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Interception of host angiogenic signalling limits mycobacterial growth

Abstract

Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis1,2. Here we examine the development and consequences of vascularization of the tuberculous granuloma in the zebrafish–Mycobacterium marinum infection model, which is characterized by organized granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis2. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated with angiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule Vegfaa. Pharmacological inhibition of the Vegf pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergize with the first-line anti-tubercular antibiotic rifampicin, as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations3. Our data indicate that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting tuberculosis therapy, particularly in extensively drug-resistant disease for which current antibiotic regimens are largely ineffective.

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Figure 1: M. marinum infection induces angiogenesis in the zebrafish infection model.
Figure 2: M. marinum induces vascularization through granuloma formation in cooperation with host leukocytes and the expression of Vegf.
Figure 3: Inhibition of VEGFR signalling reduces M. marinum pathogenicity in zebrafish larvae.
Figure 4: Inhibition of VEGFR signalling reduces M. marinum burden in adult zebrafish.

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Acknowledgements

We thank D. Sisk and J. Saelens for technical assistance, L. Ramakrishnan, P. Edelstein and C. Kontos for helpful discussions, L. Ramakrishnan, W. Britton and J. Coers for critical review of the manuscript, and J. Fuller, C. Gallione, E. Linney, H. Mao, S. Lee, D. Marchuk, H. Matsunami, A. Nixon, J. Perfect, J. Rawls, D. Silver, K. Smith, K. Takaki, J. Tenor and B. Uy for reagents and equipment. This work was funded by an Australian National Health and Medical Research Council CJ Martin Early Career Fellowship (S.H.O.); an American Cancer Society Postdoctoral Fellowship PF-13-223-01-MPC (M.R.C.); the Duke Summer Research Opportunities Program (N.R.S.); a Malaysian Ministry of Science and Technology and Innovation scholarship (K.S.O.); a New Zealand Ministry of Science and Innovation grant UOAX0813 (P.S.C.); the Duke University Center for AIDS Research (CFAR), a National Institutes of Health (NIH)-funded program (5P30 AI064518), and by a Mallinckrodt Scholar Award, a Searle Scholar Award, a Vallee Foundation Young Investigator Award and an NIH Director’s New Innovator Award 1DP2-OD008614 (D.M.T.).

Author information

Authors and Affiliations

Authors

Contributions

S.H.O. and D.M.T. designed the experiments and wrote the paper. S.H.O., N.R.S., M.I.T. and K.S.O. performed and analysed the experiments. M.R.C., E.M.W. and R.W.B. generated transgenic zebrafish lines. S.H.O., P.S.C. and D.M.T. supervised the project.

Corresponding author

Correspondence to David M. Tobin.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Angiogenesis in the zebrafish M. marinum infection model.

a, Image of 6 dpi Tg(mfap4:turquoisext27) larvae infected with M. marinum SM2 pMAP49::Venus. Blue arrowheads indicates site of granuloma with induced expression of Venus from phagocytosed M. marinum. White arrowheads indicate sites of extracellular M. marinum growth detected by constitutive DsRed expression but no macrophage-induced Venus expression. Image is representative of granulomas found in five individual animals. b, Time-lapse images of Cerulean-fluorescent M. marinum dissemination from an established granuloma into the adjacent intersegmental vessel in a Tg(flk1:eGFP, mpeg1:tdTomato-caaxxt3) double-transgenic larva where bacterial are labelled blue, blood vessels are labelled green and macrophages are labelled red. Yellow arrow tracks a single infected macrophage egressing the established granuloma and entering the vasculature. Images are representative of macrophage behaviour in three individual animals. c, Plots of vessel growth kinetics from three individual branches in individual Tg(flk1:eGFP) larvae. Videos of each larva analysed are available in Supplementary Videos 6 and 7 (left), and 8 and 9 (right). d, Time-lapse images of nuclear division during vascular growth in a single Tg(fli1a:eGFP-nls) larva. Blue arrowhead indicates nucleus of interest. Images are representative of nuclear division in ten individual animals. Video of nuclear division is available in Supplementary Video 10. e, Three-dimensional rendering of recruited blood vessels in a Tg(flk1:eGFP) larva infected with Tomato-fluorescent M. marinum originating from arterial and venous ISVs as indicated by red and blue arrows, respectively. Image is representative of ten individual animals. f, Extended exposure images of blood flow in Tg(flk1:eGFP, gata1:DsRedsd2) larvae. Blue arrows indicate blood flow through ectopic vessels. Images are representative of blood flow in 20 individual animals. Scale bars, 100 µm.

