Integration of multi-omics data and deep phenotyping enables prediction of cytokine responses.
ABSTRACT: The immune response to pathogens varies substantially among people. Whereas both genetic and nongenetic factors contribute to interperson variation, their relative contributions and potential predictive power have remained largely unknown. By systematically correlating host factors in 534 healthy volunteers, including baseline immunological parameters and molecular profiles (genome, metabolome and gut microbiome), with cytokine production after stimulation with 20 pathogens, we identified distinct patterns of co-regulation. Among the 91 different cytokine-stimulus pairs, 11 categories of host factors together explained up to 67% of interindividual variation in cytokine production induced by stimulation. A computational model based on genetic data predicted the genetic component of stimulus-induced cytokine production (correlation 0.28-0.89), and nongenetic factors influenced cytokine production as well.
Project description:Differences in susceptibility to immune-mediated diseases are determined by variability in immune responses. In three studies within the Human Functional Genomics Project, we assessed the effect of environmental and non-genetic host factors of the genetic make-up of the host and of the intestinal microbiome on the cytokine responses in humans. We analyzed the association of these factors with circulating mediators and with six cytokines after stimulation with 19 bacterial, fungal, viral, and non-microbial metabolic stimuli in 534 healthy subjects. In this first study, we show a strong impact of non-genetic host factors (e.g., age and gender) on cytokine production and circulating mediators. Additionally, annual seasonality is found to be an important environmental factor influencing cytokine production. Alpha-1-antitrypsin concentrations partially mediate the seasonality of cytokine responses, whereas the effect of vitamin D levels is limited. The complete dataset has been made publicly available as a comprehensive resource for future studies. PAPERCLIP.
Project description:Despite existing evidence for tuning of innate immunity to different classes of bacteria, the molecular mechanisms used by macrophages to tailor inflammatory responses to specific pathogens remain incompletely defined. By stimulating mouse macrophages with a titration matrix of TLR ligand pairs, we identified distinct stimulus requirements for activating and inhibitory events that evoked diverse cytokine production dynamics. These regulatory events were linked to patterns of inflammatory responses that distinguished between Gram-positive and Gram-negative bacteria, both in vitro and after in vivo lung infection. Stimulation beyond a TLR4 threshold and Gram-negative bacteria-induced responses were characterized by a rapid type I IFN-dependent decline in inflammatory cytokine production, independent of IL-10, whereas inflammatory responses to Gram-positive species were more sustained due to the absence of this IFN-dependent regulation. Thus, disparate triggering of a cytokine negative feedback loop promotes tuning of macrophage responses in a bacteria class-specific manner and provides context-dependent regulation of inflammation dynamics.
Project description:The goal of this project is to understand the etiology and pathology of host-pathogen interaction using high-throughput cellular phenotyping. By combining the expertise in pathogen biology, informatics and stem cell biology across the institute, this project will use targeted genetic modification in mouse embryonic stem cells to identify functions of host genes in innate immunity. We will also explore the biology of mouse embryonic stem cells and their differentiation into immune competent cells. To this end we will generate homozygous and inducible knockout lines in mouse embryonic stem cells, which will be differentiated into immune competent cells, such as macrophages or dendritic cells. These will be subjected to a panel of immune challenge and stimulation assays to characterise the functions of the disrupted genes. To identify new functions for mammalian genes in innate immunity we will:Generate a panel of homozygous inducible gene knockout mouse ES cell lines working from a list of ca. 80 candidate genes (MGP phenotyping and GWAS hits). Scale up differentiation protocols for immune challenge and stimulation assays.Benchmark in vitro differentiated ES cell models against primary immune cells from selected MGP mouse lines and eventually against human iPS cells.Screen a panel of 30 genetically modified cell lines in immunological challenge and stimulation assays, defining phenotypes at the cellular level by high content imaging, biochemically by assaying cytokine production, and at the molecular level by transcriptome analysis.Define novel gene functions in innate immunity through network analysis of gene expression data from all challenged cell lines.In the longer term scale up the production and phenotyping of genetically modified cell lines (mouse or human as appropriate).This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:The Bacillus Calmette-Guérin (BCG) vaccine is administered at birth in tuberculosis (TB) endemic countries. BCG vaccination is also associated with protective non-specific effects against non-tuberculous infections. This seems at least in part mediated through induction of innate immune memory in myeloid cells, a process termed trained immunity. ?-glucan, a component of the fungal cell wall from Candida albicans, induces a trained immunity phenotype in human monocytes with hyper-responsiveness against unrelated pathogens. We aimed to study the capacity of BCG and ?-glucan to induce a similar phenotype by examining cytokine production in cord blood monocytes following re-stimulation. We used a well-known model of in vitro induction of trained immunity. Adherent mononuclear cells from neonates and adults, which consist mainly of monocytes, were stimulated in vitro with BCG or ?-glucan for one day, after which the stimulus was washed away. Cells were rested for 5 days, then restimulated with LPS. Cytokine levels were measured using ELISA. Neonate and adult monocytes responded similarly in terms of cytokine production. BCG significantly increased IL-6 responses to LPS in both neonate and adult monocytes, while ?-glucan induced increases of IL-6, IL-10 and TNF production capacity. The BCG and ?-glucan induced increase in cytokine production, reminiscent of trained immunity, showed similar levelsin neonatal and adult monocytes. BCG mediated changes in cytokine production shows the feasibility of this in vitro assay for further studies regarding non-specific effects of vaccines.
