A central and critical structure in tuberculosis, the mycobacterial granuloma consists of highly organized immune cells, including macrophages that drive granuloma formation through a characteristic epithelioid transformation. Difficulties in imaging within intact animals and caveats associated with in vitro assembly models have severely limited the study and experimental manipulation of mature granulomas. Here we describe a new ex vivo culture technique, wherein mature, fully organized zebrafish granulomas are microdissected and maintained in three-dimensional (3D) culture. This approach enables high-resolution microscopy of granuloma macrophage dynamics, including epithelioid macrophage motility and granuloma consolidation, while retaining key bacterial and host characteristics. Using mass spectrometry, we find active production of key phosphotidylinositol species identified previously in human granulomas. We also describe a method to transfect isolated granulomas, enabling genetic manipulation, and provide proof-of-concept for host-directed small-molecule screens, identifying protein kinase C (PKC) signaling as an important regulator of granuloma macrophage organization.
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We are grateful to members of the Tobin laboratory and J. Stout for helpful discussions, E. Hughes for critical reading of the granuloma protocol, E. Hunt for fish care, A. Piro and J. Coers for reagents, R. Abramovitch for the HspX reporter plasmid, and D. Russell for suggesting RNA transfection of granuloma macrophages. This work was supported by an American Cancer Society Postdoctoral Fellowship PF-13-223-01-MPC (M.R.C.); an NSF Graduate Research Fellowship (M.A.M.); NIH NRSA 1F32AI124658-01A1 (A.F.R.) and NIH grants AI130236, AI125517 and AI127115 (D.M.T.) and 5P01DK094779-05 (J.F.R.), and a Vallee Scholar Award (D.M.T.).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 Multidimensional culture approaches are required for long-term maintenance of granuloma morphology and cell survival.
Phase contrast images of (a) granuloma cultured on tissue culture plastic. (b) granuloma cultured in low-melt agarose or (c) in 3D Matrigel culture conditions. Times indicate duration of culture ex vivo. Scale bars, 50 µm. (a-c) Similar results were observed in at least 2 independent experiments. (d) Hematoxylin and eosin staining of paraffin sections from either granulomas in vivo (2 weeks post infection) or granulomas grown in Myco-GEM conditions for 6 d. Both granulomas grown in vivo and granulomas cultured in Myco-GEM conditions show the characteristic spread, eosinophilic cytoplasm of epithelioid cells. Necrotic cores within the granulomas are outlined in dotted yellow lines. For Myco-GEM cultured granulomas, results are derived from a single group of 4 granulomas from one animal.
Supplementary Figure 2 CLARITY clearing enables visualization of neutrophil localization and necrotic core formation within dissected granulomas.
(a) Granulomas dissected from zebrafish were fixed, CLARITY-cleared and stained to visualize neutrophils (LysC, magenta) and nuclei (Hoechst, blue) while M. marinum (green) was visualized by fluorescent protein expression. (b) Freshly dissected, CLARITY-cleared granulomas from animals infected with fluorescent protein expressing M. marinum (green) stained with Hoechst (blue). (a,b) Similar results were found with at least 6 granulomas from a single animal.
Mycobacterial stress was detected by HspX-driven GFP induction (green), while mycobacterial localization was detected by constitutive mCherry expression (red). High-contrast images demonstrating persistent stress within individual mycobacteria outside of the necrotic core. White arrowheads denote stressed mycobacteria that express both GFP and mCherry, magenta arrowheads denote unstressed mycobacteria expressing only mCherry. Lower contrast images demonstrating the stressed populations (expressing both GFP and mCherry) within the necrotic core of the granuloma. HspX reporter fluorescence was visualized in 3 independent experiments, with similar results in each experiment. Scale bar, 50 µm.
Granulomas were cultured using Myco-GEM medium supplemented with 12C glucose (top) or 13C glucose (bottom) and analyzed by MALDI-MS. (a) After 4 d of culture in 13C, a + 3 mass shift was observed in the 885.55 m/z peak corresponding to phosphatidylinositol (PI) 38:4. (b) Granulomas were cultured for 4 d in 12C glucose followed by 2 d in 13C glucose. Following the 2-d incubation in 13C, a similar + 3 mass shift could be observed for the 885.55 m/z peak corresponding to phosphatidylinositol (PI) 38:4. (c) MS/MS analysis of ion 885.55 m/z shows that the two fatty acid chains are arachidonic acid (303.23 m/z) and stearic acid (283.26 m/z). (d) MS/MS analysis of ion 888.56 m/z, corresponding to 13C-labelled PI 38:4, and associated fragmentation patterns show incorporation of the labeled carbons in the glycerol moiety of PI 38:4.
Supplementary Figure 5 Granulomas form E-cadherin-positive adherens junctions that are maintained throughout Myco-GEM culture.
(a) Granulomas were dissected from E-cadherin-tomato knock-in animals, placed in Myco-GEM culture conditions and immediately imaged for E-cadherin (magenta) or M. marinum (green) at the indicated depths. Yellow box indicates area magnified below. Scale bar, 50 µm. (b) After 5 d of Myco-GEM culture, granulomas were imaged for E-cadherin (magenta) or M. marinum (green), showing a similar organization of E-cadherin-positive adherens junctions after culture. Depth is indicated on images. For cultured granulomas, depths of images were chosen to match landmarks visualized in freshly dissected granulomas. Yellow box indicates magnified area below. Scale bar, 50 µm. Experiments in a,b were repeated twice with similar results each time.
