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Macrophages, rather than DCs, are responsible for inflammasome activity in the GM-CSF BMDC model

Nature Immunology volume 20, pages 397–406 (2019)Cite this article

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Abstract

Inflammasomes are one of the most important mechanisms for innate immune defense against microbial infection but are also known to drive various inflammatory disorders via processing and release of the cytokine IL-1β. As research into the regulation and effects of inflammasomes in disease has rapidly expanded, a variety of cell types, including dendritic cells (DCs), have been suggested to be inflammasome competent. Here we describe a major fault in the widely used DC–inflammasome model of bone marrow–derived dendritic cells (BMDCs) generated with the cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF). We found that among GM-CSF bone marrow–derived cell populations, monocyte-derived macrophages, rather than BMDCs, were responsible for inflammasome activation and IL-1β secretion. Therefore, GM-CSF bone marrow–derived cells should not be used to draw conclusions about DC-dependent inflammasome biology, although they remain a useful tool for analysis of inflammasome responses in monocytes–macrophages.

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Fig. 1: GM-CSF-derived DCs contain a diminished inflammasome cDNA signature in comparison with GM-CSF-derived macrophages.
Fig. 2: GM-CSF-derived monocytes, rather than GM-CSF-derived DCs, express inflammasome components.
Fig. 3: GM-CSF-derived monocytes, rather than GM-CSF-derived DCs, are responsible for inflammasome activation and IL-1β secretion in the unsorted GM-CSF cultures.
Fig. 4: GM-DCs and GM-DNs are both negative for inflammasome activation and IL-1β secretion.
Fig. 5: NLRP3 inflammasome in primary splenic DCs.

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The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

This work was performed in partial fulfillment of the requirements for a Ph.D. degree (for Z.E. and I.S.), the Sackler Faculty of Medicine, Tel Aviv University, Israel.‬ The research of M.G. was supported by the Israel Science Foundation (ISF) (grants 1416/15 and 818/18), alpha-1 Foundation, Recanati Foundation (Tel Aviv University) and individual research grants from the Varda and Boaz Dotan Research Center.‬‬‬‬‬‬ The research of Y.Z. and A.M.L. was supported by the National Health and Medical Research Council (NHMRC) of Australia (grants 1037321, 1105209, 1080321 and 1143976). This work was also made possible by NHMRC project grants 1101405 (J.E.V.) and 1145788 (J.E.V. and K.E. L.) and NHMRC Fellowship 1141466 (J.E.V.).

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Authors and Affiliations

  1. Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Ziv Erlich, Inbar Shlomovitz, Liat Edry-Botzer, Hadar Cohen & Motti Gerlic

  2. Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Daniel Frank & James E. Vince

  3. Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia

    Daniel Frank, Andrew M. Lew, Yifan Zhan & James E. Vince

  4. Guangzhou Institute of Paediatrics, Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, Guangzhou, China

    Hanqing Wang

  5. Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Andrew M. Lew & Yifan Zhan

  6. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia

    Kate E. Lawlor

  7. Department of Molecular and Translational Science, Monash University, Clayton, Victoria, Australia

    Kate E. Lawlor

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  1. Ziv Erlich
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Contributions

Conceptualization: M.G. Methodology: Z.E., I.S., H.C., L.E.-B., Y.Z., J.E.V. and M.G. Investigation: Z.E., I.S., H.C., L.E.-B., D.F., H.W., K.E.L., Y.Z., J.E.V. and M.G. Writing: original draft, M.G.; review and editing, Z.E., I.S., A.M.L., K.E.L., Y.Z. and J.E.V. Funding acquisition: A.M.L., Y.Z., K.E.L., J.E.V. and M.G. Validation: L.E.-B., H.C., D.F. and K.E.L. Supervision: M.G.

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Correspondence to Motti Gerlic.

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Integrated supplementary information

Supplementary Fig. 1 Literature summary of DC–inflammasome papers.

Analysis of the last 5-years literature on DC–inflammasome according to the DC model (a) or the inflammasome type (b). From January 2013 to November 2017 published DC–inflammasome manuscripts were manually analyzed for the DC population that was used for the inflammasome study.

Supplementary Fig. 2 Schematic overview of single-cell immunoblot (scIB) technology (relevant to Fig. 2).

