STEM I

This project aims to discover a novel and innovative treatment for Crohn's disease that is cost-effective with minimal side effects. An autoimmune disease is the result of the immune system accidentally attacking the body instead of protecting it and is often treated through immunosuppressive drugs. Crohn’s disease induces inflammation in the gastrointestinal tract, hindering nutrient absorption and resulting in excessive diarrhea, ulcers, and abdominal discomfort. The inflammation also creates opportunities for activating the STING pathway. The STING pathway, in turn, exacerbates the situation through the release of pro-inflammatory chemokines and cytokines in the digestive system, causing further damage to the gastrointestinal lining. This project aims to find therapeutic agents that inhibit the cGAS-STING pathway, which is linked with inducing inflammation in Crohn’s disease. Using pro-inflammatory ELISAs and quantitative PCR, novel therapies in this field called Methotrexate (MTX) and Dimethyl fumarate (DMF) proved to be effective. In the ELISA test, DMF had significant differences for both IFNb and IL-6 inhibition, while MTX only had it for IFNb, establishing that DMF is more functional. The drug's novel application in this project suggests that the STING pathway can be inhibited, reducing excessive inflammatory responses while not completely dampening the immune system. This focused method provides a more specific treatment plan with fewer adverse effects, which is significant when considering the overall immunosuppressive effects of existing medicines. The next steps include assessing long-term adaptability, evaluating therapy side effects, and conducting immunogenicity assessments. Keywords: Inflammation, Tissue Cultures, Targeted Therapies, Immunocompromised, Crohn’s Disease

The Cleveland Clinic states that an autoimmune disease is the result of the immune system attacking the body rather than protecting it for unknown reasons (Autoimmune Diseases: Causes, Symptoms, What Is It & Treatment, 2021). As one of the forefathers of immunology, Paul R. Ehrlich came up with a theory in 1901 that an immune reaction aimed at one’s body can result in the development of antibodies that are harmful to the individual. His famous “horror autotoxicus” theory revealed that the body requires a mechanism to protect itself from the dangers of self-autoantibodies (Ahsan, 2022). This theory laid the foundations for what autoimmunity is today. Donath and Landsteiner were the first to recognize an autoimmune disease, which is now called Donath-Landsteiner hemolytic anemia (Ahsan, 2022). In the 1900s, there was a new groundbreaking theory that the body could fight off foreign antigens (invaders). This theory was previously unheard of, but after a while, scientists agreed that the body had a self-defense mechanism. In 1962, Professor Ian Mackay and Sir Frank Macfarlane Burnet first proposed that the body had an immune response not only to foreign molecules but to self-antigens as well (WEHI History: 1962 Mackay and Burnet's Autoimmunity Theory | WEHI, n.d.). Although scientists initially doubted this hypothesis, thorough research and analysis proved their hypothesis correct. These foundational works paved the way for understanding complex immune pathways such as the Stimulator of interferon genes (STING) pathway. Recent research has shed light on the STING pathway's crucial function in autoimmune illnesses like Crohn’s disease, especially regarding its activation by self and non-self antigens, causing inflammation. This thesis focuses on suppressing the STING pathway as a novel method for treating Crohn's disease, demonstrating the direct translation of historical knowledge into current treatment tactics. Recently, the significance of autoimmune diseases in the world has grown exponentially, and investigations have found a consistent 3–4% annual increase in type 1 diabetes over the last three decades (Miller, 2023). This data point is relevant as more and more people are affected by these harmful illnesses. Autoimmune diseases affect around 10% of the global population (1 in 10 People Suffer From Autoimmune Diseases, 2023), displaying the severity of the crisis at hand. This percentage is equivalent to 740 million people. In the Middle East and East Asia, the number of cases of Inflammatory Bowel Disease (IBD) is increasing exponentially (Chen et al., 2023). Many people have this harmful sickness that needs to be treated more efficiently. Crohn’s disease is a lifelong chronic disease where inflammation and irritation occur from the immune system attacking the gastrointestinal tract, making it hard to absorb nutrients from food. Those with Crohn’s disease experience pain throughout the body. Crohn’s disease has also been on the rise for the past decade (Figure One). In the past, corticosteroids were used to combat this. These drugs suppress the immune system, reducing inflammation and other pains. The immune system is suppressed, leading to patients being prone to infections and sicknesses. For these reasons, it is crucial to develop better treatment options. Patients on current treatment plans state that they still get flare-ups and symptoms, which is further evidence that there is still room