3D-printed quantitative microfluidics for bacteria and macrophage studies and the impact of indole on modulating itaconate in macrophages

Abstract

Microfluidics technology has advanced analytical capabilities, offering precise fluid manipulation across various scientific fields. While traditional microfluidic fabrication methods exist, 3D printing has revolutionized this field with its precision, customization, and versatility. However, the limited transparency and high costs associated with 3D printed microfluidic devices, hinder their application and widespread adoption. This dissertation addresses these challenges with innovative solutions. It introduces a groundbreaking approach by directly integrating optical components into 3D printed microfluidic systems, enabling direct optical measurements through these devices for the first time. Furthermore, a toolkit was designed to facilitate the rapid assembly of 3D printed microfluidic devices using low-cost microbore tubing, lowering the costs. These microfluidic systems were applied to the analysis of indole, a bacterial-released molecule, and nitrite, a pro-inflammatory molecule released by macrophages, frontline immune cells. Indole is released by bacteria, particularly in high concentrations during chronic infections, and it is known to suppress pro-inflammatory molecules in macrophages. Yet, the near-real-time release kinetics of indole in bacteria have remained unknown. Similarly, the near-real-time release kinetics of nitrite, a well-established pro-inflammatory molecule released by macrophages, have yet to be explored. Understanding release kinetics is essential for identifying unexpected events during time intervals and laying a foundation for therapeutic discoveries. Leveraging the newly developed systems with integrated optical devices, the release kinetics of both indole and nitrite were acquired in near-real time, with measurements taken every twenty seconds. Moreover, this dissertation investigates the impact of indole on macrophage metabolism. The hypothesis proposes that indole induces itaconate production, a metabolite known to suppress pro-inflammatory molecules. Using activated RAW 264.7 macrophages treated with various indole concentrations, the study quantified itaconate production and immune-responsive gene 1 (IRG1) levels, which is the protein that catalyzes itaconate production. The results confirmed that indole induces itaconate and unveiled a novel mechanism of indole-induced itaconate production through the aryl hydrocarbon receptor (AHR). In summary, this dissertation addresses microfluidics challenges and advances our understanding of the interplay between bacteria and macrophages. These findings hold potential for the development of new antibiotics and therapeutic interventions against chronic infections, marking a significant contribution to the scientific field.