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Open Access Publications from the University of California

Scholarly Works (2 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2021)

Macrophages play a key role in the innate immune system, and their activation is tightly regulated to avoid excess and harmful inflammation. Studies have revealed the roles of soluble and adhesive cues in regulating macrophage polarization. The importance of intercellular communication within a macrophage population has also been demonstrated. However, as macrophages become crowded, the increasing cell density could alter the surrounding soluble and adhesive environments, as well as the coordination within a population. How these changes in the multicellular microenvironment affect macrophage functions is not well understood.

In my dissertation, I investigated the effects of the multicellular environment and its alteration on macrophage functions, characterizing how these factors affect paracrine signaling at macroscopic and microscopic levels, as well as how it could affect the physical interaction of cells. We characterized the regulatory effects of density-dependent intercellular communication on macrophage populations by stimulating cells in bulk, in small groups, and in isolation. We also assessed how cell crowding affects the function of a mechanosensitive ion channel, Piezo1, as this channel is important for the physical sensing of macrophages. We found that macrophage activation depends on the communication among the cell population, and both seeding density and absolute cell number contribute to the resultant responses. In addition, Piezo1 is found to contribute to the crowding-dependent regulation of macrophage activation. These works demonstrated that altering cell density could have broad-spectrum effects on macrophage functions. As a result, the impact of seeding density and its impacts on the multicellular environment should not be overlooked when designing an experiment that uses macrophages.

Overall, this study expanded our knowledge on how the macrophage population could regulate cellular functions by altering their surrounding soluble and adhesive environment in a density-dependent manner. This work could help explain the discrepancy in experimental results that can occur between different studies, as well as among different culture systems. Furthermore, this density-dependent regulation of macrophage activation may represent a novel regulatory mechanism with meaningful impacts toward cellular functions in vivo. Future work would focus on understanding how cell crowding could affect other aspects of cellular processes, and how these effects could further modulate macrophage functions in both in vitro and in vivo settings.

    Cover page: Regulation of Macrophage Function by the Multicellular Microenvironment
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2019)
    Single cell analysis methods are increasingly being utilized to investigate how individual cells process information and respond to diverse stimuli. Soluble proteins play a critical role in controlling cell populations and tissues, but directly monitoring secretion is technically challenging. Microfabricated well arrays have been developed to assess secretion at the single cell level, but these systems are limited by low detection sensitivity. Semiconductor quantum dots (QD) exhibit remarkably bright and photostable luminescence signal, but to date they have not been evaluated in single cell secretion studies using microfabricated well arrays. Here, we used QDs in a sandwich immunoassay to detect secretion of the soluble cytokine tumor necrosis factor-α (TNF-α) from single cells. To enhance detection sensitivity, we employed two different strategies. First, we used a unique single QD imaging approach, which provided a detection threshold (180 attomolar) that was >100-fold lower than previously reported results using QDs. We also amplified QD binding to each captured TNF-α molecule using the bioorthogonal cycloaddition reaction between trans-cyclooctene and tetrazine, which further lowered detection threshold to 60 attomolar. This is 6 orders of magnitude more sensitive than organic fluorophores that have been used for single cell secretion studies, and far surpasses single molecule resolution within sub-picoliter microwells that are used to assess single cell secretion. Finally, single cell secretion studies were performed using phorbol 12-myristate 13-acetate (PMA) differentiated and lipopolysaccharide (LPS) activated U-937 cells. TNF-α secretion was detected from 3-fold more single cells using the QD-based method in comparison to rhodamine, which was accomplished by extending sensitivity into the range of ∼2 to 10 000 molecules captured per microwell. In future work, we will apply this technique to assess immune cell secretion dynamics under diverse stimuli and disease settings. We will also incorporate multiplexing capabilities to evaluate the secretome at the resolution of single molecules.
      Cover page: Pushing the limits of detection for proteins secreted from single cells using quantum dots
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