Abstract Background The IL-36 signaling pathway has recently been identified as a key regulator of intestinal homeostasis and inflammation. However, the role of mutations in the IL-36R signaling pathway in the pathogenesis of inflammatory bowel disease remains unclear. Methods Mutations in the IL-36 receptor antagonist (IL-36RA, IL36RN) were identified in a cohort of 86 ulcerative colitis patients, 244 Crohn’s disease patients and 45 non-inflamed controls by whole exome sequencing followed by targeted Sanger sequencing. In-depth immune cell profiling of one IL36RN-mutated patient was performed using mass cytometry and multiplexed immunoassays. Overexpression experiments and functional assays characterized identified IL36RN mutations. Clinical and molecular responses of one IL36RN-mutated patient to a combined treatment with the IL-36R-blocking antibody spesolimab and the TNF-blocker certolizumab-pegol were assessed by fecal calprotectin, endoscopy, multiplexed immunoassays and mass cytometry. Results We identified four Crohn’s disease patients with heterozygous missense mutations in IL36RN. Experimental overexpression and functional assays demonstrated that two identified mutations resulted in a reduced protein expression of IL-36RA. In-depth immune profiling of one IL36RN-mutated patient revealed an increased response of PBMCs to IL-36 stimulation and elevated serum levels of IL-36-regulated cytokines. Administration of the IL-36R-blocking antibody spesolimab in combination with certolizumab-pegol over a period of 22 weeks to this patient resulted in a reduction of intestinal inflammation and alterations in immune cell composition and function. Conclusion Our findings indicate that pathogenic IL36RN mutations may contribute to the pathogenesis of Crohn’s disease in a subset of patients and that inhibiting IL-36 signaling in combination with TNF-blockade could offer a personalized therapeutic approach for these patients. Disclaimer Spesolimab was made available based on an out-of-scope request. Boehringer Ingelheim was given the opportunity to review this abstract as a courtesy. Boehringer Ingelheim had no role in the design, analysis or interpretation of the results in this study.

Abstract Background Crohn’s disease (CD) is a chronic inflammatory disease, which can affect any part of the gastrointestinal tract. It is characterized by alterations in the innate and adaptive immune systems. Changes in both monocyte and macrophage (MΦ) populations have been reported in CD patients. However, the precise contribution of these cells to disease pathogenesis and progression remains unclear. Our hypothesis is that in CD, the local environment affects the metabolism of monocytes and MΦ. Furthermore, we believe that the cell’s metabolism is essential for the phenotypic and functional identity of these cells. The aim of this study is to analyze and compare the metabolic signatures of peripheral blood monocyte subsets and tissue MΦ in CD patients with ileitis (iCD), deep remission (rCD) and healthy controls (HC). Methods A total of 28 adults from the IBDome5 cohort were analyzed, consisting of 10 individuals with iCD, 8 with rCD and 10 HC. Suspension mass cytometry was utilized to analyze peripheral blood mononuclear cells (PBMCs) using a 34-marker panel targeting key proteins and rate-limiting enzymes across multiple metabolic pathways. In addition, we conducted further analysis on intestinal tissue obtained during ileocolonoscopy using imaging mass cytometry. This allowed for the application of a similar marker panel to compare the metabolic states of tissue-resident MΦ (ongoing analysis) with the identified signatures found in peripheral blood. Results Peripheral blood samples of CD patients and HC were analyzed. FlowSOM clustering of CD45+ events discriminated 11 major clusters of PBMCs. Reclustering based on CD14 and CD16 allowed the identification of 4 monocyte clusters, including classical monocytes. Classical monocytes exhibited unique metabolic signatures that distinguished them from other monocyte populations. Ongoing investigations targeting metabolic signatures in the monocyte-MΦ compartment will determine whether disease states differ significantly between the single disease entities. Conclusion Single-cell phenotyping using suspension and imaging mass cytometry can assess metabolic states of the monocyte-MΦ compartment in CD and HC. Such insights may provide novel markers for monitoring disease activity and reveal potential therapeutic targets.

