InnateDB – Facilitating Systems Level Analyses of the Mammalian Innate Immune Response

1. InnateDB – Facilitating Systems Level Analyses of the Mammalian Innate Immune Response David Lynn M.Sc., Ph.D., Research Associate, Brinkman Lab., Simon Fraser University & Hancock Lab., University of British Columbia. InnateDB & Data Analysis Workshop - UBC, Vancouver. April 2nd & 3rd 2008. Updated Sept. 2009.

2. Systems Biology Approaches to Investigating the Innate Immune Response: Although progress has been made in understanding the innate immune response including the detailed dissection of some of the critical signaling pathways involved. Now becoming clear that the innate immune response does not involve simple linear pathways but rather complex networks of pathways and interactions, negative feedback loops and multifaceted transcriptional responses. To better understand the complexities of the innate immune response and the cross-talk between its components, complimentary systems level analyses and more focused follow-up experimental approaches are now needed.

3. InnateDB Developed in the Context of Two Large International Systems Biology Projects Mouse Model Datasets: Cerebral Malaria mouse model (IMR, Australia) Tuberculosis mouse model (AECM) Shigella xenograft model (Pasteur) Human Clinical Datasets: Typhoid & Malaria Vietnam (OUCRU/Stanford/Sanger) Non Typhoidal Salmonella Malawi (Sanger) Chronic/Acute Helminth Ecuador (USF de Quito/Sanger) + Modulating innate immune response viaHost Defense peptides (Hancock lab, UBC) Mouse KOs (Sanger) Novel insight into host response and mechanism of peptides. Common Pathways, networks and transcriptional regulation?

4. Why Systems Approaches are Needed: Many layers of complexity: Layers of regulation: 100s – 1000s DE genes: Not simple pathways  networks of molecular interactions. Gardy*, Lynn*, Brinkman, Hancock. Enabling a systems biology approach to immunology: focus on innate immunity. Trends in Immunology June 2009.

5. The Need for InnateDB & the Manual Curation of Innate Immunity Relevant Molecular Interactions & Pathways. Quickly apparent that available resources provided poor coverage and detail of the molecular interactions and pathways relevant to innate immunity. This information is essential for the systems-orientated interpretation of large scale genomics data. TLR4  one of the most important molecules in the innate immune response, has relatively few molecular interactions annotated in the major publicly available interaction DBs. 5 of these DBs combined contained annotated molecular interactions between TLR4 and just 11 other proteins. Through a review of the literature we have curated, in detail, a further 16 unique interactions, and provided annotation of nearly 60 different lines of evidence supporting these interactions. Relatively new pathways (NLR, RLR pathways) not annotated at all in major pathway databases. Few resources available for analysis of data in a pathway/network context that were accessible to a biologist. No resources for innate immunity.

6. Overview of InnateDB Project (www.innatedb.ca) InnateDB (www.innatedb.ca) is a database of all human and mouse experimentally-verified interactions and pathways (& their component molecules – Genes/Proteins/RNAs). Particular emphasis on the contextual manual curation of interactions involved in innate immunity (10,000 intxns). InnateDB facilitates systems-level analyses of mammalian signaling through integrated bioinformatics and visualization tools: pathway & ontology analysis, network construction & analysis, orthologs, Cerebral, Cytoscape, CyOOg, etc. Manual curation project & integration of publicly available databases into InnateDB greatly increases innate immunity relevant molecular interaction networks & pathways. Enable biologists without a computational background to explore their data in a more systems-oriented, yet user-friendly, manner.

7. Contextually Curating Innate Immunity-Relevant Interactions Manual curation > 10,000 innate immune-relevant interactions (human and mouse). Involving 2,700+ genes from review of 2,600+ unique publications. We can often double # of interactions for a given gene. Pathways & interactions are curated with contextual annotations (supporting publication; participant molecules; the species; the interaction detection method; the host system; the interaction type; the cell, cell-line and tissue types etc). Developed InnateDB submission system software to allow submission of interaction annotation in an ontology-controlled and MIMIx & PSI-MI 2.5 compliant manner. Developed curator tool software to allow curators modify existing annotations.

