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Intercellular communication mediated by cytokines is critical to the development of immune responses, particularly in the context of infectious and inflammatory diseases. By releasing these small molecular weight peptides, the source cells can influence numerous intracellular processes in the target cells, including the secretion of other cytokines downstream. However, there are no readily available bioinformatic resources that can model cytokine - cytokine interactions. In this effort, we built a communication map between major tissues and blood cells that reveals how cytokine-mediated intercellular networks form during homeostatic conditions. We collated the most prevalent cytokines from literature, and assigned the proteins and their corresponding receptors to source tissue and blood cell types based on enriched consensus RNA-Seq data from the Human Protein Atlas database. To assign more confidence to the interactions, we integrated literature information on cell - cytokine interactions from two systems immunology databases, immuneXpresso and ImmunoGlobe. From the collated information, we defined two metanetworks: a cell-cell communication network connected by cytokines; and a cytokine-cytokine interaction network depicting the potential ways in which cytokines can affect the activity of each other. Using expression data from disease states, we then applied this resource to reveal perturbations in cytokine-mediated intercellular signalling in inflammatory and infectious diseases (ulcerative colitis and COVID-19, respectively). For ulcerative colitis, with CytokineLink we demonstrated a significant rewiring of cytokine-mediated intercellular communication between non-inflamed and inflamed colonic tissues. For COVID-19, we were able to identify inactive cell types and cytokine interactions that may be important following SARS-CoV-2 infection when comparing the cytokine response with other viruses capable of initiating a cytokine storm. Such findings have potential to inform the development of novel, cytokine-targeted therapeutic strategies. CytokineLink is freely available for the scientific community through the NDEx platform and the project github repository (https://github.com/korcsmarosgroup/CytokineLink).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents an unprecedented worldwide health problem. Although the primary site of infection is the lung, growing evidence points towards a crucial role of the intestinal epithelium. Yet, the exact effects of viral infection and the role of intestinal epithelial-immune cell interactions in mediating the inflammatory response are not known. In this work, we apply network biology approaches to single-cell RNA-seq data from SARS-CoV-2 infected human ileal and colonic organoids to investigate how altered intracellular pathways upon infection in intestinal enterocytes leads to modified epithelial-immune crosstalk. We point out specific epithelial-immune interactions which could help SARS-CoV-2 evade the immune response. By integrating our data with existing experimental data, we provide a set of epithelial ligands likely to drive the inflammatory response upon infection. Our integrated analysis of intra- and inter-cellular molecular networks contribute to finding potential drug targets, and suggest using existing anti-inflammatory therapies in the gut as promising drug repurposing strategies against COVID-19.

Interactions between the epithelium and the immune system are critical in the pathogenesis of inflammatory bowel disease (IBD). In this study, we mapped the transcriptional landscape of human colonic epithelial organoids in response to different cytokines responsible for mediating canonical mucosal immune responses. By profiling the transcriptome of human colonic organoids treated with the canonical cytokines interferon gamma, interleukin-13, -17A, and tumor necrosis factor alpha with next-generation sequencing, we unveil shared and distinct regulation patterns of epithelial function by different cytokines. An integrative analysis of cytokine responses in diseased tissue from patients with IBD (n = 1,009) reveals a molecular classification of mucosal inflammation defined by gradients of cytokine-responsive transcriptional signatures. Our systems biology approach detected signaling bottlenecks in cytokine-responsive networks and highlighted their translational potential as theragnostic targets in intestinal inflammation.

We describe a large-scale community effort to build an open-access, interoperable, and computable repository of COVID-19 molecular mechanisms - the COVID-19 Disease Map. We discuss the tools, platforms, and guidelines necessary for the distributed development of its contents by a multi-faceted community of biocurators, domain experts, bioinformaticians, and computational biologists. We highlight the role of relevant databases and text mining approaches in the enrichment and validation of the curated mechanisms. We describe the contents of the Map and their relevance to the molecular pathophysiology of COVID-19 and the analytical and computational modelling approaches that can be applied for mechanistic data interpretation and predictions. We conclude by demonstrating concrete applications of our work through several use cases and highlight new testable hypotheses.; We describe a large-scale community effort to build an open-access, interoperable, and computable repository of COVID-19 molecular mechanisms - the COVID-19 Disease Map. We discuss the tools, platforms, and guidelines necessary for the distributed development of its contents by a multi-faceted community of biocurators, domain experts, bioinformaticians, and computational biologists. We highlight the role of relevant databases and text mining approaches in the enrichment and validation of the curated mechanisms. We describe the contents of the Map and their relevance to the molecular pathophysiology of COVID-19 and the analytical and computational modelling approaches that can be applied for mechanistic data interpretation and predictions. We conclude by demonstrating concrete applications of our work through several use cases and highlight new testable hypotheses.

