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Abstract

Introduction: Lung alveolar macrophages (AMs) and epithelial cells form the first line of defense against inhaled pathogens. Their interactions strongly influence innate immune responses in the lung, yet the mechanisms underlying this cross-talk remain incompletely understood. Methods: In this study, we established a co-culture system using a primary model of AMs (MPI alveolar macrophage-like cells) and MLE-12 alveolar epithelial cells to investigate innate responses and cellular interactions during bacterial lipopolysaccharide (LPS)-induced TLR4 activation. Results: Cytokine and chemokine profiling revealed that co-cultures exhibited significantly enhanced proinflammatory responses to both LPS and TLR2 ligands—including IL-6, TNF-a, and MCP-1 secretion—compared with mono-cultures. Strikingly, we identified MLE-12 epithelial cells as a source of lipopolysaccharide-binding protein (LBP), which is essential for LPS recognition in AMs and MPI alveolar macrophage-like cells. LBP secretion by epithelial cells explained cytokine responses to LPS under serum-free conditions; however, additional mechanisms—apparent in the presence of serum/LBP—also contributed to the amplified co-culture responses. These mechanisms included direct cell–cell contacts, as conditioned media from unstimulated cells failed to reproduce similar effects in mono-cultures. Moreover, co-cultures of naïve MPI cells and inflamed epithelial cells (MLE-12 cells pretreated with media from activated MPI macrophages) were found to release a nonnegligible amount of chemokines, even in the absence of LPS. This demonstrated an inflammatory amplification loop mediated by both contact dependent and soluble factors. Phospho-flow cytometry further revealed coculture- specific signaling, with enhanced MAPK pathway activation in macrophages and NF-kB activation in epithelial cells. Finally, LPS-activated MPI alveolar macrophage-like cells induced TNF-a–dependent ICAM-1 expression and apoptosis in MLE-12 cells. Increased ICAM-1 expression, in turn, promoted MCP-1 production in epithelial cells in an ICAM-1–dependent and cell contact mediated manner. Discussion: Together, these findings identify cellular contacts and a TNF-a–ICAM-1–MCP-1 axis—supported by epithelial-derived LBP—as key drivers of innate immune synergy between lung alveolar macrophages and epithelial cells. Our results establish the MPI–MLE-12 co-culture as a tractable model for dissecting pulmonary innate immune mechanisms.

Publication Date

2026-01-09

Publication Title

Frontiers in Immunology

Volume

16

Acceptance Date

2025-12-15

Deposit Date

2026-03-06

Funding

The author(s) declared that financial support was received for this work and/or its publication. Institut Pasteur Korea is a member of the Institut Pasteur International Network (https://pasteur-network.org/). This research was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT), through institutional grants NRF-2017M3A9G6068246 and RS-2024-00398073, and an individual grants (NRF-2019R1A2C2084652 to VD and RS-2025–02272981 to CW). Additional support was provided by Gyeonggi Province, Korea. This work was also funded by grants from the United Kingdom NERC (NE/M011747/1 to SJ and GF) and NC3Rs (NC/V001019/1 and NC/X002446/1 to GF.) BioAirNet (NE/V002171/1 to SJ, ZN and FC). VD was supported by the French ministry of foreign affairs.

Keywords

co-culture, epithelial cells, intercellular adhesion molecule 1 (ICAM-1), LPS, lung alveolar macrophages, MPI cells, Cell Line, Acute-Phase Proteins/metabolism, Coculture Techniques, Membrane Glycoproteins/metabolism, Immunity, Innate, Lung/immunology, Lipopolysaccharides/immunology, Animals, Tumor Necrosis Factor-alpha/metabolism, Alveolar Epithelial Cells/immunology, Chemokine CCL2/metabolism, Macrophages, Alveolar/immunology, Mice, Cell Communication/immunology, Epithelial Cells/immunology, Carrier Proteins/metabolism

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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