INDIGO Work Packages
WP1 Project Management , Training and Dissemination (Leader PTP SME)
The overall scientific coordination and operation of the project is the responsibility of Cardiff University, which will be assisted for administrative management by PTP together with input from several boards/committees.
Dissemination and training activities include workshops open to all members of participant organisations and EUGOGO, the first will take place in Essen in April 2015. Events open to the public or to a wider audience (schools, patients and their relatives) are also planned. The first of these took place in Newcastle in May 2014. A 2 day closing conference will present project successes and outputs in the context of latest research and clinical practice in scientific (day1, audience of scientific experts) and layman’s terms (day 2, audience of stakeholders, including patient groups, policy makers and funders).
WP2 In vitro models (Leader CULTECH SME)
WP2 will optimise and extend in vitro models using cell lines for human or mouse gut epithelium and monocytes. The human models will compare how the gut microbiota from GD patients and healthy controls affects cytokines produced and whether a probiotic can modulate the quality and quantity of this response. The mouse systems will identify GD/GO gut organisms producing inflammatory cytokines expected to exacerbate disease (“contrabiotics”).
WP3 Patient Studies (Leader UMIL University)
Samples of serum, nasal swabs, tears and faeces will be collected to identify biomarkers and the role of the microbiome in human (and murine) disease. Lifestyle data (questionnaires for completion by all recruited patients and controls) will identify links between dietary, nutritional or microbial factors with immune responses. A trial will assess the impact of a probiotic on the microbiome of GD patients undergoing standard treatment for the disease.
WP4 In vivo mouse model (Leader UKESSEN University)
Improvements to an in vivo GD/GO model will be attempted by manipulating the mouse gut microbiome to alter the Treg and Th17 balance. This will be done by colonizing with micro-organisms (‘contrabiotics’) from GD/GO patients or from mice induced to exhibit GO following immunization with the thyrotropin receptor (TSHR) or segmented filamentous bacteria implicated in autoimmunity.
WP5 Probiotics and Contrabiotics (Leader Cultech SME)
This WP will produce the probiotic (and placebo) for the trial in WP3. It will also generate the cultures of human and mouse micro-organisms from individuals with disease (‘contrabiotics’) and healthy controls for use in the in vitro models of WP 2 and in vivo model of WP 4.
WP6 Proteomic and Genomic Analysis (Leader PTP SME)
Samples from controls, GD and GO patients will be analysed using high through-put proteomics techniques to search for protein patterns diagnostic of GD and GO. miRNA will be analysed from parallel samples to identify and quantify species present and seek correlations with disease states. Protein and miRNA profiles from patients and in vivo mouse models will be correlated with microbiome analyses to investigate the association between the microbiome-gut interaction and GO autoimmune responses. Proteomic analysis of supernatants from in vitro cell cultures exposed to different micro-organisms will reveal variations in cellular responses.
WP7 Immune response (Leader PTP SME)
WP7 will analyse GD & GO patient antibody responses to environmentally derived antigens to identify risk factors associated with the gut-microbiome or exogenous antigens expressed on common micro-organisms or in foods. Proteins from micro-organisms or food stuffs displaying an antibody response in either GO or GD will be characterised by LC-MS/MS to identify the proteins involved.
WP8 Microbiome (Leader CU University)
The microbiomes of GD/GO patients versus controls and from mouse models will be investigated by analysing the highly variable regions of bacterial 16S ribosomal RNA genes from human/murine faecal samples. A library of 16S RNA sequences of micro-organisms will be created for mice using published data and for humans using information from the human microbiome project. The V1-V3 region NGS data will be analysed with a bioinformatic pipeline to determine species present and identify statistically significant variations between the microbiomes for the mouse models and among human controls, GD and GO patients. The same approach will be used to investigate the impact of probiotics on the human microbiome and ‘contrabiotics’ on the mouse microbiome by defining changes that occur in micro-organism numbers and strains during administration and following withdrawal.
WP9 Consolidation (Leader CU)
WP9 will consolidate WP outputs, analyse correlations and define results to be carried forward in future trials or research. The 7 RTD WPs in the project use different and complementary approaches to obtain samples and produce data. WP9 will examine which of the various outputs can be combined to confirm or strengthen individual findings.
They will also undertake statistical post-analysis and modelling to explore the molecular and physiological factors that impact on disease. Models will be evaluated by simulation to identify key interactions associated with GD and progression to GO. The best models will be tested using data from a larger group of patients provided by EUGOGO.