Abstract Background Etrasimod (ETR) is an oral, once-daily, selective sphingosine 1-phosphate receptor 1,4,5 (S1PR) modulator for the treatment of moderately to severely active ulcerative colitis (UC). The mechanism of action is based on a retention of lymphocytes in lymphatic tissue hindering circulating lymphocytes from entering the gut and causing inflammation. However, the effect of ETR on immune cell function and composition is not well characterized. In addition, it is unknown whether the response of UC patients to ETR treatment is associated with specific immune cell signatures. Methods Single-cell RNA sequencing (scRNAseq), mass cytometry (CyTOF) and clinical data integration were used to characterize the effects of 12 weeks of treatment with ETR on the immune composition and function of peripheral blood mononuclear cells (PBMCs) collected from 15 UC patients at weeks 0 and 12 during the ELEVATE UC Phase 3 clinical trial. As an exploratory cohort, PBMCs from 5 UC patients reaching the primary endpoint of clinical remission at week 12 (modified Mayo score <2), 5 UC patients not responding to ETR treatment (modified Mayo score >2) and 5 placebo-treated UC patients were included. 30 samples were processed for scRNAseq; additionally, 28 blood samples were ex-vivo stimulated with ionomycin/PMA for 4 h and cytokine production was assessed by CyTOF. To determine the effects of ETR in UC patients, immune cell function and composition were compared intra-individually between weeks 0 and 12. Subsequent comparisons between ETR responders, non-responders and placebo-treated UC patients were performed to discriminate responders from non-responders. Results Significant reductions in T and B cell subsets, along with increases in the abundance of dendritic and monocytic cells, were observed in responders by week 12 of -treatment. Importantly, ETR treatment significantly reduced circulating T cells producing pro-inflammatory cytokines, including TNFα and IFNγ, and S1PR modulation led to a mitigation of naïve and effector memory CD4 and CD8 T cells in ETR-treated UC patients achieving clinical remission. In contrast, patients not responding to ETR treatment had significantly higher frequencies of both naïve and TNFα-producing CD4 T cells by week 12. Conclusion In line with the described role of S1PR in lymphocyte trafficking, single-cell analyses of circulating immune cells from ETR-treated UC patients confirmed a significant reduction in B cells and pathogenic T cell subsets after ETR treatment providing an in-depth cellular characterization of S1PR modulation in UC patients thereby contributing to a better understanding of the key mechanism of action of ETR. However, these findings require validation in larger cohorts.

Abstract Background The interleukin 36 (IL-36) family includes the agonists IL-36α, IL-36β, IL-36γ, which activate the NF-κB pathway and the natural antagonist IL-36RA, which inhibits IL-36 signaling. While missense mutations in the gene encoding IL-36RA (IL36RN) are associated with severe skin inflammation, their role in Crohn’s disease (CD) is currently unknown. Therefore, we here investigated if IL36RN mutations contribute to inflammation in CD and whether IL-36 signaling might represent a potential drug target for personalized therapy. Methods Mutations in IL36RN were identified by whole exome sequencing (WES) followed by targeted Sanger sequencing. Peripheral blood mononuclear cells (PBMCs) of affected patients were analyzed by mass cytometry and serum cytokines were measured by ELISA. Identified IL36RN mutations were characterized by overexpression experiments and functional assays. Clinical and molecular responses of one IL-36RN mutant patient to combined treatment with spesolimab and certolizumab pegol was assessed by fecal calprotectin, endoscopy, RNA sequencing and mass cytometry at week 0 and 12 of treatment. Results In a cohort of 47 ulcerative colitis patients, 177 CD patients and 34 healthy controls, we identified 3 CD patients with heterozygous missense mutations in IL36RN (IL-36RA S113L, IL-36RA