for improvement (Managing Crohn’s Disease: Flare-Ups and Treatment Options, n.d.). Despite developments from corticosteroids, existing Crohn's disease medications – such as Humira – have limitations due to their significant side effects and non-specific nature. For example, one concerning side effect is that the drugs cause the patient to be immunocompromised, reducing their ability to fight infections. This issue is compounded by the generalization of treatments, which often are not tailored to individual patient-based variations in disease growth. Standardized procedures and broad-acting drugs, such as corticosteroids and immunosuppressants, fail to address the specific characteristics of each patient's disease. Moreover, the cost to combat this disease is around $30,000 a year, reducing the accessibility of this treatment (Stewart & Breite, 2022). With such a significant cost to receive assistance with the challenges and burdens that come with Crohn’s disease, it is vital that a more accessible and effective solution is researched. However, a more specific drug could be even more expensive than the general treatment option, posing more issues to patients. Recently, work has shown that Crohn’s disease is aggravated by activating the STING pathway (Zhao et al., 2021). The STING pathway is a critical aspect of the innate immune system. It plays a role in detecting cytosolic DNA, which can be a sign of viral or bacterial infection, as well as other cell damage. An enzyme called cGAS, when detecting cytosolic DNA, activates the STING pathway. The cellular response of this signal transduction pathway is proinflammatory cytokines and interferons. Regarding Crohn’s disease, exosomal dsDNA is involved in activating the STING pathway, which promotes intestinal inflammation in Crohn’s disease and its hallmark symptoms. To control Crohn's disease effectively, therapies inhibiting the pathway must be developed while also minimizing its effect on the immune system. This project discovers a new approach to alleviating symptoms in autoimmune diseases, specifically Crohn’s disease, by using a medication that is specific but also affordable. The treatments are Dimethyl Fumarate(DMF) and Methotrexate (MTX), and they are novel approaches in this research because they show promise for inhibiting the STING pathway, so also Crohn's disease, due to their anti-inflammatory and non-suppressive properties on the immune system. Researchable Question How does the modulation of the cGAS-STING pathway by Dimethyl Fumarate (DMF) and Methotrexate (MTX) impact the expression of pro-inflammatory markers and cytokines in Crohn's Disease? Hypothesis Hyp. 1a: DMF will serve as a better therapeutic agent for inhibiting STING when compared to MTX. Hyp. 2a: When DMF is applied with an activated STING pathway, then the mRNA of CXCL10, IFNb, and TNFa and the amount of INFb and IL-6 will be decreased. Hyp. 2b: When MTX is present with an activated STING pathway, then the mRNA of CXCL10, IFNb, and TNFa and the amount of INFb and IL-6 will be decreased.

Section II: Methodology Role of Student vs. Mentor Dr. Fitzgerald has given the funds for this project. Dr. Crowthers has helped with the written portions of this project and has provided guidance. Dr. Mingqi has helped with the methodology and conducting my research and data collection. I have conducted all of the tests, data collection, and analysis and have written everything. This project has been worked on for 144 hours and 30 minutes. Equipment and Materials The cell line utilized in this project was Bone-marrow-derived macrophage (BMDM). BMDMs offer a model for studying inflammation since they are primary cells derived from the immune system. Macrophages are critical to the immunological response, especially in the presence of inflammation. In Crohn’s disease, macrophages are crucial to the gastrointestinal tract’s inflammatory process. They are known to produce pro-inflammatory cytokines in response to indications of intestinal damage and foreign DNA. BMDMs give a more accurate representation of the inflammatory milieu associated with Crohn’s disease and the functioning of the cGAS-STING pathway in this context. DiABZI, an agonist that targets the STING pathway, was used in this project. Excessive inflammation is a result of abnormal activation of the STING pathway in the context of Crohn's disease. The diABZI agonist artificially activates the STING pathway. The result of the activation simulates the inflammatory response observed in Crohn's disease, which allows the investigation of the function of the route in the illness and the consequences of blocking it. The groups of medications, DMF, MTX, and their respective controls, DMSO and PBS, were then given to different dishes and observed. A rt-qPCR and ELISA test were conducted to see the results of the inhibition, as well as a drug curve to see the maximum dosage of each group in the ELISA test. Bone-Marrow-Derived Macrophages (BMDMs) Preparation BMDM was created by removing bone marrow cells from a mouse's tibias and femurs, then using a medium enhanced with macrophage colony-stimulating factor (M-CSF) to differentiate the cells into macrophages. The bone marrow cells were grown in 10% DMEM that was enhanced with