Macrophages orchestrate antiviral defense and epithelial repair in a human iPSC-derived alveolar air-liquid interface
Declan L. Turner, Hannah Baric, Katelyn Patatsos, Sahel Amoozadeh, Michael See, Kathleen A. Strumila, Jack T. Murphy, Jeremy J. Wiyana, Liam Gubbels, Elizabeth S. Ng, Andrew G. Elefanty, Melanie R. Neeland, Shivanthan Shanthikumar, Sarah L. Londrigan, Mirana Ramialison, Fernando J. Rossello, Ed G. Stanley, Rhiannon B. Werder
Declan L. Turner, Hannah Baric, Katelyn Patatsos, Sahel Amoozadeh, Michael See, Kathleen A. Strumila, Jack T. Murphy, Jeremy J. Wiyana, Liam Gubbels, Elizabeth S. Ng, Andrew G. Elefanty, Melanie R. Neeland, Shivanthan Shanthikumar, Sarah L. Londrigan, Mirana Ramialison, Fernando J. Rossello, Ed G. Stanley, Rhiannon B. Werder
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Research Article Cell biology Infectious disease Inflammation

Macrophages orchestrate antiviral defense and epithelial repair in a human iPSC-derived alveolar air-liquid interface

Abstract

The lung alveoli are continually exposed to inhaled pathogens and environmental hazards and rely on coordinated communication between alveolar macrophages and type 2 alveolar epithelial cells (AT2s) to maintain homeostasis. Disruption of these interactions can impair immunity and repair, contributing to acute and chronic respiratory diseases. To better define these mechanisms and support therapeutic discovery, we established a human iPSC-derived air-liquid interface platform that captures key features of AT2-macrophage crosstalk. Using this system, we show that coculture enhances AT2-specific transcriptional programs including lipid synthesis, while macrophages actively phagocytose AT2-derived surfactant. iPSC-derived macrophages adopt an alveolar macrophage–like phenotype and respond to AT2-derived M-CSF. During respiratory infection, macrophages play a crucial role in modulating epithelial inflammatory responses, augmenting antiviral immunity, and limiting viral replication. We further identify a role for macrophages in epithelial repair, where VEGF-mediated signaling to macrophages increases epithelial permeability during viral infection. Together, these findings reveal dimensions of AT2-macrophage cooperation in homeostasis, infection, and repair, and demonstrate how this iPSC-derived platform can be used to dissect mechanisms that may initiate or drive the progression of respiratory diseases.

Authors

Declan L. Turner, Hannah Baric, Katelyn Patatsos, Sahel Amoozadeh, Michael See, Kathleen A. Strumila, Jack T. Murphy, Jeremy J. Wiyana, Liam Gubbels, Elizabeth S. Ng, Andrew G. Elefanty, Melanie R. Neeland, Shivanthan Shanthikumar, Sarah L. Londrigan, Mirana Ramialison, Fernando J. Rossello, Ed G. Stanley, Rhiannon B. Werder

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Figure 3

iMacs are sustained in coculture by iAT2-derived M-CSF.

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iMacs are sustained in coculture by iAT2-derived M-CSF. (A) UMAP of iMac...
(A) UMAP of iMacs alone (blue) or iMacs after coculture with iAT2s (green) for 5 days. Populations clustered distinctly based on original identity. (B) UMAPs showing expression of CD14 and CD68. (C) UMAP of cell cycle phase. (D) Volcano plot of differentially expressed genes upregulated in iMac alone (left) or iMac after coculture (right). (E) scType analysis of iMacs alone or iMacs after coculture with iAT2s. (F) CSF1 and CSF2 expression in adult human AT2s (97) and iAT2s (32, 33). (G) M-CSF and GM-CSF secretion was measured by ELISA from undifferentiated iPSCs, iAT2s in 3D conditions, and iAT2s at ALI. Statistical significance was determined by 1-way ANOVA; compared with iPSCs or as indicated. (H) CSF1 expression in iAT2 alone (pink) or iAT2 after coculture with iMacs (purple). CSF1 expression was increased in cocultured iAT2s. Statistical significance was determined by Wilcoxon rank-sum test (log2FC = 0.37, adjusted P = 1.37 × 10–6). (I) M-CSF secretion was measured in the basolateral compartment of iAT2 or iAT2-iMac cocultures at ALI. Levels normalized to iAT2 alone. (J) The CSF signaling pathway network identified by CellChat analysis of iAT2s in coculture (purple) or iMacs in coculture (green). (K) Conditioned CK-DCI from iAT2s was added to iMacs alone. Alternatively, iMacs were cultured in CK-DCI supplemented with M-CSF or GM-CSF. MFI of HLA-DR was measured by flow cytometry after 72 hours. Statistical significance was determined by ANOVA with Tukey’s post hoc test. (L) iMacs treated with conditioned CK-DCI from iAT2s were incubated with neutralizing antibodies against M-CSF, GM-CSF, or both. HLA-DR MFI was measured by flow cytometry after 72 hours. Statistical significance was determined by 1-way ANOVA with Tukey’s post hoc test; *P < 0.05, ***P < 0.001, ****P < 0.0001.