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AhR sensing of bacterial pigments regulates antibacterial defence

Abstract

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.

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Figure 1: Bacterial pigmented virulence factors activate AhR.
Figure 2: AhR activated by secreted P. aeruginosa compounds induces pyocyanin degradation.
Figure 3: AhR regulates host defence against P. aeruginosa.
Figure 4: Haematopoietic and non-haematopoietic cells contribute to AhR-mediated defence against P. aeruginosa.
Figure 5: AhR is critical for host defence against M. tuberculosis.
Figure 6: AhR mediates macrophage activation and mycobacterial growth inhibition.

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Gene Expression Omnibus

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Gene Expression Omnibus

Data deposits

Data are deposited in the GEO database under accession number GSE48133.

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Acknowledgements

The authors are highly grateful to J. Welch for his contribution in the early stage of research, and for support in AhR ligand screening, A. Zychlinsky and L. E. Dietrich for discussions, H.-G. Hoymann for support in lung function measurements and interpretation of results, U. Klemm for mouse breeding and M. L. Grossman for excellent support in preparing the manuscript. Ahr–/–mice were provided by B. Stockinger and shRNA constructs by D. Krastev and F. Buchholz. The PA14 WT2 and PA14 Δphz1/2 were a gift from D. K. Newman and L. E. Dietrich. M. Kolbe acknowledges grant support from the European Union’s Seventh Framework Programmes (EU-FP7/2007-2013). S.H.E.K. acknowledges grant support from the European Union’s Seventh Framework Programmes (EU-FP7) NEWTBVAC (Health-F3-2009-241745) and PHAGOSYS (Health-F4-2008-223451).

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Authors

Contributions

P.M.-A., E.H., K.F., A. Dorhoi and S.H.E.K. conceived and designed the study and wrote the manuscript. P.M.-A., E.H., K.F. and A. Dorhoi designed and performed experiments and data analysis. P.C. and M.D. performed Mycobacterium lipid fractionation. A.M. and B.T. designed and performed Pseudomonas spirometry studies. U.G.-B., M. Klemm, A.-B.K. and S.B. provided technical help for in vitro and in vivo experiments. R.H. performed and analysed HPLC experiments. A.K., G.K. and H.O. performed virtual docking studies. S.F. discussed experiments. T.S., V.B. and C.G. performed and discussed experiments. H.-J.M. performed and analysed microarray experiments. M. Kolbe, N.B., J.F., A. Diehl and H.O. performed binding studies. All authors commented on the paper.

Corresponding author

Correspondence to Stefan H. E. Kaufmann.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 AhR binding, activation and cell viability.

a, Chemical structures of phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN). b, In silico docking of PCA and PCN into the ligand-binding pocket (yellow surface) of AhR. Hydrophilic residues (magenta), aromatic and hydrophobic residues (green). c, Intrinsic tryptophan fluorescence quenching of purified AhR1–417 or mock transfected control titrated with increasing concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 1-hydroxyphenazine (1-HP). d–f, Luciferase activity of AhR reporter cells after 24 h stimulation. d, THP-1 cells stimulated with 50 μM of PCA or PCN. e, H358 cells stimulated with TCDD (10 nM), pyocyanin (Pyo, 50 μM), 1-HP (50 μM) or phthiocol (Pht, 50 µM). f, AhR activation of THP-1 AhR reporter cells upon stimulation with different concentrations of known AhR ligands (TCDD, 6-formylindolo[3,2-b] carbazole (FICZ), β-naphthoflavone or kynurenic acid (Kyn) or bacterial pigments (Pyo, 1-HP or Pht). g, Cell viability assessed after 24 h stimulation with 50 μM of Pyo, 1-HP and Pht. h, Luciferase activity of THP-1 AhR reporter cells stimulated for 24 h with 50 μM of Pyo, 1-HP, Pht and lipopolysaccharide (LPS, 1 μg ml−1), flagellin (100 ng ml−1) or CpG oligodeoxynucleotides (ODN2006, 5 μM). c, Representative of at least two experiments; d–h, cumulative data of at least three experiments, mean + s.e.m.; d–h, Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001.

