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

The research team employed imaging mass cytometry (IMC), a technology that uses metal-conjugated antibodies and mass spectrometry to detect dozens of proteins simultaneously on a single tissue section. Where conventional immunofluorescence is limited to roughly four or five markers because of spectral overlap, IMC allowed the researchers to build a panel of 32 antibodies covering 19 cell population markers, 3 activation markers and 10 ECM components. Critically, the team went beyond standard cellular analysis. Because matrix molecules sit outside cells, traditional cell-mask-based approaches fail to capture them. To solve this, the researchers developed DeepThresh, a novel deep-learning thresholding algorithm modelled on a U-Net architecture with a ResNet152 encoder. Trained on expert-annotated images, DeepThresh generated robust binary masks of ECM staining across all tissue regions, overcoming the variability that plagued classical thresholding methods such as Otsu’s algorithm.

With matrix staining segmented, the pipeline calculated Euclidean distances from every cell to each ECM component, and these distance profiles were clustered to define twelve distinct lung matrix environments. This is the study’s central methodological innovation: for the first time, every cell in the image carries both its canonical identity and a quantitative description of its immediate matrix surroundings. The approach was applied to lung sections from BALB/c and C57BL/6 mice, two widely used inbred strains, following chronic twice-weekly intranasal exposure to a cocktail of house dust mite, ragweed and Aspergillus extracts, a model known as DRA that reproduces key features of human allergic airway pathology, including collagen deposition, hyaluronan accumulation and steroid-resistant recruitment of eosinophils and neutrophils.

The resulting atlas revealed that the healthy lung is divided into two major matrix compartments. The alveolar parenchyma, the delicate gas-exchange region, is characterised by proximity to heparan sulphate, laminin gamma-1, type-IV collagen and fibrinogen. In contrast, the adventitial cuff, the connective tissue sheath surrounding airways and blood vessels, is enriched for type-I, type-III and type-VI collagens together with hyaluronan. During allergic inflammation, these spatially constrained environments changed in strikingly region-specific ways, and the two mouse strains responded differently, underscoring how genetic background shapes not just the magnitude of inflammation but its anatomical architecture.

One of the most visually dramatic findings concerned the adventitial cuff. In allergic animals, infiltrating immune cells accumulated in discrete, contiguous patches around the pulmonary artery and airway-adjacent vessels, and these patches were significantly more numerous and larger in BALB/c mice than in C57BL/6 mice. Three-dimensional precision-cut lung slices stained with fluorescent antibodies confirmed the IMC findings and revealed internal structure within the cuff: the region adjacent to the blood vessel was dominated by CD11b-positive CD64-positive monocyte-derived macrophages, while a more distal, densely nucleated zone resembled induced bronchus-associated lymphoid tissue (iBALT), rich in B cells and largely excluding macrophages. These iBALT-like structures formed only in BALB/c animals, suggesting that strain-specific type-2 immune skewing may provide the signals needed to seed organised lymphoid tissue within the inflamed lung.

The matrix itself shifted around these inflammatory patches in a coordinated fashion. Allergic BALB/c mice showed expansion of sparse ‘inflammatory zone’ matrix environments with increased distances to type-I, type-III and type-VI collagens, but reduced distances to laminin gamma-1 and hyaluronan. This represents a shift from a densely collagenous matrix to one dominated by laminin and hyaluronan, molecules known to regulate cell migration through tissue. Because hyaluronan-rich matrices can facilitate or direct immune cell infiltration, this remodelling may actively sculpt the localisation of B cell activation and myeloid accumulation within the cuff, offering a mechanistic hypothesis for why inflammation aggregates in these specific niches.

The alveolar parenchyma told a different story. Following allergen challenge, the ratio of alveolar type-I (ATI) to alveolar type-II (ATII) epithelial cells fell from roughly 3:1 in healthy controls to approximately 1:1 in allergic animals of both strains, a signature of injury and repair, since ATII cells are known to proliferate and replace damaged ATI cells. Immunofluorescent validation using RAGE as an ATI marker and surfactant protein C as an ATII marker confirmed the relative loss of the ATI compartment. Spatially, the alveolar region subdivided into resting alveoli enriched for ATI cells and basement membrane components, and activated alveolar regions enriched for ATII cells, alveolar macrophages and proximity to hyaluronan and chondroitin sulphate. These glycosaminoglycans are known regulators of immune cell migration, and chondroitinase treatment in fibrosis models reduces macrophage numbers, suggesting that this matrix shift may directly control macrophage retention in the damaged alveolus.

Perhaps the most translational insight concerns the airway subepithelial space, the region beneath the airway epithelium that undergoes extensive remodelling in human asthma. The study found that this region expanded significantly during allergic challenge in both strains and became a hub of immune-stromal interaction. Two fibroblast populations were identified there: alpha-smooth muscle actin-positive stromal cells, present at steady state, and S100a4-positive fibroblasts, which expanded dramatically during allergy. Neighbourhood analysis showed that S100a4-positive fibroblasts and CD11b-positive immune cells, including MerTK-positive macrophages, came into close contact specifically during allergic inflammation, and both stromal populations overlayed type-I and type-III collagen deposition. Because macrophages and fibroblasts are known to regulate each other reciprocally, and because type-VI collagen in this niche can promote fibroblast survival and migration, the authors propose that this immune-stromal network constitutes a regulatory circuit controlling ECM deposition during allergic airway pathology.

The study’s design choices also matter for future work. The entire pipeline was developed on formalin-fixed paraffin-embedded tissue, the predominant preservation method in human biobanks, meaning the approach can be translated directly to archived patient samples. All datasets and code, including the DeepThresh and MatrixIMC pipelines, have been made publicly available. By integrating cellular and matrix analysis within a single tissue section, the framework captures a dimension of tissue biology that single-cell RNA sequencing alone cannot, since transcriptomic profiles of matrix genes are often poor surrogates for the post-translationally modified proteins actually present in tissue. The authors caution that future work must clarify the temporal sequence of these changes and establish causality, but the atlas already generates testable hypotheses about how targeting specific matrix components or the macrophage-fibroblast interactions within remodelling niches could intervene in allergic airway disease. For a field that has long catalogued inflammation cell by cell, this study makes a compelling case that where a cell sits, and what it sits on, may matter as much as what the cell is.

Subject of Research: Spatial mapping of extracellular matrix and cellular environments during allergic airway inflammation using imaging mass cytometry in mouse models

Article Title: Extracellular matrix phenotyping by imaging mass cytometry defines distinct cellular matrix environments associated with allergic airway inflammation

Article References: Parkinson, J. E., Bryant, M., Ghafoor, M., Dodd, R. J., Tompkins, H. E., Fergie, M., Burgess, M. O., Rattray, M., & Sutherland, T. E. (2026). Extracellular matrix phenotyping by imaging mass cytometry defines distinct cellular matrix environments associated with allergic airway inflammation. Molecular Systems Biology. https://doi.org/10.1038/s44320-026-00234-5

Image Credits: AI Generated

DOI: 10.1038/s44320-026-00234-5

Keywords: imaging mass cytometry, extracellular matrix, allergic airway inflammation, asthma, airway remodelling, lung immunology, hyaluronan, collagen, fibroblasts, macrophages, spatial biology, mouse models