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 2

Coculture with iMacs promotes iAT2 transcriptional signature.

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Coculture with iMacs promotes iAT2 transcriptional signature. (A) UMAP o...
(A) UMAP of iAT2 cells alone (pink) and iAT2 cells after coculture with iMacs (purple) for 5 days. (B) Louvain clustering at a resolution of 0.1. (C) Proportion of iAT2 alone (pink) or iAT2 after coculture (purple) in each cluster at Louvain resolution 0.1. (D) Volcano plot of differentially expressed genes upregulated in iAT2 alone (pink, left) or iAT2 after coculture (purple, right). (E) Gene set enrichment analysis depicting upregulated pathways in iAT2s after coculture compared with iAT2s alone. (F) Violin plots of differentially expressed genes including glycosylated surfactants SFTPB (log2FC = 0. 41, adjusted P = 1.65 × 10–14) and (G) surfactant transporter ABCA3 (log2FC = 0.37, adjusted P = 9.35 × 10–5). Statistical significance for F and G determined by Wilcoxon rank-sum test. (H) Module score of AT2 maturation gene set (45) indicating that coculture enhances AT2 maturation. Statistical significance was determined by Welch’s 2-sample t test. (I) iAT2s were treated with the lipophilic dye FM4-64, washed, and then treated with a secretagogue cocktail (ATP and PMA). iMacs were immediately added and incubated for 20 or 90 minutes prior to collection and flow cytometry to measure internalized FM4-64 in iMacs. iMacs were treated with cytochalasin D to inhibit phagocytosis. Statistical significance was determined by 2-way ANOVA. n = 3 experimental replicates of independent wells of a differentiation; data shown as mean ± SD. Statistical significance tests indicated for each panel; *P < 0.05.