P76L and IL-36RA L133I). In the patient carrying the IL-36RA S113L variant, we detected elevated serum levels of IL-18 and IL-23, as well as an increased frequency of TH17 cells in PBMCs, suggesting an overactivation of the IL-36 pathway. Overexpression experiments showed that IL-36RA S113L causes decreased expression of IL-36RA, resulting in increased IL-36 signaling. The patient was therefore treated with 1200 mg of the IL-36R blocking antibody spesolimab every 4 weeks and 200 mg of certolizumab pegol biweekly on the basis of an Out Of Scope request. This approach resulted in reduced intestinal inflammation and a reduction in the frequency of pro-inflammatory myeloid cells in PBMCs. Conclusion In this study, we show that pathogenic IL36RN mutations are present in a subset of CD patients and that blocking IL-36 signaling may represent a personalized therapeutic approach for this rare subset of patients in whom conventional biologic therapies have failed. Disclaimer Boehringer Ingelheim was given the opportunity to review the abstract for medical and scientific accuracy as well as intellectual property considerations in relation to the potential mention of BI substances. Boehringer Ingelheim had no role in the design, analysis or interpretation of the results in this study. Spesolimab was made available on the basis of an out of scope request. Boehringer Ingelheim was given the opportunity to review this abstract as a courtesy.

Abstract Background Crohn’s disease (CD) is characterized by chronic inflammation that can discontinuously affect any segment of the gastrointestinal tract. New findings concerning clinical behaviour, epidemiology, genetics, and gut microbiota suggest that CD in the ileum (iCD) and the colon (cCD) should be considered as two distinct subtypes of IBD. As systemic immune cell signatures differ between CD and UC, we propose a similar strategy for the differentiation between iCD and cCD. The aim of this study is to analyse and compare peripheral blood mononuclear cell (PBMC) subsets of iCD and cCD patients. For a robust analysis of the peripheral immune compartment in inflammatory bowel diseases (IBD), PBMC subsets of ileocolonic CD (icCD), ulcerative colitis (UC) and healthy controls (HC) were included as well. Methods In this study a total of 50 adults, 10 subjects per subgroup – iCD, cCD, icCD, UC and HC – were included. From each individual, both naïve and PMA/Ionomycin stimulated samples were stained for phenotypic and functional characterization using mass cytometry. For data analysis pre-gating was performed in Cytobank followed by debarcoding, compensation, normalization, clustering, and analysis on differential abundances edge R (DA_edgeR) and differential states limma (DS_limma) with CATALYST in R. Results FlowSOM allowed us to cluster CD45+ PBMCs of all samples into 16 major cell subtypes as visualized via UMAP (Fig. 1). DA testing showed significant differences between iCD and cCD, iCD and icCD but not cCD and icCD (Fig 2). Significantly different abundances among CD subgroups and HC included cluster 1 CD33+ myeloid cells, 7 CD45RO+ CD16+ cells, 8 CD11b+ cells, 9 CD4+ IL7R+ T cells and 12 CD27+ CD38+ CD45RA+ B cells (Fig. 3). When focussing on differences between iCD, cCD and icCD, DS testing revealed significantly higher IFNgamma and TNFalpha production in cluster 9 (CD4+ IL7R+ T cells), 15 (CD8+ T cells) and 16 (CD8+ CD45RO+ T cells) in iCD compared to icCD. Moreover, cluster 9 showed significantly higher levels of IFNgamma in iCD compared to cCD, and for TNFalpha cluster 16 revealed significantly higher levels in cCD compared to icCD (Fig. 4). Fig. 1 UMAP clustering of stimulated cells. Fig. 2 Differential analysis of stimulated samples with DA_edgeR and DS_limma. Fig. 3 Relative population abundances with DA_edge R testing. Fig. 4 Relative state marker abundance within clusters with DS_limma testing. Conclusion Deep phenotyping of the peripheral immune cell compartment in CD might serve as a confirmatory marker for disease location and therefore as predictor of clinical behaviour.

