Periodic Reporting for period 5 - ImmunAID (Immunome project consortium for AutoInflammatory Disorders)
Reporting period: 2024-05-01 to 2024-10-31
The systemic autoinflammatory diseases (SAID) are a set of rare diseases with unspecific symptoms (fevers, rash, joint pain ...). Monogenic SAID carry identified genetic mutations, which is not the case for genetically undiagnosed SAID, the diagnosis being then based on the elimination of any other cause of disease.
The underlying causes of the genetically undiagnosed SAID are poorly understood, and their diagnosis is difficult and time-consuming, often leading to misdiagnosis. ImmunAID aimed to generate a unique and comprehensive set of data based on multi-omic unbiased analysis (genomics, transcriptomics, proteomics, microbiomics), and functional tests of the innate immune system. Overall, ImmunAID aimed to disentangle the spectrum of SAID and to propose a new classification based on omics and disease pathogenesis, with ultimately a clinical decision algorithm that can be implemented in daily practice.
The underlying causes of the genetically undiagnosed SAID are poorly understood, and their diagnosis is difficult and time-consuming, often leading to misdiagnosis. ImmunAID aimed to generate a unique and comprehensive set of data based on multi-omic unbiased analysis (genomics, transcriptomics, proteomics, microbiomics), and functional tests of the innate immune system. Overall, ImmunAID aimed to disentangle the spectrum of SAID and to propose a new classification based on omics and disease pathogenesis, with ultimately a clinical decision algorithm that can be implemented in daily practice.
The recruitment of ImmunAID cohort of SAID patients was performed under the work-package (WP) 1. 390 SAID patients, 56 trios (one SAID patient and his/her 2 parents) and 73 healthy controls were recruited.
All OMIC analysis (WP2) have been run. 117 new candidate genes that could play a role in the pathogenesis were identified. Among the new candidate genes, 28 are common to several patients with SAID and 35 are new variants of known genes. Functional studies to test the pathogenicity of these candidate genes are ongoing. The miRNA profile of SAID patients and healthy controls of different cell types was analysed. Distinct miRNA were found upregulated in Adult-onset Still’s disease (AOSD) compared to all other SAIDs. Using mass spectrometry and SomaScan technologies, a comprehensive dataset highlighting differential protein expression in various SAIDs was generated, with a particular focus on AOSD and Kawasaki disease. Finally, the microbiome data allowed for the evaluation of dysbiosis across different patient populations was evaluated. One specific enterotype is increased in various SAID patient groups, compared to healthy controls.
WP3 was dedicated to studying the inflammasomes in order to identify the deregulatory mechanisms leading to their abnormally intense and prolonged activation in SAID. A diagnostic test that specifically measures the deregulation of the pyrin inflammasome in patients' blood was developed. The test makes it possible to measure a patient's response to treatment and determine whether the pyrin inflammasome is functioning normally or is deregulated. Another test based on a flow cytometry was also developed to detect protein specks specifically derived from the NLRP3 inflammasome. The test proved that these NLRP3-derived specks provide an additional level of information about inflammatory biological processes. Furthermore, the test allows the categorisation of patients with AOSD three ways; the link between each category and disease outcomes or treatment responses is still under study.
WP4 was dedicated to the exploration of the role played by inflammation resolution mediators in the autoinflammatory process. The lipidomics analyses for circulating levels of ω3 and ω6-derived Specialized Proresolving lipid Mediators (SPMs) and Lipid Mediators (LMs) indicates that SAID patients show severe dysregulation in their lipidomic profile in periphery. In addition, the role of neutrophils and monocytes in the defective metabolic pathways was investigated at the molecular level. An attempt to setup in vitro assays able to test effects from a variety of stimuli specifically in monocytes and neutrophils faced technical challenges. A whole blood assays was thus developed and proved the, so far unknown, ability of a specific drug to induce SPMs which may largely account for the drug’s potent anti-inflammatory and immune regulatory activity.
