Inflammatory bowel disease (IBD) is a debilitating illness associated with the altered regulation of the gastrointestinal mucosal immune system leading to intestinal inflammation in the presence of native luminal flora. Nearly 1.5 million people in the United States are affected by IBD and the incidence and prevalence of the disease continues to increase internationally.1, 2 IBD is comprised of two distinct phenotypes; ulcerative colitis (UC) and Crohn's disease (CD), each of which have unique clinical manifestations despite sharing many genetic and consequently mechanistic features.3–5 Although the exact pathophysiology of IBD is not yet fully understood, the etiology of this disease is known to be multifactorial, driven by a number of genetic and environmental factors including loss of regulation of the host's innate immune response and defects in mucosal barrier function.6
There appears to be a correlation between IBD and autoimmune disorders. Over a quarter of IBD patients have been known to experience musculoskeletal complications resulting from various subtypes of arthritis.7, 8 Moreover, microRNA (miRNA) expression changes have high similarity in autoimmune disorders, including IBD.9 The complex host–pathogen interactions within the colon are also known to play a vital role in the pathogenesis of IBD and drive differential gene expression. Recent studies of the intestinal microbiome have shown a shift in the composition of bacterial populations in patients with UC and CD and the involvement of bacterial–host interactions in the pathogenesis of IBD.10–17 However, it still remains unclear how resident bacteria interact with the host to activate and sustain chronic activation of the intestinal immune system through genetic regulation.
A system approach has already emerged in the literature for studying IBD within the context of autoimmune disorders. A comprehensive first draft of the human disease network (HDN) was constructed recently using the disease gene information in the literature.18 This and other system models19–24 identified disease–disease associations not readily observed in the disease–phenotype-based Medical Subject Headings (MeSH) disease classification tree. Some of these models utilized text search-based data whereas others based the draft network on gene lists identified via the literature and genome-wide association studies (GWAS).25, 26 A number of other disease network drafts utilized protein–protein interaction networks in addition to gene lists to identify gene–disease modules.27, 28 In all cases, however, the starting point is a set of gene lists specific for each disease. Hu and Agarwal29 obtained such lists utilizing high-throughput microarray analysis of thousands of genomic expression profiles existent in the literature. Their draft of the human disease–drug network brought out new possibilities for further investigation such as the use of malaria drugs in treating CD and also showed the potential of known fold changes in transcript copy numbers in guiding the type of drugs (agonists, antagonists) to be targeting druggable proteins and pathways.
The present study benefits from the previous work on the HDN and studies of global gene and miRNA expression profiling of IBD. It presents gene signature analysis of IBD subtypes UC and CD in terms of altered pathways, biological processes, disease correlations, transcripts, and possible drug targets, all determined via bioinformatics analysis of integrated microarray and miRNA datasets with open-access datasets. Gene lists derived from microarray data have the advantage of having features such as statistical significance and fold changes to be used for additional annotation. However, previous disease and drug network modeling studies suggest potentially high levels of noise in predictions, the extent of which depends on the accuracy of gene lists used to represent the specific diseases.29 In that regard, our previous meta- and large-scale microarray analyses of cancer tissue datasets from hundreds of different laboratories point to accurate replication of nonmicroarray literature with microarray data.30 Our gene lists derived from integrated datasets intersected with the shorter gene lists produced by Noble et al31, 32 with highly significant P values in a hypergeometric test. Moreover, cellular pathways statistically enriched by Noble et al gene lists turned out to be a subset of our predicted pathways. Our predictions of IBD-associated genes captured nonmicroarray IBD research literature with significant P values. Taken together, these results illustrate the predictive potential of microarray datasets in identifying genes associated with CD and UC subtypes of IBD. The resulting gene lists come with statistical significance, rank, and fold changes, parameters that are invaluable in biomarker discovery and drug repositioning and have been included in the Supporting files.
