A temporal classifier predicts histopathology state and parses acute-chronic phasing in inflammatory bowel disease patients.

Previous studies have conducted time course characterization of murine colitis models through transcriptional profiling of differential expression. We characterize the transcriptional landscape of acute and chronic models of dextran sodium sulfate (DSS) and adoptive transfer (AT) colitis to derive temporal gene expression and splicing signatures in blood and colonic tissue in order to capture dynamics of colitis remission and relapse. We identify sub networks of patient-derived causal networks that are enriched in these temporal signatures to distinguish acute and chronic disease components within the broader molecular landscape of IBD. The interaction between the DSS phenotype and chronological time-point naturally defines parsimonious temporal gene expression and splicing signatures associated with acute and chronic phases disease (as opposed to ordinary time-specific differential expression/splicing). We show these expression and splicing signatures are largely orthogonal, i.e. affect different genetic bodies, and that using machine learning, signatures are predictive of histopathological measures from both blood and intestinal data in murine colitis models as well as an independent cohort of IBD patients. Through access to longitudinal multi-scale profiling from disease tissue in IBD patient cohorts, we can apply this machine learning pipeline to generation of direct patient temporal multimodal regulatory signatures for prediction of histopathological outcomes.

Deep Analysis of the Peripheral Immune System in IBD Reveals New Insight in Disease Subtyping and Response to Monotherapy or Combination Therapy.

BACKGROUND: Inflammatory bowel disease (IBD) is a complex disease with variable presentation, progression, and response to therapies. Current disease classification is based on subjective clinical phenotypes. The peripheral blood immunophenome can reflect local inflammation, and thus we measured 39 circulating immune cell types in a large cohort of IBD and control subjects and performed immunotype:phenotype associations. METHODS: We performed fluorescence-activated cell sorting or CyTOF analysis on blood from 728 Crohn's disease, 464 ulcerative colitis, and 334 non-IBD patients, with available demographics, endoscopic and clinical examinations and medication use. RESULTS: We observed few immune cell types commonly affected in IBD (lowered natural killer cells, B cells, and CD45RA- CD8 T cells). Generally, the immunophenome was distinct between ulcerative colitis and Crohn's disease. Within disease subtype, there were further distinctions, with specific immune cell types associating with disease duration, behavior, and location. Thiopurine monotherapy altered abundance of many cell types, often in the same direction as disease association, while anti-tumor necrosis factor (anti-TNF) monotherapy demonstrated an opposing pattern. Concomitant use of an anti-TNF and thiopurine was not synergistic, but rather was additive. For example, thiopurine monotherapy use alone or in combination with anti-TNF was associated with a dramatic reduction in major subclasses of B cells. CONCLUSIONS: We present a peripheral map of immune cell changes in IBD related to disease entity and therapies as a resource for hypothesis generation. We propose the changes in B cell subsets could affect antibody formation and potentially explain the mechanism behind the superiority of combination therapy through the impact of thiopurines on pharmacokinetics of anti-TNFs.

High-Throughput Identification of the Plasma Proteomic Signature of Inflammatory Bowel Disease.

BACKGROUND: The molecular aetiology of inflammatory bowel disease [IBD] and its two subtypes, ulcerative colitis [UC] and Crohn's disease [CD], have been carefully investigated at genome and transcriptome levels. Recent advances in high-throughput proteome quantification has enabled comprehensive large-scale plasma proteomics studies of IBD. METHODS: The study used two cohorts: [1] The CERTIFI-cohort: 42 samples from the CERTIFI trial of anti-TNFα-refractory CD patients; [2] the PROgECT-UNITI-HCs cohort: 46 UC samples of the PROgECT study, 84 CD samples of the UNITI I and UNITI II studies, and 72 healthy controls recruited in Mount Sinai Hospital, New York, USA. The plasma proteome for these two cohorts was quantified using high-throughput platforms. RESULTS: For the PROgECT-UNITI-HCs cohort, we measured a total of 1310 proteins. Of these, 493 proteins showed different plasma levels in IBD patients to the plasma levels in controls at 10% false discovery rate [FDR], among which 11 proteins had a fold change greater than 2. The proteins upregulated in IBD were associated with immunity functionality, whereas the proteins downregulated in IBD were associated with nutrition and metabolism. The proteomic profiles were very similar between UC and CD. In the CERTIFI cohort, 1014 proteins were measured, and it was found that the plasma protein level had little correlation with the blood or intestine transcriptomes. CONCLUSIONS: We report the largest proteomics study to date on IBD and controls. A large proportion of plasma proteins are altered in IBD, which provides insights into the disease aetiology and indicates a potential for biomarker discovery.

