Identification of Novel Therapeutic Molecular Targets in Inflammatory Bowel Disease by Using Genetic Databases

Plain Language Summary

In the era of translational and personalized medicine, Inflammatory Bowel disease (IBD) remains a complex disease. This complicated disease presents with a wide array of symptoms that arise from underlying pathophysiological alterations in the patient’s mucosal immune system, the intestinal microbiome, the patient’s genome, and environmental factors. While the predominant disease manifests with gastrointestinal symptoms, involvement of other organs, including but not limited to the skin, eyes, and bones that present as arthritis, uveitis, and pyoderma gangrenosum, is not uncommon. Though the last decade has identified possible genetic factors that confer susceptibility to IBD, the signaling pathways involved in these extra-intestinal manifestations still remain poorly understood. In this study, we use genetic databases to identify an exclusive set of genes that are involved in the extra-intestinal manifestations of IBD and propose these molecules as potential drug targets.

Introduction

Inflammatory Bowel Diseases, which primarily include Ulcerative Colitis and Crohn’s disease, are now increasingly recognized as a complex, multi-factorial constellation of diseases whose incidence has been increasing globally.^1,2 While the disease is predominantly described as a gastrointestinal disease, symptoms involving other organ systems are increasingly recognized.^3,4 In the past decade, studies conducted in both animals and human models have suggested that several genetic factors are involved in the pathogenesis of IBD.^5–10 Animal studies have argued for an immune mediated role for IBD. These studies have utilized techniques like gene deletion, gene mutation, chemical induction, and genetic engineering, where manipulation of several genes, specifically distinct immune regulators increase the genetic susceptibility for IBD; possibly by alteration of host defense mechanisms and modulation of the individual’s microbiome.^6,7,10 The human studies on the other hand posit that there is a multi-factorial basis, where genetic alterations in combination with environmental factors have been implicated. This data is derived from studies of inheritance patterns of Crohn’s in monozygotic twins, where a higher incidenceof Crohn’s has been noted.^7,8,11 Further, association studies of genomewide databases have postulated that more than 230 IBD loci are implicated in the pathogenesis of IBD.^12–14 However, understanding of the mechanistic basis of extra-intestinal manifestations of IBD still remains limited. Few of the signaling pathways known have been proposed to be shared with host response to various bacteria and other immune mediated disorders which comprise extra-intestinal manifestations. This study aims to identify genes involved in IBD and its’ extra-intestinal manifestations, ie, PSC, pyoderma gangrenosum, arthritis, and uveitis. We hypothesize a network of signaling cascades exist that form the etiological backbone of these diseases, their diverse manifestations, and can offer potential pharmaceutical targets.

Methods

We utilized four genetic databases, including the Genecards: The Human Gene Database (www.genecards.org), DisGeNET (https://www.disgenet.org/), The Comparative Toxicogenomics Database (http://ctdbase.org/) and the Universal Protein Resource (https://www.uniprot.org/), to identify genes implicated in UC, CD, uveitis, arthritis, PSC, and pyoderma gangrenosum. Subsequently, using these genes as possible nodes (aka the common genes involved), and the underlying common signaling cascade in these disease processes, we queried NDex, Network Data Exchange (http://www.ndexbio.org/), an open source bioinformatic platform to predict signaling networks.^15,16 Of note, these databases provide unique scores to each gene, based on information of curated databases and the likelihood of protein–protein interactions.

