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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Mouse innate-like B-1 lymphocytes promote inhaled particle-induced in vitro granuloma formation and inflammation in conjunction with macrophages
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Ramakrishnan, L. Revisiting the role of the granuloma in tuberculosis. Nat. Rev. Immunol. 12, 352–366 (2012).
Lenaerts, A., Barry, C. E. III & Dartois, V. Heterogeneity in tuberculosis pathology, microenvironments and therapeutic responses. Immunol. Rev. 264, 288–307 (2015).
Adams, D. O. The structure of mononuclear phagocytes differentiating in vivo. I. Sequential fine and histologic studies of the effect of Bacillus Calmette-Guerin (BCG). Am. J. Pathol. 76, 17–48 (1974).
Cronan, M. R. et al. Macrophage epithelial reprogramming underlies mycobacterial granuloma formation and promotes infection. Immunity 45, 861–876 (2016).
Mattila, J. T. et al. Microenvironments in tuberculous granulomas are delineated by distinct populations of macrophage subsets and expression of nitric oxide synthase and arginase isoforms. J. Immunol. 191, 773–784 (2013).
Wolf, A. J. et al. Mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their function in vivo. J. Immunol. 179, 2509–2519 (2007).
Volkman, H. E. et al. Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant. PLoS Biol. 2, e367 (2004).
Davis, J. M. & Ramakrishnan, L. The role of the granuloma in expansion and dissemination of early tuberculous infection. Cell 136, 37–49 (2009).
Lin, P. L. et al. Sterilization of granulomas is common in active and latent tuberculosis despite within-host variability in bacterial killing. Nat. Med. 20, 75–79 (2014).
Samstein, M. et al. Essential yet limited role for CCR2+ inflammatory monocytes during Mycobacterium tuberculosis-specific T cell priming. eLife 2, e01086 (2013).
Wolf, A. J. et al. Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs. J. Exp. Med. 205, 105–115 (2008).
Harding, J. S., Rayasam, A., Schreiber, H. A., Fabry, Z. & Sandor, M. Mycobacterium-infected dendritic cells disseminate granulomatous inflammation. Sci. Rep. 5, 15248 (2015).
Malherbe, S. T. et al. Persisting positron emission tomography lesion activity and Mycobacterium tuberculosis mRNA after tuberculosis cure. Nat. Med. 22, 1094–1100 (2016).
Coleman, M. T. et al. PET/CT imaging reveals a therapeutic response to oxazolidinones in macaques and humans with tuberculosis. Sci. Transl. Med. 6, 265ra167 (2014).
Egen, J. G. et al. Intravital imaging reveals limited antigen presentation and T cell effector function in mycobacterial granulomas. Immunity 34, 807–819 (2011).
Torabi-Parizi, P. et al. Pathogen-related differences in the abundance of presented antigen are reflected in CD4+ T cell dynamic behavior and effector function in the lung. J. Immunol. 192, 1651–1660 (2014).
Egen, J. G. et al. Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas. Immunity 28, 271–284 (2008).
Guirado, E. et al. Characterization of host and microbial determinants in individuals with latent tuberculosis infection using a human granuloma model. MBio 6, e02537-14 (2015).
Puissegur, M. P. et al. An in vitro dual model of mycobacterial granulomas to investigate the molecular interactions between mycobacteria and human host cells. Cell Microbiol. 6, 423–433 (2004).
Tezera, L. B. et al. Dissection of the host-pathogen interaction in human tuberculosis using a bioengineered 3-dimensional model. eLife 6, e21283 (2017).
Birkness, K. A. et al. An in vitro model of the leukocyte interactions associated with granuloma formation in Mycobacterium tuberculosis infection. Immunol. Cell Biol. 85, 160–168 (2007).
Davis, J. M. et al. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos. Immunity 17, 693–702 (2002).
Oehlers, S. H. et al. Interception of host angiogenic signalling limits mycobacterial growth. Nature 517, 612–615 (2015).
