Thorough assessment of primary cell cultures for murine and human alveolar macrophages - 20/03/24

Alveolar macrophages (AM) are the first-line lung defenders, but appropriate cell culture models are still limited for their study. The uses of freshly isolated macrophages are hampered by limited quantities or difficult access to human donors. Indeed, research on AM is often restricted to cells differentiated in vitro from bone-marrow progenitors or blood monocytes; however, such cells do not present the specific features of tissue-specialized macrophages, like AM. To overcome these issues, Max Planck Institute (MPI) cells were characterized as a nontransformed, self-renewing AM-like macrophage murine model. Yet, MPI cannot fully reproduce AM characteristics because they developed outside the alveolar niche. To optimize the primary cell cultures of mouse AM (mAM), recent works have proposed strategies to expand and maintain these cells ex vivo from bronchoalveolar lavage (BAL). The functional impact of the amplification process is unclear. Moreover, the heterogeneity of AM population, which is constantly remodeled during the lifespan, is poorly recapitulated in laboratory mice, therefore limiting extrapolation of mAM physiopathology to human. Here, we propose a comprehensive comparison of mice and human AM (hAM) culture models and give insight into the translational value of different mice AM models.

Ex vivo primary mAM were isolated from BAL of 3–5 mice and expanded for 3–4 weeks per passage. hAM were freshly isolated and maintained from BAL of uninfected patients. Immunophenotypes of MPI, mAM and hAM were compared. We evaluated the cells production of inflammatory mediators (IL-6, TNF-α, IL-1β) in response to different pro-inflammatory agonists such as LPS, poly (I: C), flagellin, Pam³CSK4 and curdlane. We induced cell death using LPS/ATP and measured real-time cell death using IncuCyte SX5 cell-live analysis.

Immunophenotypes of MPI and mAM were comparable despite lower SiglecF and CD₁₁c expression in MPI or mAM after expansion compared to freshly isolated mAM. MPI, mAM and hAM presented similar inflammatory signature but differences in the amplitude of response (exception for Flagellin that induced IL-1-β response in murine models but not in hAM). Overall, MPI had a more pro-inflammatory profile than mAM, as illustrated by a 10-fold production of IL-6 in response to LPS compared to mAM. Differences were observed regarding cell death in mice and human AM models: pyroptotic events were more rapid (1h) and important in murine AM models compared to hAM.

While clarifying the limits of MPI and ex vivo mAM models, our work showcased consistent inflammatory responses across AM models, albeit with differing response amplitudes. Conversely, mouse and human models exhibited variations in cell death kinetics.