Abstract
Disruption of the lung endothelial–epithelial cell barrier following respiratory virus infection causes cell and fluid accumulation in the air spaces and compromises vital gas exchange function1. Endothelial dysfunction can exacerbate tissue damage2,3, yet it is unclear whether the lung endothelium promotes host resistance against viral pathogens. Here we show that the environmental sensor aryl hydrocarbon receptor (AHR) is highly active in lung endothelial cells and protects against influenza-induced lung vascular leakage. Loss of AHR in endothelia exacerbates lung damage and promotes the infiltration of red blood cells and leukocytes into alveolar air spaces. Moreover, barrier protection is compromised and host susceptibility to secondary bacterial infections is increased when endothelial AHR is missing. AHR engages tissue-protective transcriptional networks in endothelia, including the vasoactive apelin–APJ peptide system4, to prevent a dysplastic and apoptotic response in airway epithelial cells. Finally, we show that protective AHR signalling in lung endothelial cells is dampened by the infection itself. Maintenance of protective AHR function requires a diet enriched in naturally occurring AHR ligands, which activate disease tolerance pathways in lung endothelia to prevent tissue damage. Our findings demonstrate the importance of endothelial function in lung barrier immunity. We identify a gut–lung axis that affects lung damage following encounters with viral pathogens, linking dietary composition and intake to host fitness and inter-individual variations in disease outcome.
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Data availability
Sequencing data are available in the Gene Expression Omnibus under accession codes GSE203427 and GSE225958. Source data are provided with this paper.
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Acknowledgements
We thank staff at the Crick flow cytometry, advanced sequencing, bioinformatics and animal facilities for excellent support; and J. Kohl for help and advice on RNAscope data generation and interpretation. This work was funded by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC002085), the UK Medical Research Council (FC002085) and the Wellcome Trust (FC002085).
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J.M. and A.W. conceived the idea and designed the experimental strategy. J.M., S.C., K.F., B.F., R.D., M.G. and L.M. designed and performed experiments and analysed data. P.C. performed the bioinformatics analysis. K.S. and B.S. provided key intellectual input and experimental tools. A.S.-B. and S.P. performed histopathological analyses and scoring. J.M. and A.W. wrote the manuscript. All authors edited the manuscript. All authors read and approved the final manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Gating strategies for lung cell populations.
a–c, Gating strategy for lung endothelial cell and epithelial cell populations (a), RBCs and immune cells in the BALF day 6 post influenza virus infection (b), and lung lymphoid immune cell populations (c) analysed by flow cytometry.
Extended Data Fig. 2 The AHR landscape in mouse and human lung endothelia.
a, Immunofluorescence staining of steady-state AHR-tdTomato and Cyp1a1-eYFP lung sections stained with the vascular endothelial marker endomucin or lymphatic marker LYVE-1, and Hoechst (blue). Scale bars, 100 μm (left panels), 20 μm (middle panels), 5 μm (right panels). Data are representative of three independent experiments with similar results. b, Representative histogram plots of AHR-tdTomato and Cyp1a1-eYFP expression in steady-state lung endothelial cells (CD31+PDPN–) and lymphatic endothelia (CD31+PDPN+) relative to B6 WT controls (grey). Mean fluorescence intensity (MFI). c, Frequency of lymphatic and vascular endothelial cells measured by flow cytometry. d, RNA-FISH analysis of WT steady-state lung. RNA probes for Ahr (cyan) and Cyp1a1 (yellow) and stained with E-Cadherin for epithelia. White arrowheads indicate Ahr expression in airway epithelial cells. Scale bar, 20 μm. Data are representative of four independent experiments with similar results. e, f, Expression of indicated genes in uniform manifold approximation and projection (UMAP) plots of mouse (e) and human (f) lung scRNA-seq datasets obtained from lungendothelialcellatlas.com. g, Primary human lung microvasculature endothelial cell (HMVEC-L) cultures were treated with AHR agonist FICZ or antagonist CH-223191 for 24 h and indicated gene expression was determined by qPCR (n = 6 biological replicates). Statistical analysis was performed using one-way ANOVA with Tukey’s post-test. Data are shown as mean±SEM. Data are shown as mean±SEM. ns, not significant.
Extended Data Fig. 3 Dampened pulmonary inflammation in CYP1-deficient mice.
a, b, Lung immune cell numbers were determined in the BALF on day 6 post infection (a) or in whole lung on indicated days post infection (b) in WT (n = 4) and Cyp1−/− (n = 5) mice by flow cytometry. c, d, BALF cytokine concentration in influenza virus infected WT and Cyp1−/− mice was determined on indicated days for IFN (n = 3) (c) or day 6 for remaining cytokines (d) post infection (n = 5). e, Histopathological analysis of WT (n = 4) and Cyp1−/− (n = 3) H&E lung sections on day 6 post infection. Black arrowheads indicate areas of perivascular, peribronchiolar, and intra-alveolar inflammatory cell infiltration. Scale bars, 500 μm (upper panels) and 100 μm (lower panels). All Data are representative of three to four independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t test (a, d), two-way ANOVA with Sidak’s post-test (b, c), or two-tailed Mann–Whitney U test (e) and significant P values are indicated on the graphs. Data are shown as mean±SEM. ns, not significant.
Extended Data Fig. 4 CYP1 deficiency confers protection against respiratory pathogens.
a, b, Lung damage was assessed in the BALF of X31 influenza virus infected WT (n = 8) and Cyp1a1/Cyp1b1 double-knockouts (Cyp1a2+/−) (n = 9) (a) or Cal09 H1N1 influenza virus infected WT (n = 6) and Cyp1−/− (n = 5) mice (b) by measurement of total cells, Ter119+ RBCs, total protein, and serum albumin concentrations on day 6 post infection. (c) Weight loss of influenza (X31) and Streptococcus pneumoniae coinfected WT (n = 23) and Cyp1−/− (n = 23) mice. All Data are representative of two independent experiments or pooled from three experiments (c). Statistical analysis was performed using unpaired two-tailed Student’s t test (a, b) or two-way ANOVA with Sidak’s post-test (c) and significant P values are indicated on the graphs. Data are shown as mean±SEM.
