Revisiting hypoxia therapies for tuberculosis 1

2

Stefan H Oehlers 3

4

Tuberculosis Research Program at the Centenary Institute, The University of Sydney, Camperdown, 5

NSW 2050, Australia 6

The University of Sydney, Discipline of Infectious Diseases & Immunology and Marie Bashir 7

Institute, Camperdown, NSW 2050, Australia 8

Corresponding author stefan.oehlers@sydney.edu.au 9

10

Funding: 11

This work was supported by an Australian National Health and Medical Research Council CJ 12

Martin Early Career Fellowship APP1053407 and Project Grant APP1099912; The University of 13

Sydney Fellowship G197581; NSW Ministry of Health under the NSW Health Early-Mid Career 14

Fellowships Scheme H18/31086; and the Kenyon Family Foundation Inflammation Award 15

(S.H.O.). 16

17

Abstract 18

The spectre of the coming post-antibiotic age demands novel therapies for infectious diseases. 19

Tuberculosis, caused by Mycobacterium tuberculosis, is the single deadliest infection throughout 20

human history. M. tuberculosis has acquired antibiotic resistance at an alarming rate with some 21

strains reported as being totally drug resistant. Host-directed therapies attempt to overcome the 22

evolution of antibiotic resistance by targeting relatively immutable host processes. Here I 23

hypothesise the induction of hypoxia via anti-angiogenic therapy will be an efficacious host-24

directed therapy against tuberculosis. I argue that anti-angiogenic therapy is a modernisation of 25

industrial revolution era sanatoria treatment for tuberculosis, and present a view of the tuberculosis 26

This accepted manuscript has been published https://doi.org/10.1042/CS20190415

granuloma as a “bacterial tumour” that can be treated with anti-angiogenic therapies to reduce 27

bacterial burden and spare host immunopathology. I suggest two complementary modes of action, 28

induction of bacterial dormancy and activation of host Hypoxia Induced Factor-mediated immunity, 29

and define the experimental tools necessary to test this hypothesis. 30

31

1. Introduction 32

After a period of relative success during the golden age of antibiotics in the second half of the 20th

33

century, tuberculosis (TB) rates climbed dramatically with the HIV pandemic from the 1980s. 34

Although recent advances in HIV treatment have seen a fall in the rates of HIV-related TB 35

mortality, the failure to completely eradicate TB and subsequent emergence of multidrug resistant 36

strains of M. tuberculosis has created pockets of endemic TB of global importance. 37

38

The emergence of extensively and totally drug resistant TB strains have led to the contemplation of 39

living in a post-antibiotic era with this ancient killer. Given the short lag between the deployment of 40

a new antibiotic and reports of resistance, new efforts are being directed to discover host-directed 41

therapies (HDTs) for TB. This hypothesis will discuss the history and recent evidence supporting 42

the induction of granuloma hypoxia as an intervention for difficult to treat TB. 43

44

2. TB therapies in the pre-antibiotic era 45

The high altitude sanatoria of the pre-antibiotic era sought to strengthen patients, at least for those 46

who were able to afford the fees of a sanatorium, with regimented daily regimes. It is important to 47

note that these sanatoria would not deliver a curative treatment but rather induce convalescence or 48

prolong patient survival [1, 2]. Three elements of these regimes that we recognise as biologically 49

active are better nutrition, daily sun exposure to boost vitamin D levels, and reducing the supply of 50

oxygen to the bacterial lesions. 51

52

Epidemiological studies have informed us that the burden of TB mortality, like many other 53

infectious diseases, falls disproportionally on the poor, the young, the frail and the weak. 54

Nutritional supplementation, historically achieved by increasing milk consumption, could directly 55

disrupt the infection-malnutrition cycle in favour of patient recovery [3]. Under nutrition is clearly a 56

predisposing factor for TB that remains to be solved as a public health issue [4, 5]. 57

58

The increased sun exposure prescribed for city dwellers of industrial Europe also has a modern 59

analogy in the form of vitamin D supplementation. The micronutrient vitamin D can boost anti-TB 60

immunity in pre-clinical models and is often found to be deficient in TB patients [6]. Again 61

however, systemic review of vitamin D supplementation as an interventional strategy has not 62

yielded evidence of a clinical benefit [4]. 63

64

This leaves the final element of altitude and the availability of oxygen on the outcome of infection. 65

Epidemiological studies in mountainous regions of the Americas and Himalayas have noted a 66

decrease in TB rates in high altitude populations [2, 7-11]. Although the effects of urbanisation and 67

poverty can negate the protective effects of living at high altitude, these observations provide strong 68

evidence for a protective role of hypoxia in TB biology. 69

70

Efforts to surgically bring the benefits of a high altitude retreat to the masses resulted in the short-71

lived lung collapse therapies, which peaked in usage immediately prior to the antibiotic age [12]. 72

