Inflammatory Bowel Disease (IBD) is a chronic condition characterized by inflammation in the gastrointestinal (GI) tract. The primary cause of IBD is an overreactive immune response, where immune cells mistakenly attack the gut lining, leading to prolonged inflammation and tissue damage. A key regulator of such inflammation, Interleukin-10 (IL-10), plays a fundamental role in modulating inflammation and maintaining cell homeostasis by protecting the body from an uncontrolled immune response by inhibiting pro-inflammatory cytokines like TNF-α and IL-6. However, IL-10’s therapeutic potential is severely limited by its structural instability due to its homodimer nature and easy degradation within the body. This instability reduces its effectiveness as a long-term treatment leaving patients with limited options for prolonged IBD treatments. To address this issue, we propose a probiotic-based approach, engineering a mutated more stable form of Il-10 (MIL-10). Delivered through dairy products like yogurt, this system would provide MIL-10 production in reaction to pro-inflammatory cytokines, ensuring sustained inflammation control for diseases like IBD.

Inflammatory Bowel Disease (IBD) is an autoimmune disorder in which the body’s immune system attacks healthy tissues in the GI tract, activating the immune system and leading to prolonged inflammation. The two primary ones are Crohn’s disease and ulcerative colitis (Figure 1), with symptoms including abdominal pain, diarrhea, rectal bleeding, and swelling or masses (Johns Hopkins Medicine). Available evidence suggests that this disorder results from inappropriate inflammatory response to intestinal microbes in a genetically susceptible host (Abraham & Cho, 2009) or dysfunctions of innate and adaptive immune pathways (Zhang & Li, 2014). Due to this disorder’s rapidly increasing incidence worldwide (Ng et al., 2017), U.S. prevalence being estimated between 2.4 and 3.1 million (CDC, 2024), and risks including tearing of the intestinal wall, bowel contents leaking into the bladder, and developing colon cancer (Johns Hopkins Medicine), it’s crucial to address the inflammation itself.

IMPACT ON SOCIETY

IBD is not only physically debilitating but also deeply impacts mental health and daily functioning. Patients frequently experience fatigue, malnutrition, and growth delay in pediatric cases, as well as increased rates of depression and anxiety. A study in Lancet Gastroenterology & Hepatology showed that up to 60% of IBD patients report diminished quality of life, primarily due to unpredictable flare-ups and chronic symptoms (Kaplan & Ng, 2017). Further, the financial costs of IBD are staggering. In the United States, the annual direct healthcare costs are estimated at over $30 billion, with biologic therapies (such as anti-TNF agents) being the primary driver of expense (Park et al., 2020, Inflammatory Bowel Diseases). Indirect costs—lost productivity, absenteeism, and disability—contribute billions more, underscoring IBD as not just a medical but also an economic challenge. Once considered a disease primarily of Western nations, IBD is rapidly emerging worldwide. Newly industrialized countries in Asia, the Middle East, and South America are now seeing sharp increases in incidence, likely linked to urbanization, diet changes, and reduced microbial exposure (Ng et al., 2018, Lancet). This global expansion indicates that IBD is not confined to one population but represents a growing worldwide health crisis.

Conventional treatments control symptoms of IBD through pharmacotherapy, including aminosalicylates, corticosteroids, immunomodulators, and biologics (Cai et al., 2021). Aminosalicylates (5-ASA) are compounds containing 5-aminosalicyclic acid and function as free radical scavengers that reduce production of leukotrienes (Greenfield et al., 1993), which are released by white blood cells in response to triggers like allergens and act as potent inflammatory mediators, amplifying the inflammatory response. Aminosalicylates also inhibit the release of IL-1, a group of pro-inflammatory cytokines that trigger a cascade of other inflammatory cytokines and amplify the inflammatory response (Gelfo et al. 2020). Through this mechanism, aminosalicylates reduce inflammation in the lining of the intestine (Crohn’s & Colitis Foundation, 2018). However, there are side effects including headache, nausea, abdominal pain and cramping, loss of appetite, vomiting, rash, fever, diarrhea, and rarely even decrease in sperm production and pancreatitis (Colitis Foundation, 2018).

Corticosteroids (also known as glucocorticoids or steroids) are human-made drugs similar to cortisol, slowing down the production of the chemicals that cause inflammation (Cleveland Clinic). They enter the cell and reverse histone acetylation of activated inflammatory genes by binding the liganded glucocorticoid receptors (GR) to coactivators and recruiting histone deacetylase-2 (HDAC2); however, in patients with oxidative/nitrative stress, inflammation becomes resistant to the anti-inflammatory actions of corticosteroids (Barnes, 2009). Also, though they are generally safe and useful for quickly reducing inflammation, they have side effects including unexpected weight gain, skin changes, stomach irritation, muscle weakness, mood swings, and increased risk of Cushing syndrome, diabetes, high blood pressure, osteoporosis, and infections (Cleveland Clinic).