Swaim, L. E. et al. Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity. Infect. Immun. 74, 6108–6117 (2006).
Parikka, M. et al. Mycobacterium marinum causes a latent infection that can be reactivated by gamma irradiation in adult zebrafish. PLoS Pathog. 8, e1002944 (2012).
Cronan, M. R. et al. CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections. Dis. Model Mech. 8, 1643–1650 (2015).
Flynn, J. L. et al. Tumor necrosis factor-alpha is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2, 561–572 (1995).
Lin, P. L. et al. Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model. Arthritis Rheum. 62, 340–350 (2010).
Clay, H., Volkman, H. E. & Ramakrishnan, L. Tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death. Immunity 29, 283–294 (2008).
Marjoram, L. et al. Epigenetic control of intestinal barrier function and inflammation in zebrafish. Proc. Natl Acad. Sci. USA 112, 2770–2775 (2015).
Shapouri-Moghaddam, A. et al. Macrophage plasticity, polarization, and function in health and disease. J. Cell. Physiol. 233, 6425–6440 (2018).
Tan, S., Sukumar, N., Abramovitch, R. B., Parish, T. & Russell, D. G. Mycobacterium tuberculosis responds to chloride and pH as synergistic cues to the immune status of its host cell. PLoS Pathog. 9, e1003282 (2013).
Tan, S. & Russell, D. G. Trans-species communication in the Mycobacterium tuberculosis-infected macrophage. Immunol. Rev. 264, 233–248 (2015).
Philips, J. A. & Ernst, J. D. Tuberculosis pathogenesis and immunity. Annu. Rev. Pathol. 7, 353–384 (2012).
Marakalala, M. J. et al. Inflammatory signaling in human tuberculosis granulomas is spatially organized. Nat. Med. 22, 531–538 (2016).
Zhurinsky, J., Shtutman, M. & Ben-Ze’ev, A. Plakoglobin and beta-catenin: protein interactions, regulation and biological roles. J. Cell Sci. 113, 3127–3139 (2000).
Walton, E. M., Cronan, M. R., Beerman, R. W. & Tobin, D. M. The macrophage-specific promoter mfap4 allows live, long-term analysis of macrophage behavior during mycobacterial infection in zebrafish. PLoS ONE 10, e0138949 (2015).
Martin, C. J. et al. Digitally barcoding Mycobacterium tuberculosis reveals in vivo infection dynamics in the macaque model of tuberculosis. MBio 8, e00312–e00317 (2017).
Dartois, V. The path of anti-tuberculosis drugs: from blood to lesions to mycobacterial cells. Nat. Rev. Microbiol. 12, 159–167 (2014).
Andreu, N. et al. Optimisation of bioluminescent reporters for use with mycobacteria. PLoS ONE 5, e10777 (2010).
Cosma, C. L., Klein, K., Kim, R., Beery, D. & Ramakrishnan, L. Mycobacterium marinum Erp is a virulence determinant required for cell wall integrity and intracellular survival. Infect. Immun. 74, 3125–3133 (2006).
Adams, K. N. et al. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell 145, 39–53 (2011).
Rodriguez-Boulan, E. & Macara, I. G. Organization and execution of the epithelial polarity programme. Nat. Rev. Mol. Cell Biol. 15, 225–242 (2014).
Sato, K. et al. Numb controls E-cadherin endocytosis through p120 catenin with aPKC. Mol. Biol. Cell 22, 3103–3119 (2011).
Winograd-Katz, S. E., Fässler, R., Geiger, B. & Legate, K. R. The integrin adhesome: from genes and proteins to human disease. Nat. Rev. Mol. Cell Biol. 15, 273–288 (2014).
Tobin, D. M. et al. The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans. Cell 140, 717–730 (2010).
Bafica, A. et al. Host control of Mycobacterium tuberculosis is regulated by 5-lipoxygenase-dependent lipoxin production. J. Clin. Invest. 115, 1601–1606 (2005).
