Immortalized lung macrophage cell line keeps primary-cell identity, offering new tool for respiratory research

Alveolar macrophages are the sentinels of the lung. Nestled within the delicate air sacs where oxygen exchange takes place, these immune cells patrol constantly, engulfing inhaled debris and pathogens while carefully suppressing inflammation against harmless stimuli. When a genuine threat appears, they switch gears, presenting antigen, recruiting neutrophils, and releasing inflammatory signals that coordinate the lung’s defense. Despite their central role in respiratory immunity and in diseases ranging from asthma to pulmonary fibrosis, studying them has long been an exercise in frustration, because the cells that matter most are also the hardest to obtain and keep alive in the laboratory.

A research team at Sun Yat-sen University in Guangzhou now reports a solution that could reshape how lung immunology is done. In a study published in Advanced Biotechnology, the researchers describe an immortalized mouse alveolar macrophage cell line, created not by introducing cancer-causing oncogenes but by switching on telomerase, the enzyme that naturally maintains the protective caps at the ends of chromosomes. The resulting cells, named iAMs, divide indefinitely yet, according to the team’s detailed molecular and functional profiling, retain an unmistakably primary-like identity.

The motivation is easy to appreciate. Harvesting primary alveolar macrophages requires flushing the airways of experimental animals, a procedure that yields only 200,000 to 500,000 cells per mouse. That quantity is quickly consumed by basic characterization experiments, leaving little material for the high-throughput screens, genetic manipulations, and replication studies on which modern biomedical science depends. Worse still, the cells rapidly lose their defining traits once removed from the alveolar environment, senescing quickly in culture dishes.

Existing cell lines have not filled the gap convincingly. The AMJ2-C8 and AMJ2-C11 lines, created decades ago by v-raf and v-myc oncogene transduction, respond abnormally to bacterial products such as lipopolysaccharide and muramyl dipeptide and fail to secrete adequate amounts of the inflammatory cytokines IL-1 and TNF. The widely used MH-S line, immortalized with SV40 T antigen, lacks Siglec-F, one of the most recognizable surface markers of the mouse alveolar macrophage, and does not express the receptor CD116, leaving it deaf to GM-CSF, a growth factor central to alveolar macrophage biology, and blunted in its responses to bacterial stimulation.

The Sun Yat-sen team chose a gentler strategy. Human telomerase reverse transcriptase, or hTERT, has previously been used to immortalize a range of primary cell types without dragging them toward a cancer-like state, because it extends replicative lifespan rather than rewiring growth-control pathways. The researchers packaged hTERT into a lentiviral vector and delivered it to primary alveolar macrophages isolated from C57BL/6 mice by bronchoalveolar lavage. After selection with puromycin and isolation of single-cell clones, four stable lines emerged, all confirmed free of mycoplasma contamination and indistinguishable from one another in morphology, marker expression, and growth. Short tandem repeat profiling verified that the final line traced back to the original primary cells, and all subsequent work used cells between passages 1 and 12.

The proliferative leap is dramatic. Where primary alveolar macrophages stagnate, the iAMs double roughly every 12 hours, nearly all of them staining positive for the proliferation marker Ki67 and incorporating the DNA precursor EdU at rates far above those of their primary counterparts. Crucially, this vigor did not come at the cost of appearance. Under bright-field and transmission electron microscopy, the cells resemble freshly isolated alveolar macrophages, with similar Giemsa and Oil Red O staining patterns. Scanning electron microscopy revealed one subtle difference: the immortalized cells keep the spherical shape of their primary ancestors but extend fewer pseudopodia, the membrane protrusions macrophages use to crawl and engulf.

Transcriptionally, the resemblance holds up under rigorous scrutiny. Bulk RNA sequencing compared iAMs with primary alveolar macrophages, with AM-like cells differentiated from bone marrow precursors using GM-CSF, TGF-beta and rosiglitazone, and with bone marrow-derived macrophages, the workhorse surrogate in many labs. Principal component analysis placed the iAMs closest to primary alveolar macrophages, and pairwise correlation and hierarchical clustering confirmed the same picture. Out of the whole transcriptome, differential expression analysis flagged 2,953 genes changing by at least twofold between iAMs and primary cells, concentrated in immune signaling and metabolic pathways. Functional module scoring showed the immortalized cells mirroring primary cells across lysosome, phagocytosis, oxidative phosphorylation, chemotaxis and M2 polarization programs, with antigen presentation scoring somewhat higher in the immortalized line.

Surface phenotype and function tracked the transcriptome. Flow cytometry confirmed the canonical alveolar macrophage signature of intermediate F4/80, low CD11b, high Siglec-F and high CD11c, and quantitative PCR validated expression of AM-specific, alternative activation and self-renewal genes at levels comparable to primary cells. In functional assays, the iAMs actually outperformed primary cells in some respects: they engulfed fluorescently labeled E. coli more avidly than either primary alveolar macrophages or AM-like cells, and they migrated more efficiently toward Pseudomonas aeruginosa in Transwell chemotaxis chambers. When stimulated with lipopolysaccharide, lipoteichoic acid, or heat-inactivated Pseudomonas, they mounted inflammatory cytokine responses, including Il1a, Il1b, Il6, Ccl2 and Tnfa, closely matching those of primary cells.

The most demanding test came in living animals. The team depleted resident alveolar macrophages in mice with clodronate liposomes, achieving roughly 95.7 percent depletion, and then transferred iAMs directly into the airways before challenging the animals with Pseudomonas aeruginosa. The transferred cells engrafted stably in the alveolar space and, tracked with the fluorescent dye PKH26, were still detectable 14 days later, by which point they accounted for more than half of the CD11c-positive population. Mice lacking alveolar macrophages developed severe lung injury, with blood-tinged and turbid lavage fluid, elevated protein levels, massive neutrophil infiltration and high bacterial burdens. Reconstitution with iAMs significantly eased every one of these pathological features, restoring bacterial clearance and restraining neutrophil recruitment, evidence that the immortalized cells can genuinely stand in for their natural counterparts in pulmonary host defense.

The authors are candid about the model’s limits. Immortalization and long-term culture can introduce genetic or epigenetic drift and clonal selection, and the enhanced antigen-presenting capacity and reduced pseudopod formation show the cells are not perfect copies of freshly isolated macrophages. The team recommends working within the validated passage range and monitoring key markers and functions, with critical findings confirmed in primary cells when maximum fidelity is required. Even with those caveats, the resource fills a conspicuous void. Bone marrow-derived macrophages and monocyte-derived macrophages differ fundamentally from tissue-resident macrophages in developmental origin, transcriptional wiring and functional repertoire, and oncogene-transformed lines carry distortions of their own. A telomerase-immortalized line that preserves the alveolar macrophage identity opens the door to high-throughput drug screening, CRISPR-based mechanistic dissection and reproducible functional assays that were previously impractical, and it promises to accelerate research on lung immunology, host defense and pulmonary homeostasis for years to come.

Subject of Research: Generation and characterization of an hTERT-immortalized mouse alveolar macrophage cell line

Article Title: hTERT-immortalized mouse alveolar macrophages retain primary-like transcriptional and functional programs

Article References: hTERT-immortalized mouse alveolar macrophages retain primary-like transcriptional and functional programs. (n.d.). https://doi.org/10.1007/s44307-026-00130-x

Image Credits: AI Generated

DOI: 10.1007/s44307-026-00130-x

Keywords: alveolar macrophages, hTERT, cell immortalization, telomerase, lung immunology, phagocytosis, Pseudomonas aeruginosa, RNA sequencing, host defense, macrophage cell line, pulmonary homeostasis, GM-CSF