Project description:Gut microbial dysbioses are linked to aberrant immune responses, which are often accompanied by abnormal production of inflammatory cytokines. As part of the Human Functional Genomics Project (HFGP), we investigate how differences in composition and function of gut microbial communities may contribute to inter-individual variation in cytokine responses to microbial stimulations in healthy humans. We observe microbiome-cytokine interaction patterns that are stimulus specific, cytokine specific, and cytokine and stimulus specific. Validation of two predicted host-microbial interactions reveal that TNF? and IFN? production are associated with specific microbial metabolic pathways: palmitoleic acid metabolism and tryptophan degradation to tryptophol. Besides providing a resource of predicted microbially derived mediators that influence immune phenotypes in response to common microorganisms, these data can help to define principles for understanding disease susceptibility. The three HFGP studies presented in this issue lay the groundwork for further studies aimed at understanding the interplay between microbial, genetic, and environmental factors in the regulation of the immune response in humans. PAPERCLIP.
Project description:Innate immune memory, also termed "trained immunity" in vertebrates, has been recently described in a large variety of plants and animals. In most cases, trained innate immunity is induced by pathogens or pathogen-associated molecular patterns (PAMPs), and is associated with long-term epigenetic, metabolic, and functional reprogramming. Interestingly, recent findings indicate that food components can mimic PAMPs effects and induce trained immunity. The aim of this study was to investigate whether bovine milk or its components can induce trained immunity in human monocytes. To this aim, monocytes were exposed for 24 h to ?-glucan, Toll-like receptor (TLR)-ligands, bovine milk, milk fractions, bovine lactoferrin (bLF), and bovine Immunoglobulin G (bIgG). After washing away the stimulus and a resting period of five days, the cells were re-stimulated with TLR ligands and Tumor necrosis factor (TNF-) and interleukin (IL)-6 production was measured. Training with ?-glucan resulted in higher cytokine production after TLR1/2, TLR4, and TLR7/8 stimulation. When monocytes trained with raw milk were re-stimulated with TLR1/2 ligand Pam3CSK4, trained cells produced more IL-6 compared to non-trained cells. Training with bIgG resulted in higher cytokine production after TLR4 and TLR7/8 stimulation. These results show that bovine milk and bIgG can induce trained immunity in human monocytes. This confirms the hypothesis that diet components can influence the long-term responsiveness of the innate immune system.
Project description:The specific binding of transcription factors to cognate sequence elements is thought to be critical for the generation of specific gene expression programs. The transcription factors nuclear factor kB (NF-kB) and the interferon (IFN) regulatory factors (IRFs) bind to the kB site and the interferon response element (IRE), respectively, of target genes, and they are activated in macrophages after exposure to pathogens. However, how these factors produce pathogen-specific inflammatory and immune responses remains poorly understood. Combining top-down and bottom-up systems biology approaches, we have identified the NF-kB p50 homodimer (p50:p50) as a regulator of IRF responses. First, unbiased genome-wide expression analysis revealed that p50 repressed a subset of IFN-inducible genes through a previously uncharacterized subclass of guanine-rich IRE (G-IRE) sequences, which was substantiated by biochemical and structural analyses. Second, mathematical modeling predicted that p50:p50 might enforce the stimulus-specificity of composite promoters. Indeed, the production of the antiviral regulator IFN-b was rendered stimulus-specific by the binding of p50:p50 to the G-IRE–containing IFNb enhancer to suppress cytotoxic IFN signaling. Specifically, a deficiency in p50 resulted in the inappropriate production of IFN-b in response to bacterial DNA sensed by Toll-like receptor 9. This role for NF-kB p50 in enforcing the specificity of the cellular response to pathogens by binding to a previously uncharacterized subset of IRE sequences alters our understanding of how the NF-kB and IRF signaling systems cooperate to regulate antimicrobial immunity. Overall design: [BMDM]: Total RNA extracted from wt, p50-/- or ifnar-/- bone marrow derived macrophages (BMDMs) were subjected to stimulation with LPS, CpG or IFNb [MEF]: Total RNA extracted from wt or p50KO primary mouse embryonic fibroblasts were subjected to stimulation with LPS or IFNb
Project description:The contribution of host genetic and nongenetic factors to immunological differences in humans remains largely undefined. Here, we generated bacterial-, fungal-, and viral-induced immune transcriptional profiles in an age- and sex-balanced cohort of 1,000 healthy individuals and searched for the determinants of immune response variation. We found that age and sex affected the transcriptional response of most immune-related genes, with age effects being more stimulus-specific relative to sex effects, which were largely shared across conditions. Although specific cell populations mediated the effects of age and sex on gene expression, including CD8+ T cells for age and CD4+ T cells and monocytes for sex, we detected a direct effect of these intrinsic factors for the majority of immune genes. The mapping of expression quantitative trait loci (eQTLs) revealed that genetic factors had a stronger effect on immune gene regulation than age and sex, yet they affected a smaller number of genes. Importantly, we identified numerous genetic variants that manifested their regulatory effects exclusively on immune stimulation, including a Candida albicans-specific master regulator at the CR1 locus. These response eQTLs were enriched in disease-associated variants, particularly for autoimmune and inflammatory disorders, indicating that differences in disease risk may result from regulatory variants exerting their effects only in the presence of immune stress. Together, this study quantifies the respective effects of age, sex, genetics, and cellular heterogeneity on the interindividual variability of immune responses and constitutes a valuable resource for further exploration in the context of different infection risks or disease outcomes.