Supplementary Figure 6 Deep imaging within ex vivo–cultured granulomas by spinning disk confocal microscopy and light-sheet microscopy.
Images of plakoglobin-citrine gene trap (yellow) and fluorescent M. marinum (cyan) at the indicated depths through the granuloma acquired by (a) spinning disk or (b) light-sheet microscopy. Granuloma images obtained ~ 1 h post-plating. Scale bars, 50 µm. Experiments in a were repeated independently 3 times and in b were repeated 2 times with similar results.
Supplementary Figure 7 Longitudinal imaging of granuloma adherens junctions by light-sheet microscopy.
(a) Maximum projection images of a 39-µm z-stack image showing plakoglobin-citrine (yellow) and fluorescent M. marinum (cyan). Time lapse of the same granuloma seen in z-section in Supplementary Fig. 6. Scale bar, 50 µm. (b) Light-sheet imaging of a granuloma cultured for 7 d under Myco-GEM conditions showing plakoglobin-citrine (yellow). Individual images are distinct z-planes as indicated on the image. (a,b) Similar results were obtained in 2 independent experiments.
Granulomas from tnf:gfp animals infected with fluorescent M. marinum were cultured and longitudinally imaged for 5 d and tnf transcriptional induction was monitored by GFP expression, showing similar levels throughout culture. Gamma-adjusted images are shown to visualize GFP expression in the cells surrounding the necrotic core due to the substantial concentration of GFP within the necrotic core. GFP and M. marinum images are maximum projections of 70-µm z-stacks. Phase contrast images are single-plane images from near the center of the granuloma. TNF reporter experiments were independently repeated 3 times with similar results. Scale bar, 50 µm.
(a) Images of cultured granulomas either untreated or treated with the indicated concentrations of anti-mycobacterial agents. Numbers under the images indicate the CFU count from plating and the relative light units (RLU) detected by plate reader. CFU results are representative from 3 granulomas from each group from a single experiment. (b) Graph of relative light units detected from untreated, 200 µg/ml isoniazid treated, or 80 µg/ml streptomycin treated granulomas at 3 days post-plating (dpp). Results are presented as mean ± s.d. for n = 9 granulomas. ****P < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test. Luminescence results are representative of three independent experiments.
Supplementary Figures 1–9 and Supplementary Table 1
Three-dimensional culture enables long-term visualization of granuloma dynamics: An isolated granuloma cultured in 3D Matrigel culture for 4 d was longitudinally observed by phase contrast microscopy, enabling the visualization of cellular movement within the granuloma. Time of culture is indicated in hours:minutes:seconds. Similar results were seen in two independent experiments
Neutrophil localization in a CLARITY-cleared, dissected granuloma: Granulomas from animals infected with fluorescent protein expressing M. marinum (green) were dissected and CLARITY-cleared followed by staining for the neutrophil marker LysC (magenta) and nuclei (Hoechst, blue). Movie is a z-stack of 180 µm. Similar results were seen in 6 granulomas from one animal
Groups of bacteria localize to a central necrotic core devoid of nuclei: Granulomas generated by infecting animals with fluorescent mycobacteria (green) were dissected, CLARITY-cleared and stained with Hoechst to visualize nuclei (blue) within the granuloma. Movie is a 100-µm z-stack. Microscopy of 8 granulomas from one animal; similar results were seen in all 8 granulomas
Macrophage dynamics within a mature granuloma during Myco-GEM culture: Macrophages were sparsely labeled by macrophage lineage tracing (red) and tracked overnight after plating. Top, macrophages alone visualized by macrophage lineage tracing. Middle, overlay of macrophage labelling with DIC. Bottom, Macrophage labeling overlaid with dots and lines denoting the migration of individual macrophages. Time of culture is indicated in hours:minutes:seconds, Scale bar, 50 µm. Similar results were observed in 3 independent experiments
Visualization of granuloma consolidation at cellular resolution: Two granulomas—one in which the cells ubiquitously express tomato fluorescent protein (Tg(ubb:tomato), red), infected with cerulean expressing M. marinum (cyan), and one isolated from wild-type animals infected with wasabi expressing M. marinum (green)—were placed adjacent to one another and allowed to undergo consolidation over the course of 6 d. Left, Phase contrast image. Center, Tomato fluorescence. Right,
Longitudinal imaging of transfected granuloma macrophages: Granuloma transfected with Tomato RNA and cultured for 3 d in Myco-GEM conditions. Transfected macrophages (red) continue to maintain viability and motility after transfection and 3 d of culture. Left, Phase contrast images of the transfected granuloma, Center, Tomato expression, Right, M. marinum within the granuloma. Similar results were observed in 3 independent experiments
Vehicle-treated granulomas maintain adherens junctions after treatment: Light-sheet microscopy of a DMSO-treated granuloma showing long-term maintenance of granuloma adherens junctions (yellow) during the course of imaging. Similar results were seen in 3 independent experiments
Organization of granuloma adherens junctions is disrupted by Gö6983 treatment: Light-sheet microscopy of a granuloma treated with 10 µM Gö6983. During treatment, granuloma adherens junctions (yellow) become disrupted and reorganize. 3 independent experiments showed similar results
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Cronan, M.R., Matty, M.A., Rosenberg, A.F. et al. An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas. Nat Methods 15, 1098–1107 (2018). https://doi.org/10.1038/s41592-018-0215-8
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