Source data

Extended Data Figure 2 Formation of ectopic vasculature is dependent on granuloma formation.

a, Length of abnormal vasculature in Tg(flk1:eGFP) larvae injected with PBS, live M. marinum, heat-killed M. marinum and E. coli. One-way ANOVA with Tukey’s post-test, data are representative of two biological replicates. b, Recruitment of vasculature by intracellular and extracellular foci of M. marinum. Total number of foci analysed: 4 dpi, 221 intracellular, 105 extracellular; 5 dpi, 71 intracellular, 26 extracellular; and 6 dpi, 131 intracellular, 50 extracellular. Fisher’s exact test. c, Comparative images of 5 dpf control and Pu.1 morphant Tg(mpeg1:tdTomato-caaxxt3) larvae. White arrowhead indicates comparative locations within the caudal haematopoietic tissue. Blue arrowhead indicates intestinal and yolk sac autofluorescence. Scale bar, 100 µm. Images are representative of transgene expression in 20 animals per treatment group. d, e, Bacterial burden in 5 dpi control and Pu.1 morphant larvae (d), and 4 dpi larvae infected with wild-type (WT) or ΔESX1 Tomato-fluorescent M. marinum (e). Student’s t-test with Welch’s correction, all data are pooled from two biological replicates. Error bars represent mean ± s.d. **P < 0.01, ***P < 0.001.

Source data

Extended Data Figure 3 Granuloma vascularization correlates with granuloma size.

a, Plot of abnormal vasculature length and bacterial burden for individual foci of infection measured by fluorescent pixel count (FPC) in Tg(flk1:eGFP) larvae. Slope significantly not zero, P < 0.0001 linear regression, data are pooled from three biological replicates. b, Whole-mount in situ hybridization detection of phd3 expression. Images are representative of phd3 staining in uninfected (20/20), caudal vein (CV)-infected (20/20) and trunk-infected (7/20) zebrafish. c, Left, images of Tg(lyzC:ntr-p2A-lanYFPxt14) larvae treated with metronidazole as indicated. Green arrowheads indicate comparative locations within caudal haematopoietic tissue. Images are median images from experimental groups: control, n = 21; 100 μM, n = 22; 1 mM, n = 24; and 10 mM, n = 19. Right, quantification of neutrophil numbers by area of fluorescence in Tg(lyzC:ntr-p2A-lanYFPxt14) larvae treated with metronidazole from 2 dpf to 6 dpf. Error bars represent mean ± s.d.

Source data

Extended Data Figure 4 M. marinum infection induces expression of vegfaa.

a, Whole-mount in situ hybridization detection of vegfaa expression in uninfected, caudal vein (CV)-injected and trunk-injected larvae. Red arrow indicates sites of infection with vegfaa expression. Images are representative of 20 animals per treatment group. b, Representative histological sections of whole-mount in situ hybridization detected vegfaa expression in control infected larvae and a Pu.1 morpholino (MO)-treated infected larva. Black arrows indicate sites of infection identified by increased nuclear fast red staining density. Images are representative of ten animals per treatment group. c, Microangiography of Tg(flk1:eGFP) larvae imaged at 1, 5 and 10 min post-injection (mpi). Top panels are representative of uninfected larvae, bottom panels are representative of larvae infected with unlabelled M. marinum. Images are representative of ten animals per treatment group. Scale bars, 100 µm.