Immunomodulators are drug treatments that change the body’s immune response. The ones commonly used for IBD kill T cells, which are part of the immune system and stimulate inflammation; block their ability to build DNA and thus prevent their production; or block calcineurin, an enzyme responsible for immune response (Crohn’s and Colitis Canada). However, these may affect the liver condition, bone marrow, and white blood cell activity (Crohn’s and Colitis Canada).

Biologics are medicines that are derived from living organisms, such as proteins and genes (Cleveland Clinic, 2024). Existing biologics for IBD include TNF-α blockers, integrin blockers that prevent white blood cells that cause inflammation from entering the GI track, and interleukin blockers that target IL-12 and IL-23, two proteins associated with inflammation in the GI track (UChicago Medicine). However, blocking these molecules raises concerns of interference with the human physiology and loss of effect over time.

Overall, conventional treatments for IBD inhibit and reduce the production of molecules and cells that cause inflammation. However, suppressing the immune system can pose risks on the body against other pathogens, since the immune system is meant to protect the body against outside harm.

Another part of the immune system are anti-inflammatory cytokines, which naturally regulate immune response and suppress inflammation (Opal & DePalo, 2000). Notable among these is interleukin-10 (IL-10), due to its association with Crohn’s disease and ulcerative colitis with its function of regulating intestinal inflammation (Abraham & Cho, 2009). It works mainly by activating Signal Transducer and Activator of Transcription 3 (STAT3), which plays a critical role in regulating inflammation—driving pro-inflammatory responses while also promoting anti-inflammatory processes in different contexts (Yu, Pardoll & Jove, 2009). By activating STAT3, IL-10 induces the expression of anti-inflammatory mediators such as SOCS3, which act as a feedback inhibitor for the expression of pro-inflammatory cytokines, preventing overactivation (Cevey et al., 2019). IL-10 also inhibits the production of pro-inflammatory mediators such as TNF by inhibiting NF-KB, a crucial transcription factor for pro-inflammatory gene expression (Cyktor & Turner, 2011).

IL-10’s role in regulating inflammation also suggests association with IBD. For instance, evidence shows that IBD is the most prominent phenotype in patients with IL-10 receptor deficiency, and that patients with loss of function mutations in the IL-10 receptor resulted in a Crohn’s disease prominent phenotype (Glocker et al., 2009). Moreover, polymorphism in a region of chromosome containing the IL-10 gene was strongly associated with ulcerative colitis (Franke et al., 2008) and mice deficient in IL-10 spontaneously developed ulcerative colitis (Abraham & Cho, 2009), suggesting that defective IL-10 function is central to the pathogenesis of IBD.

IL-10's THERAPEUTIC POTENTIAL

IL-10’s significant role in suppressing intestinal inflammation hints at its potential to be used as a therapeutic to regulate hyperinflammation. However, a significant drawback is IL-10’s instability and short half-life, stemming from its non-covalent homodimer structure, meaning that two identical subunits of this protein are held together by weak forces, allowing it to dissociate easily (Acuner-Ozbabacan et al., 2014).

Figure 8. Homodimer structure of IL-10. From Interleukin-10 (IL-10) Family, by R&D Systems. Retrieved September 16, 2025, from https://www.rndsystems.com/cn/resources/articles/interleukin-10-il-10-family

Thus, we aim to modify the amino acid sequence of IL-10 to create more interactions between R-groups within the protein, making it less prone to degradation and more stable in high-temperature environments similar to hyperinflammation. By engineering a more stable and enhanced version of a molecule already in our system, we reduce the risks that conventional treatments can cause, such as over-suppressing the immune system. In these attempts, we utilize AI software tools to identify certain mutations that could increase the cytokine’s stability, then put it to the test with wet lab techniques (running an ELISA assay) that test the binding and stability after being put in high temperatures.

