Immunometabolic Properties of Tissue-Resident Alveolar Macrophages Depend on ex vivo Culturing Conditions

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Olga A. Namakanova^1,2,a*, Anastasia S. Yakovleva^1,2,3, Ekaterina O. Gubernatorova^1,2, Tamara V. Pukhalskaia^4,5, Sergei A. Nedospasov^1,2,3,4, and Marina S. Drutskaya^1,2,3,4,b*

¹Laboratory of Molecular Mechanisms of Immunity, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia

²Center for Precision Genome Editing and Genetic Technologies for Medicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia

³Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

⁴Department of Immunobiology and Biomedicine, Scientific Center for Genetics and Life Sciences, Sirius University of Science and Technology, 354349 Sirius Federal Territory, Russia

⁵Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia

^* To whom correspondence should be addressed.

Received: November 10, 2025; Revised: December 4, 2025; Accepted: December 25, 2025

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Alveolar macrophages (AMs) comprise the predominant immune cell population in the lungs, maintaining homeostasis and providing the first line of immune defense against various respiratory diseases. Most studies focus on macrophages differentiated from bone marrow precursors in vitro. However, the ontogeny of the tissue-resident macrophages and the lung microenvironment significantly determine their properties and functions, fundamentally distinguishing AMs from the cells derived in vitro. The use of AMs ex vivo, which maximally preserves their original phenotype and proliferative potential, is the most physiological and informative approach for studying various aspects of innate immune responses in lung diseases. Improving methods for their isolation and culturing remains an important task for obtaining relevant data on the functions of these cells. In this study, we evaluated the effect of enzymatic treatment, the most common method for detaching cultured AMs, on their phenotype. It was found that enzymatic treatment led to the decrease in production of GM-CSF and VEGF by the AMs mediators of myeloid cell differentiation and maturation, and angiogenesis, respectively, both in the non-activated state and in response to one of the most common allergens – house dust mite extract (HDM). Enzymatic treatment promoted the formation of a pro-inflammatory phenotype, manifested by the increased production of IL-6, as well as chemokines CCL4 and CCL5, which attract monocytes and lymphocytes to the site of inflammation, along with the trend toward increased expression of the M1-associated genes such as Nos2 and Cd38, in response to HDM. Thus, enzymatic detachment of the tissue-resident AMs promotes M1 polarization, which predetermines a more pronounced response to the allergen in vitro. The presented data highlight disadvantages of the enzymatic treatment of AMs, increasing the risk of artifacts, and affecting reliability of the experimental results.

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KEY WORDS: tissue-resident cells, alveolar macrophages, HDM, accutase, IL-6

DOI: 10.1134/S0006297925603867

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