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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - Inflammostrome (Stromal cells as primary drivers of immunopathology: towards targeted disease modification in spondyloarthritis)

Teaser

Summary

Over the last 2 decades, we have made a huge progress in the treatment of inflammatory diseases such as chronic arthritis by developing treatments that target immune cells and their products. A striking example of this is the major improvement in signs and symptoms of disease as well as in quality of life of patients with spondyloarthritis, a prototypical form of rheumatic inflammation affecting mainly the spine, when treated ith so-called biologic treatments (monoclonal antibodies blocking important inflammatory molecules such as TNF and IL-17). Despite these advances, however, a significant fraction of patients do not respond to these treatments, do only partially respond, or even when to do respond well still continue to accumulate organ damage over time. These clinical observations indicate that we have not yet found the primary cause of the disease.

In this project, we hypothesize that not only immune cells but also resident tissue cells contribute to the disease process. We propose that in same disorders tissue cells may even be the primary driver of chronic inflammation. Therefore, understanding the mechanisms by which tissue cells can drive these conditions is essential to find new targets for causal treatment.

Using spondyloarthritis as a model disease, this project aims to unravel the role of stromal cells in chronic tissue inflammatiom by defining a) the molecular profile of the stromal alterations, b) the mechanisms of pro-inflammatory cytokine production by stromal cells, c) the activation of downstream effector patwhays drivin inflammation as well as tissue remodeling, and d) the functional contribution of the tissue cells to experimental and human spondyloarthritis.

With out sigificant advances in symptomic treatment of many inflammatory conditions, this project is part of a large effort to move towards real disease-modifying treatments. The relevance relates to our ambition to modulate the natural course of those disabling conditions and thereby to maintain health in our patients.

Work performed

Objective 1: Molecular characterization of the primary stromal alternations in SpA

The basic hypothesis of this work-package is that specific stromal alterations rather than immune dysregulation underlies the immunopathology of SpA, based on the strong myofibroblast signature seen in SpA but not RA synovitis (Yeremenko, Arthritis Rheum 2013). This signature, once established, appeared to be independent of inflammation as it was not modulated by anti-inflammatory treatment in vivo or exposure to inflammatory stimuli in vitro (Yeremenko, in preparation).
Screening for epigenetic factors that may impact the Thy-1 positive synovial myofibroblasts indicated HDACs as potential modulators. Extensive in vitro studies showed that, whereas HDAC5 may suppress the inflammatory stromal phenotype, HDAC3 is an important driver of pro-inflammatory mediator production by synovial fibroblasts (Angiolilli, Ann Rheum Dis 2016; Angiolilli, Ann Rheum Dis 2017; Angiolilli, Epigenetics 2017). Interestingly, the impact of these HDACs was much more profound on synovial fibroblasts than on prototypical inflammatory cells such as macrophages.
Investigating which factor may trigger such epigenetic changes, we focused our attention on ER stress as this type of stress, but not mechanical stress or hypoxia, was able to recapitulate the myogene signature in vitro (Yeremenko, in preparation). ER stress alone was not sufficient to activate the synovial fibroblasts, but there was a clear synergy with TLR stimulation leading to significant upregulation upregulation of the inflammation potential of these cells through mRNA stabilization (Kabala, Arthritis Res Ther 2017). These findings are consistent with the fact that TLR signalling is crucially modulated by â€˜co-activationâ€™, with as examples not only ER stress but also FcR signalling (Hansen, J Immunol 2017; Hansen, Nat Commun 2018). We also found that ER stress abrogates the anti-inflammatory effects of IL-10 on myeloid cells by inhibiting STAT3 phosphorylation (Hansen, Front Immunol in press).
As to the origin of ER stress in SpA, it has been suggested to non-conventional forms of HLA-B27 could induce this type of cellular stress. Investigating these nc HLA-B27 forms, we found there expression in synovial and gut tissue of human SpA patients; more importantly however, they were not only expressed by infiltrating cells but also by tissue-resident cells, supporting our hypothesis of stromal contribution to the disease process (Rysnik, J Autoimmun 2016). Moreover, using our HLA-B27 tg rat model, we could demonstrate the these nc HLA-B27 forms were already present prior to disease onset (Rysnik, Data Brief 2016). Also in this model where a fraction of the male rats spontaneously develop disease after 3 to 6 months, we demonstrated that innate immune stimulation using heat-killed M Tub lead to accelerated and synchronized disease in >90% of both males and female animals, confirming the role of synergy between ER stress and innate activation in the disease pathophysiology (van Tok, Front Immunol 2017).

Objective 2: Analysis of IL-23 and tmTNF production by stressed stromal cells
In our experiments with ER stress on stromal cells, in particular synovial fibroblasts, we noticed that one of the strongest increases in mRNA was for the IL-23 sub-unit p19. As described above, this was mainly observed upon co-stimulation with inflammatory cytokines or TLR-ligands together with ER stress. We could also demonstrate that p19 mRNA was paralleled by increased expression levels of p19 protein. However, we could not demonstrate p40 production and/or the presence of IL-23 protein, the p19/p40 heterodimer. This contrast sharply with what we see in macrophages, eg our recent demonstration of an increase in IL-23+ inflammatory macrophages in human SpA (Ciccia, Arthritis Rheum 2018).
We therefore assessed two other molecular pathways that could be related to p19 in stromal cells: a) detailed analysis of stressed synovial fibrob