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What is IBD?
Inflammatory bowel disease (IBD) is a non-specific chronic inflammatory disease, mainly including Crohn's disease (CD) and ulcerative colitis (UC), which is caused by long-term uncontrolled intestinal mucosal inflammation. CD can occur in any part of the intestine, and the most common sites are in the terminal ileum and the area around the anus. In contrast, the inflammatory lesions of UC only occur in the mucosa and submucosa, with rectal-centered diffusion and development, often involving the surrounding area.
Figure 1. Pathological diagram of CD and UC, CD (left), UC (right)
The clinical manifestations of IBD patients are long-term abdominal pain, diarrhea, gastrointestinal bleeding and malabsorption, and often accompanied by some extraintestinal complications, which have a serious impact on the lives of patients[1]. Moreover, the occurrence of some diseases such as colorectal cancer is also closely related to IBD[2].
Figure 2. Abdominal pain caused by IBD
IBD is more common in high-income countries in the West, but epidemiological studies over the past two decades have reported a shift, indicating that the incidence of IBD has reached a plateau in high-income countries[3]. At the beginning of the 21st century, IBD has become a global disease, and its prevalence is increasing in newly industrialized countries in Asia, South America and the Middle East[4]. In China, the incidence of IBD has also been increasing in recent years. From 1990 to 2019, the incidence of IBD increased from 1.45 per 100,000 people to 3.62 per 100,000 people, with an overall increase of 14.97%[5]. Due to the large population base and serious aging problems, the burden of IBD in China is expected to continue to grow in the next 25 years[6].
So far, the exact cause of IBD is not very clear. A series of studies have shown that individual genetic factors, intestinal microorganisms, external environment, intestinal mucosal barrier function, oxidative stress, autophagy dysfunction and adaptive immune response are all involved in the pathogenesis of IBD[7]. At present, the common clinical drugs for relieving diseases include salicylic acid, glucocorticoids, and immunosuppressive agents. However, long-term or overdose use of these drugs will cause more adverse reactions and consume a lot of time and money, resulting in many patients unable to maintain treatment[8]. In a word, it is very important to develop a new convenient, targeted and less side effects treatment.
Reactive oxygen species affect IBD progression
Reactive oxygen species (ROS) include highly active free radicals (such as superoxide anions) and non-free radicals (such as hydrogen peroxide)[9]. Although the pathogenesis of IBD is complex and poorly understood, many studies have pointed out that ROS plays an important role in the progression of IBD[10,11]. Proinflammatory cytokines can activate immune cells and amplify inflammation and generate ROS when IBD occurs. At the same time, excessive ROS production will further activate the inflammatory/immune response through the NF-κB signaling pathway, resulting in increased expression and secretion of pro-inflammatory cytokines. High ROS exposure can lead to mitochondrial oxidative damage, thereby inducing apoptosis of intestinal epithelial cells [12]. Therefore, the removal of ROS in the intestine is necessary to prevent the progression of IBD.
Figure 3 . The role of ROS in inflammation
Unfortunately, several non-enzymatic and enzymatic antioxidants are currently ineffective and can cause some adverse immune responses [13,14]. Superoxide dismutase (SOD) and catalase (CAT) are two kinds of antioxidant enzymes that are ubiquitous in various organisms. They can scavenge superoxide anions and hydrogen peroxide, and are key enzymes in the biological defense system [15,16]. This year, we chose to transform plasmids containing SOD and CAT genes into probiotics, and overexpress these two enzymes to reduce the concentration of ROS in the inflammatory site, thereby alleviating inflammatory bowel disease.
Elafin's therapeutic potential
Elafin is an endogenous human protein that inhibits neutrophil-derived serine proteases elastase (NE) and protease-3 (PR3) through a competitively tight binding mechanism[17,18].
Figure 4 . Elafin protein spatial structure map, derived from https://en.wikipedia.org/wiki/Elafin
Neutrophil activation in inflammation stimulates elastase and protease-3 to be transferred to the phagosome or exported to the plasma membrane and released into the extracellular matrix. Elastase and protease-3 play an important role in the initiation and maintenance of inflammatory response, including degradation of extracellular matrix proteins, phagocytosis of bacterial proteins, induction of proinflammatory cytokines and proteolytic activity. Therefore, inhibition of elastase and protease-3 by Elafin can attenuate several key processes in the inflammatory cascade[19].
Figure 5 . Elafin effect diagram
Moreover, Elafin is also a protective factor of IBD. The possible mechanism of its protective effect is to exert anti-inflammatory effects by inhibiting neutrophil elastase, reducing tissue proteolysis, protecting intestinal mucosal barrier function, regulating the release of inflammatory cytokines, and reducing inflammatory cell infiltration. It plays a role in regulating the intestinal innate immune disorder of IBD by inhibiting the activity of pro-inflammatory transcription factor activator protein 1 and NF-κB. Through its inherent disulfide bonds, cationic charge and so on, it can kill a variety of microorganisms including Gram-positive and Gram-negative bacteria, fungi and some viruses, and play a regulatory role in the intestinal microecological imbalance of IBD [20].
In view of the above, Elafin may develop into a new and natural therapeutic drug for IBD in the future. Therefore, we chose to transform the plasmid containing Elafin gene into probiotics this year to reduce the content of elastase and protease-3 in the inflammatory site and alleviate IBD.
Our Solution: Engineering Probiotics
This year, our team NWU-CHINA-A found a novel solution to solve the problem of IBD treatment, hoping to improve the daily life of patients through synthetic biology. We constructed non-pathogenic edible probiotics E.coli Nissle 1917 (EcN) into engineering probiotics. Through the hydrogen peroxide-induced promoter, probiotics would be induced by hydrogen peroxide at the inflammatory site, specifically expressing CAT, SOD and Elafin to alleviate the inflammatory response. In addition, we will construct a suicide switch to prevent engineering probiotics from leaking into the environment. We are also expected to prepare our products in the form of oral capsules for IBD patients. In brief, our engineering probiotics treatment method has the advantages of high safety, strong curative effect, harmless, cheap and convenient compared with traditional treatment.
Figure 6 . Modification of probiotics to treat IBD
Inspiration
Our determination of this project is a tortuous process, because we found that there are many problems that can be solved by synthetic biology. We are committed to solving problems related to human health. Initially inspired by sugar-free Coke, we wanted to produce D-tagatose using maltodextrin as raw material through a multi-enzyme co-expression system. For the first few months, we had been exploring the feasibility of this project and designing an emoji package, but eventually abandoned the project because we were worried about the uncertainty of the multi-enzyme co-expression system and the safety of sugar substitutes. Fortunately, at the beginning of the project, some members of our team expected to continue the general direction of last year's(NWU-CHINA-A – iGEM Project) to engage in intestinal-related projects. In the process of searching for information, we inadvertently found a family with two patients with Crohn 's disease. Through their private prosecution, we admired their positive and optimistic attitude towards the disease. At the same time, it also inspired us to focus on the increasingly serious IBD, hoping to provide new ideas for the treatment of IBD through synthetic biology and help improve the quality of life of IBD patients. Moreover, in the survey, we found that there was a lack of awareness of synthetic biology and IBD. We also hope that through our actions, we can make more people know about IBD, give more care to IBD patients, and also let them know that we are trying to use the way of thinking and tools provided by synthetic biology to change the status quo and synthetic biology is changing the world!
Reference
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