Research ArticleINFECTIOUS DISEASE

Alveolar macrophages generate a noncanonical NRF2-driven transcriptional response to Mycobacterium tuberculosis in vivo

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Science Immunology  26 Jul 2019:
Vol. 4, Issue 37, eaaw6693
DOI: 10.1126/sciimmunol.aaw6693
  • Fig. 1 AMs provide a replication niche for M.tb. through the first 10 days of infection.

    (A) Percentage of total mEmerald+ cells, (B) percentage of mEmerald+ for each cell population, and (C) total number of mEmerald+ cells in the lung between 2 hours and 14 days after high-dose aerosol infection with mEmerald-tagged H37Rv (n = 3 mice per time point). (D) Microscopy of BAL samples 1 and 10 days after high-dose aerosol infection with mEmerald-tagged H37Rv and quantitation of bacteria per AM (n = 3 replicates per time point, each replicate was pooled from three mice). Eosin, eosinophils. Data are means ± SEM and are representative of two independent experiments.

  • Fig. 2 M.tb.-infected AMs up-regulate an NRF2-associated antioxidant gene signature.

    (A) Gating scheme to sort naïve, bystander, and M.tb.-infected AMs from BAL samples after high-dose aerosol infection with mEmerald-tagged H37Rv. (B) Heatmap of gene expression (log2 fold change over average of naïve AMs) for 196 DE genes between naïve and M.tb.-infected AMs (filtering criteria: average CPM > 1, |fold change| > 2, and FDR < 0.01, Benjamini-Hochberg calculated). Columns are independent experiments (pooled mice), and rows are genes. Genes called out are known NRF2 target genes of interest as well as down-regulated pro-inflammatory genes. Colored bars to the left indicate NRF2 association as determined by three different methods: (C) IPA, (D) transcription factor promoter binding motif enrichment analysis (HOMER), and (E) ChIP-Seq peak analysis. (F) Quantitative reverse transcription PCR (RT-qPCR) validation of NRF2-associated genes for naïve, bystander, and M.tb.-infected AMs 24 hours after infection. Values are relative to Ef1a. Data are presented from three independent experiments with one-way ANOVA with Dunnett’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 Up-regulation of the NRF2-associated signature does not require virulent M.tb. infection.

    (A) Heatmap of log2 fold change gene expression over average of naïve AMs for H37Rv-infected, ΔRD1-infected, and bead-positive AMs 24 hours after treatment. Columns represent averages of three independent experiments. Rows represent 196 DE genes described in Fig. 2. Colored bars to the left indicate NRF2 association as described in Fig. 2. (B and C) Scatterplots comparing gene expression values [log2 fold change (FC) over average of naïve AMs] for H37Rv-infected versus ΔRD1-infected AMs (B) or H37Rv-infected versus bead-positive AMs (C) 24 hours after infection, with significant DE genes highlighted (average CPM > 1, |fold change| > 2, and FDR < 0.01, Benjamini-Hochberg calculated). Data are presented from three independent experiments.

  • Fig. 4 M.tb.-infected AMs induce both NRF2-associated and pro-inflammatory responses 10 days after infection.

    (A) Heatmap of gene expression (log2 fold change over naïve AMs) for 252 genes up-regulated in M.tb.-infected AMs compared with naïve AMs for at least one of five time points (filtering criteria: average CPM > 1, |fold change| > 2, and FDR < 0.01, Benjamini-Hochberg calculated). Top 131 genes are significantly up-regulated in M.tb.-infected AMs at 1 day after infection. Bottom 121 genes are not significantly up-regulated in M.tb.-infected AMs at 1 day after infection. Colored bars indicate NRF2 association as described in Fig. 2. (B and C) GSEA and top 50 ranked leading edge genes in the “TNFA signaling via NFkB” pathway for M.tb.-infected AMs at 10 days after infection. Data are presented from three independent experiments per time point.