B Cell Defects Observed in Nod2 Knockout Mice Are a Consequence of a Dock2 Mutation Frequently Found in Inbred Strains.

Phenotypic differences among substrains of laboratory mice due to spontaneous mutations or pre-existing genetic variation confound the interpretation of targeted mutagenesis experiments and contribute to challenges with reproducibility across institutions. Notably, C57BL/6 Hsd mice and gene-targeted mice that have been backcrossed to this substrain have been reported to harbor a duplication in exons 28 and 29 of Dock2 In this study, we demonstrate the presence of this Dock2 variant in the widely used Nod2-/- mice. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a cytosolic innate immune receptor associated with inflammatory bowel disease susceptibility. Consistent with a role of NOD2 in an immunological disorder, Nod2-/- mice bred at our institution displayed multiple B cell defects including deficiencies in recirculating B cells, marginal zone B cells, and B1a cells in vivo, as well as defects in class switch recombination in vitro. However, we found that these effects are due to the Dock2 variant and are independent of Nod2 deletion. Despite originating from the same gene-targeted founder mice, Nod2-/- mice from another source did not harbor the Dock2 variant or B cell defects. Finally, we show that Dock2-/- mice display the same B cell defects as mice harboring the Dock2 variant, confirming that the variant is a loss-of-function mutation and is sufficient to explain the alterations to the B cell compartment observed in Nod2-/- mice. Our findings highlight the effects of confounding mutations from widely used inbred strains on gene-targeted mice and reveal new functions of DOCK2 in B cells.

A functional genomics predictive network model identifies regulators of inflammatory bowel disease.

A major challenge in inflammatory bowel disease (IBD) is the integration of diverse IBD data sets to construct predictive models of IBD. We present a predictive model of the immune component of IBD that informs causal relationships among loci previously linked to IBD through genome-wide association studies (GWAS) using functional and regulatory annotations that relate to the cells, tissues, and pathophysiology of IBD. Our model consists of individual networks constructed using molecular data generated from intestinal samples isolated from three populations of patients with IBD at different stages of disease. We performed key driver analysis to identify genes predicted to modulate network regulatory states associated with IBD, prioritizing and prospectively validating 12 of the top key drivers experimentally. This validated key driver set not only introduces new regulators of processes central to IBD but also provides the integrated circuits of genetic, molecular, and clinical traits that can be directly queried to interrogate and refine the regulatory framework defining IBD.

High-Throughput Characterization of Blood Serum Proteomics of IBD Patients with Respect to Aging and Genetic Factors.

