Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids (original) (raw)

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Acknowledgements

We thank S. Falkow and P. Edelstein for sharing their knowledge and insights, P. Donald for discussions about human infectivity in tuberculosis, B. Cormack for manuscript review and editing, J. Bubeck-Wardenberg for the fluorescent S. aureus strain, K. Hicks for initial MyD88 experiments, T.-Y. Chen, B. Moody, P. Manzanillo and J. Cox for help with lipid analyses, and J. Cameron for fish facility management. Supported by a National Science Foundation predoctoral fellowship to C.J.C., a Senior Research Training Fellowship from the American Lung Association and the National Institutes of Health (NIH) Training Grant “Training Clinical and Basic Immunologists” to R.P.L., a NIH “Academic Pediatric Infectious Disease” Training Grant award to R.E.H., an American Cancer Society Postdoctoral Fellowship and NIH Bacterial Pathogenesis Training Grant Award to D.M.T., and NIH grants to K.B.U. and L.R. D.M.T. is a recipient of the NIH Director’s New Innovator Award and L.R. is a recipient of the NIH Director’s Pioneer Award.

Author information

Authors and Affiliations

  1. Department of Immunology, University of Washington, Seattle, 98195, Washington, USA
    C. J. Cambier, Ryan P. Larson, Kevin B. Urdahl & Lalita Ramakrishnan
  2. Department of Microbiology, University of Washington, Seattle, 98195, Washington, USA
    Kevin K. Takaki, David M. Tobin, Christine L. Cosma & Lalita Ramakrishnan
  3. Seattle Biomedical Research Institute, Seattle, 98109, Washington, USA
    Ryan P. Larson & Kevin B. Urdahl
  4. Department of Pediatrics, University of Washington, Seattle, 98195, Washington, USA
    Rafael E. Hernandez & Kevin B. Urdahl
  5. Department of Medicine, University of Washington, Seattle, 98195, Washington, USA
    Lalita Ramakrishnan

Authors

  1. C. J. Cambier
  2. Kevin K. Takaki
  3. Ryan P. Larson
  4. Rafael E. Hernandez
  5. David M. Tobin
  6. Kevin B. Urdahl
  7. Christine L. Cosma
  8. Lalita Ramakrishnan

Contributions

C.J.C., C.L.C., K.K.T., D.M.T., R.E.H. and L.R. conceived and designed M. marinum/zebrafish experiments and analysed data; C.J.C., C.L.C., K.K.T., D.M.T. and R.E.H. performed these experiments; R.P.L. and K.B.U. designed the M. tuberculosis/mouse experiments and analysed the data; R.P.L. performed the mouse experiments; C.J.C., C.L.C. and L.R. wrote the paper; C.J.C., R.P.L. and K.K.T. prepared the figures; and all authors edited the paper.

Corresponding author

Correspondence toLalita Ramakrishnan.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Coordinate use of PDIM-mediated immune evasion and PGL-mediated recruitment by pathogenic mycobacteria.

Models for infection with wild-type (WT) and PDIM-deficient mycobacteria are shown in the context of the relatively sterile lower airway versus the upper airway, with its higher levels of resident microflora and inhaled environmental organisms.

Extended Data Figure 2 ΔmmpL7 bacteria are attenuated in zebrafish larvae.

a, Kaplan–Meier graph showing daily survival of larvae infected via caudal vein injection with medium (mock), 29 wild-type or 70 Δ_mmpL7 M. marinum_. N = 25 (mock), 31 (wild-type), or 29 (Δ_mmpL7_) larvae per group. Mean time to death (days): mock (11), wild type (7.6) and Δ_mmpL7_ (11.2). Survival was compared by log-rank test: wild type versus mock and wild type versus Δ_mmpL7_, P < 0.0001; mock versus Δ_mmpL7_, P = 0.5601. b, c, Larvae were infected via caudal vein injection 1 dpf with 550 wild-type, 650 ΔmmpL7, or 700_Δerp_, fluorescent M. marinum. b, Infection burdens were measured by Fluorescent Pixel Count (FPC; mean ± s.e.m.). c, Representative images at 7 dpi. N = 29 (wild-type and Δ_mmpL7_) or 30 (Δ_erp_) larvae per group. Scale bar, 500 μm. At 3, 5 and 7 dpi, log10 FPC was compared by ANOVA, with Dunnett’s post-test. ***P < 0.001. d, e, Representative images from wild-type (d) and Δ_mmpL7_ (e) M. marinum HBV infections quantified in Fig. 1d. N = 18 (wild-type) or 16 (Δ_mmpL7_) larvae per group. HBVs are outlined with a dashed white line. Scale bar, 100 µm.

Extended Data Figure 3 Knockdown of MyD88 results in a late, dose-dependent hypersusceptibility to M. marinum systemic infection.

a, RT–PCR for actin (top) and myd88 (bottom), demonstrating that that the majority of myd88 transcripts at 7 dpf are abnormal in MyD88 morphants. Lanes marked ‘b’ and ‘c’ correspond to morphants from the same experiments depicted in panels b and c, respectively. The abnormal larger transcript (indicated by an asterisk) results from the inclusion of intron 2 in the final transcript, incorporating a premature stop codon that truncates the protein before the TIR (Toll/interleukin receptor) domain. b, c, Caudal vein infection of MyD88 morphants with 141 (b) or 325 (c) c.f.u. M. marinum/larva. Bacterial burden was assessed by FPC, values plotted represent the mean ± s.e.m. Time points were compared by one-way ANOVA and Bonferroni’s post-tests. *** P < 0.001. d, Representative images of larvae at 5 dpi from experiment in c, N = 30 control, 15 MyD88 morphant. Scale bar, 500 μm.

