Notes From the 6th Global Arthritis Research Network (GARN) Meeting

Session 1

Friday, May 11, 2007 (am)
Molecular biology and immunology
Chairs: Jean-Michel Dayer, Geneva, Switzerland and David M. Lee, Boston, Massachusetts

Josef Jiricny, Zurich, Switzerland
The multifaceted mismatch DNA repair system

In his presentation, Josef Jiricny explained the diverse roles of the DNA mismatch repair (MMR) system by highlighting five facets of its function. As the first facet, Jiricny described how the MMR machinery recognizes base-base mismatches and directs the repair machinery to the newly synthesized DNA strand. Failure in this facet increases microsatellite instability, a condition found in familial as well as sporadic colon cancer. In facet two, the role of the MMR system in processing modified DNA bases was addressed, and possible mechanisms for methylating chemotheurapeutical agents were explained. It was suggested that, since the MMR system continually repairs the newly made strand, the defective, modified base persists in the template, which leads to repetitive futile cycles of repair. Finally, G2 arrest of the cells is induced. Consequently, inactivation of MMR prevents damage processing and apoptosis induction in treated cells, leading to resistance to methylating agents. Another proposed hypothesis assumes that no base partner for the modified base can be found, which leads to a single base gap, and in consequence a double strand break during the next cell cycle induces apoptosis. Facet three described how the MMR system is involved in recombination. In this respect it was mentioned that MMR proteins can interact with the tumor suppressor genes BRCA1 and BRCA2 forming a complex that is necessary but not absolutely required for suppressing somatic recombination in mammalian cells. Facet four highlighted the connection of MMR and error prone DNA polymerases in the context of somatic hypermutation in B cells. Since somatic hypermutation functions differently in the absence of MMR, it was postulated that during the processing of uracil residues, the MMR actually triggers a mutagenic process by binding of error-prone DNA polymerases. The last facet addressed how MMR activity is affected by the structure of chromatin. Jiricny showed that the MMR protein hMSH6 contains a PWWP domain that probably links MMR to chromatin. In conclusion, a variety of events such as repair of damaged DNA, replication, DNA damage signalling, control of homologous recombination, class switch recombination, somatic hypermutation, and chromatin interaction were shown to be affected by the status of the MMR.

Witold Filipowicz, Basel, Switzerland
Function of miRNA in gene regulation

Filipowicz started his talk by highlighting the similarities and differences of microRNA and RNAi pathways. Both can induce sequence-specific mRNA silencing and both are processed by dicer, an RNase III family endonuclease. But while RNAi is perfectly matched to the target sequence, in miRNA only some of the 21 nucleotides actually correspond to the target. miRNA precursors are encoded in the genome. Clusters that can contain up to 50 independent miRNA can be found in the exon or in the intron regions of protein coding sequences. Up to now, 500 miRNA have been described, but more than one thousand encoded miRNA are predicted. Fifty-three percent of the genome is targeted by miRNA, and almost every biological process has been found to be regulated by them. In order to inhibit protein synthesis, miRNA are loaded onto an Argonaute-containing effector ribonucleoprotein (RNP) complex, referred to as miRNP. In general, multiple miRNA repress the synthesis of a single protein, and the repression gets stronger the more miRNA are involved. A single miRNA can probably bind to more than 100 different mRNA and in addition also control the activity of other miRNA. Therefore, Filopowicz defined networking and fine tuning as well as fast switching from one gene expression profile to another as the major role of miRNA. As two major challenges for miRNA research, the identification of miRNA targets, as well as the analysis of the mechanisms of inhibition by miRNA were mentioned. Up to now several mechanisms for miRNA-mediated repression of target mRNAs have been proposed. Supposedly, miRNA repress translation by initiation arrest. Thereby, they bind to 3'UTR regions of target transcripts and cause ribosomal run-off. This hypothesis is supported by the finding of a polysomal shift, suggesting that the message cannot be translated, and by the fact that mRNA cap independent translation is not inhibited. If it is assumed that miRNA work by postinitiation arrest, the repression of translation occurs at the elongation or termination step by promotion of ribosome drop-off. Postinitiation mechanisms could also involve miRNA-mediated deadenylation and subsequent decay of target mRNA. Several recent reports indicate that Argonaute proteins, miRNA and also repressed mRNA accumulate in discrete cytoplasmic foci, so-called P-bodies. Therefore, P-bodies are regarded as sites of translational repression and mRNA decay. Interestingly, it has been found that specific mRNA can be relieved from miRNA repression and relocated out of the P-body, implying that miRNA repression is reversible. In the last part of his presentation, Filipowicz described changes in the expression of miRNA that relate to different malignancies. He stressed the importance of profiling miRNA to study the origin of tumors and finally pointed to the great potency of miRNA as therapeutic targets.

