The lab has previously established that TNF is of pivotal importance in the development of inflammatory arthritis, Crohn’s inflammatory bowel disease , systemic inflammation and multiple sclerosis . Through the early establishment of TNF-driven transgenic and mutant animal models for these diseases, the lab was also involved in the development of anti-TNF therapies currently used in the clinical management of rheumatoid arthritis, Crohn’s disease and psoriasis. Additional early work led to the generation and characterization of TNF-deficient mice and to the establishment of the importance of TNF in secondary lymphoid organ structure and function , in host defence against intracellular bacteria and in the suppression of systemic and organ-specific autoimmunity .

The lab is now continuing research aiming at delineating the identity of the specific molecular and cellular signals and their mode of function in TNF-mediated disease, as a means to gain better understanding of such mechanisms and to contribute to the development of more selective molecular therapies for these diseases in humans . To this end, the lab has been developing and using technologies for the constitutive or inducible tissue-specific inactivation or re-activation of gene expression in mutant mice as well as high-throughput functional genomic approaches (gene expression profiling and random mutagenesis screens) in order to discover novel genes contributing to disease.

Molecular and cellular mechanisms in disease pathogenesis:

(Maria Apostolaki, post-doc; Darren Plant, post-doc (Marie Curie RTN fellow); Marietta Armaka, PhD student; Christina Eftychi, PhD student (Marie Curie early stage researcher); Alexandra Amaral-Psarris, (Marie Curie early-stage researcher)

Our recent studies addressing cellular targets of TNF in transgenic models developing arthritis or IBD pathology provided a first evidence for redundant cellular pathways of disease induction operating downstream of TNF . Analysis of effector kinase signaling operating downstream of TNF has identified Cot/Tpl2 and JNK2 kinases as dominantly contributing in the pathogenesis of disease, while in contrast, MAPKAP kinase-2 (MK2) showed an overall anti-inflammatory role . At present we focus on the identification of the specific cell types and signaling pathways with which the Tpl2 and MK2 kinases interfere to modulate disease development. This issue is addressed by the generation of cell-specific Tpl2 and MK2 deficient animals. In a further attempt to understand mechanisms with which MAP-kinases may modulate disease, we have become interested in delineating the role of a newly identified and poorly characterized kinase. TAK1 is a MAP3K that has been shown to have important roles in innate immune responses and in inflammation. It has a key position in MAPK signaling pathways triggering activation of Jnk, p38 signaling as well as the transcription factor NFκB upon stress (LPS) and inflammatory (TNFα, IL1) signals. To study the in vivo function of TAK1 in physiology and disease, we are generating a conditional TAK1 knockout mouse with the aim to gain further insight into the potential cell-specific role of this kinase in the immune processes and disease models employed in our lab.

Role of endothelial NF-kB in acute sepsis:

(Elena Kotsaki, PhD student)

In parallel studies, we are addressing the role of endothelial-specific deregulation of NF-κB in inflammatory conditions, in transgenic mice expressing the dominant-negative IκBα mutant (IκBαS32A) under the control of the endothelial-specific tie2 promoter. These mice develop chronic spontaneous liver necrosis and inflammation and most interestingly a lethal hypersensitivity towards low doses of administered TNF with vasodilatation and shock characteristics of acute sepsis (manuscript in preparation). Deriving mechanistic insights into this phenotype may reveal important mechanisms underlying development of this lethal syndrome in humans.

Role of HO-1 in the regulation of inflammation:

(Sotiria Tzima, post-doc)

Disbalances in the regulation of inflammatory and anti-inflammatory cascades during development and regression of inflammation may explain disease pathogenesis. To gain insight in the temporal regulation of inflammatory processes we have been using Heme Oxygenase-1 (HO-1) as a paradigm of an anti-inflammatory modulator of disease. The evolving paradigm of HO-1 mediated protection of cells and tissues is supported by several animal models of oxidant injury (endotoxic shock, ischemia, hyperoxia etc.) and acute inflammation. HO-1 elevation has been proposed to confer potent resistance to stress, cell injury and LPS-induced death whereas blocking of HO-1 activity with specific inhibitors abrogates cytoprotection, resulting in severe tissue damage. To elucidate the mechanisms underlying the anti-inflammatory and cytoprotective properties of HO-1 in different disease models we are developing conditional HO-1 knock out mice (Cre-loxP system) and inducible, tissue-specific HO-1 over-expressing mice (TET ON). Using these tools we attempt to delineate the molecular mechanisms of HO-1 mediated protection in inflammation and autoimmunity, and to define specific cell types mediating this protection.