Extended Data Figure 2 Bacterial pigments induce transcription of canonical AhR pathway genes.

a, qRT–PCR of CYP1A1, AHRR and CYP1B1 in different cells after stimulation with 50 μM of Pyo, 1-HP and Pht. b, AhR gene knockdown (KD) efficiency following infection with lentivirus encoding a pool of AhR-specific shRNAs. Cells transduced with a non-targeting Scramble shRNA were considered as reference control. c, qRT–PCR of CYP1A1 in H358 Scramble and AhR-KD cells after 24 h stimulation with 50 μM of Pyo, 1-HP and Pht. a–c, Cumulative data of at least three experiments, mean + s.e.m.; a–c, Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Extended Data Figure 3 Bacterial pigments induce global AhR signalling in pneumocytes.

Microarray analysis of A549 Scramble cells upon stimulation with virulence factors. Cells were treated with 50 μM of the different bacterial pigmented virulence factors or DMSO as control for 24 h. RNA was collected and subjected to microarray analysis. a, List of genes differentially expressed (P < 0.00001) upon stimulation of cells with the different ligands, as compared to DMSO. b, Top 20 canonical pathways predicted by Ingenuity pathway analysis software to be differentially regulated upon stimulation. Up, upregulated. Down, downregulated. Blue bars (left, y axis) depict –log P values calculated by Fisher’s exact test whereas yellow line (right, y axis) represents the ratio between the number of genes in a given pathway compared to the total number of genes in that pathway.

Extended Data Figure 4 AhR activation in THP-1 cells in the absence of Trp or CYP1A1.

a, Luciferase activity of AhR reporter in THP-1 cells stimulated with TCDD (10 nM), FICZ (20 nM), Pyo (50 μM), 1-HP (50 μM) or Pht (50 µM) for 24 h in the absence of Trp. b, CYP1A1 gene KD efficiency in THP-1 cells. Cells were transduced using lentivirus encoding a pool of CYP1A1-specific shRNAs. Cells transduced with a non-targeting Scramble shRNA were considered as reference control. c, d, qRT–PCR of AHRR in THP-1 in Scramble control (c) and CYP1A1-KD (d) cells after 24 h stimulation with 10 nM TCDD or 50 μM of Pyo, 1-HP and Pht. a–d, Cumulative data of at least three experiments, mean + s.e.m.; a–d, Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Extended Data Figure 5 P. aeruginosa PA14 bacterial growth, phenazine concentrations and AhR activation.

Different PA14 mutants (09480 and Δphz1/2) and parental WT controls (WT1 and WT2, respectively) were tested. a, Bacterial density and Pyo concentration in the filtered supernatants were determined at different time points. Means of bacterial density and Pyo concentration are shown. Full lines represent bacterial growth, and dashed lines depict Pyo concentration for each strain. b, HPLC analysis of different phenazines (Pyo, 1-HP, PCA and PCN) present in the supernatants of P. aeruginosa PA14 WT1 and mutant PA14 09480 strains. c, d, Filtered supernatants from PA14 mutants and parental WT controls were used to stimulate THP-1 AhR reporter cell line for 24 h. Luciferase activity was measured and normalized to non-stimulated cells (control). a, Representative of at least three experiments; b, representative of at least three experiments, mean + s.d.; c, d, cumulative data of three experiments, mean + s.e.m. c, d, Student’s t-test. **P < 0.01.

Extended Data Figure 6 Degradation of bacterial pigments upon AhR activation.

a, b, HPLC of Pyo (a) and Pht (b) degradation in supernatants of A549 Scramble and AhR-KD cells 48 h after stimulation. a, Arrows depict new peaks emerging at 368 nm at 1.21, 1.31 and 1.42 min with phenazine-like characteristics, suggesting formation of a new metabolite(s) from Pyo. Higher levels of these metabolite(s) are observed in supernatants of scramble cells challenged with Pyo, suggesting an AhR dependent role. These peaks were not detected in supernatants of unstimulated cells. b, Decreased levels of native Pht (arrow) are detected in supernatants of A549 Scramble cells, 48 h after challenge with Pht. c, Expression of the AhR gene battery: Phase I and Phase II xenobiotic metabolizing enzymes (XME)2,33,66,67. A549 cells (Scramble and AhR-KD) were stimulated for 24 h with different bacterial ligands or DMSO as control. RNA was extracted and microarray analysis was performed. The table depicts fold induction of different XME upon stimulation, as compared to DMSO control in different cell lines tested. The remaining differently regulated genes in A549 cells (Scramble and AhR-KD) upon stimulation are depicted in the Supplementary Tables 4 and 5. n.d., not detected. a, b, Representative of three experiments.

