Abstract

Introduction

Macrophages, integral to the immune system, play diverse roles shaped by their origin, tissue context, and environmental cues. Due to the limited availability of lung macrophages and the inadequacies of current models in faithfully replicating the physiological characteristics of macrophages in their natural in vivo state, the primary objective of this study was to establish optimal culture conditions for in vitro differentiation and study of macrophages, particularly human and porcine alveolar-like macrophages (AMs). While previous murine models provided valuable insights, interspecies differences in innate immune responses necessitate the creation of specific human and porcine models.

Methods

We established an in vitro model of GM-CSF-dependent human AM-like macrophages (GMDMs) by culturing peripheral blood mononuclear cells (PBMCs) in Teflon bags under the influence of granulocyte-macrophage colony-stimulating factor (GM-CSF), crucial for the development and maintenance of AMs. Simultaneously, the porcine PLTA58 cell line was derived from splenic embryonic tissue, cultured with GM-CSF, and immortalised with pLenti-CMV-SV40 Large T (tsA58)-GFP-Puro vector encoding SV40 Large T (LTAg), a temperature-sensitive protein. The phenotypic characteristics of these models were explored.

Results

The study successfully established GMDMs and a porcine macrophage cell line, offering reliable in vitro models for human and porcine AMs, respectively. The GMDMs resembled BAL-derived macrophages in the RNA profile. Stimulation with various ligands, investigation of LPS signalling, and examination of responses to OC43 coronavirus and influenza A virus (IAV) contributed novel insights into inflammatory cytokine production and the unique requirement for CD14 and independency of LPS binding protein (LBP) demonstrated so far unknown mechanisms of innate immune signalling in GMDMs. PLTA58 cells resembled the parental non-transformed cells in morphology, phagocytic ability, macrophage surface marker expression and innate responses to stimuli. The cells showed enhanced proliferation through increased cell cell contact. The proteomic analysis demonstrated the influence of the SV40 Large T antigen on crucial macrophage functions.

Conclusion

Our results provide essential insights into the biology of macrophages from different species and demonstrate that these human and porcine alveolar-like macrophage systems provide good AM models to study general and specific macrophage functions. Both macrophage models can be employed to investigate lung diseases while reducing the need to kill animals.

Keywords

Macrophages, Alveolar macrophages, Innate immunity, Respiratory infection

Document Type

Thesis

Publication Date

2024

Embargo Period

2024-07-17

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