Abstract Background Genetics plays a key role in the pathogenesis of inflammatory bowel disease (IBD). With the expanding use of next-generation sequencing, >70 different monogenic disorders associated with IBD have been identified, and most of them present in the first years of life. Recently, several patients with severe IBD were identified to harbor pathogenic mutations in Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) gene, which regulates necroptosis, a necrotic cell death mechanism. We present the clinical features, genetic analysis and immune work-up of three patients with infantile-onset IBD resulting from novel RIPK1 mutations. Methods Whole exome sequencing was performed in three patients with severe infantile-onset IBD, along with sanger sequencing for conformation. Mass cytometry time of flight was conducted for in-depth immunophenotyping, including cytokine secretion analysis following lipopolysaccharide (LPS) or Phorbol myristate acetate with ionomycin (PMA-I), on one of the patient’s peripheral blood mononuclear cells, and compared to control subjects and patients with Crohn’s disease. Results All patients, born to consanguineous Muslim families, presented with severe colitis and multiple perianal fistulas in the first months of life, without severe or atypical infections. One of the patients had a partial response to high doses of inlfliximab and azathioprine, while another one failed to respond to adaliumab and later to low dose anakinra, an IL-1 receptor antagonist. Genetic studies identified novel and pathogenic genetic variants in the RIPK1 gene in all patients, that were confirmed by Sanger sequencing. Using mass cytometry time of flight unbiased clustering analysis, we identified peripheral immune dysregulation in one of these patients, characterized by an increase in IFNγ CD8+ T cells along with a decrease in monocytes, dendritic cells and B cells. Moreover, RIPK1-deficient patient’s immune cells exhibited decreased IL-6 production in response to LPS across multiple cell types including T cells B cells and innate immune cells. Conclusion Mutations in RIPK1 should be considered in very young patients presenting with colitis and perianal fistulas. Given RIPK1’s role in both immune cells (and specifically in inflammasome activation), and epithelial cells, it is unclear whether immunosuppressive medications (including IL1 blockade) as well as allogeneic hematopoietic stem cell transplantation can suppress or cure the hyper-inflammatory response in these patients. Additional studies in humans are required to better define the role of RIPK1 in regulating intestinal immune responses, and how treatment can be optimized for patients with RIPK1 deficiency.

More than 50 different monogenic disorders causing intestinal inflammation have been identified in recent years. Activated phosphoinositide 3-kinase δ syndrome (APDS) is a recently described primary immunodeficiency resulting from PIK3CD and PIK3R1 mutations that manifests in the first years of life. Our aim was to characterise the gastrointestinal manifestations and immunological alterations of patients with APDS who presented with a severe IBD-like phenotype. Detailed immune work-up was performed in three patients with APDS (two with PIK3CD mutation and one with PIK3R1 mutation) who presented with very early-onset IBD (VEOIBD). Whole exome sequencing was completed to identify pathogenic variants. In a single patient and two healthy controls paired blood and intestinal immune cells were analysed by Mass CyTOF and high-throughput sequencing of the T- and B-cell receptor gene segments. All patients manifested with recurrent sinopulmonary infections, bronchiectasis and splenomegaly, as well as chronic diarrhoea due to severe intestinal lympho-nodular hyperplasia (Figure 1) and an IBD-like phenotype in the first years of life. Two patients developed intestinal diffuse large B-cell lymphomas in adulthood. Immune work-up displayed T-cell lymphopenia and increased IgM levels. Whole exome sequencing identified deleterious genetic variants in PIK3CD or in PIK3R1. Mass CyTOF analysis showed a marked decrease in B cells in the intestine and blood accompanied with an increase in memory CD4 and CD8 T cells. Moreover, patient’s circulating CD4 and CD8 T cells produced very high quantities of IFNγ and TNFα. Finally, marked alterations in the T and B cell receptor repertoire patterns were observed in these patients with expansion of specific clones in both the blood and intestine. Intestinal lymphonodular hyperplasia seen in a patient with a PIK3CD mutation. APDS should be included in the differential diagnosis of VEOIBD, especially in patients with intestinal lympho-nodular hyperplasia. Given the profound proliferation in the gut and tendency to develop lymphomas these patients should be monitored closely. Novel PI3K inhibitors should be evaluated as a potential targeted therapeutic modality in these patients.

Background: Loss-of-function mutations in the IL10 receptor (IL10R) genes cause severe infantile-onset IBD. Intact IL10R-dependent signals have been shown to be important for innate immune cell function and for regulation of effector and regulatory T cell function in mice. We have previously reported a key role of IL10 is the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4+T cell function. Methods: Peripheral blood mononuclear cells and intestinal lamina propria cells were extracted from IL10R-deficient patients and controls. Frequencies of CD4+ T cell subsets, naïve T cell proliferation and regulatory T cell (Treg)-mediated suppression and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling of these populations was performed by quantitative real-time PCR and nanoString platforms. Results: Analysis of 12 IL10R-deficient patients demonstrated similar frequencies of peripheral blood and colonic lamina propria Tregs, compared to healthy control subjects. Moreover, in vitro Treg suppression of naïve CD4+ T cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naïve cells exhibited significantly higher proliferative capacity, a strong TH17 signature and a marked increase in polarization towards TH17 cells, compared to controls. Moreover, the frequency of TH17 cells was increased in the colon of an IL10R-deficient patient vs. controls. Conclusions: IL10R signaling regulates TH17 polarization and T cell proliferation in humans, but is not required for the generation of Tregs in blood and mucosal compartments, and is dispensable for in vitro Treg suppression. Therapies targeting the TH17 axis might be beneficial for IL10R-deficient patients as a bridge to hematopoietic stem cell transplantation.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that plays a critical role in cytokine and growth factor signaling and is frequently activated in human tumors. Human telomerase reverse transcriptase (hTERT) is also often overexpressed in tumor cells and mediates cellular immortalization. Here we report that STAT3 directly regulates the expression of hTERT in a variety of human cancer cells. Moreover, STAT3 activity is required for the survival of many human tumors, and hTERT expression contributes to the survival of STAT3-dependent tumor cells. In addition, we find that growth factors and cytokines stimulate hTERT expression in primary human cells in a STAT3-dependent manner. Thus, STAT3 is a key regulator of hTERT expression in both normal and tumor cells.

The Down syndrome cell adhesion molecule (Dscam) is a protein overexpressed in the brains of Down syndrome patients and implicated in mental retardation. Dscam is involved in axon guidance and branching in Drosophila, but cellular roles in vertebrates have yet to be elucidated. To understand its role in vertebrate development, we cloned the zebrafish homolog of Dscam and showed that it shares high amino acid identity and structure with the mammalian homologs. Zebrafish dscam is highly expressed in developing neurons, similar to what has been described in Drosophila and mouse. When dscam expression is diminished by morpholino injection, embryos display few neurons and their axons do not enter stereotyped pathways. Zebrafish dscam is also present at early embryonic stages including blastulation and gastrulation. Its loss results in early morphogenetic defects. dscam knockdown results in impaired cell movement during epiboly as well as in subsequent stages. We propose that migrating cells utilize dscam to remodel the developing embryo.