Extended Data Figure 7 Ahr–/– mice are more susceptible to P. aeruginosa infection.

WT and AhR-deficient (Ahr–/–) mice were infected intratracheally (i.t.) with 107 c.f.u. (a), 2 × 105 c.f.u. (b) or 4 × 106 c.f.u. (c) of P. aeruginosa PAO1. a, Body temperature. b, Lung function evaluated by non-invasive head-out spirometry. Spirometric curves depict time course of tidal volume (total volume inspired and expired in one breath), expiratory flow at 50% expiration (EF50) and rate (breaths per min). c, Bacterial loads. a, Cumulative data of two experiments, mean ± s.e.m.; b, representative of at least two experiments, mean ± s.d.; c, representative of at least two experiments, median; a, b two-way ANOVA; c, Mann–Whitney U- test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Extended Data Figure 8 Cellular, cytokine and microarray analysis of P. aeruginosa-infected mice.

a, b, d–h, WT and Ahr–/– mice were infected i.t. with 4 × 106 c.f.u. of PAO1. a, b, Frequencies and absolute numbers of myeloid cells from lung and bronchoalveolar lavage fluid (BALF). c, Frequencies and absolute numbers of myeloid cells from lungs of naive animals. d, e, Frequencies of lymphoid cells in lungs from naive mice. f, Cytokine and chemokine abundance in BALF from P. aeruginosa-infected mice. g, h, Microarray analysis of BALF samples from infected WT mice, as compared to PBS control. g, List of genes differentially expressed (P < 0.00001) upon infection of mice with P. aeruginosa PAO1. h, Top 20 canonical pathways predicted by Ingenuity pathway analysis software to be differentially regulated upon infection. Up, upregulated; down, downregulated. Blue bars (left, y axis) depict –log P values calculated by Fisher's exact test. Yellow line (right, y axis) represents the ratio between number of genes in a given pathway compared to total number of genes in that pathway. a, b, d, e, Representative of two experiments, mean + s.d.; c, cumulative data of two experiments, mean + s.e.m.; f, cumulative data of two experiments, median + interquartile range; a–d, two-way ANOVA; e, f, (Mann–Whitney U- test). *P < 0.05; **P < 0.01; ****P < 0.0001.

Extended Data Figure 9 M. tuberculosis infection of mice and human primary macrophages.

a–d, WT and Ahr–/–mice were aerosol-infected with M. tuberculosis H37Rv (low-dose, 100 c.f.u.). a, b, Tissue damage. a, Lactate dehydrogenase (LDH) activity in serum. b, Hematoxylin and eosin staining of lung. Scale bars, 500 μm. c, Flow cytometry analysis of lymphoid cells. d, Cytokine and chemokine abundances in lung homogenates. e, AhR activation by M. tuberculosis in human primary macrophages. qRT–PCR of CYP1A1 and AHRR after infection with H37Rv (m.o.i. = 5). a, c Representative of two experiments, mean + s.d.; b, representative of two experiments; d, cumulative data of two experiments, median + interquartile ranges; e, n = 3 donors, mean + s.d.; a, c, two-way ANOVA; d, Mann–Whitney U-test; e, Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001.

Extended Data Figure 10 AhR activation by M. tuberculosis lipid fractions.

a, Luciferase activity of AhR reporter in THP-1 cells stimulated with different M. tuberculosis preparations (cell wall, membrane and cytosolic fractions, total lipids, mannosilated lipoarabinomannan (ManLAM), trehelose dimycolate (TDM), antigen 85 (Ag85), Ag85B, early secretory antigenic target 6 (ESAT6) and 10 kDa culture filtrate antigen (CFP10)). b, Flow chart of the sequence of M. tuberculosis lipid fractionation by chromatography on a Florisil column and identification of AhR-activating fractions. c, Luciferase activity of AhR reporter in THP-1 cells stimulated for 24 h with different M. tuberculosis lipid fractions. d, Gas chromatography coupled mass spectrometry (GC/MS) identification of Pht in AhR-activating versus -nonactivating lipid fractions. In the active fraction, an elution peak was observed at the same retention time as Pht, showing the same mass fragmentation pattern (m/z 188, 160, 131, 105, 77).

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Moura-Alves, P., Faé, K., Houthuys, E. et al. AhR sensing of bacterial pigments regulates antibacterial defence. Nature 512, 387–392 (2014). https://doi.org/10.1038/nature13684

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