WP5 encompasses all the functional analysis of the cytokines and their effectors. Cytokines play a pivotal role in the pathogenesis of SAID by modulating inflammatory responses and immune dysregulation. Upon analysis of ImmunAID’s samples, significant variations were observed in the expression levels of several cytokines and a dysregulation of pro-inflammatory cytokine could be clearly demonstrated. The profiles in AOSD and Systemic-Onset Juvenile Idiopathic Arthritis (SOJIA) share similarities but also exhibit distinct differences. Other interesting results were found for Recurrent Pericarditis, Schnitzler syndrome, Inflammation of Unknown Origin (IUO) and Chronic Recurrent Osteitis. Plasma concentration of chemokines was also measured, displaying significant increase in patients with AOSD compared to adult healthy controls.
The profiling of alarmin expression by neutrophils in SAID patients also proved the validity of some S100 proteins as biomarkers for SAIDs. A crystal structure of Interleukin-18 (IL-18) in complex with naturally occurring IL-18 binding protein (IL-18BP; a decoy receptor for circulating IL-18), was first solved. A concomitant study showed that IL-18 can discriminate AOSD from other SAID, and free IL-18 levels may be relevant to assess response to therapy in these patients. A functional assay measuring peptidylarginine deiminase (PAD) activity was also optimized and validated, giving insight into arthritic disorders.
Another angle of the work focused on specific immune cell profiling. In particular, the exact role of natural killer (NK) cells in the pathogenesis of AOSD was investigated. The proportion of NK cells from AOSD patients is significantly decreased, displaying a unique profile while most AOSD-associated features are absent in the other evaluated SAIDs. Also, deep immunophenotyping was used to identify a distinct immunological state for IUO patients, with similarity to AOSD.
A series of data analysis and data integration strategies and algorithms have been developed and used in WP6. Both supervised and unsupervised approaches were applied, based on single data modalities at first (i.e RNAseq-based model, proteomics-based model), and subsequently on multi-omics datasets. We were able to identify signatures specific of SAID as compared with healthy volunteers (pan-SAID signature), as well as signatures and mechanisms underlying specific SAID subgroups, notably AOSD, Behçet disease and recurrent pericarditis. Signatures and biomarkers of IUO were identified as well as classification of existing SAID in novel subgroups.
Up to know, ImmunAID has generated 49 peer-reviewed scientific articles. More will be published and listed on the project website (www.immunaid.eu).
All OMIC analysis (WP2) have been run. 117 new candidate genes that could play a role in the pathogenesis were identified. Among the new candidate genes, 28 are common to several patients with SAID and 35 are new variants of known genes. Functional studies to test the pathogenicity of these candidate genes are ongoing. The miRNA profile of SAID patients and healthy controls of different cell types was analysed. Distinct miRNA were found upregulated in Adult-onset Still’s disease (AOSD) compared to all other SAIDs. Using mass spectrometry and SomaScan technologies, a comprehensive dataset highlighting differential protein expression in various SAIDs was generated, with a particular focus on AOSD and Kawasaki disease. Finally, the microbiome data allowed for the evaluation of dysbiosis across different patient populations was evaluated. One specific enterotype is increased in various SAID patient groups, compared to healthy controls.
WP3 was dedicated to studying the inflammasomes in order to identify the deregulatory mechanisms leading to their abnormally intense and prolonged activation in SAID. A diagnostic test that specifically measures the deregulation of the pyrin inflammasome in patients' blood was developed. The test makes it possible to measure a patient's response to treatment and determine whether the pyrin inflammasome is functioning normally or is deregulated. Another test based on a flow cytometry was also developed to detect protein specks specifically derived from the NLRP3 inflammasome. The test proved that these NLRP3-derived specks provide an additional level of information about inflammatory biological processes. Furthermore, the test allows the categorisation of patients with AOSD three ways; the link between each category and disease outcomes or treatment responses is still under study.