Rationalizing the advantages of anti-p55TNFR versus anti-TNF therapies:

(Niki Karagianni, post-doc; Ksanthi Kranidioti, PhD student)

Following our involvement and expertise gained in the development of anti-TNF therapies, we have remained interested in building further rationales for the use of more selective anti-TNF therapies in chronic inflammation and autoimmunity . Recent work we performed in experimental autoimmune encephalomyelitis (EAE), has revealed specific advantages in blocking of the p55TNFR instead of TNF in organ-specific autoimmunity . Our data indicated that inhibiting the p55TNFR in autoimmune disease may inhibit the proinflammatory and tissue damaging activities of TNF without compromising its immune suppressive properties. The therapeutic application of this statement is further explored through (a) generation of mutant mice humanized for the p55TNFR gene, (b) development of anti-hup55TNFR neutralizing antibodies (in academic collaboration with Prof. G. Georgiou University of Texas, Austin and two more interested European companies), (c) generation of p55TNFRI conditional knockout mice, and (d) exploitation of the RNAi technology for the tissue specific knockdown of p55TNFR expression in transgenic mice and/or in situ inactivation of p55TNFR through in vivo delivery of siRNAs.

Role of the p75TNFR in demyelination – remyelination balances:

(Maria Denis, Marie Curie fellow)

In contrast to the well-studied function of p55TNFR the role of p75TNFR still remains largely unknown in many pathological conditions. Our goal is to utilize genetically modified animals to better understand the role of p75TNFR in autoimmunity, with a particular focus in Experimental Autoimmune Encephalomyelitis (EAE) where the function of this receptor is predicted to be protective both in the inflammatory/autoimmune phase and most interestingly in tissue repair and remyelination. Our objectives are to better understand: (a) the cellular targets of p75TNFR signaling and (b) the contribution of p75TNFR in the recovery phase of EAE. These objectives are currently addressed through the study of EAE-affected control and p75-/- animals. We are also in the process of generating conditional p75TNFR knock out animals by employing the Cre-LoxP system. This will allow us to eliminate p75TNFR from specific cells of the immune system and the CNS that are suspected to be key players in the progression of the disease.

TNF receptor pathways in lymphoid organ structure and function:

(Panayiotis Victoratos, post-doc)

In continuation of earlier studies where we have established the importance of TNF signaling in lymphoid organ structure and function we are now dissecting the cellular basis of this phenomenon using tissue-specific p55TNFR reactivation in mutant mice. First, a non-functional p55TNFR allele was engineered to be re-activated specifically in FDCs using TgCD21Cre-loxP mediated recombination. In addition, the FDC-specific role of IKK2 is investigated by conditional ablation of IKK2 in FDCs (in collaboration with M. Pasparakis, EMBL Monterotondo). Such mechanistic insights are important for the detailed understanding of the germinal center reaction and its potential therapeutic manipulation during pathogenic humoral responses in autoimmunity.

Random genome mutagenesis and identification of novel genes contributing to disease:

(Eleni Douni, operational scientist, Eleni Makrinou, post-doc)

Novel information from the various genome projects provides outstanding opportunities to take multidisciplinary approaches and understand the wide spectrum of diseases in terms of genetic, molecular and cellular networks. Reverse-genetic approaches such as those using conventional transgenesis and constitutive or conditional gene knockout technologies are gene-specific and usually hypothesis-driven approaches and have proven extremely useful in modelling genetic disorders, assigning functions to genes, evaluating drugs and toxins, and by helping to answer fundamental questions in basic and applied research. However, these types of approaches are at the moment largely unsuitable for high throughput functional annotation of the genome. More classical forward genetic approaches (phenotype-driven approaches) have recently also gained momentum. Random mutagenesis on a genome-wide scale has become an attractive method to track the role of virtually any gene in a particular phenotype. In particular, chemical (ENU) mutagenesis of disease sensitized animals offers unique opportunities to discover gene functions directly associated with prevention or therapy of diseases. We have thus initiated a programme of sensitized ENU mutagenesis screen applied on our established TNFΔARE model of arthritis and IBD , to identify novel genes associated with development of these diseases. Thus far, we have identified mutant mice resistant to disease development and our work currently progresses towards detailed identification of the responsible mutant alleles. Once identified these novel gene functions may constitute novel pharmaceutical targets for disease neutralization.