Abstract Background The interleukin 36 (IL-36) family consists of three agonists (IL-36α, IL-36β, IL-36γ), that activate the NF-κB pathway and lead to the production of pro-inflammatory cytokines and the natural antagonist (IL-36RA), which inhibits IL-36 signaling. While it is known that IL-36 signaling is important in the skin, it has been only recently reported that IL-36 might play an important role in maintaining gut homeostasis and in the development of fibrosis (Scheibe et al., 2019). Here, we aim to understand how IL-36RA mutations contribute to development and maintenance of intestinal inflammation in Crohn’s disease. Methods By whole-exome sequencing (WES) and subsequently targeted Sanger sequencing we identified a Crohn’s disease (CD) patient with a heterozygous missense mutation in IL-36RA (p.Ser113Leu). To understand how the mutation affects immune cells composition and function, we characterized peripheral blood mononuclear cells (PBMCs) of the patient by mass cytometry and measured cytokine levels in the serum by ELISA. In an available WES dataset of 177 CD patients, 47 ulcerative colitis (UC) patients and 34 healthy donors (HD), we searched for additional patients with IL-36RA mutations. Finally, we overexpressed the identified IL-36RA mutations in HEK cells and produced mutated recombinant proteins to study the effect of the mutations on protein expression and function. Results Our IL-36RA mutation-carrying patient presented with a severe course of Crohn’s disease and consecutive of fibrosis, which was resistant to all commonly used treatment options. In the patient, we detected increased levels of IL-36RA, IL-18 and IL-23 in the serum and an increased frequency of TH17 cells in the blood. These findings could hint towards an over-activation of the IL-36 signaling pathway in the IL-36RA mutated patient. In the available WES data, we identified two more CD patients with IL-36RA mutations (p.Leu133Ile, p.Pro76Leu), indicating that IL-36RA mutations are enriched in CD patients. By overexpressing the three identified mutations in HEK cells, we could demonstrate that two mutations (p.Ser113Leu and p.Pro76Leu) lead to a reduced protein expression. Also, our current data shows that the p.Ser113Leu mutant has a reduced capacity to antagonize IL-36 stimulation. However, further testing is required for the two other IL-36RA mutants. Conclusion Our data suggests that IL-36RA mutations are enriched in Crohn’s disease patients and should be considered as a possible cause for therapy-refractory and fibrotic Crohn’s disease.

Abstract Background The interleukin 36 (IL-36) family consists of three agonists (IL-36α, IL-36β, IL-36γ), which activate the NF-κB pathway and lead to the production of pro-inflammatory cytokines and the natural antagonist (IL-36RA), which inhibits IL-36 signaling. While it is known that IL-36 signaling is important in the skin, it has been only recently reported that IL-36 might play an important role in maintaining gut homeostasis and in the development of fibrosis (Scheibe et al., 2019). However, it is not clear how IL-36 signaling contributes to the development of intestinal inflammation and which cell types are involved in this process. To shed more light on the role of IL-36 in intestinal inflammation, we took the opportunity to perform an in-depth characterization of a Crohn’s disease patient with a mutation in IL36RA and identify cell subsets responding to IL-36 stimulation. Methods By whole exome-sequencing of EDTA blood we identified in a Crohn’s disease patient a heterozygous mutation in IL36RA (p.Ser113Leu), which we confirmed by Sanger sequencing. By ELISA, we quantified IL-36RA serum levels in comparison to other Crohn’s disease (CD) patients and healthy donors (HD). Finally, we stimulated peripheral blood mononuclear cells (PBMCs) of the IL-36RA patient, CD patients, and HDs with ionomycin/PMA, LPS or IL-36α and performed deep immune profiling by mass cytometry with 35 markers. Results Our IL36RA mutation carrying patient presented with a severe course of Crohn’s disease and consecutive of fibrosis, which was resistant to all commonly used treatment options. As an individual treatment, autologous stem cell transplantation was performed, but did not lead to long-term improvement in symptoms. By ELISA, we found strongly elevated IL-36RA levels in the serum of our patient compared to CDs and HDs. Mass cytometry analysis revealed that the IL36RA mutation does not lead to major shifts in the immune cell composition of the blood, as the frequencies of B, T, NK, and myeloid cells in PBMCs of our patient were comparable to those in HDs and CDs. Furthermore, our data revealed that IL-36α stimulation mainly induced TNFα and IL-6 expression in myeloid cells as well as IFNγ expression in NK cells but did not affect T and B cells. Conclusion Our data is the first description of a Crohn’s disease patient with an IL36RA mutation and indicate that defects in IL-36 signaling should be considered as a possible cause for therapy-refractory and fibrotic Crohn’s disease. Furthermore, we identified NK cells and myeloid cells as main responders to IL-36α stimulation. As next step, we plan to use recombinant produced, mutant IL-36RA in in-vitro assays with immune cells and fibroblasts to study the effect of the mutation on the IL-36RA function in more depth.

