Loss of IL-10 signaling in macrophages (Mφs) leads to inflammatory bowel disease (IBD). Induced pluripotent stem cells (iPSCs) were generated from an infantile-onset IBD patient lacking a functional IL10RB gene. Mφs differentiated from IL-10RB−/− iPSCs lacked IL-10RB mRNA expression, were unable to phosphorylate STAT3, and failed to reduce LPS induced inflammatory cytokines in the presence of exogenous IL-10. IL-10RB−/− Mφs exhibited a striking defect in their ability to kill Salmonella enterica serovar Typhimurium, which was rescuable after experimentally introducing functional copies of the IL10RB gene. Genes involved in synthesis and receptor pathways for eicosanoid prostaglandin E2 (PGE2) were more highly induced in IL-10RB−/− Mφs, and these Mφs produced higher amounts of PGE2 after LPS stimulation compared with controls. Furthermore, pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade enhanced bacterial killing in Mφs. These results identify a regulatory interaction between IL-10 and PGE2, dysregulation of which may drive aberrant Mφ activation and impaired host defense contributing to IBD pathogenesis.
Interferon regulating factor 5 (IRF5) is a multifunctional regulator of immune responses, and has a key pathogenic function in gut inflammation, but how IRF5 is modulated is still unclear. Having performed a kinase inhibitor library screening in macrophages, here we identify protein-tyrosine kinase 2-beta (PTK2B/PYK2) as a putative IRF5 kinase. PYK2-deficient macrophages display impaired endogenous IRF5 activation, leading to reduction of inflammatory gene expression. Meanwhile, a PYK2 inhibitor, defactinib, has a similar effect on IRF5 activation in vitro, and induces a transcriptomic signature in macrophages similar to that caused by IRF5 deficiency. Finally, defactinib reduces pro-inflammatory cytokines in human colon biopsies from patients with ulcerative colitis, as well as in a mouse colitis model. Our results thus implicate a function of PYK2 in regulating the inflammatory response in the gut via the IRF5 innate sensing pathway, thereby opening opportunities for related therapeutic interventions for inflammatory bowel diseases and other inflammatory conditions.
CD4+ T cells are pivotal cells playing roles in the orchestration of humoral and cytotoxic immune responses. It is known that CD4+ T cell proliferation relies on autophagy, but identification of the autophagosomal cargo involved is missing. Here we create a transgenic mouse model, to enable direct mapping of the proteinaceous content of autophagosomes in primary cells by LC3 proximity labelling. Interleukin-7 receptor-α, a cytokine receptor mostly found in naïve and memory T cells, is reproducibly detected in autophagosomes of activated CD4+ T cells. Consistently, CD4+ T cells lacking autophagy show increased interleukin-7 receptor-α surface expression, while no defect in internalisation is observed. Mechanistically, excessive surface interleukin-7 receptor-α sequestrates the common gamma chain, impairing the interleukin-2 receptor assembly and downstream signalling crucial for T cell proliferation. This study shows that key autophagy substrates can be reliably identified in this mouse model and help mechanistically unravel autophagy’s contribution to healthy physiology and disease.
Identifying individuals at high risk of chronic diseases via easily measured biomarkers could improve public health efforts to prevent avoidable illness and death. Here we present nuclear magnetic resonance blood metabolomics from half a million samples from three national biobanks. We built metabolomic risk scores that identify a high-risk group for each of 12 diseases that cause the most morbidity in high-income countries and show consistent cross-biobank replication of the relative risk of disease for these groups. We show that these metabolomic risk scores are more strongly associated with future disease onset than polygenic scores for most of these diseases. In a subset of 18,000 individuals with metabolomic biomarkers measured at two time points we show that people whose scores change have dramatically different future risk of disease, suggesting that repeat measurements capture the benefits of lifestyle change. We show cross-biobank calibration of our scores. Since metabolomics can be measured from a standard blood sample, we propose such tests can be feasibly implemented today in preventative health programs.