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- MATERIALS AND METHODS
- Supporting Information
IBD is a complex disorder involving commensal bacteria, multiple human cell types, and a variety of cellular networks including those related to adaptive and innate immunity. Our integrated microarray analysis of inflamed human colonic mucosa captures the widespread genetic perturbations of IBD phenotypes as associated with a variety of cell types, pathways, and biological processes, a system-level perspective of transcriptional regulation attributable to the heterogeneous nature of colonic biopsy samples. By combining raw microarray data from six independent studies, we were able to increase the statistical power of our analysis while minimizing laboratory-specific bias and experimental noise. A total of 118 unique patient samples were used including 48 UC, 41 CD, and 29 HCs. Differentially expressed miRNA obtained through literature curation were also incorporated into our analysis in order to investigate possible mechanisms of posttranscriptional regulation through validated interactions with differentially expressed genes related to IBD.63 Bioactive therapeutic small molecules targeting differentially expressed genes as well as gene involvement in other autoimmune diseases were also investigated to facilitate the identification of druggable pathways and therapeutic targets that may be candidates for a wide variety of related illnesses or existing therapeutics that may be repositioned for the treatment of IBD.
Our analysis of microarray data showed as much as a 40-fold increase in transcript number for some of the genes in IBD relative to HCs. The most upregulated gene in IBD is REG1A, coding a protein typically secreted by the exocrine pancreas and associated with islet cell regeneration. A survey of the literature shows upregulated REG1A as associated with pancreatic cancer, celiac disease, and Type I diabetes.64–66 The top upregulated genes for IBD in our list were abundant with CXC chemokines involved in inflammation, and were previously linked to such diseases as lupus, prostate cancer, and rheumatoid arthritis.68–70 The MMP family proteins are also abundant among the top 40 most highly upregulated genes in IBD. MMP proteins were previously shown to be involved in arthritis and metastasis.88 The third most highly upregulated gene in our list, S100A8, codes a soluble IBD biomarker protein calprotectin.71–74 High levels of expression of this protein is also linked to cystic fibrosis.75 Commonly upregulated pathways in IBD included the previously recognized Toll-like and NOD-like receptor signaling pathways, cytokine-cytokine interaction, and chemokine signaling. Cellular processes activated by IBD include immune defense response, cellular response to wounding, leukocyte activation, and regulations of immune response and cell proliferation as well as response to molecules of bacterial origin. Some of the pathways activated in IBD appear to be druggable, such as the calcium signaling pathway, endocytosis, cytokine–cytokine receptor interaction, and leukocyte transendothelial migration pathways. In our gene signatures, the genes UGT1A, HSD11B2, HSD17B2, HSD3B2, and LCMT1 are all significantly downregulated and are known to play a role in androgen and estrogen metabolism. The specific role the sex hormones play in the pathology of IBD is yet to be explored extensively in the literature.89, 90 All these pathways contain at least eight drug targets each, which are also significant genes in our HC to IBD comparison.
KEGG disease pathways enriched by IBD significantly upregulated genes included those with an autoimmune component, namely, autoimmune thyroid disease, asthma, primary immunodeficiency, systemic lupus, viral myocardidatis, Type I diabetes mellitus, autograft rejection, and graft-vs.-host disease. Patients with IBD were previously associated with a higher prevalence of rheumatoid arthritis, lupus, and hypothyroidism, with increased prevalence of asthma, eczema, allergic rhinitis, and diabetes.91 Moreover, the literature also contains links with already recognized IBD-associated diseases (cancer and malaria) and the diseases statistically enriched by our IBD genes cited above. A polymorphism in FCGR2B, an IBD significant gene, is associated with protection against malaria but susceptibility to systemic lupus erythematosus.92 Histone deacetylase inhibitors are currently being harnessed as a potential treatment for malaria, systemic lupus erythematosus, a wide range of neurodegenerative conditions, and asthma.93 In addition, the beneficial effects of malaria drugs have been recognized in the management of systemic lupus erythematosus and rheumatoid arthritis.94 Add to these other examples listed in the Results section, it appears that many of the top upregulated genes in IBD are also upregulated in autoimmune disorders. For this reason, we investigated the upregulated genes shared by IBD and other autoimmune diseases as possible drug targets by intersecting our gene list with the genes associated with specific diseases in the GAD.26 The genes known as drug targets and upregulated in multiple autoimmune diseases provide clues for the multidimensional nature of IBD. For example, the ATP binding transporter gene ABCC1—significantly upregulated in a number of diseases including, IBD, and rheumatoid arthritis, and is known to bind to HIV Tat protein—is targeted by drugs/supplements such as abacavir, tenovir, nevirapin, and quercetin, all with antiviral effects, as well as by the anticancer supplement apigenin and the transplant rejection medicine cyclosporine. Other significantly upregulated genes shared by rheumatoid arthritis and IBD include TLR8 (a target of the immune response modifier amiquimod); MMP3 (a target of failed drug marimastat); ICAM-1 (targeted indirectly by cholesterol-lowering lovastatin); IL-8 (targeted by tolmetin and ibuprofen); and IL2RA (targeted by ascomysin and budesonide, used for treating CD and asthma). The intersection of IBD significant genes with known drug targets provides valuable information for possible repositioning of existing drugs in the treatment and management of IBD.