(a) Schematic overview of scIB technology data analysis using Scout software (ProteinSimple) and exported as a FCS file for final data analysis using FlowJo software (Tree Star, Ashland, OR). (b) Analysis of the migration distribution of the different proteins as detected by scIB (relative to Fig. 2g; representative of 2 repeats, symbols represent individua; cells and results are presented as mean ± sd). (c) Representative picture of a ~10% of scIB chip (relative to Fig. 2h) after developing (>2000 cells were detected); the green arrow indicates ASC protein; the black arrow indicates Tubulin; the red arrow indicates IL-1β.

Supplementary Fig. 3 Inflammasome detection in BM-derived DCs (relevant to Fig. 3).

(a-d) Approximately 105 unsorted and sorted cells from 2 mice were seeded into 96-well plates in triplicate for inflammasome activation assays and primed using LPS (100 ng/ml, 3 hours (a-c) and 24 hours (d)). Cells were stimulated for inflammasome activity by poly(dA:dT) (1μg/ml, 4 hours) and ATP (5 mM, 2 hours). Where indicated, VX-765 (25 μM, caspase-1 inhibitor) was added to the cells 30 minutes before inflammasome stimulation. About 105 cells (a, b) or their supernatant (c-d) were collected at the indicated times after inflammasome stimulation. Inflammasome components, caspase-1, GSDMD and IL-1β cleavage were detected by IB or ELISA (symbols represent independent wells (n=3) and results are presented as mean ± sd). *non-specific bands. (a) and (d) were repeated once, while (b-c) are representative from 2 independent experiments. (e) Representative FACS plots showing the gating strategy for the OT-I proliferation assay. Proliferation of naïve CD8+ T cells (OT-I) in response to priming by the different DCs was assessed by FACS, according to the reduction in the dye intensity of T cells. Briefly, T cells were cultured with the indicated DC populations (2:1 ratio) with or without soluble OVA Ag, as indicated in 96-well U-bottom plates for 66 hours (OT-I). Cell numbers and proliferating T cells were determined using FACS and PE-conjugated calibration beads (BD Biosciences). Flow cytometry data analyses were performed using FlowJo software (Tree Star, Ashland, OR). (f) Representative FACS plots showing the gating strategy used to isolate the 10-day Flt3L BM-derived DCs (from 4 pooled mice; 6 independent repeats). (g-j) About 105 sorted cells or 5x104 BMDMs were seeded into 96-well plates in triplicate for inflammasome activation assays and primed using LPS (100 ng/ml, 3 hours) or CpG (100 ng/ml, 3 hours). Cells were stimulated for inflammasome activity either by ATP (5 mM, 2 hours) or nigericin (10 μM, 3 hours). About 105 cells (g-h) were collected at the indicated times after inflammasome stimulation, and inflammasome components and GSDMD cleavage were detected using IB. *non-specific bands All IBs represent 2 independent experiments. (i-j) Supernatants were collected at the indicated times after inflammasome stimulation, and IL-1β or IL-12p40 secretion was measured using commercial ELISA kits (symbols represent 1 (CpG) or 4 (LPS) independent experiments and results are presented as mean ± sd). All images from (a-c), (g) and (h) were cropped and their full-size images are presented in Supplementary Fig. 10-11.

Supplementary Fig. 4 Sorted GM-DCs and GM-DNs are functionally active cells (relevant to Fig. 4).