With great interest, we have read the research letter by Jamilloux et al. recently reporting three cases of patients with auto-inflammation and concomitant ectodermal dysplasia and progressive loss of vision due to mutations in the α-kinase 1 (ALPK1) gene [1].We here describe the case of a 47-year-old Caucasian female presenting herself in our outpatient clinic with a history of therapy-refractory, early-onset arthritis, progressive loss of vision, splenomegaly, leukopenia, anemia, chronic pain syndrome, disseminated atopic dermatitis, and anhidrosis.The clinical course of the patient is graphically summarized in Fig. 1A.Briefly, the patient first developed progressive loss of visual acuity at the age of 11 due to bilateral chronic uveitis posterior, inflammatory retinitis, and optic nerve edema and shortly after displayed signs of destructive arthritis starting at the proximal interphalangeal joints subsequently progressing to metacarpophalangeal joints and knees.The patient tested negative for infections, known immunodeficiencies, autoantibodies, and the HLA-B27 antigen.Initial immunosuppressive treatment consisted of methotrexate, which was discontinued when the patient developed a benign bladder tumor at the age of 17.The patient was subsequently treated for several years with sulfasalazine, under which the loss of vision gradually deteriorated.Numerous treatment approaches were subsequently initiated including rituximab, baricitinib, tofacitinib, and ustekinumab as well as inter-current applications of corticosteroids during flairs.Treatment with adalimumab was induced but discontinued briefly after due to the development of recurrent infections (Table S3 summarizes the duration of the single therapies and lists the reasons for discontinuation).Suspecting a genetic syndrome, we performed wholeexome sequencing of peripheral blood mononuclear cells (PBMCs) from the patient as well as her un-affected parents and brother and detected a de novo heterozygous missense mutation (c.710C > T, [p.Thr237Met]) in the ALPK1 gene.This mutation was subsequently confirmed by targeted Sanger sequencing (Fig. 1B,C).Remarkably, this ALPK1 p.Thr237Met mutation has previously been described to cause retinal dystrophy, optic nerve edema, splenomegaly, anhidrosis, and migraine headache (ROSAH) [2] and is furthermore detectable in patients with ALPK1-associated auto-inflammatory disorders and concomitant ectodermal dysplasia reported by Jamilloux et al. [1].Based on these findings, we concluded that our ALPK1-mutated patient is suffering from ROSAH syndrome with combined early-onset arthritis and systemic auto-inflammation.Since ALPK1 has been described to act as an important mediator of NF-κB activation in response to Gram-negative bacteria [3], we hypothesized that the identified ALPK1 mutation might affect immune cell function, thereby facilitating the development of systemic auto-inflammation.We therefore compared the immune cell composition and activation of PBMCs obtained from our ALPK1-mutated patient to PBMCs of healthy donors (HDs) by applying mass cytometry (Fig. 1D).