Results

Our initial analysis from the Genecards: The Human Gene Database, DisGeNET, The Comparative Toxicogenomics Database and the Universal Protein Resource, identified 3783 genes in UC, 3980 genes in CD, 1043 genes in uveitis, 5583 genes in arthritis, 1313 genes in PSC, and 119 genes in pyoderma gangrenosum. Of note, genes identified from these databases do not directly implicate casualty, but are a summation of altered genes in the respective disease state, from studies of animal models, tissue culture, and human databases. We then identified a unique array of 20 genes, that had the highest probability of involvement in UC, CD, uveitis, arthritis, pyoderma gangrenosum, and PSC. A signaling network or interactome (Figure 1) was then formulated using the NDex, Network Data Exchange.^15,16 Of note, this genetic array had a strong emphasis on immune modulators, further arguing for an immune basis in the extra-intestinal presentations of IBD (Table 1 and Figure 1). As expected, we noted Tumor necrosis factor (TNF) involvement in CD, UC, PSC, and arthritis. Further, C-C motif chemokine ligand 2 (CCL2), a chemokine essential for recruitment of monocytes, memory T-cells, and dendritic cells, and an important agent in Protein kinase B (AKT) signaling and Pigment epithelium derived factor (PEDF) pathways, was also involved in CD, UC, arthritis and uveitis.¹⁷ Interestingly, unique modulators were also identified as common nodes in distinctive disease processes. We noted that two immune modulators (a) Interleukin-17A (IL-17A), a proinflammatory cytokine, and (b) Interleukin-21 (IL-21), a regulator of Natural Killer (NK) cells and cytotoxic T cells, are both implicated in CD, UC, uveitis, PSC, and arthritis.^18,19 Arachidonate 5-lipoxygenase (ALOX5), an important member of the lipoxygenase gene family, was exclusively involved in UC, CD, and arthritis.²⁰ Fas Ligand (FASLG), an important player in apoptosis and death receptor, and CCR5, a known HIV receptor, were detected in UC, CD, PSC, uveitis, and arthritis, but not in pyoderma gangrenosum.^21,22 MON2, a regulator of endosome to golgi trafficking and IL-25, a mediator in IL-17 and PEDF signaling, were only noted in CD, UC, pyoderma gangrenosum, and arthritis.^23,24 In contrast, SLCO1B3, which encodes for organic anion transporter, was exclusively involved in Pyoderma gangrenosum, UC and CD.²⁵ (Table 1)

Table 1 Genes Overlapping in IBD (UC and CD) and Its Extra-Intestinal Manifestations (Uveitis, Arthritis, Primary Sclerosing Cholangitis and Skin Manifestations – Pyoderma Gangrenosum); with the Individual Gene, Specific Disease Processes, and Gene Function Listed

Figure 1 Signaling map of genes (interactome) overlapping in inflammatory bowel disease (UC and CD) and its extra-intestinal manifestations.

Discussion

IBD is a complex group of heterogeneous disorders, with an underlying multifactorial pathophysiological basis. A dysregulation between environmental, underlying human microbiome, and genetic susceptibility factors, is hypothesized to play a crucial role in varying manifestation of IBD.^{1,–2,–5–13} In our study, we have used genetic data sets to look for overlapping genes involved in IBD and its’ extra-intestinal disease forms (like arthritis, uveitis, and pyoderma gangrenosum). Our study supports the theory that immune modulators are critical mediators in extra-intestinal manifestations of IBD. These genes allude towards cross-sectional nodes, that could further implicate to involvement of different signal transduction molecule cascades in different manifestations of IBD.

We hypothesize that these overlapping molecules act as focal points of intersecting signal transduction pathways and are involved in distinctive clinical presentations of IBD and hence are potential therapeutic targets. While our in silico analysis is limited by a lack of wet-lab experiments, it highlights interesting candidate molecules and possible network pathways, which can be utilized in future more focused experimental designs. We believe our analysis consolidates existing information and lays the groundwork for future in vitro and in vivo studies, dedicated towards understanding the pathophysiology of this complex disease. In vitro tissue culture experiments looking at overexpression, underexpression, and protein–protein interactions are key in elucidating the specific function of these molecules; which in turn would offer more insights towards in vivo studies, either in animal models or towards clinical trials in patients with IBD.

Of particular interest in this regard, and noteworthy here is IL-17A, which was identified as a potential target in our analysis.^29,30 Secukinumab, an anti-IL-17A monoclonal antibody, was found to be safe and efficacious in psoriasis and rheumatoid arthritis.^29,30 However, higher adverse events were noted in patients with moderate-to-severe CD.³⁰ This highlights an inherent limitation of in silico database analyses and in vitro studies, where there is an inability to re-create the unique human intestinal immunological environment and the complex interactions of the host immune system with the gut microbes, which is perhaps critical in the pathophysiology of IBD. This could, in turn, be a limitation in direct translation of data retrieved from in silico analyses to animal model studies and then in therapeutic clinical trials.

Lastly, databases are ever changing, and comprise information from both animal and human tissues. Thus, we anticipate advancement in cell–cell networking, genomewide association studies to keep evolving over time. Though these ever-growing database repositories do offer the advantage of high throughput screening, we acknowledge that this information still has to be used in conjunction with wet lab data to advance our understanding of IBD.^31–33

Conclusion

Our work identifies a unique set of 20 genes involved in IBD and associated extra-intestinal diseases. These genes are involved in various aspects of cellular processes and signal transduction like processes of apoptosis, inflammation, and immune response. We propose that bioinformatics and system immunology is a potent tool to dissect the complex signaling networks in IBD, and further exploration of upstream and downstream effectors of these candidate genes may help in greater understanding of IBD and its extra-intestinal manifestations.