Apical-out airway organoids as a pathophysiological model to study macrophage-mediated epithelial responses to microplastics.

Abstract

Respiratory exposure to airborne microplastics is increasingly implicated in airway inflammation, but human-relevant in vitro systems that separate direct epithelial particle contact from immune-cell-mediated paracrine effects remain limited. Here, we generated human airway basal stem cells (hABSCs)-derived apical-out airway organoids (AOAOs) with outward-facing ciliated surfaces and used them to evaluate operationally size-fractionated, polydisperse polystyrene microplastic (PS-MP) fractions. PS-MPs were characterized by laser diffraction, SEM, FT-IR, Nile red labeling, endotoxin testing, and culture-media stability/protein-adsorption assays. AOAOs were exposed under two submerged configurations: direct application of PS-MPs to the organoid surface or indirect exposure to conditioned media from PS-MP-treated THP-1-derived macrophages. CCK-8, MTT, and LDH range-finding assays identified 1 and 100 μg/mL as non-cytotoxic nominal submerged doses for macrophage-organoid experiments. Direct exposure of AOAOs to small, medium, or large PS-MP fractions did not induce marked TNF-α expression, mucin secretion, epithelial barrier disruption, or goblet cell hyperplasia. In contrast, the small PS-MP fraction showed the strongest macrophage association and induced extracellular IL-1β release; conditioned media from small PS-MP-exposed macrophages increased TNF-α expression and Muc5ac-positive goblet cell populations in AOAOs. Selective caspase-1 activation in the small 100 μg/mL condition, together with cleaved GSDMD immunoreactivity and LDC7559-sensitive IL-1β release, supported a caspase-1-associated, GSDMD-sensitive component of IL-1β secretion, but did not establish definitive canonical inflammasome activation. This AOAO platform provides a submerged mechanistic proof-of-concept for dissecting macrophage-conditioned airway epithelial remodeling by PS-MPs, while not representing aerosol deposition or inhalation-equivalent dosimetry.