To date, no large scale, systematic description of the blood serum proteome has been performed in inflammatory bowel disease (IBD) patients. By using microarray technology, a more complete description of the blood proteome of IBD patients is feasible. It may help to achieve a better understanding of the disease. We analyzed blood serum profiles of 1128 proteins in IBD patients of European descent (84 Crohn's Disease (CD) subjects and 88 Ulcerative Colitis (UC) subjects) as well as 15 healthy control subjects, and linked protein variability to patient age (all cohorts) and genetic components (genotype data generated from CD patients). We discovered new, previously unreported aging-associated proteomic traits (such as serum Albumin level), confirmed previously reported results from different tissues (i.e., upregulation of APOE with aging), and found loss of regulation of MMP7 in CD patients. In carrying out a genome wide genotype-protein association study (proteomic Quantitative Trait Loci, pQTL) within the CD patients, we identified 41 distinct proteomic traits influenced by cis pQTLs (underlying SNPs are referred to as pSNPs). Significant overlaps between pQTLs and cis eQTLs corresponding to the same gene were observed and in some cases the QTL were related to inflammatory disease susceptibility. Importantly, we discovered that serum protein levels of MST1 (Macrophage Stimulating 1) were regulated by SNP rs3197999 (p = 5.96E-10, FDR<5%), an accepted GWAS locus for IBD. Filling the knowledge gap of molecular mechanisms between GWAS hits and disease susceptibility requires systematically dissecting the impact of the locus at the cell, mRNA expression, and protein levels. The technology and analysis tools that are now available for large-scale molecular studies can elucidate how alterations in the proteome driven by genetic polymorphisms cause or provide protection against disease. Herein, we demonstrated this directly by integrating proteomic and pQTLs with existing GWAS, mRNA expression, and eQTL datasets to provide insights into the biological processes underlying IBD and pinpoint causal genetic variants along with their downstream molecular consequences.

Intranasal delivery of a protein subunit vaccine using a Tobacco Mosaic Virus platform protects against pneumonic plague.

Yersinia pestis, one of history's deadliest pathogens, has killed millions over the course of human history. It has attributes that make it an ideal choice to produce mass casualties and is a prime candidate for use as a biological weapon. When aerosolized, Y. pestis causes pneumonic plague, a pneumonia that is 100% lethal if not promptly treated with effective antibiotics. Currently, there is no FDA approved plague vaccine. The current lead vaccine candidate, a parenterally administered protein subunit vaccine comprised of the Y. pestis virulence factors, F1 and LcrV, demonstrated variable levels of protection in primate pneumonic plague models. As the most likely mode of exposure in biological attack with Y. pestis is by aerosol, this raises a question of whether this parenteral vaccine will adequately protect humans against pneumonic plague. In the present study we evaluated two distinct mucosal delivery platforms for the intranasal (IN) administration of LcrV and F1 vaccine proteins, a live bacterial vector, Lactobacillus plantarum, and a Tobacco Mosaic Virus (TMV) based delivery platform. IN administration of L. plantarum expressing LcrV, or TMV-conjugated to LcrV and F1 (TMV-LcrV+TMV-F1) resulted in the similar induction of high titers of IgG antibodies and evidence of proinflammatory cytokine secretion. However, only the TMV-conjugate delivery platform protected against subsequent lethal challenge with Y. pestis. TMV-LcrV+TMV-F1 co-vaccinated mice had no discernable morbidity and no mortality, while mice vaccinated with L. plantarum expressing LcrV or rLcrV+rF1 without TMV succumbed to infection or were only partially protected. Thus, TMV is a suitable mucosal delivery platform for an F1-LcrV subunit vaccine that induces complete protection against pneumonic infection with a lethal dose of Y. pestis in mice.

Blood and Intestine eQTLs from an Anti-TNF-Resistant Crohn's Disease Cohort Inform IBD Genetic Association Loci.

