. The fine balance, proper timing and specific cellular expression of their action can affect the outcome of the disease process. We have generated conditional knock out animals by employing the Cre-LoxP system for TNFR2 and Tpl2. This allows us to eliminate TNFR2 and Tpl2 from specific cells of the immune system, CNS and Blood Brain Barrier that are suspected to be key players in the progression of the disease. Moreover we develop inducible conditional models, for spatial and temporal specific deletion of the key molecules in EAE. Additional transgenic mouse line as the tmTNF (transmembrane TNF which signals exclusively via TNFR2), are used for the dissection of the pathways involved. Our objectives are to better understand: (a) the cellular sources and targets of TNF, TNFR2 and Tpl2 signalling and (b) their contribution in the exacerbation and recovery phase of EAE.

Selected Publications
Tsakiri N, Papadopoulos D, Denis MC, Mitsikostas DD, Kollias G. (2011) TNFR2 on non-haematopoietic cells is required for Foxp3+ regulatory T-cell function and disease suppression in experimental autoimmune encephalomyelitis. Eur J Immunol. 42(2):403-12

Alexopoulou L, Kranidioti K, Xanthoulea S, Denis M, Kotanidou A, Douni E, Blackshear PJ, Kontoyiannis DL, Kollias G. (2006) Transmembrane TNF protects mutant mice against intracellular bacterial infections, chronic inflammation and autoimmunity. Eur. J. Immunol. 36:2768-80.

Akassoglou K., Douni E., Bauer J., Lassmann H, Kollias G. and Probert L. (2003) Exclusive tumor necrosis factor (TNF) signaling by the p75TNF receptor triggers inflammatory ischemia in the CNS of transgenic mice. Proc. Natl. Acad. Sci. USA 100:709-714

Kassiotis G, Kollias G. (2001) Uncoupling the proinflammatory from the immunosuppressive properties of tumor necrosis factor (TNF) at the p55 TNF receptor level: implications for pathogenesis and therapy of autoimmune demyelination. J Exp Med. 193(4):427-34.

Akassoglou K., Bauer J., Kassiotis G., Pasparakis M., Lassmann H., Kollias G. and Probert L. (1998) Oligodendrocyte apoptosis and primary demyelination induced by local TNF/p55TNF receptor signaling the CNS of transgenic mice: models for Multiple Sclerosis with primary oligodendrogliopathy. Am. J. Pathol. 153:801-813

Akassoglou K., Probert L., Kontogeorgos G. and Kollias G. (1997) Astrocyte-specific but not neuron-specific transmembrane TNF triggers inflammation and degeneration in the central nervous system of transgenic mice. J. Immunol. 158:438-445.

Probert L., Akassoglou K., Kontogeorgos G. and Kollias G. (1995) Spontaneous inflammatory demyelinating disease in transgenic mice showing CNS-specific TNFα expression. Proc. Natl. Acad. Sci. USA 92, 11294-11298

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