NYMC Student Theses and Dissertations

Date of Award

8-31-2024

Document Type

Master's Thesis - Open Access

Degree Name

Master of Science

Department

Basic Medical Sciences

First Advisor

Xiu-Min Li

Second Advisor

Chandra Shekhar Bakshi

Third Advisor

Jan Geliebter

Abstract

Staphylococcus aureus is a versatile Gram-positive bacterium commonly found on human skin. While often harmless, it can cause various infections when the skin is compromised, ranging from minor issues to life-threatening conditions in immunocompromised individuals.

The bacterium’s ability to resist antibiotics, particularly methicillin, underscores its clinical challenge. Staphylococcus aureus can also acquire vancomycin resistance. It evades immune responses through multiple mechanisms, including capsule formation, interference with immune proteins like staphylococcal protein A, and secretion of toxins such as Panton-Valentine Leukocidin. Understanding these mechanisms is crucial for developing effective strategies against Staphylococcus aureus infections.

The immune response to Staphylococcus aureus infections is orchestrated through a complex interplay of innate and adaptive immune mechanisms. Upon encountering Staphylococcus aureus, innate immune cells such as neutrophils, monocytes/macrophages, dendritic cells, and natural killer cells are mobilized to the infection site. Neutrophils play a crucial role in the initial defense by phagocytosing bacteria and releasing antimicrobial peptides and reactive oxygen species through an oxidative burst. Monocytes differentiate into macrophages that engulf bacteria and secrete cytokines and chemokines to recruit and activate other immune cells. Dendritic cells process and present Staphylococcus aureus antigens to T cells, initiating adaptive immune responses. T cells, including CD4+ and CD8+ subsets, differentiate into various effector and memory cells to coordinate long-term immune defense. B cells produce antibodies that neutralize toxins and facilitate bacterial clearance. The inflammatory cytokines IL-1β, IL-6, TNF-α, and IFN-γ orchestrate immune responses, regulating inflammation and coordinating immune cell communication. These immune components form a robust defense system against Staphylococcus aureus, although the bacterium’s ability to evade immune detection poses challenges in treatment and vaccine development.

In this study, berberine, a natural compound derived from plants like goldenseal and barberry, was explored for its potential as an inhibitor of Staphylococcus aureus mediated inflammation. Known for its broad antimicrobial properties and therapeutic benefits in traditional medicine systems such as Traditional Chinese Medicine and Ayurveda, berberine has been extensively studied for its ability to combat various infections and inflammatory conditions without causing liver damage. It acts by disrupting bacterial cell membranes, inhibiting biofilm formation, and reducing virulence factors, making it a promising candidate against antibiotic-resistant strains like methicillin-resistant Staphylococcus aureus. Furthermore, berberine exhibits potent anti- inflammatory effects by modulating cytokine production and inflammatory pathways, which could mitigate excessive inflammation during Staphylococcus aureus infections.

We hypothesized that berberine can significantly inhibit TNF-α production, gene expression involved in inflammatory pathways, and reactive-oxygen species production in response to Staphylococcus aureus stimulation in both mouse macrophage and human monocyte cell lines. The results demonstrated that berberine effectively inhibited TNF-α production in RAW 264.7 mouse macrophage and U937 human monocyte cell lines when stimulated with heat-killed Staphylococcus aureus (ATCC strain #33591). This inhibition was dose-dependent, with 20 µg/mL of berberine suppressing over 90% of TNF-α production compared to controls (p < 0.001), highlighting its robust anti-inflammatory activity without inducing cytotoxic effects.

Moreover, when tested against clinical isolates of Staphylococcus aureus (EC01-EC04) isolated from the skin of eczema patients, berberine at concentrations ranging from 2.5 to 20 µg/mL consistently inhibited TNF-α production by over 90% (p < 0.001) in U937 cells, reinforcing its potential clinical relevance. Berberine also significantly downregulated key genes involved in inflammatory pathways in U937 cells and inhibited reactive oxygen species production in both RAW 264.7 and U937 cells at 20 µg/mL (p < 0.01), indicating its capacity to reduce oxidative stress associated with bacterial infections.

Overall, these results suggest that berberine possesses dual therapeutic actions against Staphylococcus aureus infections by suppressing inflammatory responses and oxidative stress. This study provides a strong foundation for further exploration of berberine’s clinical applications, potentially as an adjunct therapy alongside conventional antibiotics, addressing critical clinical needs in infectious and inflammatory diseases.

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