Collapse of a lung was hypothesised to rest the organ allowing the body time to contain the 73

infection by fibrosis of the lesions and by starving the bacteria of oxygen [13]. Although successful 74

enough to be widely utilised as a treatment for an otherwise fatal condition, lung collapse surgeries 75

are inherently risky and were rendered obsolete with the appearance of antibiotic therapy. 76

77

Conversely, hyperoxia has been identified as a risk factor for TB disease. Analysis of a Taiwanese 78

comorbidity-controlled cohort of hyperbaric chamber users revealed an increased rate of TB 79

implying reactivation of subclinical TB infection by the hyperoxic treatment [14]. The 80

“summering” of sanatoria patients at sea level is anecdotally reported to cause relapse, even those 81

within the consolidation phase of immune control [1]. Experimentally, the pivotal experiments of 82

Rich & Follis and Sever & Youmans in the mid 20th

century demonstrated the host-protective effect 83

of hypoxia and a detrimental effect of hyperoxia in experimental TB infection (reviewed in [15]). 84

85

3. A “bacterial tumour”: Tuberculosis granulomas are structurally and molecularly similar to 86

tumours 87

Granulomas formed during TB are remarkably similar to solid tumours (Figure 1). Both lesions can 88

form in every organ system of the body, cause chronic diseases requiring lengthy treatment, and are 89

prone to relapse or reactivation. The lesions can even be difficult to differentiate by modern clinical 90

imaging techniques as both take the form of inflamed, metabolically active nodules in 91

fluorodeoxyglucose PET scans. This consumption of glucose to is caused by the engagement of 92

aerobic glycolysis in both lesions [16]. Cellular-level similarities such as a encapsulation of a 93

nodule, the development of a necrotic core during disease progression, an ineffective inflammatory 94

response that exacerbates disease [17, 18], and vascularisation are characteristic of both lesions 95

[19]. Below this cellular similarity are molecular similarities including hypoxia and hypoxia 96

induced factor (HIF) signalling [20], and the destruction of surrounding tissue by matrix 97

metalloproteinases [20, 21]. 98

99

Metabolic syndrome, encompassing diabetes and obesity, is an important co-morbidity of TB, 100

cancer, and other diseases driven by inflammation. Elevated blood glucose and cholesterol 101

associated with metabolic syndrome provide core cellular stressors in TB and tumours. Poorly 102

controlled diabetes impairs protective immunity and exacerbates immunopathology [22, 23], while 103

cholesterol can be preferentially utilised by mycobacteria as a carbon source, contributes to 104

mycobacterial evasion of host immunity, and encourages the survival of transformed cells in 105

tumours [24, 25]. Interestingly, control of either blood glucose by metformin or cholesterol 106

metabolism by statins has positive effects on immune control of TB [24, 26]. 107

108

T-cell immunity is essential for immune control of TB and tumours as evidenced by the impact of 109

HIV infection comorbidity. Two important shared aspects of immune dysfunction that are 110

characteristic of both diseases are the appearance of immune exhaustion and lack of lesion 111

penetration that reduces protective T-cell immunity (reviewed in [27]). The immune exhaustion 112

phenotype is characterised by the expression of surface molecules such as PD-L1 and cytokines 113

such as IL-10 that suppress immunity. Immune checkpoint inhibitors targeting CTLA-4 or the PD-114

1/PD-L1 axis have had remarkable success in stimulating protective immunity against a range of 115

tumours and a similar approach has been proposed for the treatment of TB where a similar 116

immunosuppressive profile can develop [28, 29]. Tumours and granuloma-resident mycobacteria 117

are both adept at feeding off inflammation. In respect to T cells, it is penetration of T cells into 118

tumour masses or into granuloma cores is an important correlate of protective immunity, as the 119

number of T cells distal to the lesion does not affect immune control [30-32]. 120

121

The conserved vascular pathologies of TB granulomas and tumours have also been well described, 122

and open the door to “Robin Hood” well-known vascular-targeting strategies from the cancer field 123

into HDTs for TB. At a molecular level, both lesions produce the vasoactive cytokines VEGF and 124

