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In vivo imaging of inflammasome activation reveals a subcapsular macrophage burst response that mobilizes innate and adaptive immunity

Abstract

The inflammasome is activated in response to a variety of pathogens and has an important role in shaping adaptive immunity, yet the spatiotemporal orchestration of inflammasome activation in vivo and the mechanisms by which it promotes an effective immune response are not fully understood. Using an in vivo reporter to visualize inflammasome assembly, we establish the distribution, kinetics and propagation of the inflammasome response to a local viral infection. We show that modified vaccinia Ankara virus induces inflammasome activation in subcapsular sinus (SCS) macrophages, which is immediately followed by cell death and release of extracellular ASC specks. This transient inflammasome signaling in the lymph node generates a robust influx of inflammatory cells and mobilizes T cells from the circulation to increase the magnitude of T cell responses. We propose that after infection, SCS macrophages deliver a burst response of inflammasome activity and cell death that translates into the broadening of T cell responses, identifying an important aspect of inflammasome-driven vaccination strategies.

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Figure 1: MVA infection induces inflammasome activation, speck formation and rapid loss of SCS macrophages in draining lymph nodes.
Figure 2: Dynamic imaging of inflammasome activation in ASC-GFP retrogenic mice reveals rapid kinetics of cellular inflammasome signaling.
Figure 3: Subcapsular macrophages provide a spatiotemporally defined inflammasome response to local viral infection.
Figure 4: ASC aggregation precedes rapid cell death releasing long-lived extracellular ASC specks in vivo.
Figure 5: Inflammasome activation regulates the magnitude of the inflammatory innate infiltrate into draining lymph nodes.
Figure 6: Inflammasome signals recruit circulating antigen-specific T cells to the draining lymph node.

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Acknowledgements

We wish to thank O. Schwartz, N. Manel and members of P.B.'s laboratory for critical review of the manuscript; A. Cumano (Institut Pasteur) for helpful discussions in generating BM chimeric mice; and the Centre d'Immunologie Humaine (Institut Pasteur) for support with flow cytometry. This work was supported by Institut Pasteur (P.B.), Institut National de la Santé et de la Recherche Médicale (P.B.), the Vaccine Research Institute (P.B.), Fondation pour la Recherche Médicale (P.B.) and a Starting Grant from the European Research Council (P.B.).

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Authors and Affiliations

Authors

Contributions

P.B. and P.S. designed the experiments, analyzed the data and wrote the manuscript; P.S. and B.B. conducted the experiments with essential support from Z.G., F.L.; D.M., M.L.A. and Y.L. provided scientific input and reagents.

Corresponding author

Correspondence to Philippe Bousso.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1557 kb)

Supplementary Movie 1

Time-lapse videomicroscopy showing ASC speck formation in BM derived macrophages stably transduced with ASC-GFP following in vitro infection with MVA. (MOV 135 kb)

Supplementary Movie 2

Intravital microscopy of popliteal lymph node in an ASC-GFP BM chimeric mouse, showing a compilation of two examples of ASC speck formation occurring in macrophages located at the lymph node capsule following subcutaneous infection with MVA. (MOV 2905 kb)

Supplementary Movie 3

Intravital microscopy of the subcapsular area of the popliteal lymph node in an ASC-GFP BM chimeric mouse injected with PBS showing GFP+ cells do not undergo ASC speck formation. (MOV 2159 kb)

Supplementary Movie 4

Intravital microscopy of deeper lymph node regions relative to the collagen capsule (>50 μm) of ASC-GFP BM chimeric mice following MVA infection, showing GFP+ cells do not undergo ASC speck formation. (AVI 4305 kb)

Supplementary Movie 5

Time-lapse videomicroscopy of BM-derived macrophages stably transduced to express ASC-mCherry and infected with MVA-GFP, showing that cell death, indicated by nuclear uptake of SytoxBlue, occurs rapidly following ASC speck formation. (AVI 9385 kb)

Supplementary Movie 6

Compilation movie showing examples of various ASC speck fates detected in the popliteal lymph nodes of ASC-GFP BM chimeric mice injected with MVA. Example of a long-lived ASC speck and of an ASC speck that forms and fragments. (MOV 1421 kb)

Supplementary Movie 7

Examples of ASC speck acquisition in vivo. Example of a formed ASC speck that becomes acquired by an adjacent ASC-GFP+ cell. Speck uptake induces the formation of a new speck within the acquiring cell, resulting in complete speck formation. Two examples of ASC specks that form and become phagocytized by another ASC-GFP+ cell. Movies acquired by intravital imaging of popliteal lymph nodes of ASC-GFP BM chimeric mice after MVA injection. (MOV 2249 kb)

Supplementary Movie 8

Inflammasome activation induces localized cell recruitment in the lymph node. Rapid localized recruitment induced by ASC speck formation after MVA treatment, visualized by intravital microscopy of macrophages in ASC-GFP BM chimeras following in situ staining of lymphoid cells with Hoechst 33342. Additional movie of intravital microscopy in ASCGFP BM chimeric mice treated with MVA, showing local recruitment of myeloid-derived (GFP+) cells near sites of ASC speck formation. (MOV 3606 kb)

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Sagoo, P., Garcia, Z., Breart, B. et al. In vivo imaging of inflammasome activation reveals a subcapsular macrophage burst response that mobilizes innate and adaptive immunity. Nat Med 22, 64–71 (2016). https://doi.org/10.1038/nm.4016

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