Summary Programmed necrosis or necroptosis is an inflammatory form of cell death that critically requires the receptor interacting protein kinase 3 (RIPK3). Here we showed that RIPK3 controls a separate, necrosis-independent pathway of inflammation through regulating dendritic cells (DCs) cytokine expression. Ripk3−/− bone marrow derived dendritic cells (BMDCs) were highly defective in lipopolysaccharide (LPS)-induced expression of inflammatory cytokines. These effects were caused by impaired NF-κB subunit RelB and p50 activation and caspase 1-mediated processing of interleukin-1β (IL-1β). This DC-specific function of RIPK3 was critical for injury-induced inflammation and tissue repair in response to dextran sodium sulfate (DSS). Ripk3−/− mice exhibited an impaired axis of injury-induced IL-1β, IL-23 and IL-22 cytokine cascade, which was partially corrected by adoptive transfer of wild type DCs, but not Ripk3−/− DCs. These results reveal an unexpected function of RIPK3 in NF-κB activation, DC biology, innate inflammatory cytokine expression, and injury-induced tissue repair.

SUMMARY How innate lymphoid cells (ILCs) in the thymus and gut become specialized effectors is unclear. The prototypic innate-like γδ T cells (Tγδ17) are a major source of interleukin-17 (IL-17). We demonstrate that Tγδ17 cells are programmed by a gene regulatory network consisting of a quartet of High Mobility Group box (HMG) transcription factors, SOX4, SOX13, TCF1 and LEF1, and not by conventional TCR signaling. SOX4 and SOX13 directly regulated the two requisite Tγδ17 cell-specific genes, Rorc and Blk, whereas TCF1 and LEF1 countered the SOX proteins and induced genes of alternate effector subsets. The T cell lineage specification factor TCF1 was also indispensable for the generation of IL-22 producing gut NKp46+ ILCs and restrained cytokine production by Lymphoid Tissue inducer-like effectors. These results indicate that similar gene network architecture programs innate sources of IL-17, independent of anatomical origins.

Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that target down-regulators of the anti-cancer immune response: Cytotoxic T-lymphocyte antigen-4, programmed cell death protein-1, and its ligand programmed death-ligand 1. ICIs have revolutionized the treatment of a variety of malignancies. However, many immune-related adverse events have also been described which mainly occurs as the immune system becomes less suppressed, affecting various organs including the gastrointestinal tract and causing diarrhea and colitis. The incidence of immune-mediated colitis (IMC) ranges from 1%-25% depending on the type of ICI and if used in combination. Endoscopically and histologically there is a significant overlap between IMC and inflammatory bowel disease, however more neutrophilic inflammation without chronic inflammation is usually present in IMC. Corticosteroids are recommended for grade 2 or more severe colitis while holding the immunotherapy. About one third to two thirds of patients are steroid refractory and benefit from infliximab. Recently vedolizumab has been found to be efficacious in steroid and infliximab refractory cases. While in grade 4 colitis, the immunotherapy is permanently discontinued, the decision is controversial in grade 3 colitis.

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Transforming growth factor ␤ (TGF␤) is an immune modulatory cytokine that is critical for the development and homeostasis of the immune system (1). The absence of TGF␤ activity in mice leads to deregulated, hyperactive T cell activations resulting in early onset fatal multiorgan autoimmunity. Recent studies have highlighted the dominant function of TGF␤ in the differentiation of T cell subsets. TGF␤ can initiate transcription of Foxp3 in naive conventional CD4 ϩ T (Tconv) 2 cells to generate induced regulatory T (iTreg) cells. Moreover, in conjunction with the pro-inflammatory cytokine IL-6, TGF␤ promotes differentiation of Tconv cells to Th17 cells (2-4) that contribute to the pathogenesis of autoimmune disorders. However, Th17 cells are also necessary for combating infectious diseases such as oral candidiasis and enteritis, caused by Candida albicans and Citrobacter rodentium, respectively (5). Hence, for CD4 ϩ T cells, TGF␤ controls the balance between inflammation and quiescence.Although extensive progress has been made in understanding the effect of TGF␤ on T cell differentiation and function, detailed characterization of the biochemical basis of TGF␤ signaling in T cells has lagged significantly. In common with other cell types, TGF␤ binds to TGF␤ receptor II on T cells, which leads to the recruitment of TGF␤ receptor I to the complex and activation of cytoplasmic transcription factors SMAD2 and SMAD3. In most cells, activated SMAD2 and SMAD3 translocate to the nucleus in a complex with SMAD4 to regulate target gene transcription (6). Some genes in the TGF␤ pathway are redundantly regulated by SMAD2 and SMAD3, but unique target genes of specific SMADs also exist, suggesting that each possesses distinct functions. Hence, differential activation of SMADs may biochemically account for context-dependent pleiotropic effects of TGF␤.Using mice deficient in individual SMADs, it has been shown that Smad3 and Smad4 are necessary for efficient TGF␤-mediated generation of FOXP3 ϩ T cells but not for the differentiation of naive CD4 ϩ T cells to the Th17 lineage (7-9). However, results from a recent study led to the contrasting conclusion that receptor SMADs are not necessary for TGF␤ signaling in T cells (10). Here, analysis of T cellspecific Smad2-deficient mice indicates that in contrast to SMAD3 and SMAD4, SMAD2 plays a non-redundant role in the generation of Th17 cells in vitro and in vivo. The diminution in IL-17 production by CD4 ϩ T cells correlates with accelerated loss of Il6ra expression and a corresponding decrease in STAT3 activation in Smad2-deficient T cells, suggesting that SMAD2 specifically modulates the cross-talk between TGF␤ and IL-6 in Th17 cell differentiation. EXPERIMENTAL PROCEDURES Mice-Smad2fl/fl mice (11) generated in the Robertson laboratory were provided by Dr. Richard Flavell. These mice were backcrossed six times to the C57BL/6 background before analysis. Smad2 conditional KO (CKO) mice were generated using hCD2 Cre Tgϩ mice (C57BL/6 background). Rag1 Ϫ/Ϫ and C57BL/6 mice were purchased...