Ulf Muller-Ladner, Giessen, Germany
Adipocytokines: a novel link between inflammation and organ function

Ulf Muller-Ladner structured his talk by pointing out links between adipocytes and inflammation. The first link he mentioned, related to the differentiation of mesenchymal cells into adipocytes under the influence of determined growth factors. Many of these factors, in particular insulin growth factor-1, have also been found to be active in inflammatory diseases like rheumatoid arthritis (RA). Also, similar signalling pathways like PPAR pathways seem to be activated in the differentiation of adipocytes and in inflammation. Furthermore, Muller-Ladner brought out a structural link between adipocytokines and proinflammatory factors by showing how similar the structures of adiponectin, C1q and TNF are. Regarding adiponectin, two receptors have been described. AdipoR1 is mainly found in muscle tissue where it mediates glucose uptake by signaling through AMP-k and p38, whereas AdipoR2 is present in the liver and regulates FFA oxidation via PPAR-α. Adiponectin is also produced in so-called 'forgotten' compartments like the fat tissue in the peri-/pararticular compartment. Its levels are elevated in the synovial fluids of RA patients and correlate with disease activity. Ulf Muller-Ladner data showed that adiponectin can stimulate the production of IL-6 and proMMP1 in synovial fibroblasts, but has no effect on IL-1 or IL-4 levels. The regulation of IL-6 and MMP1 by adiponectin was dependent on p38 kinase and could be amplified by addition of TNF and IL-1β. Interestingly, the low molecular weight isoform was less potent in inducing IL-6 in THP1 cells, compared to the high molecular weight isoform. Another adipocytokine mentioned by Muller-Ladner is resistin. Levels of resistin are higher in RA patients compared to patients with osteoarthritis. Furthermore, it was found to directly induce arthritis when injected into mouse joints. The adipocytokine CORS-26 (cartducin) has only recently been described in mice and humans and seems to be specifically related to the development of cartilage. Furthermore, Visfatin (PBEF) is up regulated by TNF and IL-1, and has been described to play a role in the pathogenesis of RA and IBD. Finally, omentin (intelectin), which enhances insulin-mediated glucose uptake was related to the pathogenesis of IBD. In conclusion, it became clear that adipocytes and adipocytokines take an active role in inflammation and cannot be seen as innocent bystanders anymore.

Chairs: Florence Apparailly, Montpellier, France, and Iain B. McInnes, Glasgow, UK

Manfred Kopf, Zurich, Switzerland
The regulation of the immune system and the lipid environment

Also, the talk by Manfred Kopf linked fat tissue (obesity/dyslipidemia) to inflammation. There is strong evidence that the lipid environment influences the immune response. Manfred Kopf's group in particular studied how the balance of Th1/Th2 is affected by lipids. They could show that Apo-/- mice increase Th2 responses after immunization and that their dendritic cells are refractory to TLR stimulation, demonstrating that dyslipidemic mice have an altered immune response. Thereby, oxidation of lipids seems to play a crucial role. Increased lipid oxidation was found in a variety of diseases such as atherosclerosis, asthma, reperfusion injury, emphysema, and rheumatoid arthritis. Oxidized cholesterol was found to inhibit Th1 and to promote Th2 cell polarization by stimulation of IL-4 and inhibition of IFNgamma. In addition, oxidized phospholipids (oxPLs) were detected to induce an anti-inflammatory situation as shown by the inhibition of the inflammatory response (IL-6, IL-12, TNF) to TLR ligands in DCs. These effects involved inhibition of RelA/p50 nuclear translocation. Furthermore, Manfred Kopf pointed out that DCs sense oxPLs through a specific mechanism. In contrast to LDL uptake which is mediated through PPAR pathways, the uptake of oxPLs by DCs is regulated by Nrf-2, a transcription factor regulating cytoprotective and antioxidant enzymes in response to cellular stress. All in all, Manfred Kopf showed that oxidized lipids can shift the immune response from Th1 to Th2 by down regulation of IL-12 and up regulation of IL-10 in DCs.

Burkhard Becher
Induction of primary T-cell immunity in the absence of secondary lymphoid tissue

At the start of the lecture Burkhard Becher introduced the main take-home message of his talk: T cells are probably evolved earlier than assumed since they don't require secondary lymphoid structures to be primed. As a model to prove this hypothesis, experimental autoimmune encephalomyelitis (EAE) was applied. During EAE, auto-aggressive, reactive T-lymphocytes migrate into the CNS, where they recognize their cognate target antigen and initiate an inflammatory cascade leading to tissue damage. Strikingly, alymphoplasia (aly) mice, which are characterized by the complete absence of lymph nodes and Peyer's patches through a point mutation in the NF-kB-inducing kinase (NIK), are resistant to the development of EAE. This finding suggests that lymph nodes are essential for T-cell priming. However, Becher's work shows that radiated aly mice that received bone marrow from wild-type (wt) mice develop EAE, proving that lymph nodes are not after all required for T-cell priming. Furthermore, it was shown that the loss of NIK leads to the resistance to autoimmunity, since radiated wt mice that receive aly bone marrow become resistant to EAE induction. Increased size of the spleen after development of the disease in aly mice reconstituted with wt bone marrow, suggested that T cells are probably primed in the spleen. Surprisingly, these mice still developed EAE after splenectomy. Experiments with fluorescent antigen finally defined the liver as the site of T-cell priming in this model. In conclusion, the work of Becher's group clearly shows that, in contrast to B cells, T cells don't require lymphoid structures to be primed, but can also be primed in the liver.

Session 1 summarised by Caroline Ospelt and Yvonne Rengel

6th Global Arthritis Research Network Meeting

• Introduction
• Notes From Session 1
• Notes From Session 2
• Notes From Session 3
• Notes From Session 4