The SARS-CoV-2 pandemic of 2020 has mobilised scientists around the globe to research all aspects of the coronavirus virus and its infection. For fruitful and rapid investigation of viral pathomechanisms, a collaborative and interdisciplinary approach is required. Therefore, we have developed ViralLink: a systems biology workflow which reconstructs and analyses networks representing the effect of viruses on intracellular signalling. These networks trace the flow of signal from intracellular viral proteins through their human binding proteins and downstream signalling pathways, ending with transcription factors regulating genes differentially expressed upon viral exposure. In this way, the workflow provides a mechanistic insight from previously identified knowledge of virally infected cells. By default, the workflow is set up to analyse the intracellular effects of SARS-CoV-2, requiring only transcriptomics counts data as input from the user: thus, encouraging and enabling rapid multidisciplinary research. However, the wide-ranging applicability and modularity of the workflow facilitates customisation of viral context, a priori interactions and analysis methods. Through a case study of SARS-CoV-2 infected bronchial/tracheal epithelial cells, we evidence the functionality of the workflow and its ability to identify key pathways and proteins in the cellular response to infection. The application of ViralLink to different viral infections in a cell-type specific manner using different available transcriptomics datasets will uncover key mechanisms in viral pathogenesis. The workflow is available on GitHub (https://github.com/korcsmarosgroup/ViralLink) in an easily accessible Python wrapper script, or as customisable modular R and Python scripts.Author summaryCollaborative and multidisciplinary science provides increased value for experimental datasets and speeds the process of discovery. Such ways of working are especially important at present due to the urgency of the SARS-CoV-2 pandemic. Here, we present a systems biology workflow which models the effect of viral proteins on the infected host cell, to aid collaborative and multidisciplinary research. Through integration of gene expression datasets with context-specific and context-agnostic molecular interaction datasets, the workflow can be easily applied to different datasets as they are made available. Application to diverse SARS-CoV-2 datasets will increase our understanding of the mechanistic details of the infection at a cell type specific level, aid drug target discovery and help explain the variety of clinical manifestations of the infection.

Interactions between the epithelium and the immune system are critical in the pathogenesis of inflammatory bowel disease (IBD). In this study, we provide new insights into the gut immune-epithelial interactome by mapping the transcriptional landscape of human colonic epithelial organoids in response to different cytokines responsible for mediating canonical mucosal immune responses. By profiling the transcriptome of human colonic organoids treated with the canonical cytokines: interferon gamma, interleukin 13, 17A and tumour necrosis factor alpha with next generation sequencing we unveil previously unrecognised levels of shared and distinct regulation of epithelial function by different cytokines. An integrative analysis of cytokine responses in diseased tissue from IBD patients (n=1,084) reveals a new molecular stratification of IBD defined by gradients of cytokine responsive transcriptional signatures. Utilising a systems biology approach, we also detect signalling bottlenecks in cytokine responsive networks and highlight their translational potential as theragnostic targets in intestinal inflammation.