ANG-2 [33-35]. VEGF is the canonical pro-angiogenic signalling molecule and acts through the 125

cognate receptor VEGFR on endothelial cells to trigger endothelial proliferation and direct 126

migration. ANG-2 is also produced under similar conditions to VEGF and acts as a context-127

dependent antagonist of the TIE-2 receptor to antagonise the vascular-stabilizing effects of 128

physiological ANG-1/TIE-2 signalling [36]. These molecular similarities underpin the tumour-like 129

vasculature seen around mycobacterial granulomas: angiogenesis [19, 37-39], permeability and 130

inconsistent perfusion [34, 35], and haemostasis [40-42]. 131

132

4. Angiogenesis fuels the growth of mycobacterial granulomas 133

The observation that tumours must recruit vasculature to continue growing beyond a critical 134

threshold when oxygen can no longer permeate into their core has led to decades of research 135

targeting tumour angiogenesis as a universal cancer therapy [43]. The VEGF-VEGFR signalling 136

axis-driven is required for angiogenesis and inhibition of the axis by neutralising VEGF, blockading 137

the VEGF binding site on VEGFR, or inhibiting the tyrosine kinase activity of VEGFR indeed 138

slows the growth of many tumours in in vivo models, however efficacious translations to human 139

oncology has been limited. 140

141

Mycobacterial granulomas are known to be highly vascularised during the active stages of infection 142

across a range of host and bacterial species [19, 20, 34, 38]. In TB, extensive angiogenesis has been 143

observed in active granulomas but is mainly absent in the immune-controlled latent form of the 144

disease [19]. Furthermore, serum VEGF levels correlate with active disease and may even 145

constitute a biomarker for disease progression [33]. 146

147

Mycobacteria intrinsically induce VEGF expression by innate immune cells and subsequent 148

angiogenesis through recognition of cyclopropanated trehalose dimycolate, a cell wall component, 149

leading to activation of innate immune cells [44-46]. Furthermore, mycobacterial ESX-1-dependent 150

macrophage aggregation concentrates pro-angiogenic macrophages to direct the growth of blood 151

vessels towards nascent granulomas [38]. This requirement of multiple bacterial effectors, 152

conserved across species of mycobacteria, to elicit the angiogenic response suggests host 153

angiogenesis is an important core function of mycobacterial virulence. 154

155

The correlation between angiogenesis and bacterial growth state prompted us to investigate a 156

mechanistic link using the zebrafish-Mycobacterium marinum infection model [38]. Using small 157

molecule inhibitors of VEGFR, we found inhibition of angiogenesis reduced growth of M. 158

marinum, reduced bacterial dissemination away from primary granulomas, and increased survival 159

of drug-treated animals. VEGFR inhibition increased granuloma hypoxia in infected zebrafish but 160

was ineffective when granulomas formed in highly vascularised tissues in oxygen permeant tissues 161

of the embryo suggesting a mechanism of action dependent largely on the ability to induce hypoxia 162

within granuloma. Additional work by Walton et al. used a dominant negative VEGF isoform to 163

elegantly demonstrate the necessity of the VEGF ligand in the VEGF-VEGFR axis that promotes 164

mycobacterial growth [46]. 165

166

Interestingly, we observed an increased rate of granuloma conversion from a highly cellular 167

phenotype to cuffed acellular core with low-burden phenotype was observed following VEGFR 168

inhibition in zebrafish (Figure 2). This histological shift is reminiscent of the fibrotic walling off of 169

granulomas seen after lung collapse therapy and may represent a conserved aspect of natural host 170

containment of mycobacterial infection. 171

An important paper by Polena et al. utilised SCID mice to show 1) human macrophage-derived M. 172

tuberculosis granulomas recruit vasculature in vivo, and 2) reduced M. tuberculosis dissemination 173

in animals treated with VEGFR-blockading antibodies [39]. This important advance showed for the 174

first time that experimental M. tuberculosis infection of mammals could be controlled by targeting 175

infection-induced angiogenesis. 176

177

Complementary to the vascular-directed effects of anti-VEGF therapy, there is an emerging body of 178

literature addressing the non-angiogenic effects of VEGF blockers in mycobacterial infection. It has 179

become apparent from the cancer literature that immune cells are responsive to VEGF and respond 180

in an immunosuppressive manner. The VEGFR inhibitors pazopanib and VEGFR2 kinase inhibitor 181

I (SU 5408) were detected as protective in a macrophage-based in vitro infection screen, 182

independent of any effect on the vasculature [47]. A recent paper by Harding et al. has elegantly 183

demonstrated a role for granuloma-derived VEGF in promoting harmful granulomatous 184

inflammation in several mouse-M. tuberculosis models [48]. Together these papers provide 185

evidence that targeting the VEGF-VEGFR axis has a net positive effect on the outcome of 186

infection. 187

188

5. Case report evidence for the efficacy of anti-angiogenic drugs against TB 189

Anti-angiogenic therapies for cancer aim to induce sufficient tumour hypoxia to cause growth 190

arrest. Technologies have largely focused on inhibiting the VEGF-VEGFR axis by neutralising 191