Chen, M. et al. Lipid mediators in innate immunity against tuberculosis: opposing roles of PGE2 and LXA4 in the induction of macrophage death. J. Exp. Med. 205, 2791–2801 (2008).
Mayer-Barber, K. D. et al. Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk. Nature 511, 99–103 (2014).
Subbian, S. et al. Chronic pulmonary cavitary tuberculosis in rabbits: a failed host immune response. Open Biol. 1, 110016 (2011).
Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A. & Lieschke, G. J. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood 117, e49–e56 (2011).
Prideaux, B. et al. The association between sterilizing activity and drug distribution into tuberculosis lesions. Nat. Med. 21, 1223–1227 (2015).
Zimmerman, M. et al. Ethambutol partitioning in tuberculous pulmonary lesions explains its clinical efficacy. Antimicrob. Agents Chemother. 61, e00924–17 (2017).
Irwin, S. M. et al. Bedaquiline and pyrazinamide treatment responses are affected by pulmonary lesion heterogeneity in Mycobacterium tuberculosis infected c3heb/fej mice. ACS Infect. Dis. 2, 251–267 (2016).
Mor, N., Simon, B., Mezo, N. & Heifets, L. Comparison of activities of rifapentine and rifampin against Mycobacterium tuberculosis residing in human macrophages. Antimicrob. Agents Chemother. 39, 2073–2077 (1995).
Bonventre, P. F., Hayes, R. & Imhoff, J. Autoradiographic evidence for the impermeability of mouse peritoneal macrophages to tritiated streptomycin. J. Bacteriol. 93, 445–450 (1967).
Tulkens, P. M. Intracellular distribution and activity of antibiotics. Eur. J. Clin. Microbiol. Infect. Dis. 10, 100–106 (1991).
Hawn, T. R., Matheson, A. I., Maley, S. N. & Vandal, O. Host-directed therapeutics for tuberculosis: can we harness the host? Microbiol. Mol. Biol. Rev. 77, 608–627 (2013).
Datta, M. et al. Anti-vascular endothelial growth factor treatment normalizes tuberculosis granuloma vasculature and improves small molecule delivery. Proc. Natl Acad. Sci. USA 112, 1827–1832 (2015).
Napier, R. J. et al. Imatinib-sensitive tyrosine kinases regulate mycobacterial pathogenesis and represent therapeutic targets against tuberculosis. Cell Host Microbe 10, 475–485 (2011).
Xu, Y. et al. Matrix metalloproteinase inhibitors enhance the efficacy of frontline drugs against Mycobacterium tuberculosis. PLoS Pathog. 14, e1006974 (2018).
Gautam, U. S. et al. In vivo inhibition of tryptophan catabolism reorganizes the tuberculoma and augments immune-mediated control of Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 115, E62–E71 (2018).
VanderVen, B. C. et al. Novel inhibitors of cholesterol degradation in Mycobacterium tuberculosis reveal how the bacterium’s metabolism is constrained by the intracellular environment. PLoS Pathog. 11, e1004679 (2015).
Huang, L. et al. The deconstructed granuloma: A complex high-throughput drug screening platform for the discovery of host-directed therapeutics against tuberculosis. Front. Cell. Infect. Microbiol. 8, 275 (2018).
Kwan, K. M. et al. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev. Dyn. 236, 3088–3099 (2007).
Mosimann, C. et al. Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish. Development 138, 169–177 (2011).
Balciunas, D. et al. Harnessing a high cargo-capacity transposon for genetic applications in vertebrates. PLoS Genet. 2, e169 (2006).
Takaki, K., Davis, J. M., Winglee, K. & Ramakrishnan, L. Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish. Nat. Protoc. 8, 1114–1124 (2013).
Auer, T. O., Duroure, K., De Cian, A., Concordet, J. P. & Del Bene, F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res. 24, 142–153 (2014).
Moreno-Mateos, M. A. et al. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat. Methods 12, 982–988 (2015).
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