OBJECTIVES: Genome-wide association studies (GWAS) have identified loci reproducibly associated with inflammatory bowel disease (IBD) and other immune-mediated diseases; however, the molecular mechanisms underlying most of genetic susceptibility remain undefined. Expressional quantitative trait loci (eQTL) of disease-relevant tissue can be employed in order to elucidate the genes and pathways affected by disease-specific genetic variance. METHODS: In this study, we derived eQTLs for human whole blood and intestine tissues of anti-tumor necrosis factor-resistant Crohn's disease (CD) patients. We interpreted these eQTLs in the context of published IBD GWAS hits to inform on the disease process. RESULTS: At 10% false discovery rate, we discovered that 5,174 genes in blood and 2,063 genes in the intestine were controlled by a nearby single-nucleotide polymorphism (SNP) (i.e., cis-eQTL), among which 1,360 were shared between the two tissues. A large fraction of the identified eQTLs were supported by the regulomeDB database, showing that the eQTLs reside in regulatory elements (odds ratio; OR=3.44 and 3.24 for blood and intestine eQTLs, respectively) as opposed to protein-coding regions. Published IBD GWAS hits as a whole were enriched for blood and intestine eQTLs (OR=2.88 and 2.05; and P value=2.51E-9 and 0.013, respectively), thereby linking genetic susceptibility to control of gene expression in these tissues. Through a systematic search, we used eQTL data to inform 109 out of 372 IBD GWAS SNPs documented in National Human Genome Research Institute catalog, and we categorized the genes influenced by eQTLs according to their functions. Many of these genes have experimentally validated roles in specific cell types contributing to intestinal inflammation. CONCLUSIONS: The blood and intestine eQTLs described in this study represent a powerful tool to link GWAS loci to a regulatory function and thus elucidate the mechanisms underlying the genetic loci associated with IBD and related conditions. Overall, our eQTL discovery approach empirically identifies the disease-associated variants including their impact on the direction and extent of expression changes in the context of disease-relevant cellular pathways in order to infer the functional outcome of this aspect of genetic susceptibility.

Variants in TRIM22 That Affect NOD2 Signaling Are Associated With Very-Early-Onset Inflammatory Bowel Disease.

BACKGROUND & AIMS: Severe forms of inflammatory bowel disease (IBD) that develop in very young children can be caused by variants in a single gene. We performed whole-exome sequence (WES) analysis to identify genetic factors that might cause granulomatous colitis and severe perianal disease, with recurrent bacterial and viral infections, in an infant of consanguineous parents. METHODS: We performed targeted WES analysis of DNA collected from the patient and her parents. We validated our findings by a similar analysis of DNA from 150 patients with very-early-onset IBD not associated with known genetic factors analyzed in Toronto, Oxford, and Munich. We compared gene expression signatures in inflamed vs noninflamed intestinal and rectal tissues collected from patients with treatment-resistant Crohn's disease who participated in a trial of ustekinumab. We performed functional studies of identified variants in primary cells from patients and cell culture. RESULTS: We identified a homozygous variant in the tripartite motif containing 22 gene (TRIM22) of the patient, as well as in 2 patients with a disease similar phenotype. Functional studies showed that the variant disrupted the ability of TRIM22 to regulate nucleotide binding oligomerization domain containing 2 (NOD2)-dependent activation of interferon-beta signaling and nuclear factor-κB. Computational studies demonstrated a correlation between the TRIM22-NOD2 network and signaling pathways and genetic factors associated very early onset and adult-onset IBD. TRIM22 is also associated with antiviral and mycobacterial effectors and markers of inflammation, such as fecal calprotectin, C-reactive protein, and Crohn's disease activity index scores. CONCLUSIONS: In WES and targeted exome sequence analyses of an infant with severe IBD characterized by granulomatous colitis and severe perianal disease, we identified a homozygous variant of TRIM22 that affects the ability of its product to regulate NOD2. Combined computational and functional studies showed that the TRIM22-NOD2 network regulates antiviral and antibacterial signaling pathways that contribute to inflammation. Further study of this network could lead to new disease markers and therapeutic targets for patients with very early and adult-onset IBD.

Unifying immunology with informatics and multiscale biology.

The immune system is a highly complex and dynamic system. Historically, the most common scientific and clinical practice has been to evaluate its individual components. This kind of approach cannot always expose the interconnecting pathways that control immune-system responses and does not reveal how the immune system works across multiple biological systems and scales. High-throughput technologies can be used to measure thousands of parameters of the immune system at a genome-wide scale. These system-wide surveys yield massive amounts of quantitative data that provide a means to monitor and probe immune-system function. New integrative analyses can help synthesize and transform these data into valuable biological insight. Here we review some of the computational analysis tools for high-dimensional data and how they can be applied to immunology.