The SARS-CoV-2 pandemic of 2020 has mobilised scientists around the globe to research all aspects of the coronavirus virus and its infection. For fruitful and rapid investigation of viral pathomechanisms, a collaborative and interdisciplinary approach is required. Therefore, we have developed ViralLink: a systems biology workflow which reconstructs and analyses networks representing the effect of viruses on intracellular signalling. These networks trace the flow of signal from intracellular viral proteins through their human binding proteins and downstream signalling pathways, ending with transcription factors regulating genes differentially expressed upon viral exposure. In this way, the workflow provides a mechanistic insight from previously identified knowledge of virally infected cells. By default, the workflow is set up to analyse the intracellular effects of SARS-CoV-2, requiring only transcriptomics counts data as input from the user: thus, encouraging and enabling rapid multidisciplinary research. However, the wide-ranging applicability and modularity of the workflow facilitates customisation of viral context,a prioriinteractions and analysis methods. Through a case study of SARS-CoV-2 infected bronchial/tracheal epithelial cells, we evidence the functionality of the workflow and its ability to identify key pathways and proteins in the cellular response to infection. The application of ViralLink to different viral infections in a cell-type specific manner using different available transcriptomics datasets will uncover key mechanisms in viral pathogenesis. The workflow is available on GitHub (https://github.com/korcsmarosgroup/ViralLink) in an easily accessible Python wrapper script, or as customisable modular R and Python scripts.

Summary The underlying health-driving mechanisms of Bifidobacterium during early life are not well understood, particularly how this microbiota member may modulate the intestinal barrier via programming of intestinal epithelial cells (IECs). We investigated the impact of Bifidobacterium breve UCC2003 on the transcriptome of neonatal murine IECs. Small IECs from two-week-old neonatal mice administered B. breve UCC2003 or PBS (control) were subjected to global RNA sequencing, and differentially expressed genes, pathways, and affected cell types were determined. We observed extensive regulation of the IEC transcriptome with ∼4,000 genes significantly up-regulated, including key genes linked with epithelial barrier function. Enrichment of cell differentiation pathways was observed, along with an overrepresentation of stem cell marker genes, indicating an increase in the regenerative potential of the epithelial layer. In conclusion, B. breve UCC2003 plays a central role in driving intestinal epithelium homeostatic development during early life and suggests future avenues for next-stage clinical studies., Graphical Abstract, Highlights • B. breve administration significantly alters the murine neonatal IEC transcriptome • Genes/pathways involved in epithelial barrier function are particularly impacted • Bifidobacterium may target the IEC stem cell compartment to induce regeneration, Microbiology; Microbiome; Transcriptomics

Background: Bifidobacterium represents an important early life microbiota member. Specific bifidobacterial components, exopolysaccharides (EPS), positively modulate host responses, with purified EPS also suggested to impact microbe&ndash, microbe interactions by acting as a nutrient substrate. Thus, we determined the longitudinal effects of bifidobacterial EPS on microbial communities and metabolite profiles using an infant model colon system. Methods: Differential gene expression and growth characteristics were determined for each strain, Bifidobacterium breve UCC2003 and corresponding isogenic EPS-deletion mutant (B. breve UCC2003del). Model colon vessels were inoculated with B. breve and microbiome dynamics monitored using 16S rRNA sequencing and metabolomics (NMR). Results: Transcriptomics of EPS mutant vs. B. breve UCC2003 highlighted discrete differential gene expression (e.g., eps biosynthetic cluster), though overall growth dynamics between strains were unaffected. The EPS-positive vessel had significant shifts in microbiome and metabolite profiles until study end (405 h), with increases of Tyzzerella and Faecalibacterium, and short-chain fatty acids, with further correlations between taxa and metabolites which were not observed within the EPS-negative vessel. Conclusions: These data indicate that B. breve UCC2003 EPS is potentially metabolized by infant microbiota members, leading to differential microbial metabolism and altered metabolite by-products. Overall, these findings may allow development of EPS-specific strategies to promote infant health.

The epithelial lining of the small intestine consists of multiple cell types, including Paneth cells and goblet cells, that work in cohort to maintain gut health. 3D in vitro cultures of human primary epithelial cells, called organoids, have become a key model to study the functions of Paneth cells and goblet cells in normal and diseased conditions. Advances in these models include the ability to skew differentiation to particular lineages, providing a useful tool to study cell type specific function/dysfunction in the context of the epithelium. Here, we use comprehensive profiling of mRNA, microRNA and long non-coding RNA expression to confirm that Paneth cell and goblet cell enrichment of murine small intestinal organoids (enteroids) establishes a physiologically accurate model. We employ network analysis to infer the regulatory landscape altered by skewing differentiation, and using knowledge of cell type specific markers, we predict key regulators of cell type specific functions: Cebpa, Jun, Nr1d1 and Rxra specific to Paneth cells, Gfi1b and Myc specific for goblet cells and Ets1, Nr3c1 and Vdr shared between them. Links identified between these regulators and cellular phenotypes of inflammatory bowel disease (IBD) suggest that global regulatory rewiring during or after differentiation of Paneth cells and goblet cells could contribute to IBD aetiology. Future application of cell type enriched enteroids combined with the presented computational workflow can be used to disentangle multifactorial mechanisms of these cell types and propose regulators whose pharmacological targeting could be advantageous in treating IBD patients with Crohn’s disease or ulcerative colitis.Table of contentsWe demonstrate the application of network biology techniques to increase understanding of intestinal dysbiosis through studying transcriptomics data from Paneth and goblet cell enriched enteroids.