L-929 cell-conditioned media (L-sup), which supplied the M-CSF required for the development of macrophages. Bone marrow-derived macrophages (BMDM) are guaranteed a favorable environment for development and maturation by adding L-sup to the medium. The BMDM was performed in order to gather a cell model that closely resembles the macrophages found in the human immune system and is medically useful, especially for the investigation of inflammatory reactions in Crohn's disease. Creation of the Groups and Dosage Concentrations This phase entailed dividing the cells made from the BMDMs into three separate groups for treatment. Each plate had a ratio of 1 million cells to 1 uM. A distinct substance was assigned to each group: DMSO and PBS were used as negative controls, and DMF and MTX were used as the experimental medications. To create a medication dose-response curve, the therapy comprised exposing each group of BMDM to various concentrations of their individual drugs. First, the final concentration, after conversion from stock, that was desired was made, so for DMF, it was 12.5uM, and for MTX, it was 10uM. Then those were divided by two, and then that number was divided by two for another concentration, and this was conducted for both groups one more time. Furthermore, the final concentrations initially made were also doubled to see if there was a higher impact with a higher concentration. This procedure is essential for comprehending how the various chemical concentrations affect macrophage activity, particularly when considering the inflammatory responses associated with diseases such as Crohn's disease, and was conducted only for ELISA. More can be learned about the toxicity and potency of methotrexate, as well as the baseline reactions to DMSO and DMF, by examining the dose-response relationships. This information will help evaluate the drug's safety record and potential for therapeutic use. Enzyme-Linked Immunosorbent Assay (ELISA) The assay was essential to help determine how BMDM responded to stimulating and inhibiting the STING pathway by analyzing the release of pro-inflammatory cytokines. The ELISA test was used to determine the concentrations of IFNb and IL-6 in the BMDM cultures. IFNb and IL-6 are detrimental markers for Crohn’s disease (Ghosh et al., 2006) (Langer et al., 2019). Series of washes and a colorimetric detection of the bound antibodies were then carried out after incubating the supernatant with certain antibodies affixed to a microplate. The knowledge of the inflammatory mechanisms underlying Crohn's disease and the potential benefit of blocking the STING pathway as a therapeutic strategy has been enhanced by this research. Reverse Transcription-Quantitative Polymerase Chain Reaction (rt-qPCR) The rt-qPCR test was used to measure the expression levels of certain genes associated with the cGAS-STING pathway and the inflammatory response. This technique was done after the ELISA test because all of the wells can be used at once for the ELISA test, whereas for the rt-qPCR, they go one by one. That is why the ELISA test showed the best wells, and then those wells were used for the rt-qPCR test. The test detected IFNb, CXCL10, and TNFa mRNA. CXCL10, TNFa, and IFNb mRNA are all pro-inflammatory cytokine in Crohn’s disease (Singh et al., 2016) (Adegbola et al., 2018) (Ghosh et al., 2006). In the setting of Crohn's disease, this method proved crucial for assessing the impact of STING pathway activation and inhibition on the expression of inflammatory cytokines and other pertinent genes. Statistical Tests The t-test was conducted for the ELISA results to determine if there was a significant difference between DMF and DMSO or MTX and PBS. In analyzing the data from ELISA samples that measure IFNb and IL-6 protein levels, a t-test is an appropriate statistical method. Unlike the bootstrap test, which is advantageous for non-normal distributions or small sample sizes, the t-test assumes that the data are approximately normally distributed and that the variances are equal between the two groups being compared. Given that ELISA data often produce results that are normally distributed due to the large number of molecules being measured, the central limit theorem supports the use of a t-test. The rt-qPCR assays do not need a t-test. Rt-qPCR is a very sensitive technology that may detect even subtle changes in gene expression. The results obtained by rt-qPCR have a distinct distribution due to the amplification of the genetic material. Furthermore, rt-qPCR data are frequently reported in terms of fold changes and relative expression levels, which do not necessarily follow the assumptions of normality or equal variances (in this research instead of fold change it was transcript/housekeeping gene (tbp)). In such circumstances, using relative quantification and transcript/housekeeping gene (tbp) analysis can offer a more accurate depiction of gene expression levels without requiring hypothesis testing. The nature of the data, which is consistent with the t-test assumptions, informs the decision to apply a t-test for ELISA samples. In contrast, the intrinsic features of rt-qPCR data provide other analytical methodologies that do not require the assumptions underpinning the t-test.