Gut Microbial Inhibition for Atherosclerosis Treatment Name, institution, course, professor, date Gut Microbial Inhibition for Atherosclerosis Treatment Gut microbes are participants in atherosclerosis, as suggested by recent studies. In particular, phosphatidylcholine, choline, and carnitine-trimethylamine (TMA)-containing nutrients are plentiful in protein-rich foods such as egg yolks, meat and meat products, and high-fat dairy products that usually serve as precursors for TMA N-Oxide (TMAO) generation in humans and mice. (TMAO) is a metabolite that usually speeds up atherosclerosis in animal models (Wang et al., 2015). The risk for both prevalent atherosclerotic heart disease and incident major adverse cardiac events in multiple independent cohorts is mostly associated with blood TMAO levels. Atherosclerosis is a disease that is brought about by the accumulation of lipids and fibrous elements in the large arteries. Interventions that are usually mitigated for lowering TMAO levels are of considerable therapeutic value. In this paper, we review how gut microbial inhibition can enable treatment for various diseases since it can control the bioavailability of metabolites by biologically metabolizing active molecules, drugs, and their precursors. Various methods can be used to offer therapeutics, but our main focus will be on protein therapy. Protein Therapy Therapeutic proteins are highly successful in clinics. They are rarely administered orally due to their low oral bioavailability. This is due to the short physiological half-life of the gastric and intestinal fluids and enzyme degradation; because of this factor, they are rapidly cleared before successful penetration into the tissues. However, spray drying is a well-established method that is successfully applied to administer this therapy. This method entails four fundamental steps: atomization of feed into spray, spray air contact, drying of spray, and separation of dried product from the drying air. This method is useful for the preparation of proteins intended for the pulmonary and nasal. Hypothesis: Research suggests that dysregulation of neuroinflammation is brought about by microglia. Plays a critical role in the pathogenesis of Alzheimer's disease (AD). Signals such as dystrophic axons, dead neurons, amyloid plaques, and phosphorylated TMAO all alter the functional phenotype of microglia from a homeostatic to a neurodegenerative state (De-Paula et al. 2012). This, in turn, drives neuroinflammation and promotes disease. This approach to targeting microglia activation can be applied to the treatment of AD. In vitro, approaches were used to understand microglial alterations at the genetic and protein level and synaptic function and plasticity in CA1 hippocampal neurons, each about both age and stage of amyloid beta pathology. Here, we show that nasally administered anti-CD3 monoclonal antibody in the 3xTg AD mouse model reduced microglial activation and, in turn, improved cognitive independence of amyloid beta deposition. Gene expression analysis shows increased oxidative stress and metabolic changes in the hippocampus and cortex of nasal anti-CD3 treated animals. Our findings suggest that nasal anti-CD3 is a better way of immune therapy in the treatment of AD that is independent of amyloid beta targeting. The major component of AD is neuroinflammation. Activated microglia and astrocytes surrounding alpha and beta plaques are involved in the inflammatory pathways of the disease. Following CNS inflammation, microglia change from a homeostatic phenotype to a neurodegenerative state. Regulatory T cells (Tregs) are the key modulators of immune responses. We found that adaptive transfer of Tregs into the 3xTg mouse model of AD enhanced cognition and reduced Aβ deposition, while depletion of Tregs aggravated spatial learning deficits in the same mice. This was found to be similar in APP/PS1 mice. Nasal anti-CD3 mAb usually dampens microglia and astrocyte activation by inducing IL-10-producing Tregs and ameliorates disease, usually in mouse models of AD. Treatment of mice with nasal anti-CD3 was shown to improve cognition and dampen microglial activation. The mechanism by which disease is ameliorated in 3xTg mice usually relates to the expansion of IL10+. Tregs migrate to the brain to dampen microglial activation and modulate the CNS environment. We identified increased numbers of IL10-secreting T cells in the spleen of mice at 6 months, and in the brain, we were able to locate CD3+ T cells that were in close contact with microglial dendrites. Gene profiling of microglia after nasal anti-CD3 treatment demonstrated a switch from an MGnD or DAM phenotype to a molecular homeostatic phenotype. We further established that nasal anti-CD3 altered the transcriptional landscape in the cortex and hippocampus of 3xTg mice in a sex-dependent fashion. This mechanism is still unknown, but it is believed that sex differences could be the ones playing a significant role in the mechanism by which nasal anti-CD3 modulates gene expression in the hippocampus, cortex, and microglia, and this could be the reason for the short-term memory observed between females and males treated with nasal anti-CD3. The number of mice used for gene expression analysis (3 to 4 per group) might have contributed to the lack of overlapping pathways in the cortex and hippocampus of female vs. male 3xTg mice treated with nasal anti-CD3. A previous study reported that a single intravenous administration of anti-CD3 mAb induced the upregulation of vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells, which favored T cell adhesion to the endothelium and subsequent

migration to the targeted organ (Benitez et al., 2021). We managed to establish that nasal anti-CD3 down-regulates several proinflammatory genes in microglia in 6-month-old 3xTg mice; this may suggest a benefit of nasal anti-CD3 through limiting microglial activation. Antiamyloid therapy has been applied to treat early AD. Because anti-CD3 decreases CNS inflammation and usually acts independently of effects on Aβ, this could be beneficial at other stages of diseases when anti-amyloid therapy is no longer effective. Conclusion Our results suggest that nasal anti-CD3 has the potential to be a great immunotherapy to treat AD that targets microglial cells. A fully human anti-CD3 mAb has been effectively given to human subjects and has proven immune effects with negligible toxicity. The in-depth characterization of pathological hallmarks of AD in this novel and open-access mouse model can serve as a resource for the scientific community to investigate disease-relevant biology. References Wang, Z., Roberts, A. B., Buffa, J. A., Levison, B. S., Zhu, W., Org, E.,... & Hazen, S. L. (2015). Non-lethal inhibition of gut microbial trimethylamine production for the treatment of atherosclerosis. Cell, 163(7), 1585–1595. De-Paula, V. J., Radanovic, M., Diniz, B. S., & Forlenza, O. V. (2012). Alzheimer's disease. Sub-cellular biochemistry, 65, 329–352. https://doi.org/10.1007/978-94-007-54 Benitez, D.P., Jiang, S., Wood, J., et al. Knock-in models related to Alzheimer's disease: synaptic transmission, plaques, and the role of microglia. Mol Neurodegeneration 16, 47 (2021). https://doi.org/10.1186/s13024-021-00457-016-4_14