Extended Data Figure 5 Pazopanib and SU5416 reduce M. marinum pathogenicity in zebrafish larvae.

a, Left, comparative images of Tg(flk1:eGFP) larvae infected with Tomato-fluorescent M. marinum and treated with DMSO, pazopanib or SU5416. Top panels depict Tomato-fluorescent M. marinum and labelled vasculature. Bottom panels depict only Tg(flk1:eGFP)-labelled vasculature. Blue arrowheads indicate somites with ectopic vasculature. Images are representative of 20 animals per treatment group. Right, length of abnormal vasculature in pazopanib- or SU5416-treated larvae. Student’s t-test, data are pooled from two or three biological replicates, respectively. b, Growth curve of Tomato-fluorescent M. marinum in 7H9 broth culture supplemented with pazopanib or SU5416. Data are representative of two biological replicates. c, Bacterial burden in caudal-vein-infected larvae treated with either pazopanib or SU5416. Student’s t-test, data are pooled from two biological replicates. d, Longitudinal bacterial burden from 2 to 6 dpi in trunk-infected larvae treated with pazopanib. One-way ANOVA with Tukey’s post-test. NS, not significant; n = 14 individuals per group. e, Comparison of M. marinum foci between control and pazopanib-treated larvae scored by association with macrophages. Fisher’s exact test, n = 40 individuals per group. f, Left, microangiography of larvae infected with cerulean-fluorescent M. marinum, injected with high-molecular-weight dextran-Texas Red at 6 dpi and imaged at 5 minutes post dextran injection (mpi). Top panels depict Cerulean-fluorescent M. marinum and dextran-Texas Red, bottom panels depict only dextran-Texas Red in vasculature and leakage around sites of infection. Green arrowheads indicate somites with the highest leakage signals in infected larvae. Images are median images from graph on right. Right, quantification of vascular leakage in uninfected, DMSO- and pazopanib-treated larvae. One-way ANOVA with Tukey’s post-test, data are representative of two biological replicates. g, Dissemination of Wasabi-fluorescent M. marinum in larvae treated with DMSO or pazopanib. Red arrowheads indicate contained foci of infection that remain in the same location throughout the course of infection, blue arrowheads indicate disseminated foci of infection. Images are representative of data in Fig. 3b. h, Bacterial burden (left), length of abnormal vasculature (middle) and dissemination (right) in 5 dpi control and Lta4h morphant larvae. i, Whole-mount in situ hybridization detection of phd3 expression in uninfected (white arrow) and M. marinum-infected zebrafish larvae. Blue arrows indicate phd3-expression-positive larvae with purple staining, red arrow indicates site of bacterial infection with no purple staining, indicating phd3-expression-negative larva. Image is representative of data in Fig. 3c. Scale bars, 100 µm. Error bars represent mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Figure 6 Effects of pazopanib treatment are reproduced in adult zebrafish infections.

a, Images of non-necrotic (left) and necrotic (right) Tomato-fluorescent M. marinum granulomas stained with DAPI (top) and haematoxylin and eosin (bottom). White arrows indicate non-necrotic granuloma, yellow arrows indicate necrotic granuloma. Images are representative of granulomas found in eight individual animals. b, Representative image of a necrotic granuloma from a negative control, not injected with pimonidazole, 2 wpi adult Tg(flk1:eGFP) zebrafish infected with cerulean-fluorescent M. marinum (cyan), and stained for hypoxyprobe (red) and with DAPI (blue). Images are representative of granulomas found in two individual animals. c, Left, representative image of Tomato-fluorescent M. marinum granuloma in Tg(flk1:eGFP) zebrafish stained with DAPI. White arrow indicates granuloma, yellow line indicates path measured for distance between granuloma and nearest vasculature (indicated by green arrow). Image is representative of data presented on the right, in panel d and Extended Data Fig. 7a. Right, distance between granulomas and nearest vasculature measured in 2 wpi adult Tg(flk1:eGFP) zebrafish. Total number of zebrafish analysed: 4 (control), 4 (pazopanib). d, Left, distance between granulomas and nearest vasculature measured in 2 wpi adult Tg(flk1:eGFP) zebrafish treated with pazopanib for 1 week. Total number of zebrafish analysed: 2 (control), 2 (pazopanib). Right, bacterial burden in 2 wpi adult zebrafish treated with pazopanib for 1 week. Student’s t-test, data are pooled from three biological replicates.