POINT MUTATION DESIGN

In order to identify the mutations that would make IL-10 protein more stable, we began with structure-guided inspection in PyMOL, a software that allows us to visualize, manipulate, and create high-quality images of molecular structures. We loaded available IL-10 homodimer structures and mapped candidates sites from our stability screen onto the 3D model. We also attained 3000 mutations that were thermally favored, given by ThermoMPNN, a software that predicts how mutations will affect a protein’s stability. Out of the 3000, we focused on the 400 most thermally favored mutations and plotted them and checked if the change would disrupt the protein structure and thus its ability to bind to its receptor, which is critical in its function within the body once integrated into the human system. Our guidelines for whether the mutation would disrupt the structure too much or not were: ensuring the mutation causes no steric clashes, no loss of key H-bond/salt bridges, no disruption of secondary structure, no loss of receptor interactions (if applicable), and that the rotamer is favorable and not strained. After examining each of the 400 most thermally favored mutations with these guidelines, we narrowed down to two mutations with the highest potential to be more stable than the original IL-10 while still maintaining its structure and thus function. We refer to them as MIL-10 1 and MIL-10 2, for “Mutated IL-10.”

WET LAB

With the amino sequence of the MIL-10 1 and MIL-10 2, we converted them into a DNA sequence and integrated it into a separate plasmid that we constructed with vectors that would work with an E. coli system. The vector backbone was sourced from Addgene, pLp_3050sNuc, Plasmid #122030, and it includes a selectable marker erythromycin and the promoter sppA, which can be induced by sodium acetate. We had three plasmids constructed containing different mutations of the following: original IL-10, M1IL-10 1, and M2IL-10 (with M1 being mutation 1 and M2 being mutation 2). However, technical difficulties required us to pivot into a different plasmid system due to unsuccessful attempts, leading to the design of our protein in a pET 21 vector aimed to express our proteins of interest in E. coli strains of DHalpha and Bl21(DE3). The designs of the backbone are provided below:

Figure 9. Plasmid backbone used in our wet lab for production of IL-10. Sourced from Twist Biosciences, https://www.twistbioscience.com/products/genes/vectors?tab=catalog-vectors

Notes about the plasmid: T7 RNA polymerase driven transcription vector for expression in E. coli. The vector, which lacks the ribosome binding site and ATG start codon, is designed for protein expression from translation signals carried by the cloned DNA. Vector features: C-terminal His Tag sequence lac repressor/lac operator to inhibit transcription in E. coli. Expression can be induced by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG) Production of virions containing single-stranded DNA corresponding to the coding strand upon co-transfection with helper phage. We used this plasmid to insert our sequences in through codon optimization tools, adding in a ribosomal attachment site and a start codon as well.

To verify the expression of our protein, we conducted both Western Blots and SDS pages, showing bands that proved the expression successful. We then ran a series of protocols that led up to ELISA, where the functionality of our newly designed proteins were tested under different temperature conditions. Results can be found in our tab with graphical analysis and data.

FUTURE STEPS

We hope that MIL-10 can be expanded beyond the lab, imaging a daily supplement that patients have easy access to—similar to yogurts or probiotic drinks. In practice, MIL-10 would be manufactured through scaled fermentation of Lactobacillus plantarum under GMP conditions, then freeze-dried into stable probiotic cultures. These cultures could be formulated into yogurt, capsules, or powders, depending on patient needs. Unlike biologics, which require infusion centers and strict cold chains, MIL-10 could be shelf-stable and widely distributed through pharmacies and supermarkets. Enhanced stability through mutation ensures that the protein remains functional despite the digestive process, allowing it to reach the colon intact. In future steps, our roadmap includes transitioning to L. plantarum chassis systems, validating performance in gut-on-chip models, and working toward classification as a biotherapeutic probiotic under FDA and EFSA guidelines.

Figure 10. Fermentation of Dietary Fibre-Added Milk with Yoghurt Bacteria and L. rhamnosus and Use in Ice Cream Production which can be studied to mirror later fermented processes taken to create a probiotic. Sourced from MDPI, by Elif Sezer, Ahmet Ayar, and Suzan Öztürk Yılmaz https://www.mdpi.com/2311-5637/9/1/3#

THE MARKET

The financial and social impact of MIL-10 has enormous potential to be transformative in the probiotic industry. Whereas biologic treatments can cost more than $40,000 per patient annually, a probiotic-based therapy could be produced for under $500, opening access to millions who are currently underserved. The global supplement industry, valued at over $150 billion and projected to double by 2032, provides a natural entry point for MIL-10 as a medically validated probiotic. By aligning with the UN Sustainable Development Goals—specifically Good Health & Well-Being (SDG 3) and Reduced Inequalities (SDG 10)—our project demonstrates not only scientific innovation but also global accessibility. We envision MIL-10 bridging the gap between pharmaceutical therapeutics and consumer health, offering an affordable, effective solution for patients worldwide.

Ultimately, we hope to reinvent therapeutic treatment in the supplements industry, creating a product that is not only effective but also accessible. Engaging in community feedback, professional opinions, and researching deeper into entrepreneurship and patenting all bring us closer to this goal. The science of stability, the magic of relief—scroll through our pages to learn more.

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