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The JAK-STAT pathway critically regulates T-cell differentiation, and STAT1 is postulated to regulate several immune-mediated diseases by inducing proinflammatory subsets. Here we show that STAT1 enables CD4 + T-cell-mediated intestinal inflammation by protecting them from natural killer (NK) cell-mediated elimination. Stat1 −/− T cells fail to expand and establish colitis in lymphopenic mice. This defect is not fully recapitulated by the combinatorial loss of type I and II IFN signaling. Mechanistically, Stat1 −/− T cells have reduced expression of Nlrc5 and multiple MHC class I molecules that serve to protect cells from NK cell-mediated killing. Consequently, the depletion of NK cells significantly rescues the survival and spontaneous proliferation of Stat1 −/− T cells, and restores their ability to induce colitis in adoptive transfer mouse models. Stat1 −/− mice however have normal CD4 + T cell numbers as innate STAT1 signaling is required for their elimination. Overall, our findings reveal a critical perspective on JAK-STAT1 signaling that might apply to multiple inflammatory diseases. The JAK-STAT signaling pathway is important for cytokine responses and CD4 T-cell differentiation. Here the authors show that Stat1 also serves to protect CD4 T cells from natural killer cell-mediated killing, potentially by promoting the expression of Nlrc5 and MHC-I, to preserve the induction of experimental colitis via the adoptive transfer of CD4 T cells.

PurposeReceptor-interacting serine/threonine-protein kinase 1 ( RIPK1) is an important regulator of necroptosis and inflammatory responses. We present the clinical features, genetic analysis and immune work-up of two patients with infantile-onset inflammatory bowel disease (IBD) resulting from RIPK1 mutations.MethodsWhole exome and Sanger sequencing was performed in two IBD patients. Mass cytometry time of flight (CyTOF) was conducted for in-depth immunophenotyping on one of the patient’s peripheral blood mononuclear cells, and compared to control subjects and patients with Crohn’s disease.ResultsThe patients presented with severe colitis and perianal fistulas in the first months of life, without severe/atypical infections. Genetic studies identified pathogenic genetic variants in RIPK1 (Patient 1, A c.1934C>T missense mutation in Exon 11; Patient 2, c.580G>A missense mutation residing in Exon 4). Protein modeling demonstrated that the mutation in Patient 1 displaces a water molecule, potentially disrupting the local environment, and the mutation in Patient 2 may lead to disruption of the packing and conformation of the kinase domain. Immunofluorescence RIPK1 staining in rectal biopsies demonstrated no expression for Patient 1 and minimal expression for Patient 2, compared to controls and patients with active Crohn’s disease. Using CyTOF 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 lipopolysaccharide (LPS) across multiple cell types including T cells, B cells and innate immune cells.ConclusionsMutations in RIPK1 should be considered in very young patients presenting with colitis and perianal fistulas. Given RIPK1’s role in inflammasome activation, but also in epithelial cells, it is unclear whether IL1 blockade or 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.

Genetic analyses showed that mutations affecting the interleukin-10 receptor are associated with early-onset colitis. Further molecular analyses showed that the mutations abrogated interleukin-10 signaling. Treatment of one of the affected children by means of allogeneic hematopoietic stem-cell transplantation was successful. Genetic analyses showed that mutations affecting the interleukin-10 receptor are associated with early-onset colitis. Treatment of an affected child by means of allogeneic hematopoietic stem-cell transplantation was successful. Inflammatory bowel disease is a heterogeneous group of disorders, classified as Crohn's disease, ulcerative colitis, and indeterminate colitis. 1 , 2 In most patients, these disorders are manifested in adolescence or adulthood; however, they may present in infancy and may be inherited as an autosomal recessive trait. 3 – 6 The genetic causes of inflammatory bowel disease are only partly understood. Studies in transgenic murine models 7 and genomewide genetic-linkage and association studies have provided insights into the genetic complexity underlying these inflammatory conditions. 8 Investigators using these approaches have implicated several genes in the pathogenesis of inflammatory bowel disease; the identity of these genes suggests . . .