Source data

Extended Data Figure 7 Pazopanib increases the frequency of hypoxic and low-burden granulomas.

a, Distance between granulomas and nearest vasculature measured in 6 wpi adult Tg(flk1:eGFP) zebrafish. Total number of zebrafish analysed: 4 (control), 4 (pazopanib). Green dot indicates outlier that was omitted from statistical analysis. b, Images of low burden/hypoxic (left) and high burden/non-hypoxic (right) granulomas in zebrafish that were injected with pimonidazole. Asterisks indicate Tomato-fluorescent M. marinum, arrows indicate areas of hypoxia in granuloma. Images are representative of data in c, d and Fig. 4d. c, Comparison of granulomas between control and pazopanib-treated adult zebrafish scored for pimonidazole staining. Total number of zebrafish analysed: 4 (control), 4 (pazopanib). d, Comparison of granulomas between non-hypoxic and hypoxic granulomas in control and pazopanib-treated adult zebrafish scored for M. marinum burden. Total number of zebrafish analysed: 4 (control), 4 (pazopanib). Scale bars, 100 µm. Error bars represent mean ± s.d.

Source data

Supplementary information

Dissemination of M. marinum-tomato into vascular rich areas of the larva including the intestine and CHT

Video depicting the dissemination of M. marinum-tomato into vascular rich areas of the larva including the intestine and CHT. Vascular recruitment in this larva is depicted in Supplementary Video 6. (MOV 4818 kb)

Interstitial dissemination of M. marinum-cerulean within a macrophage from a trunk granuloma in a Tg(flk1:EGFP, mpeg1:tomato-caaxtd3) double transgenic larva

Video depicting interstitial dissemination of M. marinum-cerulean within a macrophage from a trunk granuloma in a Tg(flk1:EGFP, mpeg1:tomato-caaxtd3) double transgenic larva where bacterial are blue labelled, blood vessels are green labelled and macrophages are red labelled. (MOV 8872 kb)

Transfer of M. marinum-cerulean between trunk granulomas in a Tg(flk1:EGFP) larva.

Video depicting transfer of M. marinum-cerulean between trunk granulomas in a Tg(flk1:EGFP) larva. (MOV 3184 kb)

Coalescence of M. marinum-tomato from a distal site in the trunk to a central trunk granuloma in a Tg(flk1:EGFP) larva

Video depicting coalescence of M. marinum-tomato from a distal site in the trunk to a central trunk granuloma in a Tg(flk1:EGFP) larva. (MOV 4948 kb)

M. marinum-cerulean dissemination from an established granuloma into the adjacent intersegmental vessel in a Tg(flk1:EGFP, mpeg1:tomato-caaxtd3) double transgenic larva

Video depicting M. marinum-cerulean dissemination from an established granuloma into the adjacent intersegmental vessel in a Tg(flk1:EGFP, mpeg1:tomato-caaxtd3) double transgenic larva. Stills of this Video are displayed in Figure 1D. (MOV 321 kb)

M. marinum-tomato and blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi

Video depicting M. marinum-tomato and blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi. Red arrows indicate sites of vessel sprouting. Supplementary Video 7 depicts only green channel. (MOV 2901 kb)

Blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi

Video depicting blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi. Red arrows in indicate sites of vessel sprouting. Supplementary Video 6 depicts both red and green channels. Vessel was traced from initial sprouting through to connection with other vasculature in Extended Data 1Ci. (MOV 2741 kb)

M. marinum-tomato and blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi

Video depicting M. marinum-tomato and blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi. Red arrow indicates the origin of a vessel that sprouts to the bottom right of the field of view before retreating during the course of the Video. Supplementary Video 9 depicts only green channel. (MOV 3375 kb)

Blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi

Video depicting blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi. Red arrow indicates the origin of a vessel that sprouts to the bottom right of the field of view before retreating during the course of the Video. Supplementary Video 8 depicts both red and green channels. Vessel was traced from initial sprouting through to the end of the recording period in Extended Data 1Ciii. (MOV 3179 kb)

M. marinum-cerulean growth and endothelial cell division in a Tg(fli1a:nlsEGFPy7, flk1:mCherryis5) larva

Video depicting M. marinum-cerulean growth and endothelial cell division in a Tg(fli1a:nlsEGFPy7, flk1:mCherryis5) larva. Red arrow indicates previously sprouted endothelial cell nucleus in the somite that divides during the course of the Video. (MOV 27920 kb)

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Oehlers, S., Cronan, M., Scott, N. et al. Interception of host angiogenic signalling limits mycobacterial growth. Nature 517, 612–615 (2015). https://doi.org/10.1038/nature13967

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