  • Fig. 5 Bystander AMs express a unique transcriptional signature 10 days after infection.

    (A and B) Scatterplots comparing gene expression values (log2 fold change over average of naïve AMs) for H37Rv-infected versus bystander AMs at 1 day (A) and 10 days (B) after infection, with significant DE genes highlighted (|fold change| > 2 and FDR < 0.01, Benjamini-Hochberg calculated). Data are presented from three independent experiments. (C to E) Heatmaps of log2 fold change gene expression at 10 days over average of naïve AMs. Colored bars indicate NRF2 association as described in Fig. 2. (C) Twenty-eight genes DE by both bystander and M.tb.-infected AMs. (D) Two hundred genes DE only by M.tb.-infected AMs. (E) One hundred seventy-seven genes DE only by bystander AMs. Columns represent the average of three independent experiments. Genes of interest are noted to the right. (F) IPA comparing gene expression from bystander AMs and M.tb.-infected AMs. Canonical pathways with |z scores| > 1 and P values < 0.05 were reported.

  • Fig. 6 The AM response to M.tb. is driven by both cell type and environment.

    In vitro H37Rv infection of AMs and BMDMs. (A) RT-qPCR gene expression analysis of pro-inflammatory genes 8 hours after infection. (B) RT-qPCR analysis of NRF2-associated genes 8 hours after infection. (C) CFU assay to measure bacterial burden in each cell type over 5 days. Data are representative of three independent experiments with three technical replicates each. Multiple t tests with Holm-Sidak correction. *P < 0.05, ***P < 0.001.

  • Fig. 7 Modulation of NRF2 activity alters macrophage response and control of M.tb.

    (A) Heatmap of gene expression (log2 fold change over average of respective naïve AMs) for WT (first column) and Nrf2−/− (second column) M.tb.-infected AMs, averaged from at least two independent experiments. Rows depict all genes that are DE between naïve and M.tb.-infected WT AMs, as shown in Fig. 2B (181 genes expressed out of original 196 gene list). Groups 1 and 2 are defined in the text. Colored bars indicate NRF2 association as described in Fig. 2. (B) Lung bacterial burden measured by CFU assay at 10 days after infection with low-dose H37Rv from Nrf2LysM, Nrf2CD11c, and their respective Nrf2fl littermate controls. (C) MHC II mean fluorescence intensity (MFI) of Zombie Violet (ZV) M.tb.-infected AMs (top) and percentage of dead (Zombie Violet+) M.tb.-infected AMs (bottom) as measured by flow cytometry at 10 days after infection with high-dose mEmerald-H37Rv from Nrf2LysM, Nrf2CD11c, and their respective Nrf2fl littermate controls. Data are presented from two independent experiments (A) or are representative of two independent experiments with five mice per group (B and C). Two-tailed unpaired Student’s t test. *P < 0.05.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/37/eaaw6693/DC1

    Fig. S1. Lung flow cytometry gating scheme for myeloid cells.

    Fig. S2. Lung flow cytometry gating scheme for detection of M.tb.-infected cells with overlay of mEmerald+ populations.

    Fig. S3. BAL and lung flow cytometry gating scheme for cell sorting.

    Fig. S4. Bacterial burden at the time of AM cell sorting.

    Fig. S5. Gene expression heatmaps of naïve, bystander, and M.tb.-infected AMs 24 hours after aerosol infection.

    Fig. S6. Gating strategy for sorting of ΔRD1-infected AMs and bead-positive AMs.

    Fig. S7. Gene expression of BMDMs after treatment with fluorescent beads.

    Fig. S8. Gene expression heatmaps of M.tb.-infected AMs 0.5, 1, 2, 4, and 10 days after aerosol infection.

    Fig. S9. AM response to in vitro M.tb. infection with and without GM-CSF treatment.

    Fig. S10. AM response to in vitro M.tb. infection at 8, 12, and 24 hours after infection.