Gastric emptying of liquids correlates well with gastric emptying of solids. When evaluating patients for gastroparesis, assessment of gastric emptying of liquids in addition to solids may help identify additional patients with delayed gastric emptying, particularly non-diabetic patients where 26% with normal solid emptying may have delayed emptying of liquids. Symptoms of gastroparesis, however, are primarily associated with delayed gastric emptying of solids.

To determine the breadth and underpinning of changes in immunocyte gene expression due to genetic variation in mice we performed, as part of the Immunological Genome Project, gene expression profiling for CD4+ T cells and neutrophils purified from 39 inbred strains of the Mouse Phenome Database. Considering both cell types, a large number of transcripts showed significant variation across the inbred strains, 22% of the transcriptome varying by two-fold or more. These included 119 loci with apparently complete loss-of-function, where the corresponding transcript was not expressed in some of the strains, representing a useful resource of “natural knockouts”. We identified 1,222 cis- expression quantitative trait loci (cis-eQTL) that control some of this variation. Most (60%) cis-eQTLs were shared between T cells and neutrophils, but a significant portion uniquely impacted one of the cell types, suggesting cell-type specific regulatory mechanisms. Using a conditional regression algorithm we predicted regulatory interactions between transcription factors and potential targets, and demonstrated that these predictions overlap with regulatory interactions inferred from transcriptional changes during immunocyte differentiation. Finally, comparison of these and parallel data from CD4+ T cells of healthy humans demonstrated intriguing similarities in variability of a gene's expression: the most variable genes tended to be the same in both species, and there was an overlap in genes subject to strong cis-acting genetic variants. We speculate that this “conservation of variation” reflects a differential constraint on intra-species variation in expression levels of different genes, either through lower pressure for some genes, or by favoring variability for others.

Background and study aims: Current diagnostic testing is inadequate to determine the malignant potential of pancreatic cysts, resulting in overcautious patient management. Integrated molecular pathology (IMP) testing combines molecular analysis with first-line test results (cytology, imaging, and fluid chemistry) to assess the malignant potential of pancreatic cysts. This multicenter study aimed to determine the diagnostic accuracy of IMP for pancreatic adenocarcinoma, and the utility of IMP testing under current guideline recommendations for managing pancreatic cysts. Patients and methods: Patients who had undergone previous IMP testing as prescribed by their physician and for whom clinical outcomes were available from retrospective record review were included (n?=?492). Performance was determined by correlation between clinical outcome and previous IMP diagnosis (?benign?/?statistically indolent? vs. ?statistically higher risk [SHR]?/ ?aggressive?) or an International Consensus Guideline (Sendai 2012) criteria model for ?surveillance? vs. ?surgery.? The Cox proportional hazards model determined hazard ratios for malignancy. Results: Benign and statistically indolent IMP diagnoses had a 97?% probability of benign follow-up for up to 7 years and 8 months from initial IMP testing. SHR and aggressive diagnoses had relative hazard ratios for malignancy of 30.8 and 76.3, respectively (both P??93?% probability of benign follow-up, with relative hazard ratios for SHR and aggressive IMP diagnoses of 16.1 and 50.2, respectively (both P?