Intercellular communication mediated by cytokines is critical to the development of immune responses, particularly in the context of infectious and inflammatory diseases. By releasing these small molecular weight peptides, the source cells can influence numerous intracellular processes in the target cells, including the secretion of other cytokines downstream. However, there are no readily available bioinformatic resources that can model cytokine–cytokine interactions. In this effort, we built a communication map between major tissues and blood cells that reveals how cytokine-mediated intercellular networks form during homeostatic conditions. We collated the most prevalent cytokines from the literature and assigned the proteins and their corresponding receptors to source tissue and blood cell types based on enriched consensus RNA-Seq data from the Human Protein Atlas database. To assign more confidence to the interactions, we integrated the literature information on cell–cytokine interactions from two systems of immunology databases, immuneXpresso and ImmunoGlobe. From the collated information, we defined two metanetworks: a cell–cell communication network connected by cytokines, and a cytokine–cytokine interaction network depicting the potential ways in which cytokines can affect the activity of each other. Using expression data from disease states, we then applied this resource to reveal perturbations in cytokine-mediated intercellular signalling in inflammatory and infectious diseases (ulcerative colitis and COVID-19, respectively). For ulcerative colitis, with CytokineLink, we demonstrated a significant rewiring of cytokine-mediated intercellular communication between non-inflamed and inflamed colonic tissues. For COVID-19, we were able to identify cell types and cytokine interactions following SARS-CoV-2 infection, highlighting important cytokine interactions that might contribute to severe illness in a subgroup of patients. Such findings have the potential to inform the development of novel, cytokine-targeted therapeutic strategies. CytokineLink is freely available for the scientific community through the NDEx platform and the project github repository.

Members of the genusBifidobacteriumrepresent an important bacterial group for promoting health during early life. Previous studies have indicated that bifidobacterial exopolysaccharides (EPS) are involved in host interactions, with purified EPS also suggested to modulate microbe-microbe interactions by acting as a nutrient substrate. To further explore the role of EPS as a potential dietary component, we determined the longitudinal effects of bifidobacterial EPS on microbial communities and metabolite profiles using an infant model colon system.Bifidobacterium breveUCC2003 was utilised as a representative early life bifidobacterial strain, and a corresponding isogenic EPS-deletion mutant (B. breveUCC2003 EPS-). Initial transcriptomics analysis of the EPS mutant vs. parentB. breveUCC2003 strain highlighted differential expression in a discrete number of genes, including theepsbiosynthetic cluster, though overall growth dynamics between the two strains were unaffected. Model colon vessels were inoculated withB. brevestrains and microbiome dynamics were monitored using metataxonomic (via 16S rRNA sequencing) and metabolomic (via1H NMR) approaches. Baseline early life microbiota profiles were similar between vessels, with persistence ofB. breve(EPS+ and EPS-) observed between 0-36h. Within the EPS-positive vessel there was a significant shift in microbiome and metabolite profiles until the end of the study (405h); we observed increases ofEscherichiaandTyzzerella, and short-chain fatty acids including acetate, propionate and formate, including further correlations between taxa and metabolites which were not observed in the EPS-negative vessel. These data indicate that theB. breveUCC2003 EPS is potentially being metabolised by members of the infant microbial community, leading to differential microbial metabolism and altered metabolite by-products. Overall, these findings may allow for development of EPS-specific strategies to beneficially alter the early life microbiota to promote infant health.