8. Going Beyond Innate Immunity – A Centralized Resource for Interactions & Pathways Aside from the well known signalling pathways  a range of other disparate processes, including apoptosis, ubiquitination, endocytosis, cell activation and recruitment  all required to mount effective innate immune response. Adding to this complexity  borders between the innate and adaptive immune responses are becoming increasingly blurred. Furthermore, if we hope to identify new networks or pathways involved in innate immunity, analyzes must include genes and proteins that are, as yet, not known to play specific roles in the innate immune response. To address these issues  InnateDB also incorporates data on the entire human and mouse interactomes.

9. Going Beyond Innate Immunity – An Integrative Biology Resource 115,000+ human and mouse interactions extracted & loaded from BIND, INTACT, DIP, BIOGRID & MINT DBs. Cross-referenced genes to >3,000 pathways from KEGG, PID, BIOCARTA, INOH, NetPath & Reactome DBs. Allows one to visualize/analyze interactions associated with specific pathway. Pathway ORA. Annotation from Ensembl provides details of human & mouse genes, transcripts and proteins. UniProt, Entrez, Gene Ontology  rich protein & gene annotation.

10. Through manual curation & integration of existing data from publicly available databases we can greatly increase innate immunity relevant networks TLR4 direct and secondary interactions annotated by InnateDB TLR4 direct and secondary interactions annotated by MINT Database

11. Direct and Secondary Interactions of TLR4 in InnateDB(~20% of these interactions unique to InnateDB)

12. www.innatedb.ca

13. InnateDB – Advanced Yet User-Friendly Searching – Find & Analyze Relevant Interactions, Pathways & Genes/Proteins. Search for interactions by: Gene, Pathway, Interaction type (phosphorylation), Detection method (GST Pull Down), Cell/Tissue type, Protein-Protein, Protein-DNA/RNA etc etc. Rich contextual annotation for each interaction. Find orthologous interactions in other species. Download in variety of formats (e.g. PSI XML) - Visualize & Analyze interactions in Cerebral - Cytoscape plugin - Uses subcellular localization annotation to generate more biologically intuitive pathway-like layouts of networks.

14. InnateDB – Facilitating Systems-Level Analyses of Gene Expression Data: Upload Your Own Gene Expression Data - Up to 10 conditions/timepoints at 1 time. Overlay Gene Expression Data from Multiple Conditions on Networks/Pathways Pathway, Gene Ontology & TF ORA tools Find – DE Pathways/Functionally Related Genes/TFs Go Beyond Pathway Analysis – Differentially Expressed Sub-networks – New Pathways? How Are DE Genes Actually Inter-connected? Central Regulators (Network Hubs)

15. InnateDB  3,000+ human & mouse pathway annotations  all processes not just immunity. Pathway Analysis – Any type of Quantitative Data. Orthologous Pathways GWA Candidate Associated Genes • InnateDB pathway analysis: • identify OR pathways. • highlight potentially unknown relationships between makers on different chromosomes.

16. Constructing & Analyzing Networks Using InnateDB Pathway analysis can be very powerful in determining which annotated pathways are most significantly associated with DE genes. Network analysis  move from simple view of the signaling response to a more comprehensive analysis of the molecular interactions between DE genes and their encoded proteins & RNAs. Potentially uncover as yet unknown signaling cascades or pathways, functionally relevant sub-networks and the central molecules, or hubs, of these networks.

17. Results: Visualize Gene Expression Data in an Interaction Network Context Click here to visualize interactions in Cerebral with overlaid gene expression data.

18. Multi-experiment View in Cerebral

19. Robust Orthology & Gene Order Predictions – Facilitating Comparative Analysis Majority of mammalian interaction data available in InnateDB and other interaction databases primarily refers to human genes and proteins. To facilitate comparative network-based analysis of the human, mouse and bovine interactomes, detailed orthology predictions have been integrated into InnateDB. Orthology predictions generated using an in-house method, Ortholuge, which provides accurate predictions of orthology using a phylogenetic distance-based approach. Orthology predictions are further supported through the development of a human and mouse gene order and synteny browser.