Here, the presentation of lipid antigens by CD1d is shown to induce retrograde anti-inflammatory signalling in intestinal epithelial cells, resulting in the production of IL-10. Anti-inflammatory IL-10 in the intestinal mucosa Intestinal epithelial cells (IECs) are crucial to mucosal homeostasis, serving as a physical barrier and regulating the responses of mucosal immune cells to environmental factors. This paper shows that CD1d, a glycoprotein involved in the presentation of lipid antigens, induces a self-reinforcing pathway of protective mucosal immunity within the intestinal epithelium. The pathway is mediated by regulatory cytokines and heat shock proteins, and interference with any molecules involved (including CD1d, IL-10 and HSP110) is associated with uncontrolled intestinal inflammation. These findings could have relevance for inflammatory bowel disease and similar conditions. The mechanisms by which mucosal homeostasis is maintained are of central importance to inflammatory bowel disease. Critical to these processes is the intestinal epithelial cell (IEC), which regulates immune responses at the interface between the commensal microbiota and the host 1 , 2 . CD1d presents self and microbial lipid antigens to natural killer T (NKT) cells, which are involved in the pathogenesis of colitis in animal models and human inflammatory bowel disease 3 , 4 , 5 , 6 , 7 , 8 . As CD1d crosslinking on model IECs results in the production of the important regulatory cytokine interleukin (IL)-10 (ref. 9 ), decreased epithelial CD1d expression—as observed in inflammatory bowel disease 10 , 11 —may contribute substantially to intestinal inflammation. Here we show in mice that whereas bone-marrow-derived CD1d signals contribute to NKT-cell-mediated intestinal inflammation, engagement of epithelial CD1d elicits protective effects through the activation of STAT3 and STAT3-dependent transcription of IL-10, heat shock protein 110 (HSP110; also known as HSP105), and CD1d itself. All of these epithelial elements are critically involved in controlling CD1d-mediated intestinal inflammation. This is demonstrated by severe NKT-cell-mediated colitis upon IEC-specific deletion of IL-10, CD1d, and its critical regulator microsomal triglyceride transfer protein (MTP) 12 , 13 , as well as deletion of HSP110 in the radioresistant compartment. Our studies thus uncover a novel pathway of IEC-dependent regulation of mucosal homeostasis and highlight a critical role of IL-10 in the intestinal epithelium, with broad implications for diseases such as inflammatory bowel disease.

A cell’s phenotype and function are influenced by dynamic interactions with its microenvironment. To examine cellular spatiotemporal activity, we developed SPACECAT—Spatially PhotoActivatable Color Encoded Cell Address Tags—to annotate, track, and isolate cells while preserving viability. In SPACECAT, samples are stained with photocaged fluorescent molecules, and cells are labeled by uncaging those molecules with user-patterned near-UV light. SPACECAT offers single-cell precision and temporal stability across diverse cell and tissue types. Illustratively, we target crypt-like regions in patient-derived intestinal organoids to enrich for stem-like and actively mitotic cells, matching literature expectations. Moreover, we apply SPACECAT to ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model. Lastly, we provide a computational framework to identify spatially-biased transcriptome patterns and enriched phenotypes. This minimally perturbative and broadly applicable method links cellular spatiotemporal and/or behavioral phenotypes with diverse downstream assays, enabling insights into the connections between tissue microenvironments and (dys)function. Spatial transcriptomics aims to pair omic data with tissue structure. Here the authors report Spatially PhotoActivatable Colour Encoded Cell Address Tags (SPACECAT) to track and isolate live cells by location; this enables spatially informed downstream assays like scRNA-seq and flow cytometry.

Intestinal stem cells (ISCs) reside in regionally variable niches that provide diverse microenvironmental cues such as tissue oxygen status, and morphogen signaling. Integration of these cues with ISC metabolism and fate remains poorly understood. Here, we show that cellular redox balance orchestrates niche factors with metabolic state to govern cell fate decisions. We demonstrate that hypoxia and Wnt signaling synergistically restrict the reactive oxygen species generating enzyme NADPH oxidase 1 (NOX1) regionally to the crypt base in the distal colon. NOX1 enables maintenance of an oxidative cell state that licenses cell cycle entry, altering the balance of asymmetric ISC self-renewal and lineage commitment. Mechanistically, cell redox state directs a self-reinforcing circuit that connects hypoxia inducible factor 1α-dependent signaling with post-translational regulation of the metabolic enzyme isocitrate dehydrogenase 1. Our studies show redox balance acts as a cellular rheostat that is central and causative for metabolic control of the ISC cell-cycle. Intracellular redox state orchestrates a self-reinforcing circuit connecting hypoxia inducible factor 1α-dependent signalling with post-translational regulation of the metabolic enzyme isocitrate dehydrogenase 1 to govern intestinal stem cell fate.

Macrophages are a heterogeneous population of mononuclear phagocytes abundantly distributed throughout the intestinal compartments that adapt to microenvironmental specific cues. In adult mice, the majority of intestinal macrophages exhibit a mature phenotype and are derived from blood monocytes. In the steady-state, replenishment of these cells is reduced in the absence of the chemokine receptor CCR2. Within the intestine of mice with colitis, there is a marked increase in the accumulation of immature macrophages that demonstrate an inflammatory phenotype. Here, we asked whether CCR2 is necessary for the development of colitis in mice lacking the receptor for IL10. We compared the development of intestinal inflammation in mice lacking IL10RA or both IL10RA and CCR2. The absence of CCR2 interfered with the accumulation of immature macrophages in IL10R-deficient mice, including a novel population of rounded submucosal Iba1 + cells, and reduced the severity of colitis in these mice. In contrast, the absence of CCR2 did not reduce the augmented inflammatory gene expression observed in mature intestinal macrophages isolated from mice lacking IL10RA. These data suggest that both newly recruited CCR2-dependent immature macrophages and CCR2-independent residual mature macrophages contribute to the development of intestinal inflammation observed in IL10R-deficient mice.