Background Intestinal homeostasis depends on complex interactions between the microbiota, the intestinal epithelium and the host immune system. Paneth cells play an important role thanks to their release of antimicrobial peptides and their dysfunction has been shown to contribute to Crohn’s disease. The human gut commensal Bifidobacteria has been highlighted as a protective agent in IBD. Whilst the specific modulating factors are largely unidentified, evidence suggests that Bifidobacteria can interact with host intestinal cells. Such interactions can alter host molecular pathways, resulting in a modified intestinal cell function. Understanding these effects is crucial to unravel the potential benefits of bifidobacteria for IBD. In this study, we developed a workflow to investigate the beneficial effects of the probiotic bifidobacteria in modulating gene regulation and function of different intestinal epithelial cells in the gut. Methods We gavaged either conventionally raised (SPF) or germ-free mice (n = 5) with Bifidobacterium breve UCC2003 (1–5 × 108 CFU), and sorted intestinal epithelial cell types (Lgr5+ stem cells, Paneth cells and transit-amplifying cells) from the mouse intestinal tissue by flow cytometry using a panel of known antibodies. Each isolated cell population (n = 200) was subsequently profiled by low input RNA sequencing and DNA methylation. Results Intracellular lysozyme staining for Paneth cells and initial gene expression data exploration suggests successful separation of the different populations from the mouse intestinal tissue using flow cytometry. Gene expression data were further analysed using network biology approaches to generate cell-type-specific molecular regulatory networks indicating genes regulation by bifidobacteria in each cell types. Overlaying the methylation data will elucidate the role of epigenetics played during this regulation. Together, these networks will improve our understanding of how Bifidobacteria affects these different cells types through communication with precursor stem cells and/or with direct effects on the fully differentiated cells, ultimately impacting their function. Conclusion In the future, we aim to use a similar workflow using two-dimensional human-derived organoids monolayers co-cultured with bifidobacteria to investigate the effects on host intestinal epithelial cell function in a high-throughput and patient-specific manner. This will increase our understanding of the complex role played by host genetics and microbial factors in the pathogenesis of gastrointestinal diseases such as IBD, and hopefully pave the way for translational developments in the prevention these pathologies.

Background Interactions between the immune system and the intestinal epithelium play an important role in the pathogenesis of chronic immune mediated inflammatory diseases, including inflammatory bowel disease (IBD). In IBD, debilitating symptoms and complications including abscesses and cancer are associated with aberrant cytokine production and resulting intestinal epithelial damage. Despite the advent of biological therapies targeting key pathogenic cytokines, like tumour necrosis α (TNF), only 18% of IBD patients will achieve complete disease control and mucosal healing. Here, we provide new insights into the epithelial response to cytokines using network analysis of transcriptomics data from colonic organoids (colonoids). Methods We generated an atlas of the transcriptomic effects of cytokines by treating human-derived colonoids with IFNg, IL13, IL9, IL17A and TNF (independently). By integrating the observed transcriptional changes with previously published signalling and regulatory interactions, we generated causal networks to elucidate the effect of cytokine cues on epithelial cells. These networks comprised experimentally verified protein–protein and transcription factor (TF)–target gene interactions, forming signalling pathways linking cytokines to TFs and from TFs to differentially expressed genes. Results With this analysis, we identified previously unrecognised levels of shared and distinct transcriptional regulation of colonic epithelial function by different cytokines. While IL9 had a negligible impact on the transcriptome, the transcripts with differential expression induced by IFNg, IL13, IL17A or TNF were consistent with their recognised function in other tissues. IFNg and TNF exhibited similar magnitude and directional effects on key immune pathways while IL13 had the opposite effect. Using a network approach, we found that regulatory effects of cytokines are primarily transduced through unique signalling routes, some of which converge on the same key transcription factors; CEBPA, E2F1, E2F2, ETS1, FOS, IRF1 and MAZ. We observed independent regulatory mechanisms of the different cytokines as well as complementarity in the epithelial responses regulated by different canonical cytokines. Conclusion The generated cytokine transcriptional atlas provides a unique insight into the immune-epithelial interactome by allowing the identification of shared and distinct transcriptional pathways across different types of immunity at the mucosal barrier. In addition, it provides the unique opportunity to study cytokine responses in the context of human disease and generate novel hypotheses.