Signalling crosstalk between NOD2 and IL-31

Dendritic cells are highly specialized antigen-presenting cells which represent the first line of defense against invading pathogens. Depending on the type of pathogen they encounter, dendritic cells induce appropriate immune- responses by providing distinct signals to cells of the adaptive immune system. While bacteria, viruses and fungi mainly instruct dendritic cells to induce T helper cell 1 (Th1) and T helper cell 17 (Th17)-mediated immune responses, nematodes and other parasites activate the differentiation of type 2 T-helper cells (Th2 cells). Full activation of dendritic cells first of all requires sensing of the pathogen by pattern recognition receptors. Yet, activation of dendritic cells can be further enhanced or suppressed by additional signals, like the stimulation by specific cytokines. In the current project we propose an entirely novel crosstalk between the cytosolic pattern recognition receptor NOD2 (nucleotide-binding oligomerization domain 2) and signals provided by the Th2-derived cytokine IL-31. In general, Th2 cytokines like IL-4, IL-5 and IL-13 are involved in the defense against helminthes. Notably, it was shown that IL-4 and IL-13 can also suppress pathogen-induced Th1-mediated immunity. This indicates that Th1- and Th2-mediated immune-responses can partially antagonize each other. Here we show that the Th2 cytokine IL-31 has opposite effects. Signalling induced by IL-31 does not inhibit, but rather enhances the activation of dendritic cells that have been stimulated via NOD2. Consequentially, these dendritic cells promote an increase in Th cell activity as shown by the enhanced release of IL-17 and IFN-?. Moreover, we speculate that IL- 31-mediated effects involve a specific isoform of the IL-31 receptor that has so far never been investigated in detail. This isoform may activate signals which differ considerably from signals that are induced by the commonly expressed receptor isoform. Taken together, the present project gives completely new insights into signalling pathways induced by IL-31 and the interplay of IL-31- and NOD2-mediated signalling. In addition, we propose that the Th2 cytokine IL-31 has entirely unexpected functions in supporting the manifestation of a Th17 response that has been induced via NOD2 activation. This allows for a better understanding of fine-tuning mechanisms which are involved in the regulation of NOD2-mediated Th cell responses.

Interleukin (IL)-31 is a cytokine that plays a key role especially in skin diseases associated with severe itching (e.g. atopic dermatitis). Therefore, new drugs blocking IL-31 signaling seem to be very promising. By interrupting the cycle of itching and scratching, such drugs are expected to improve skin inflammation and pruritic skin eczema. Indeed, therapeutic antibodies against IL-31 and the IL-31 receptor are already in development and are currently being tested in clinical studies. Besides its important role in skin inflammation, IL-31 is also found in many other tissues and organs, especially in those which are strongly infiltrated by T cells. A likely explanation for this co-localization is that T cells are the main source of IL-31. Indeed, especially under pro- allergic conditions, T cells secrete high concentrations of IL-31. However, the role of IL-31 in organs other than the skin is relatively unclear. To complicate matters further, IL-31 mediates its biological effects via the IL-31 receptor, of which seven different isoforms have been described. But little is known about which isoforms occur in which tissues and organs, how they are regulated, and what function IL-31 signaling has in organs other than skin. Therefore, we first investigated the signaling capacities of the seven IL-31 receptor isoforms in detail. While all tested IL-31 receptor isoforms where capable of transducing IL-31- mediated signals, the signals differed in their intensity and strength. In addition, we identified SOCS3 as an inhibitory molecule which regulates IL-31-mediated signals via a negative feedback loop. The second part of the study dealt with the interplay between the effects of IL- 31 and the signals triggered by the pattern recognition receptor NOD2. Contrary to our working hypothesis, we could not elucidate significant interactions between IL-31 and NOD2 that improve our understanding of the role of IL-31 in allergy. However, in the course of this study we discovered a completely unknown interaction between two proteins closely related to IL-31 and NOD2, namely IL-10 and NOD1. Our study shows that NOD1 has a significant effect on the anti-inflammatory cytokine IL-10. More specifically, downregulation of NOD1 leads to hyperactivation of IL-10 and thus to increased immunosuppression. Since lowered NOD1 concentrations were found in cancer cells, the mechanism we describe could be a further explanation for why tumor cells are able to escape the immune system. We also investigated why the data on which our original hypothesis was based could no longer be reproduced. Our detailed research showed that the commercial IL-31 batch we originally used was contaminated with endotoxin (LPS). Unfortunately, this contamination was not detectable by conventional endotoxin tests. However, because undetectable endotoxin contamination is a major problem, especially for work on human immune cells, we studied the phenomenon of endotoxin masking in more detail. We were able to describe various procedures that can lead to endotoxin masking and we proposed a cell-based assay with which masked endotoxin can be reliably detected. Finally, the last part of the project was focused on the role of IL-31 in allergic lung inflammation. Since IL-31 is an interesting therapeutic target for the treatment of itchy skin diseases, the question was raised, could IL- 31-blocking antibodies be used to treat allergic diseases in general? Thus, we investigated the role of IL-31 in allergic asthma. Although IL-31 serum levels are significantly elevated in patients with pollen allergy or allergic asthma, our in vivo models show that mice overexpressing IL-31 have less severe lung inflammation compared to wild-type mice. Thus, IL-31 might be part of a negative feedback mechanism to limit allergic inflammation in the lung. Taken together, our findings demonstrate that IL-31 signaling induces a complex network of signaling events. These trigger pruritic skin inflammation, but may also have additional, underappreciated functions in other organs, including regulation of allergic lung inflammation. However, further studies will be needed to fully understand the role of IL-31 in allergy.