Inflammatory bowel disease (IBD) are heterogenous disorders of the gastrointestinal tract caused by a spectrum of genetic and environmental factors. In mice, overlapping regions of chromosome 3 have been associated with susceptibility to IBD-like pathology, including a locus called Hiccs . However, the specific gene that controls disease susceptibility remains unknown. Here we identify a Hiccs locus gene, Alpk1 (encoding alpha kinase 1), as a potent regulator of intestinal inflammation. In response to infection with the commensal pathobiont Helicobacter hepaticus ( Hh ), Alpk1-deficient mice display exacerbated interleukin (IL)-12/IL-23 dependent colitis characterized by an enhanced Th1/interferon(IFN)-γ response. Alpk1 controls intestinal immunity via the hematopoietic system and is highly expressed by mononuclear phagocytes. In response to Hh , Alpk1 −/− macrophages produce abnormally high amounts of IL-12, but not IL-23. This study demonstrates that Alpk1 promotes intestinal homoeostasis by regulating the balance of type 1/type 17 immunity following microbial challenge. The Hiccs locus has been associated with susceptibility to colitis in mice. Here the authors identify a Hiccs locus gene encoding Alpha kinase 1 as a potent regulator of intestinal inflammation via modulation of the IL-12/Th1 axis.

There is extensive evidence implicating the intestinal microbiota in inflammatory bowel disease [IBD], but no microbial agent has been identified as a sole causative agent. Bacteroidales are numerically dominant intestinal organisms that associate with the mucosal surface and have properties that both positively and negatively affect the host. To determine precise numbers and species of Bacteroidales adherent to the mucosal surface in IBD patients, we performed a comprehensive culture based analysis of intestinal biopsies from pediatric Crohn's disease [CD], ulcerative colitis [UC], and control subjects. We obtained biopsies from 94 patients and used multiplex PCR or 16S rDNA sequencing of Bacteroidales isolates for species identification. Eighteen different Bacteroidales species were identified in the study group, with up to ten different species per biopsy, a number higher than demonstrated using 16S rRNA gene sequencing methods. Species diversity was decreased in IBD compared to controls and with increasingly inflamed tissue. There were significant differences in predominant Bacteroidales species between biopsies from the three groups and from inflamed and uninflamed sites. Parabacteroides distasonis significantly decreased in inflamed tissue. All 373 Bacteroidales isolates collected in this study grew with mucin as the only utilizable carbon source suggesting this is a non-pathogenic feature of this bacterial order. Bacteroides fragilis isolates with the enterotoxin gene [bft], previously associated with flares of colitis, were not found more often at inflamed colonic sites or within IBD subjects. B. fragilis isolates with the ability to synthesize the immunomodulatory polysaccharide A [PSA], previously shown to be protective in murine models of colitis, were not detected more often from healthy versus inflamed tissue.

Owing to advances in genomics that enable differentiation of molecular aetiologies, patients with monogenic inflammatory bowel disease (mIBD) potentially have access to genotype-guided precision medicine. In this Expert Recommendation, we review the therapeutic research landscape of mIBD, the reported response to therapies, the medication-related risks and systematic bias in reporting. The mIBD field is characterized by the absence of randomized controlled trials and is dominated by retrospective observational data based on case series and case reports. More than 25 off-label therapeutics (including small-molecule inhibitors and biologics) as well as cellular therapies (including haematopoietic stem cell transplantation and gene therapy) have been reported. Heterogeneous reporting of outcomes impedes the generation of robust therapeutic evidence as the basis for clinical decision making in mIBD. We discuss therapeutic goals in mIBD and recommend standardized reporting (mIBD REPORT (monogenic Inflammatory Bowel Disease Report Extended Phenotype and Outcome of Treatments) standards) to stratify patients according to a genetic diagnosis and phenotype, to assess treatment effects and to record safety signals. Implementation of these pragmatic standards should help clinicians to assess the therapy responses of individual patients in clinical practice and improve comparability between observational retrospective studies and controlled prospective trials, supporting future meta-analysis.In this Expert Recommendation, Uhlig and colleagues review the therapeutic landscape for monogenic inflammatory bowel disease and propose recommendations for standardized reporting of clinical outcomes.