(a) Representative FACS plots showing the gating strategy used to isolate the GM-DCs, GM-DNs and GM-Macs from the loosely adherent seven-day GM-CSF BM-derived heterogeneous cell populations, pooled from 2 mice (8 independent repeats). (b-f) Approximately 105 unsorted and sorted cells were seeded into 96-well plates for inflammasome activation assays and primed using LPS (100 ng/ml) (c, e-f) for 3 hours or CpG (100 ng/ml) for 3 (b) or 6 hours (d). Cells were stimulated for inflammasome activity by ATP (5 mM, 2 hours). (b) Cell lysate was collected post inflammasome stimulation, and inflammasome components, caspase-1, GSDMD and IL-1β cleavage were detected by IB (representative of 3 independent biological experiments; All images were cropped and their full-size images are presented in Supplementary Fig. 12). (c) PI was added to triplicated-sorted cells post-LPS priming before inflammasome stimulation. PI uptake was measured using real-time microscopy (Incucyte ZOOM) and graphs as a means of determining PI-positive cells (repeated once; this is the graph that is presented in Fig. 4d with longer kinetic). (d-f) Supernatants were collected at the indicated times after inflammasome stimulation, IL-1β, TNFα and IL-6 secretion was measured using commercial ELISA kits (mean + sd). Symbols in (d) represent independent wells (n=3) while symbols in (e-f) represent independent experiments (n=3); results are presented as mean ± sd. (g-i) Different GM-CSF BM cell populations (106/ml) were (g-h) co-cultured at a 10:1 ratio with BD-beads for 3, 5 and 18 hours or (i) infected with GFP–P.Vibrio (MOI 20) for one hour in triplicate. Cells were collected, washed three times with PBS and analyzed for phagocytosis by flow cytometry. (g) Representative FACS plots of 2 independent experiments showing the gating strategy for the bead phagocytosis assay. Symbols in (h) and (i) represent independent samples (n=3 and 1, respectively) and results are presented as mean ± sd. Representative FACS plots of 3 independent animal replicates showing the gating strategy for the OT-I proliferation assay. Proliferation of naïve CD8+ T cells (OT-I) in response to priming by the different DCs was assessed by FACS, according to the reduction in the dye intensity of T cells. Briefly, T cells were cultured with the indicated DC populations (2:1 ratio) with or without soluble OVA Ag as indicated in the 96-well U-bottom plates for 66 h (OT-I). Cell numbers and proliferating T cells were determined using FACS and PE-conjugated calibration beads (BD Biosciences). Flow cytometry data analyses were performed using FlowJo software (Tree Star, Ashland, OR).

Supplementary Fig. 5 NLRP3 inflammasome in primary splenic DCs (relevant to Fig. 5).

(a) Representative FACS plots showing the purity of the isolated primary splenic pDCs; cDC1, cDC2 from Fig. 5a,c. (b, d) FACS plots showing the gating strategy used to isolate primary splenic pDCs, cDC1, cDC2 (b) and moDCs or CD11c+ monocytes (d) for determining the data in panels c and e. (c, e) About 105 sorted splenic DCs from 3 mice were seeded into 96-well plates in triplicate for inflammasome activation assays and primed using CpG or LPS (100 ng/ml, 2–3 hours). Primed cells were stimulated for NLRP3 inflammasome activity by nigericin (10 μM). At 3 hours post-NLRP3 inflammasome stimulation, the cells were collected and (c) Inflammasome components were detected using IB (numbers on the right of each blot indicate the blot number: All images were cropped and their full-size images are presented in Supplementary Fig. 12). (e) Supernatants were collected after inflammasome stimulation, and IL-1β secretion was measured using commercial ELISA kits (symbols represent independent samples (n=3) and results are presented as mean ± sd). (f) FACS plots showing the comparison of in vivo GMtg “moDCs” with the ex-vivo BM-derived GM-DCs. Red boxes indicate a direct comparison between the pure populations. All FACS plots represent at least 3 independent biological experiments. (c), and (e) are representative from 2 independent experiments.

Supplementary Fig. 6

Whole-gel scans of immunoblots presented in Fig. 2c and 2d.

Supplementary Fig. 7

Whole-gel scans of immunoblots presented in Fig. 3f,g.

Supplementary Fig. 8

Whole-gel scans of immunoblots presented in Fig. 4b,e.

Supplementary Fig. 9

Whole-gel scans of immunoblots presented in Fig. 5b,f,g.

Supplementary Fig. 10

Whole-gel scans of immunoblots presented in Supplementary Fig. 3a–c.

Supplementary Fig. 11

Whole-gel scans of immunoblots presented in Supplementary Fig. 3g,h.

Supplementary Fig. 12

Whole-gel scans of immunoblots presented in Supplementary Figs. 4b and Fig. 5c.

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Erlich, Z., Shlomovitz, I., Edry-Botzer, L. et al. Macrophages, rather than DCs, are responsible for inflammasome activity in the GM-CSF BMDC model. Nat Immunol 20, 397–406 (2019). https://doi.org/10.1038/s41590-019-0313-5

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