88 Modelling the alveolar niche using mono-, co-, and triple-culture systems to mimic the inflammatory response to inhaled nano materials

Abstract Carbon-based nanomaterials (CBN), including carbon nanotubes (CNT), are increasingly used in industry, yet inhalation of soot-like carbon nanoparticles (CNP) and engineered CNT is linked to lung inflammation and interstitial lung disease. Prior single-cell RNA sequencing identified material- and cell-specific inflammatory circuits in vivo, but monocultures fail to capture the multicellular crosstalk that drives these responses. We modelled the alveolar niche using mono-, co-, and triple-culture systems with MLE-12 (epithelial), J774.1 (macrophage), and CCL206 (fibroblast) cells. Mitochondrial activity and membrane integrity were assessed 24 h post-exposure (WST-1, LDH). J774.1 macrophages were most sensitive, with IC50 ≈ 25 µg/mL for CNP and 35 µg/mL for MWCNT; LDH release was negligible. qPCR at 6 h revealed particle- and cell-specific induction of inflammatory mediators consistent with in vivo profiles. In monocultures, Cxcl1 was strongly induced in MLE-12 by high-dose CNP and MWCNT, while J774.1 upregulated Ccl2 and Ccl3 at both CNT exposure levels. MLE12/J774.1 co-cultures amplified cytokine signalling, particularly Csf2, indicating strong epithelial–macrophage interaction. Triple co-culture maintained broad chemokine expression (Cxcl1, Ccl2, Ccl3) and high Csf2 but did not exceed levels observed in the MLE-12/J774.1 co-culture, suggesting fibroblasts provide supportive rather than amplifying effects under these conditions. These findings demonstrate that multicellular systems are essential to reproduce cytokine networks triggered by CBN exposure. Aligning these in vitro responses with macrophage–epithelial–fibroblast circuits observed in vivo provides a mechanistic basis for physiologically relevant models in nanomaterial safety assessment.