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.
Your institute does not have access to this article
Open Access articles citing this article.
NLRP12 collaborates with NLRP3 and NLRC4 to promote pyroptosis inducing ganglion cell death of acute glaucoma
Molecular Neurodegeneration Open Access 15 April 2020
BMC Microbiology Open Access 08 February 2019
Molecular Cancer Open Access 17 November 2018
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Monie, T.P., Moncrieffe, M.C. & Gay, N.J. Structure and regulation of cytoplasmic adapter proteins involved in innate immune signaling. Immunol. Rev. 227, 161–175 (2009).
Franchi, L., Muñoz-Planillo, R. & Núñez, G. Sensing and reacting to microbes through the inflammasomes. Nat. Immunol. 13, 325–332 (2012).
Bryan, N.B., Dorfleutner, A., Rojanasakul, Y. & Stehlik, C. Activation of inflammasomes requires intracellular redistribution of the apoptotic speck-like protein containing a caspase recruitment domain. J. Immunol. 182, 3173–3182 (2009).
Lu, A. et al. Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell 156, 1193–1206 (2014).
Fernandes-Alnemri, T. et al. The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ. 14, 1590–1604 (2007).
Stutz, A., Horvath, G.L., Monks, B.G. & Latz, E. ASC speck formation as a readout for inflammasome activation. Methods Mol. Biol. 1040, 91–101 (2013).
Mariathasan, S. et al. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430, 213–218 (2004).
Franklin, B.S. et al. The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation. Nat. Immunol. 15, 727–737 (2014).
Baroja-Mazo, A. et al. The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat. Immunol. 15, 738–748 (2014).
Compan, V. et al. A genetically encoded IL-1β bioluminescence resonance energy transfer sensor to monitor inflammasome activity. J. Immunol. 189, 2131–2137 (2012).
Compan, V. et al. Cell volume regulation modulates NLRP3 inflammasome activation. Immunity 37, 487–500 (2012).
Kindermann, M. et al. Selective and sensitive monitoring of caspase-1 activity by a novel bioluminescent activity-based probe. Chem. Biol. 17, 999–1007 (2010).
Bartok, E. et al. iGLuc: a luciferase-based inflammasome and protease activity reporter. Nat. Methods 10, 147–154 (2013).
Dinarello, C.A. Immunological and inflammatory functions of the interleukin-1 family. Annu. Rev. Immunol. 27, 519–550 (2009).
Rider, P. et al. IL-1α and IL-1β recruit different myeloid cells and promote different stages of sterile inflammation. J. Immunol. 187, 4835–4843 (2011).
Kastenmüller, W., Torabi-Parizi, P., Subramanian, N., Lämmermann, T. & Germain, R.N. A spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread. Cell 150, 1235–1248 (2012).
Rowe, S.J., Allen, L., Ridger, V.C., Hellewell, P.G. & Whyte, M.K. Caspase-1–deficient mice have delayed neutrophil apoptosis and a prolonged inflammatory response to lipopolysaccharide-induced acute lung injury. J. Immunol. 169, 6401–6407 (2002).
Gross, O. et al. Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence. Nature 459, 433–436 (2009).
Ichinohe, T., Lee, H.K., Ogura, Y., Flavell, R. & Iwasaki, A. Inflammasome recognition of influenza virus is essential for adaptive immune responses. J. Exp. Med. 206, 79–87 (2009).
Shio, M.T. et al. Malarial hemozoin activates the NLRP3 inflammasome through Lyn and Syk kinases. PLoS Pathog. 5, e1000559 (2009).
Lima-Junior, D.S. et al. Inflammasome-derived IL-1β production induces nitric oxide-mediated resistance to Leishmania. Nat. Med. 19, 909–915 (2013).
Ghiringhelli, F. et al. Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β-dependent adaptive immunity against tumors. Nat. Med. 15, 1170–1178 (2009).
Chung, Y. et al. Critical regulation of early Th17 cell differentiation by interleukin-1 signaling. Immunity 30, 576–587 (2009).
Ben-Sasson, S.Z. et al. IL-1 enhances expansion, effector function, tissue localization, and memory response of antigen-specific CD8 T cells. J. Exp. Med. 210, 491–502 (2013).
Delaloye, J. et al. Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome. PLoS Pathog. 5, e1000480 (2009).
Waibler, Z. et al. Modified vaccinia virus Ankara induces Toll-like receptor-independent type I interferon responses. J. Virol. 81, 12102–12110 (2007).
Hornung, V. et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458, 514–518 (2009).
Gross, O., Thomas, C.J., Guarda, G. & Tschopp, J. The inflammasome: an integrated view. Immunol. Rev. 243, 136–151 (2011).
Gross, O. Measuring the inflammasome. Methods Mol. Biol. 844, 199–222 (2012).
Iwasaki, A. A virological view of innate immune recognition. Annu. Rev. Microbiol. 66, 177–196 (2012).
Junt, T. et al. Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature 450, 110–114 (2007).
Kastenmüller, W. et al. Peripheral prepositioning and local CXCL9 chemokine-mediated guidance orchestrate rapid memory CD8+ T cell responses in the lymph node. Immunity 38, 502–513 (2013).
Garcia, Z. et al. Subcapsular sinus macrophages promote NK cell accumulation and activation in response to lymph-borne viral particles. Blood 120, 4744–4750 (2012).