    Fig. S11. Gene expression differences between naïve WT and Nrf2−/− AMs.

    Fig. S12. Baseline lung conditions of WT and Nrf2−/− mice.

    Fig. S13. Gene expression and protein confirmation of NRF2 knockout in AMs from Nrf2LysM, Nrf2CD11c, and Nrf2fl littermate controls; WT; and Nrf2−/− mice.

    Fig. S14. Baseline lung conditions of Nrf2LysM, Nrf2CD11c, and Nrf2fl mice.

    Fig. S15. Gene expression heatmaps of M.tb.-infected Nrf2LysM, Nrf2CD11c, Nrf2fl, WT, and Nrf2−/− AMs 24 hours after aerosol infection.

    Fig. S16. Viability of bystander Nrf2LysM, Nrf2CD11c, and Nrf2fl AMs 10 days after high-dose aerosol infection.

    Table S1. Summary table accounting for M.tb.-infected cells in the lung 1 to 14 days after high-dose mEmerald-H37Rv infection.

    Table S2. IPA and HOMER results tables.

    Table S3. RNA-Seq data for WT AMs.

    Table S4. RNA-Seq data for Nrf2−/− AMs.

    Table S5. RNA-Seq data for Nrf2fl, Nrf2LysM, and Nrf2CD11c AMs.

    Table S6. Raw data file.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Lung flow cytometry gating scheme for myeloid cells.
    • Fig. S2. Lung flow cytometry gating scheme for detection of M.tb.-infected cells with overlay of mEmerald+ populations.
    • Fig. S3. BAL and lung flow cytometry gating scheme for cell sorting.
    • Fig. S4. Bacterial burden at the time of AM cell sorting.
    • Fig. S5. Gene expression heatmaps of naïve, bystander, and M.tb.-infected AMs 24 hours after aerosol infection.
    • Fig. S6. Gating strategy for sorting of ΔRD1-infected AMs and bead-positive AMs.
    • Fig. S7. Gene expression of BMDMs after treatment with fluorescent beads.
    • Fig. S8. Gene expression heatmaps of M.tb.-infected AMs 0.5, 1, 2, 4, and 10 days after aerosol infection.
    • Fig. S9. AM response to in vitro M.tb. infection with and without GM-CSF treatment.
    • Fig. S10. AM response to in vitro M.tb. infection at 8, 12, and 24 hours after infection.
    • Fig. S11. Gene expression differences between naïve WT and Nrf2−/− AMs.
    • Fig. S12. Baseline lung conditions of WT and Nrf2−/− mice.
    • Fig. S13. Gene expression and protein confirmation of NRF2 knockout in AMs from Nrf2LysM, Nrf2CD11c, and Nrf2fl littermate controls; WT; and Nrf2−/− mice.
    • Fig. S14. Baseline lung conditions of Nrf2LysM, Nrf2CD11c, and Nrf2fl mice.
    • Fig. S15. Gene expression heatmaps of M.tb.-infected Nrf2LysM, Nrf2CD11c, Nrf2fl, WT, and Nrf2−/− AMs 24 hours after aerosol infection.
    • Fig. S16. Viability of bystander Nrf2LysM, Nrf2CD11c, and Nrf2fl AMs 10 days after high-dose aerosol infection.
    • Legends for tables S1 to S6

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Summary table accounting for M.tb.-infected cells in the lung 1 to 14 days after high-dose mEmerald-H37Rv infection.
    • Table S2 (Microsoft Excel format). IPA and HOMER results tables.
    • Table S3 (Microsoft Excel format). RNA-Seq data for WT AMs.
    • Table S4 (Microsoft Excel format). RNA-Seq data for Nrf2−/− AMs.
    • Table S5 (Microsoft Excel format). RNA-Seq data for Nrf2fl, Nrf2LysM, and Nrf2CD11c AMs.
    • Table S6 (Microsoft Excel format). Raw data file.

    Files in this Data Supplement:

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