Extended Data Figure 4 Characteristics of macrophages recruited to wild-type and PDIM-deficient bacteria.

a, Mean Mpeg1-positive macrophages recruited at 3 hpi into the HBV of wild-type fish after infection with 80 wild-type or Δ_mmpL7 M. marinum._ b, Data from Fig. 2c expressed as mean numbers of total infected macrophages and iNOS-expressing infected macrophages after HBV infection with 80 wild-type, Δ_mmpL7_, or Δ_erp M. marinum._ c, Bacterial burdens after L-NAME treatment. Mean bacterial burdens of 2 dpf control (CTRL)- or iNOS inhibitor (L-NAME)-treated fish after HBV infection with 80 wild-type or Δ_mmpL7 M. marinum_. NS, not significant.

Extended Data Figure 5 Wild-type bacterial burdens after co-infection with wild-type or Δ_mmpL7_ bacteria.

Representative images from the HBV co-infections quantified in Fig. 2e. a, b, Red fluorescent wild-type (WT) M. marinum co-infected with green fluorescent wild-type (a) or Δ_mmpL7_ (b) M. marinum. N = 18 (wild-type) and 19 (Δ_mmpL7_) larvae per group. Scale bar, 50 µm. c, Wild-type bacterial burdens after co-infection with wild-type or Δ_mmpL7 M. marinum_ with and without L-NAME treatment. Significance tested by one-way ANOVA with Bonferroni’s post-test for comparisons shown.

Extended Data Figure 6 MyD88-dependent macrophage recruitment occurs in response to PDIM deficiency rather than being due to loss of another MmpL7-exported product.

a, Mean macrophage recruitment at 3 hpi into the HBV of wild-type or MyD88-morphant (MO) larvae after infection with 80 Δ_mas M. marinum._ Student’s unpaired t_-test. b, Mean surviving bacterial volume of red fluorescent wild-type M. marinum (initial infection dose of 30–40 c.f.u.) when co-infected with 30–40 green fluorescent wild-type, Δ_mmpL7 or Δ_mas M. marinum_ at 3 dpi. Representative of two separate experiments. Significance tested by one-way ANOVA with Tukey’s post-test.

Extended Data Figure 7 Gating strategy and isotype controls for iNOS staining of mouse lung.

a, Representative gating strategy for isolation of inflammatory monocytes. A dump channel containing anti-CD4, CD8 and CD11c was plotted against a channel exhibiting autofluorescence and also containing anti-Ly6G. Using these markers, T cell, dendritic cell, alveolar macrophage, and neutrophil cell populations were excluded from the double-negative gate. Inflammatory monocytes were identified within the double-negative population by their co-expression of Ly6C and CD11b. These cells were then evaluated for intracellular iNOS expression. a, N = 4 per group (Fig. 3a, b) or b, with isotype control antibodies, N = 4 per group.

Extended Data Figure 8 Specificity of CCL2-mediated macrophage recruitment in wild-type and CCR2-morphant larvae

a, Mean macrophage recruitment at 3 hpi into the HBV of control (ctrl), or CCR2-morphant (CCR2) larvae after injection of vehicle control (‘mock’; 0.1% BSA in PBS), human CCL2 (hCCL2), human CCL4 (hCCL4), or human CCL5 (hCCL5). b, c, Mean macrophage (b) and neutrophil (c) recruitment at 3 hpi into the HBV of control (CTRL), CCR2-morphant (CCR2), or MyD88-morphant (MyD88) larvae after injection of vehicle control (mock), murine CCL2 (mCCL2), human CCL2 (hCCL2), human IL-8 (hIL-8), or human LTB4 (hLTB4). Representative of three separate experiments. Significance assessed by one-way ANOVA with Bonferroni’s post-test for the comparisons shown. *P < 0.05; ***P < 0.001.

Extended Data Figure 9 Identification of zebrafish CCL2 orthologue.

a, mRNA levels of potential CCL2 orthologues (mean ± s.e.m. of four biological replicates) induced at 3 h after caudal vein infection of 2 dpf larvae with 250–300 wild-type M. marinum. These assays were performed on the same cDNA pools as the data presented in Fig. 4b. b, Mean macrophage recruitment at 3 hpi into the HBV of wild-type or CCL2 morphant (MO) fish following infection with 80 M. marinum. Representative of two separate experiments.

Extended Data Figure 10 Infectivity assay.

a, b, Representative 5 hpi images from Fig. 4d following HBV infection with one (a) or three (b) M. marinum. Scale bar, 100 µm. N values for fish represented in a and b (that is, those found to be infected with 1–3 bacteria) are presented in Fig. 4d (18, 22, 28, 28, 28, 28, 22, 22 for the respective conditions as specified in the figure). c, Mean bacterial burdens 5 h after HBV infection with 1–3 wild-type (WT), Δ_mmpL7_ or Δ_pks15 M. marinum_.

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Cambier, C., Takaki, K., Larson, R. et al. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.Nature 505, 218–222 (2014). https://doi.org/10.1038/nature12799

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