Balci-Peynircioglu, B. et al. Expression of ASC in renal tissues of familial mediterranean fever patients with amyloidosis: postulating a role for ASC in AA type amyloid deposition. Exp. Biol. Med. (Maywood) 233, 1324–1333 (2008).
Phan, T.G., Green, J.A., Gray, E.E., Xu, Y. & Cyster, J.G. Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation. Nat. Immunol. 10, 786–793 (2009).
Miao, E.A., Rajan, J.V. & Aderem, A. Caspase-1–induced pyroptotic cell death. Immunol. Rev. 243, 206–214 (2011).
Fernandes-Alnemri, T. & Alnemri, E.S. Assembly, purification, and assay of the activity of the ASC pyroptosome. Methods Enzymol. 442, 251–270 (2008).
Lehmann, M.H. et al. Modified vaccinia virus ankara triggers chemotaxis of monocytes and early respiratory immigration of leukocytes by induction of CCL2 expression. J. Virol. 83, 2540–2552 (2009).
Dvoriantchikova, G. et al. Genetic ablation of Pannexin1 protects retinal neurons from ischemic injury. PLoS One 7, e31991 (2012).
Mezzaroma, E. et al. The inflammasome promotes adverse cardiac remodeling following acute myocardial infarction in the mouse. Proc. Natl. Acad. Sci. USA 108, 19725–19730 (2011).
Tran, H.B. et al. Immunolocalization of NLRP3 inflammasome in normal murine airway epithelium and changes following induction of ovalbumin-induced airway inflammation. J. Allergy (Cairo) 2012, 819176 (2012).
Shirasaki, Y. et al. Real-time single-cell imaging of protein secretion. Sci. Rep. 4, 4736 (2014).
Liu, T. et al. Single-cell imaging of caspase-1 dynamics reveals an all-or-none inflammasome signaling response. Cell Rep. 8, 974–982 (2014).
Cho, J.S. et al. Neutrophil-derived IL-1β is sufficient for abscess formation in immunity against Staphylococcus aureus in mice. PLoS Pathog. 8, e1003047 (2012).
Seo, S.U. et al. Distinct commensals induce interleukin-1β via NLRP3 inflammasome in inflammatory monocytes to promote intestinal inflammation in response to injury. Immunity 42, 744–755 (2015).
Chtanova, T. et al. Dynamics of neutrophil migration in lymph nodes during infection. Immunity 29, 487–496 (2008).
Gaya, M. et al. Host response. Inflammation-induced disruption of SCS macrophages impairs B cell responses to secondary infection. Science 347, 667–672 (2015).
Lämmermann, T. et al. Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo. Nature 498, 371–375 (2013).
McDonald, B. et al. Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 330, 362–366 (2010).
Lotze, M.T. et al. The grateful dead: damage-associated molecular pattern molecules and reduction/oxidation regulate immunity. Immunol. Rev. 220, 60–81 (2007).
Zhang, Q. et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 464, 104–107 (2010).
Amaral, F.A. et al. NLRP3 inflammasome-mediated neutrophil recruitment and hypernociception depend on leukotriene B4 in a murine model of gout. Arthritis Rheum. 64, 474–484 (2012).
Kumar, V. et al. Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway. Blood 115, 4725–4733 (2010).
Dostert, C., Ludigs, K. & Guarda, G. Innate and adaptive effects of inflammasomes on T cell responses. Curr. Opin. Immunol. 25, 359–365 (2013).
Dixit, V.D. Nlrp3 inflammasome activation in type 2 diabetes: is it clinically relevant? Diabetes 62, 22–24 (2013).
Yang, M., Hearnden, C.H., Oleszycka, E. & Lavelle, E.C. NLRP3 inflammasome activation and cytotoxicity induced by particulate adjuvants. Methods Mol. Biol. 1040, 41–63 (2013).
O'Donnell, H. et al. Toll-like receptor and inflammasome signals converge to amplify the innate bactericidal capacity of T helper 1 cells. Immunity 40, 213–224 (2014).
Dunne, A. et al. Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis. J. Immunol. 185, 1711–1719 (2010).
Ben-Sasson, S.Z. et al. IL-1 acts directly on CD4 T cells to enhance their antigen-driven expansion and differentiation. Proc. Natl. Acad. Sci. USA 106, 7119–7124 (2009).
Kuida, K. et al. Altered cytokine export and apoptosis in mice deficient in interleukin-1β–converting enzyme. Science 267, 2000–2003 (1995).
Brandler, S. et al. Preclinical studies of a modified vaccinia virus Ankara-based HIV candidate vaccine: antigen presentation and antiviral effect. J. Virol. 84, 5314–5328 (2010).
Nakano, T., Kodama, H. & Honjo, T. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265, 1098–1101 (1994).
Celli, S., Breart, B. & Bousso, P. Intravital two-photon imaging of natural killer cells and dendritic cells in lymph nodes. Methods Mol. Biol. 415, 119–126 (2008).
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.).
The authors declare no competing financial interests.
Supplementary Figures 1–7 (PDF 1557 kb)
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)
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)
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)
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)
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)
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)
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)
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)
About this article
Cite this article
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
Effect of Curcumol on NOD-Like Receptor Thermoprotein Domain 3 Inflammasomes in Liver Fibrosis of Mice
Chinese Journal of Integrative Medicine (2021)
NLRP12 collaborates with NLRP3 and NLRC4 to promote pyroptosis inducing ganglion cell death of acute glaucoma
Molecular Neurodegeneration (2020)
BMC Microbiology (2019)
Nature Immunology (2019)
Molecular Cancer (2018)