Project description:Meniscus injury increases the risk of osteoarthritis; however, the biologic mechanism remains unknown. We hypothesized that pro-inflammatory stimulation of meniscus would increase production of matrix-degrading enzymes, cytokines and chemokines which cause joint tissue destruction and could contribute to osteoarthritis development.Meniscus and cartilage tissue from healthy tissue donors and total knee arthroplasties (TKAs) was cultured. Primary cell cultures were stimulated with pro-inflammatory factors [IL-1?, IL-6, or fibronectin fragments (FnF)] and cellular responses were analyzed by real-time PCR, protein arrays and immunoblots. To determine if NF-?B was required for MMP production, meniscus cultures were treated with inflammatory factors with and without the NF-?B inhibitor, hypoestoxide.Normal and osteoarthritic meniscus cells increased their MMP secretion in response to stimulation, but specific patterns emerged that were unique to each stimulus with the greatest number of MMPs expressed in response to FnF. Meniscus collagen and connective tissue growth factor (CTGF) gene expression was reduced. Expression of cytokines (IL-1?, IL-1?, IL-6), chemokines (IL-8, CXCL1, CXCL2, CSF1) and components of the NF-?B and tumor necrosis factor (TNF) family were significantly increased. Cytokine and chemokine protein production was also increased by stimulation. When primary cell cultures were treated with hypoestoxide in conjunction with pro-inflammatory stimulation, p65 activation was reduced as were MMP-1 and MMP-3 production.Pro-inflammatory stimulation of meniscus cells increased matrix metalloproteinase production and catabolic gene expression. The meniscus could have an active biologic role in osteoarthritis development following joint injury through increased production of cytokines, chemokines, and matrix-degrading enzymes.
Project description:Genetically identical cells exhibit extensive phenotypic variation even under constant and benign conditions. This so-called nongenetic heterogeneity has important clinical implications: within tumors and microbial infections, cells show nongenetic heterogeneity in growth rate and in susceptibility to drugs or stress. The budding yeast, Saccharomyces cerevisiae, shows a similar form of nongenetic heterogeneity in which growth rate correlates positively with susceptibility to acute heat stress at the single-cell level. Using genetic and chemical perturbations, combined with high-throughput single-cell assays of yeast growth and gene expression, we show here that heterogeneity in intracellular cyclic AMP (cAMP) levels acting through the conserved Ras/cAMP/protein kinase A (PKA) pathway and its target transcription factors, Msn2 and Msn4, underlies this nongenetic heterogeneity. Lower levels of cAMP correspond to slower growth, as shown by direct comparison of cAMP concentration in subpopulations enriched for slower vs. faster growing cells. Concordantly, an endogenous reporter of this pathway's activity correlates with growth in individual cells. The paralogs Msn2 and Msn4 differ in their roles in nongenetic heterogeneity in a way that demonstrates slow growth and stress tolerance are not inevitably linked. Heterogeneity in growth rate requires each, whereas only Msn2 is required for heterogeneity in expression of Tsl1, a subunit of trehalose synthase that contributes to acute-stress tolerance. Perturbing nongenetic heterogeneity by mutating genes in this pathway, or by culturing wild-type cells with the cell-permeable cAMP analog 8-bromo-cAMP or the PKA inhibitor H89, significantly impacts survival of acute heat stress. Perturbations that increase intracellular cAMP levels reduce the slower-growing subpopulation and increase susceptibility to acute heat stress, whereas PKA inhibition slows growth and decreases susceptibility to acute heat stress. Loss of Msn2 reduces, but does not completely eliminate, the correlation in individual cells between growth rate and acute-stress survival, suggesting a major role for the Msn2 pathway in nongenetic heterogeneity but also a residual benefit of slow growth. Our results shed light on the genetic control of nongenetic heterogeneity and suggest a possible means of defeating bet-hedging pathogens or tumor cells by making them more uniformly susceptible to treatment.