Cypel, M. | bioRxiv Science

bioRxiv2025

Bioactive Flavonoid Extract Suppresses NLRP3 Activation and Inflammation in 2D and 3D Lung Models

Inflammasome activation plays a critical role in lung inflammation, with the NLRP3 (NOD-, LRR-and pyrin domain-containing protein 3) inflammasome serving as a key mediator of inflammatory cytokine release and pyroptotic cell death. This study investigates the effects of a bioactive flavonoid extract (BFE), as a potential modulator of NLRP3-mediated inflammation using THP-1 derived macrophages, A549 lung cells (2D cells), and lung organoids (3D cells). Cells were primed with lipopolysaccharide (LPS), treated with BFE, and then activated with nigericin to induce NLRP3 activation and examine BFEs effects. Markers of inflammation, including presence of ASC specks (3D models), reactive oxygen species production (ROS) (2D models), caspase-1 activity (2D and 3D models), and IL-1{beta} release (2D and 3D models) were measured to assess the extent of inflammasome activation in several treatment conditions. By integrating 2D and 3D lung models, this work provides insight into the NLRP3 inflammasome axis in lung inflammation and explores BFE as a potential therapeutic strategy for inflammasome-driven pulmonary inflammatory processes.

molecular biology↗

bioRxiv2021

Ex vivo delivery of regulatory T cells for control of alloimmune priming in the donor lung

Survival after lung transplantation (LTx) is hampered by uncontrolled inflammation and alloimmunity. Regulatory T cells (Tregs) are being studied for post-implantation cell therapy in solid organ transplantation. Whether these systemically administered Tregs can function at the appropriate location and time is an important concern. We hypothesized that in vitro expanded, recipient-derived Tregs can be delivered to donor lungs prior to LTx via ex vivo lung perfusion (EVLP), maintaining their immunomodulatory ability. In a rat model, Wistar Kyoto (WKy) CD4+CD25high Tregs were expanded in vitro prior to EVLP. Expanded Tregs were administered to Fisher 344 (F344) donor lungs during EVLP; left lungs were transplanted into WKy recipients. Treg localization and function post-transplant were assessed. In a proof-of-concept experiment, cryopreserved expanded human CD4+CD25+CD127low Tregs were thawed and injected into discarded human lungs during EVLP. Rat Tregs entered the lung parenchyma and retained suppressive function. Expanded Tregs had no adverse effect on donor lung physiology during EVLP; lung water as measured by wet- to-dry weight ratio was reduced by Treg therapy. The administered cells remained in the graft at 3 days post-transplant where they reduced activation of intragraft effector CD4+ T cells; these effects were diminished by day 7. Human Tregs entered the lung parenchyma during EVLP where they expressed key immunoregulatory molecules (CTLA4+, 4-1BB+, CD39+, and CD15s+). Pre-transplant Treg administration can inhibit alloimmunity within the lung allograft at early time points post- transplant. Our organ-directed approach has potential for clinical translation.

immunology↗