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Background Retinoic acid receptor related Orphan Receptor gamma t (RORγt) is a nuclear hormone receptor expressed in a subset of pathogenic T cells and innate lymphoid cells. RORγt regulates the transcription of key pro inflammatory cytokines such as IL-17 and IL-22 in response to multiple activation signals including cytokines and T cell receptor engagement. Antagonism of RORγt is hypothesised to block transcription of these pathogenic cytokines resulting in reduced tissue inflammation and aberrant joint remodelling. Spondyloarthropathy (SpA) is characterised by a peripheral oligoarthritis and enthesitis. A key feature of SpA is the imbalance between bone resorption and formation leading to aberrant bone formation and ankylosis. The contribution of the IL-23/IL-17 axis to the pathogenesis of SpA is supported by several lines of evidence. However, the specific role of RORγt in regulating the cellular and molecular pathways contributing to entheseal and synovial inflammation remains incompletely understood. Objectives The goal of this study was to determine the role of RORγt antagonism in both an IL-23 minicircle in vivo model and ex vivo tissue culture model. Methods Model 1: Mice were administered an IL-23 minicircle via hydrodynamic injection to induce systemic IL-23 expression on day 1. Starting on day 2, mice were dosed p.o. for 28 days. Mice were monitored daily for signs of arthritis. Upon sacrifice tissue was collected for mRNA biomarker analysis and histologic assessment. Model 2: Patients undergoing total hip replacement were recruited into 2 groups: SpA (either psoriatic arthritis or ankylosing spondylitis) or OA (osteoarthritis). Entheseal and synovial tissues were collected and cultured ±RORγt inhibitor and cytokine stimulation (hTNFa/hIL23) for 24 and 48 hours and then analysed for gene expression. Results Model 1: Overexpression of IL-23 induced SpA like phenotype including enthesitis, synovitis and aberrant bone formation. RORγt inhibition resulted in significant reduction of inflammation and arthritic score, histologic parameters of bone remodelling, and tissue biomarkers associated with the IL-23 axis (IL17A, IL17F, IL22). Model 2: Both entheseal and synovial cultures from SpA patients expressed increased Th17 axis genes relative to OA after cultures. RORγt inhibition reduced the expression of these Th17 axis genes (IL17A, IL17F, IL22) as well as reducing tissue remodelling related genes (FN1, MMP1/3, BMP5), in alignment with the results seen in model 1. Conclusions IL-23 induced Th17 axis gene expression induced a SpA- like phenotype in mice,-including arthritis and aberrant bone formation, that was reversed upon RORγt inhibition. Similarly, SpA patient explants showed increased expression of Th17 axis cytokines and tissue remodelling related genes that were decreased with RORγt inhibition. Together, these complementary approaches support the hypothesis that RORγt treatment could block inflammation as well the underlying pathologies associated with SpA. Disclosure of Interest K. Hoyt Employee of: Boehringer Ingelheim, J. Galdamez: None declared, M. Panzenbeck Employee of: Boehringer Ingelheim, D. Souza Employee of: Boehringer Ingelheim, E. Glynn Employee of: Boehringer Ingelheim, J. Pelletier Employee of: Boehringer Ingelheim, M. Lewis Employee of: Boehringer Ingelheim, G. Nabozny Employee of: Boehringer Ingelheim, E. Purdue: None declared, L. Mandl Consultant for: Boehringer Ingelheim, J. Wahle Employee of: Boehringer Ingelheim

NK cells have important functions in cancer immunosurveillance, BM allograft rejection, fighting infections and reproduction. NK cell-based therapies are promising blood cancer treatments. Overcoming their limited efficacy requires a better understanding of the molecular mechanisms dampening their effector functions. NK cells recognize and kill pathogen-infected or tumor cells through invariant NK cell receptors (NKR). Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3-generation by PI3K, and generation of DAG and IP3 by PLCγ. IP3 plays a key signaling role by mobilizing Calcium, but can also be converted into soluble IP4. We and others previously showed that IP4 can act as a soluble analog of the PI3K lipid-product PIP3 and control PIP3-mediated protein membrane recruitment and activation in thymocytes and granulocytes. Here, we show data which suggest a novel IP4 function in promoting NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR induced IFNγ secretion, granule exocytosis and target-cell clearance, in part by inhibiting the PIP3 effector Akt. This identifies IP4 as an important novel regulator of NK cell development and function, and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.

NK cells have important functions in cancer immunosurveillance, bone marrow allograft rejection, fighting infections and reproduction. NK cell-based therapies are promising blood cancer treatments. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize pathogen-infected or tumor cells through invariant NK cell receptors (NKR), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3-generation by PI3K, and generation of DAG and IP3 by PLCγ. IP3 plays a key signaling role by mobilizing Calcium, but can also be converted into soluble inositol(1,3,4,5)tetrakisphosphate (IP4). We and others previously showed that IP4 can act as a soluble analog of the PI3K lipid-product phosphatidylinositol(3,4,5) trisphosphate (PIP3) and control PIP3-mediated signaling protein membrane recruitment and activation in thymocytes and granulocytes. Here, we show data which suggest a novel IP4 function in promoting NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR induced IFNγ secretion, granule exocytosis and target-cell clearance, in part by inhibiting the PIP3 effector-kinase Akt. This identifies IP4 as an important novel regulator of NK cell development and function, and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.

Background and Aims: Despite the negative results of blocking IL-17 in Crohn's disease (CD) patients, selective modulation of Th17-dependent responses warrants further study. Inhibition of retinoic acid-related orphan receptor gamma (RORγt), the master regulator of the Th17 signature, is currently being explored in inflammatory diseases. Our aim was to determine the effect of a novel oral RORγt antagonist (BI119) in human CD and on an experimental model of intestinal inflammation. Methods: 51 CD patients and 11 healthy subjects were included. The effects of BI119 were tested on microbial-stimulated peripheral blood mononuclear cells (PBMCs), intestinal crypts and biopsies from CD patients. The ability of BI119 to prevent colitis in vivo was assessed in the CD4+CD45RBhigh T cell transfer model. Results: In bacterial antigen-stimulated PBMCs from CD patients, BI119 inhibits Th17-related genes and proteins, while upregulating Treg and preserving Th1 and Th2 signatures. Intestinal crypts cultured with supernatants from BI119-treated commensal-specific CD4+ T cells showed decreased expression of CXCL1, CXCL8 and CCL20. BI119 significantly reduced IL17 and IL26 transcription in colonic and ileal CD biopsies and did not affect IL22. BI119 has a more profound effect in ileal CD with additional significant downregulation of IL23R, CSF2, CXCL1, CXCL8, and S100A8, and upregulation of DEFA5. BI119 significantly prevented development of clinical, macroscopic and molecular markers of colitis in the T-cell transfer model. Conclusions: BI119 modulated CD-relevant Th17 signatures, including downregulation of IL23R while preserving mucosa-associated IL-22 responses, and abrogated experimental colitis. Our results provide support to the use of RORγt antagonists as a novel therapy to CD treatment.