Engineering Immunocompetent Human Tissues to Define Inflammatory State Transitions

About This Grant

Project Summary Chronic inflammation is a central factor in a major fraction of all human disease burden. In chronic inflammation, immune cells mediate a dysregulated inflammatory response that persists despite the removal of the initial insult, suggesting stable, steady-state dynamics between immune and tissue cells. Macrophages are key players in this process, mediated by their status as central effectors of tissue inflammation and their high plasticity for both inflammatory and anti-inflammatory states, as well as tissue-specific functionality. This process of transition from a healthy to a chronically inflamed tissue steady state involves competition between trafficking and invading macrophages from the periphery vs. resident ones, conversion between inflammatory states, and crosstalk with neighboring tissue cells that is hypothesized to be tissue-specific. Existing tools for studying chronic inflammation are limited in their ability to provide a mechanistic understanding of these processes. They use overly high doses of inflammatory agents, which cause non- specific responses with little predictive value. The macrophages used are not authentic resident cells; they lack the correct epigenetic, immune effector, and metabolic characteristics, and their polarized states are poorly defined. Furthermore, these models fail to account for tissue-specific inflammatory responses, leading to inaccurate, non-physiological results that misrepresent the therapeutic window for chronic diseases. The overarching goal of this project is to develop tools that allow us to manipulate and study inflammatory state space in complex human tissues with a high degree of control. Aim 1 focuses on assembling immunocompetent human brain, liver, and adipose microtissues from pluripotent stem cell-derived progenitors. We will quantify resident macrophage expansion versus monocyte infiltration during inflammation initiation while benchmarking tissue structure and immune responsiveness. Aim 2 will establish advanced optical and genetic tools to continuously track transitions between basal, inflammatory, and reparative tissue states. We will combine fluorescence and label-free imaging techniques (e.g., fluorescence-lifetime and second harmonic generation) with engineered genetic reporters to generate high-density time-series data, identifying links between macrophage functions and inflammation divergence. Aim 3 will solve the problem of nonphysiological inflammatory cues by creating stable and inducible genetic switches to control macrophage activation states orthogonally and in situ within human microtissues. We will assess how these cells influence tissue structure, signaling, and the trajectory of inflammatory state, as well as test whether chronic tissue remodeling can be driven or reversed through specific pathway regulations. The successful development of this platform will provide the first human system to distinguish the roles of resident and infiltrating macrophages, track their real-time impact on tissue trajectories, and define the requisite physiological parameters needed to stabilize or reverse chronic inflammation.