A combination of genetic susceptibility and environmental exposure is thought to cause inflammatory bowel disease (IBD), but the non-genetic component remains poorly characterized. We therefore undertook a search for environmental variables and gene-environment interactions associated with future IBD diagnosis in a large UK cohort. Using self-report and electronic health records, we identified 1946 Crohn’s disease (CD) and 3715 ulcerative colitis (UC) patients after quality control in the UK Biobank. Based on prior literature and biological plausibility , we tested 38 candidate environmental variables for association with CD, UC, and overall IBD using Cox proportional hazard regressions. We also tested whether these variables interacted with polygenic risk in predicting disease, following up significant (FDR < 0.05) results with tests for SNP-environment associations. We performed robustness analyses on all significant results. As in previous reports, appendectomy protected against UC, smoking (both current and previous) elevated risk for CD, current smoking protected against UC, and previous smoking imparted a risk for UC. Childhood antibiotic use associated with IBD, as did sun exposure during the winter. Socioeconomic deprivation was conferred a risk for IBD, CD, and UC. We uncovered negative interactions between polygenic risk and previous oral contraceptive use for IBD and UC. Polygenic risk also interacted negatively with previous smoking in predicting UC. There were no individually significant SNP-environment interactions. Thus, for a limited set of environmental variables, there was strong evidence of association with IBD diagnosis in the UK Biobank, and interaction with polygenic risk was minimal.
17Inflammatory bowel disease (IBD) is a group of inflammatory disorders of the gastro-intestinal 18 tract caused by a complex combination of genetic and environmental factors. Interferon 19 regulating factor 5 (IRF5) is a multifunctional regulator of immune responses, which plays a 20 key pathogenic role in mouse colitis models and is a genetic risk factor for IBD. A screen of a 21 protein kinase inhibitor library in macrophages revealed a list of putative IRF5 kinases. Among 22 MAIN 36A recent single-cell transcriptomic analysis of colon biopsies from patients with ulcerative 37 colitis (UC) provided a framework for linking GWAS risk loci with specific cell types and 38 functional pathways and helped to nominate causal genes across GWAS loci 3 , amongst them 39Interferon regulatory factor 5 (IRF5). IRF5 is a multifunctional regulator of immune 40 responses 4-6 . The IRF5 risk variant has consistent effects across monocytes and macrophage 41 conditions, but also impacts gene expression and splicing across a wide range of other immune 42 cells and tissues 7 . 43Recent studies using IRF5-deficient mice have established a critical role of this transcription 44 factor in the pathogenesis of mouse models of colitis 8,9 . IRF5 is proposed to exert its molecular 45 function via a cascade of events involving its phosphorylation, ubiquitination, dimerisation, 46 nuclear translocation and selective binding to its target genes to enable their expression 10 . 47 PYK2-deficient cells (Fig. 2f, Supplementary Fig. 4a, b). Interestingly, we could only detect 130 Y171 phosphorylation in WT cells (Fig. 2f, g Supplementary Fig. 4b), while S56 and Y312 131 residues were modified in PYK2-deficient cells (Fig. 2f, Supplementary Fig. 4a, b), possibly 132 reflecting on modification by other enzymes. In fact, Src tyrosine kinase Lyn was capable of 133 phosphorylating IRF5 at orthologues of Y312 and Y334 sites in in vitro co-expression 134 system 19 . We individually mutated these sites (Y171, Y312, Y334) as well as the published 135
T cell activation pathways have been repeatedly implicated by genetic studies as being enriched for risk genes for immune and inflammatory diseases. Many of these risk genes code for costimulatory receptors or ligands. Costimulatory receptors are cell surface proteins on T cells, which are engaged by costimulatory ligands on antigen-presenting cells. Both costimulation and antigen binding are required to trigger T cell activation. In order to study the different pathways activated by these costimulatory risk molecules, and the role they may play in inflammatory disease genetics, we carried out gene expression (RNA-seq) and chromatin accessibility (ATAC-seq) profiling of naive and memory CD4+ T cells (N=5 donors) activated via four different costimulatory receptors: CD28 (the standard molecule used for in vitro activation studies), along with alternative costimulatory molecules ICOS, CD6, and CD27. Most, but not all, activation genes and regions are shared by different costimulation conditions. Alternative costimulation induced lower proliferation and cytokine production, but higher lysosome production, altered metabolic processing, and indications of “signal seeking” behaviour (homing and expression of costimulatory and cytokine receptors). We validated a number of these functions at the surface protein level using orthogonal experimental techniques. We found the strongest enrichment of heritability for inflammatory bowel disease in shared regions upregulated by all costimulatory molecules. However, some risk variants and genes were only induced by alternative costimulation, and the impact of these variants on expression were less often successfully mapped in studies of T cells activated by traditional CD28 costimulation. This suggests that future genetics studies of gene expression in activated T cells may benefit from including alternative costimulation conditions.
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