Comparison of different alveolar macrophage in vitro models to recapitulate inhaled carbon nanomaterial-induced cellular injury

Ren, Hongyu (2026): Comparison of different alveolar macrophage in vitro models to recapitulate inhaled carbon nanomaterial-induced cellular injury. Dissertation, LMU München: Faculty of Medicine

Abstract

Engineered carbon nanomaterials (CNMs), such as carbon nanotubes and carbon black, are used in many industrial and biomedical applications. Owing to their respirable size and biopersistence, inhaled CNMs deposit in the distal lung and may persist there for long periods. Alveolar macrophages (AMs), as the first line of defence in the alveolar space, are a main cellular target for these particles. Previous research from our group has shown that inhalation of CNMs induces persistent AM depletion without restoration, suggesting AM cell death in a shape dependent manner in which fiber - shaped multi-walled carbon nanotube (MWCNT) having the strongest effect, followed by tangled double-walled carbon nanotubes (DWCNT), whereas spherical carbon nanoparticles (CNP) do not reduce AM numbers (Voss C et al., 2024). Yet how CNM shape and agglomeration state together affect AM cellular homeostasis, cell death and subsequent inflammatory responses has not been systematically studied. A stable and reproducible CNM dispersion system relevant to biological conditions, as well as an in vitro AM culture system that recapitulates the phenotype and functions of in vivo resident alveolar macrophages (resAMs), is a prerequisite in CNM toxicology research. Therefore, surfactant-based suspensions were established that were more stable and showed fewer large aggregates than other dispersants. In parallel, transcirptomic and phenotypic comparisons between several AM models including AMs cultured for seven days with a cytokine cocktail (AMs GTR), bone marrow-derived AM-like macrophages (BM-AMs) and immortalised AM-like cell lines demonstrated that AMs GTR and BM-AMs are the best suited cell models. To study underlying mechanisms at organelle-level. AMs GTR, BM-AMs and MHS cells were exposed to MWCNT, DWCNT and CNP, and lysosomal, mitochondrial changes as well as cellular uptake and cell migration were investigated. Here, we found that MWCNT induced significant lysosomal membrane destabilization with cathepsinB leakage, mitochondrial membrane depolarization, followed by reduction in cellular migration velocity and inflammatory cytokine release in both AMs GTR and BM-AMs. In contrast, DWCNT and CNP mainly reduced lysosomal acidity without extensive membrane rupture, mild mitochondrial changes whereas cellular movement and inflammatory cytokine release was largely unaffected in both AMs GTR and BM-AMs. Further pharmacological inhibition of cysteine cathepsins mitigated MWCNT and DWCNT but not CNP triggered cell viability loss at higher doses in vitro. Notably, pretreatment of AMs GTR and BM-AMs with cathepsin inhibitors attenuated MWCNT and CNP uptake at low doses. Taken together, our findings suggest that MWCNT induce similar lysosomal and mitochondrial damage, cathepsin-dependent cell death and inflammatory cytokine release in both AMs GTR and BM-AMs that can be potentially mapped with in vivo sterile lung inflammation induced by MWCNT. In addition, pharmacological inhibition of cysteine cathepsins reduces CNM internalization and alleviates cathepsin-related cell death providing a potential therapeutic target for diseases and exacerbations driven by environmental and engineered nanomaterials.

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: alveolar macrophages, carbon-based nanomaterials, AMs GTR, BM-AMs, MWCNT
• Subjects: 600 Technology, Medicine; 600 Technology, Medicine > 610 Medical sciences and medicine
• Faculties: Faculty of Medicine
• Language: English
• Date of oral examination: 15. June 2026
• 1. Referee: Rehberg, Markus
• Signature of the printed copy: 0700/UMD 22872