WP4 was dedicated to the exploration of the role played by inflammation resolution mediators in the autoinflammatory process. The lipidomics analyses for circulating levels of ω3 and ω6-derived Specialized Proresolving lipid Mediators (SPMs) and Lipid Mediators (LMs) indicates that SAID patients show severe dysregulation in their lipidomic profile in periphery. In addition, the role of neutrophils and monocytes in the defective metabolic pathways was investigated at the molecular level. An attempt to setup in vitro assays able to test effects from a variety of stimuli specifically in monocytes and neutrophils faced technical challenges. A whole blood assays was thus developed and proved the, so far unknown, ability of a specific drug to induce SPMs which may largely account for the drug’s potent anti-inflammatory and immune regulatory activity.
WP5 encompasses all the functional analysis of the cytokines and their effectors. Cytokines play a pivotal role in the pathogenesis of SAID by modulating inflammatory responses and immune dysregulation. Upon analysis of ImmunAID’s samples, significant variations were observed in the expression levels of several cytokines and a dysregulation of pro-inflammatory cytokine could be clearly demonstrated. The profiles in AOSD and Systemic-Onset Juvenile Idiopathic Arthritis (SOJIA) share similarities but also exhibit distinct differences. Other interesting results were found for Recurrent Pericarditis, Schnitzler syndrome, Inflammation of Unknown Origin (IUO) and Chronic Recurrent Osteitis. Plasma concentration of chemokines was also measured, displaying significant increase in patients with AOSD compared to adult healthy controls.
The profiling of alarmin expression by neutrophils in SAID patients also proved the validity of some S100 proteins as biomarkers for SAIDs. A crystal structure of Interleukin-18 (IL-18) in complex with naturally occurring IL-18 binding protein (IL-18BP; a decoy receptor for circulating IL-18), was first solved. A concomitant study showed that IL-18 can discriminate AOSD from other SAID, and free IL-18 levels may be relevant to assess response to therapy in these patients. A functional assay measuring peptidylarginine deiminase (PAD) activity was also optimized and validated, giving insight into arthritic disorders.
Another angle of the work focused on specific immune cell profiling. In particular, the exact role of natural killer (NK) cells in the pathogenesis of AOSD was investigated. The proportion of NK cells from AOSD patients is significantly decreased, displaying a unique profile while most AOSD-associated features are absent in the other evaluated SAIDs. Also, deep immunophenotyping was used to identify a distinct immunological state for IUO patients, with similarity to AOSD.
A series of data analysis and data integration strategies and algorithms have been developed and used in WP6. Both supervised and unsupervised approaches were applied, based on single data modalities at first (i.e RNAseq-based model, proteomics-based model), and subsequently on multi-omics datasets. We were able to identify signatures specific of SAID as compared with healthy volunteers (pan-SAID signature), as well as signatures and mechanisms underlying specific SAID subgroups, notably AOSD, Behçet disease and recurrent pericarditis. Signatures and biomarkers of IUO were identified as well as classification of existing SAID in novel subgroups.
Up to know, ImmunAID has generated 49 peer-reviewed scientific articles. More will be published and listed on the project website (www.immunaid.eu).
No prior study has generated such a large and comprehensive set of omics data of diverse nature for SAID. ImmunAID major competitive advantage, and extent beyond the state-of-the-art, lies in its large collection of data, from a large and heterogeneous SAID patient cohorts, with high standards for sample and data management.
The ImmunAID project results already delivered a collection of original biomarkers and signatures spanning biological areas not currently associated to disease pathogenesis. Innovative classification work (rather pathogenic pathway-based than phenotype-based) is still ongoing. In addition, the different assays developed open new avenues to better investigate pathogenesis, disease heterogeneity i.e. understanding the mechanisms underlying disease complications (prognosis/severity), and development or repositioning of targeted immunomodulating agents.
The ImmunAID project results already delivered a collection of original biomarkers and signatures spanning biological areas not currently associated to disease pathogenesis. Innovative classification work (rather pathogenic pathway-based than phenotype-based) is still ongoing. In addition, the different assays developed open new avenues to better investigate pathogenesis, disease heterogeneity i.e. understanding the mechanisms underlying disease complications (prognosis/severity), and development or repositioning of targeted immunomodulating agents.