20. A Guide to Using InnateDB

21. InnateDB – User Friendly Interface www.innatedb.ca

23. Not sure what you want to search for?… Browse InnateDB by Interaction Type, Pathway or Various Immune Gene Lists

24. All InnateDB Interactions Can be Downloaded in Proteomics Standards Initiative (PSI) 2.5 XML Format

25. Resources Page: Details of Relevant Software, Databases, and Immune gene Lists

26. Statistics on Curated Interactions & Interactions from other Databases

27. Use contact form or send email to innatedb-mail@sfu.ca to report bugs, errors or to get involved in curation.

28. Documentation, Tutorials & Help

29. Searching InnateDB

30. Do a simple search for genes, proteins or interactions of interest on the InnateDB hompage e.g. IRAK genes.

31. Search using a wide variety of terms Construct more complex queries using Boolean operators Advanced Search for Genes & Proteins

32. Return direct interactors or their secondary interacting neighbors. Allows users search for interactions in particular cell/tissue types, particular interaction types e.g. phosphorylation, or experimental type e.g. co-immunoprecipitation. Advanced Search for Interactions InnateDB contains detailed information for more than 115,000 human and mouse molecular interactions integrated from several of the major public interaction databases along with 10,000+ manually-curated innate immunity relevant interactions. To reduce redundancy, interactions in InnateDB that have the same participants and interaction type are grouped together by default. Choose 'No' to return all redundant interactions separately.

33. Select a pathway from the list or search more than 3,000 pathways by typing the pathway name in the box. Pathways and the interactions in them are species specific. By default, a list of molecular interactions are returned which is restricted to interactions only between annotated members of the pathway. If "No" is selected, a more comprehensive list of interactions is returned, displaying interactions between pathway members and all other molecules with which they interact.. Search for Particular Interactions or Genes that are in a Specific Pathway

34. Allows users to download results to Excel file or Tab/CSV text files. Allows users to sort large set of results by available fields Display columns not shown by default. Search Results: searching for genes of interest e.g. IRAK

35. View interactions involving this gene and its encoded proteins Search Results: searching for genes of interest e.g. IRAK

36. View evidence supporting this interaction & contextual details e.g. cell type etc Interaction Results Page.

37. View evidence supporting this interaction & contextual details e.g. cell type etc There may be multiple evidence references for an interaction.

38. Click here to visualize interactions in Cerebral You must have a recent version of Java installed. Visualize Interactions in a subcellular localization-based layout using the Cerebral plugin for Cytoscape.

39. How a biologist thinks of a pathway ….

40. Pathway Visualization in Cytoscape

41. Pathway Visualization using Cerebralwww.pathogenomics.ca/cerebral (Bioinformatics 2007)

42. A Quick Guide to Using Cerebral in InnateDB • Cerebral can be used to visualize interaction networks from a set of interactions from InnateDB. • Cerebral uses subcellular localization annotations to provide more biologically intuitive pathway-like lay-outs of interaction networks. • Note: the subcellular localizations in Cerebral should only be used as a guide. There are many proteins with no annotated subcellular localizations and many others that have multiple possible localizations (only 1 will be shown, nuclear, extracellular and membrane localizations will take precedence over cytoplasm if there are multiple). • InnateDB batch searching allows users to upload a list of genes along with associated gene expression data from up to 4 different conditions. • Gene expression data can be overlaid on network data and you can visualize this in Cerebral.

43. You will be prompted to open a .jnlp file. You are recommended to save this file to your computer and then open it – this will allow you save a copy of this dataset. Opening the .jnlp file directly without saving sometimes causes Cerebral to hang when loading large datasets. Note: to use Cerebral you need to install Java version 6 or greater. You can get this from http://java.com/en/download/index.jsp Opening Interaction Data in Cerebral from an Interaction Results page in InnateDB.

44. Opening Cerebral • Cerebral is a Java plugin for the Cytoscape Visualization software. • When you open the .jnlp file Cytoscape will begin downloading. • You will then be prompted – “Do you want to run the application” – click Run.

45. Cerebral is Now Open and Displays Interactions Based on Protein Subcellular Localizations

46. Re-size the Network Click here to re-size the network display to full-screen.

47. Right click and push your mouse forward or back to zoom. Hold middle button of your mouse and drag to navigate around the network. Grey nodes do not have an annotated subcellular localization (from Gene Ontology data in InnateDB). Lines connecting nodes represent interactions. Dashed lines have only 1 supporting publication in InnateDB. The thicker the line the more publications support the interaction. Navigating in Cerebral

48. Right-click on a node (protein/gene) or edge (interaction line) to link to the relevant gene or interaction details page in InnateDB. Interactively Link back to InnateDB to Look up Information on Particular Genes/Interactions of Interest.

49. Click on & drag nodes to other layers. Click “Node Attribute Browser” and select from list to see node attributes. Nodes Can be Dragged to Other Layers as Desired.

50. Do a simple search for genes, proteins or interactions of interest on the InnateDB hompage e.g. IRAK genes.