Extended Data Fig. 5 Endothelial-specific AHR deletion.
a, Endothelial-specific AHR deletion was determined by measuring expression of Ahr and AHR-target gene Cyp1a1 in isolated lung CD31+ endothelial cells in Cdh5Cre-ERT2Rosa26-LSL-YFP; Ahrflox/flox mice (ECΔAhr) and WT control (Cdh5Cre−Rosa26-LSL-YFP; Ahrflox/flox) mice by qPCR (n = 8) (a) or transcripts per million (TPM) from bulk RNA-seq analysis (n = 3) (b). c, YFP-expressing lung endothelial cells as a measurement of Cre induction was determined in CD31+ lung endothelial cells by flow cytometry in ECΔAhr mice (n = 5). All Data are representative of two independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t test and significant P values are indicated on the graphs. Data are shown as mean±SEM.
Extended Data Fig. 6 AHR deletion in endothelial cells does not drastically alter influenza-induced pulmonary inflammation.
a, b Immune cell numbers were determined in the BALF of WT (n = 6) and ECΔAhr (n = 5) mice (a) and whole lung (n = 4) (b) of on day 6 post infection by flow cytometry. c, BALF cytokine concentration in WT and ECΔAhr mice was determined on day 2 (IFN) or day 6 (remaining cytokines) post infection (IL-6 and IFN-λ: WT n = 6, ECΔAhr n = 8; remaining cytokines n = 4). All Data are representative of two to three independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t test and significant P values are indicated on the graphs. Data are shown as mean±SEM. ns, not significant.
Extended Data Fig. 7 AHR signalling in endothelia prevents airway epithelial apoptosis and dysplastic repair.
a, b, Heatmaps of indicated genes from bulk RNA sequencing data comparing naïve WT and ECΔAhr CD31+ lung endothelial cells (a) and EpCam+ lung epithelial cells on day 6 post infection (b) (fold change > 1.5, padj < 0.05). c, Frequency (% of total EpCam+) and proliferation (Ki67+) of distal airway stem cells (EpCamhighCD24lowMHC-II–) in the lungs of WT and ECΔAhr mice was measured by flow cytometry in naïve (n = 3) mice and on day 6 post influenza infection (WT n = 6; ECΔAhr n = 5). d, Frequency of apoptotic (Annexin-V+) and necrotic (TO-PRO-3+) lung endothelial cells (CD31+), progenitor airway epithelial cells (EpCamhighCD24lowMHC-II–), mature airway epithelial cells (EpCamhighCD24highMHC-II–), and type II alveolar epithelial cells (EpCamlowMHC-II+) was assessed by flow cytometry in WT and ECΔAhr mice on day 6 post influenza infection (n = 4). All Data are representative of two independent experiments. Statistical analysis was performed using one-sided Wald test with Benjamini–Hochberg correction (a, b) or two-way ANOVA with Sidak’s post-test (c) and significant P values are indicated on the graphs. Data are shown as mean±SEM. ns, not significant.
Extended Data Fig. 8 AHR-dependent regulation of the apelin signalling pathway in lung endothelia.
a, Heatmap of indicated genes from RNA sequencing data comparing CD31+ lung endothelial cells on day 6 post infection (fold change > 1.5, padj < 0.05). b, Expression of indicated genes in isolated CD45+ immune cell, EpCam+ epithelial cell, and CD31+ endothelial cell was determined by qPCR in naïve WT mice (n = 4). c, d, Expression of indicated genes in UMAP plots of mouse (c) and human (d) lung scRNA-seq datasets obtained from lungendothelialcellatlas.com. e, Lung vascular leakage was assessed in PBS (n = 5) and apelin (n = 6) treated ECΔAhr mice by quantification of total cells Ter119+ RBCs in the BALF on day 6 post infection. f, Dot plot of hallmark pathways enriched or downregulated in MM54-treated WT mice (relative to PBS-treated controls) by GSEA. Comparisons are between MM54-treated and untreated from influenza infected mice, for endothelia and epithelia (two pairwise comparisons total). Dot size relates to statistical significance. All Data are representative of at least two independent experiments. Statistical analysis was performed using one-sided Wald test with Benjamini–Hochberg correction (a) or followed by Tukey’s post-test (b), or unpaired two-tailed Student’s t test (e). NES were generated with GSEA using a two-sided Kolmogorov Smirnov statistic with Hallmark genesets on genelists ranked by the Wald t statistic from DESeq2 (f) and significant P values are indicated on the graphs. Data are shown as mean±SEM. ns, not significant.
Extended Data Fig. 9 Dietary AHR ligands do not disrupt pulmonary inflammation.
a, Immune cell numbers were determined in the whole lung of WT mice fed purified or I3C-enriched diet on day 6 post infection by flow cytometry (n = 4). b, BALF IFN (day 2) and cytokine (day 6) concentrations in WT mice fed purified or I3C-enriched diet (n = 5). Statistical analysis was performed using unpaired two-tailed Student’s t test and significant P values are indicated on the graphs. Data are shown as mean±SEM. ns, not significant.
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Major, J., Crotta, S., Finsterbusch, K. et al. Endothelial AHR activity prevents lung barrier disruption in viral infection. Nature 621, 813–820 (2023). https://doi.org/10.1038/s41586-023-06287-y
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DOI: https://doi.org/10.1038/s41586-023-06287-y
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