The data on significantly altered miRNA expression in IBD, when integrated with the literature and our significant gene lists, indicate a strong role for miRNA in post transcriptional regulation gene expression related to IBD. Increased expression of miR-16 has been shown in the literature to downregulate CNTN3, C1QTNF3, ACVR2A, and CYCS, while Let-7f-1 has been shown to downregulate CNTN3, ANPEP, NAAA, PDCD, UGDH, HSDL2, and ACVR2A. These two miRNAs are highly expressed in UC and the genes shown are highly downregulated. Similarly, increased expression of mir-23b is linked to downregulation of PRAP1 in both CD and UC and increased miR-21 is associated with downregulated CTN3, PDK4, and PDCD genes in CD. The aforementioned proteins play roles in a multitude of events ranging from mitochondrion electron transport (CYCS), antiautoimmune processes (PDCD), regulation of glucose metabolism (PDK4), small intestinal microvillar events (ANPEP), neurite outgrowth-promoting activity (CNTN3), signaling through serine kinases (ACVR2A), and induction of glycosylation (UGDH). These results suggest the restorative potential of miRNA-based therapies on IBD-specific diminished biological processes. The results of our study can be put in context with mouse studies, emphasizing the effect of B lymphocytes on immunity and metabolism in the gut. A recent study in mice95 showed that the lack of B-cell immunity was associated with upregulation of genes involved in other aspects of immune defense, inflammatory, and interferon-inducible responses. The gene list provided in supplemental table 1 of Shulzhenko et al95 contains 14 significant genes from our CD/HC and UC/HC comparisons. Among these genes, DUOX2 and DUOXA2, involved in synthesis of hydrogen peroxide, are highly upregulated in CD. The genes significantly upregulated in both UC and CD with fold changes greater than 2.5 include CFI, which codes complement factor I. This protein is involved in the destruction of foreign invaders (such as bacteria and viruses), triggers inflammation, and removes debris from cells and tissues. Also upregulated in protein-deficient mouse and human IBD are interferon-induced proteins IFIT3, which potentiates antiviral signaling, and IFITM1, with antiproliferative effects; and chemokine CXCL9, thought to be involved in T-cell trafficking. The genes downregulated in B-cell deficient mice and in CD and UC consist of CYP27A1 in the synthesis of cholesterol, steroids, and other lipids, and EDN3, essential for generation of enteric neurons and was previously linked to Hirschsprung disease and Waardenberg syndrome. NR1H4 is a gene significantly downregulated both in B-cell-deficient mouse and in UC. It encodes a ligand-activated transcription factor, involved in bile acid synthesis and transport. These observations suggest the role of ineffective B-cell-mediated immunity in IBD, possibly causing activation of innate immunity and a reduction in the ability to process lipids.
In conclusion, this study presents a portrayal of the molecular rewiring of the human gut mucosa altered by IBD. Our statistical analysis reveals cellular processes altered in IBD, with a focus on biomarker selection, and possible repositioning of currently available drugs for the treatment of IBD. Our results show gene signature commonalities between IBD and asthma, lupus, and rheumatoid arthritis, among others. The results raise the question of compromised B-cell immunity in IBD patients along with overcompensation of other components of the immune system, possibly facilitating research on new treatment protocols for this chronic disorder.