VEGF, blockading the VEGF binding site on VEGFR, and inhibiting the tyrosine kinase activity of 192

VEGFR. Ocular inflammation-induced angiogenesis can cause loss of vision and is typically treated 193

by oral anti-inflammatories such as steroids or intravitreal injection of VEGF-neutralising agents 194

such as bevacizumab (Avastin) and ranibizumab. Ocular TB thus presents an important 195

manifestation of disseminated TB where TB treatment overlaps with anti-angiogenic therapy. 196

197

A total of 11 case reports have utilised VEGF neutralisation during TB. Injection of bevacizumab, 198

or the derivative ranibizumab, has been safely used as an adjunctive treatment to improve vision in 199

conjunction with antibiotics [49-52], to clear granuloma-associated angiogenesis after successful 200

antibiotic treatment [53, 54], as a component of treatment for paradoxical TB-associated immune 201

reconstitution inflammatory syndrome [55, 56], and even as monotherapy [57-59]. 202

203

The three monotherapy reports of VEGF neutralisation leading to ocular disease remission provide 204

the clearest evidence yet for an anti-TB effect of anti-angiogenic therapy in human TB. The 4 case 205

reports of anti-angiogenic therapy used as an adjunct to antibiotics demonstrate that adjunctive use 206

does not impair antibiotic efficacy, albeit only in the context of ocular infection. Together, these 207

reports provide some evidence of the efficacy of anti-angiogenic therapy in human TB and 208

demonstrate safety of anti-angiogenic therapy during active ocular TB. 209

210

6. Molecular level protective effects of hypoxia 211

6.1. Effects on the bacterium 212

The most obvious effect of hypoxia is that the lack of oxygen starves the mycobacteria and stops 213

proliferation. Indeed, low oxygen pressures do halt the growth of M. tuberculosis in vitro, causing 214

the bacteria to enter a relatively metabolically dormant lifestyle that is also observed in 215

experimental models and clinically in non-progressing latent infection [60]. Hypoxia causes the 216

build-up NADH, an indicator of cellular stress, in mycobacteria. This metabolic block exposes the 217

bacterial electron transport chain as an antibiotic target in the absence of cellular division [61]. 218

219

M. tuberculosis responds to hypoxic conditions through activation of the DosR regulon (well-220

reviewed in [62]). Expression of DosR regulon genes is exquisitely correlated with hypoxia in vivo 221

and is maximal in the hypoxic granulomas characteristic of the latent TB classification [60]. 222

Mutants lacking components of the DosR regulon are unable to established latent infection in 223

animal models that form granulomas with significant hypoxia but remain relatively virulent in 224

mouse models that do not recapitulate a hypoxic infection environment [63, 64]. Thus induction of 225

granuloma hypoxia is likely to drive a non-replicative bacterial state that can be at least partially 226

controlled by host immunity. 227

228

The intrinsic resistance of dormant mycobacteria to antibiotics compounds the hypoxia-induced 229

shift of mycobacteria to a dormant phenotype. It is conceivable that in drug-sensitive TB cases, 230

adjunctive hypoxia-inducing therapy could counterproductively promote drug resistance by 231

intrinsically limiting the sensitivity of TB to antibiotics. 232

233

6.2. Host protective effects of hypoxia 234

Granuloma hypoxia may also stimulate host immunity to mycobacterial infection. Complementary 235

to the findings of reduced TB incidence in humans living at altitude, whole blood restriction of 236

mycobacterial growth was increased in donors living at high altitude [65]. Furthermore, 237

macrophages cultured in low oxygen show increased bactericidal activity compared with 238

macrophages cultured in atmospheric oxygen [66-68]. Recent analyses of host-mycobacterium 239

interactions have revealed how exposure to hypoxia may augment anti-TB immunity at the 240

molecular level. 241

242

IFNgamma activation is required for optimal macrophage killing of mycobacteria and loss of 243

IFNgamma signalling is responsible for the heritable cluster of diseases termed Mendelian 244

Susceptibility to Mycobacterial Diseases in humans [69]. HIF1alpha signalling has recently been 245

demonstrated to potentiate IFNgamma-dependent immunity against TB [70]. Important anti-TB 246

HIF1alpha-dependent immune pathways include effectors that modulate further immune activation 247

such as PGE2, and directly bactericidal compounds such as nitric oxide [70, 71]. While HIF1alpha-248

dependent immune pathways have been shown to be directly induced by mycobacterial infection 249

[71], we have found anti-angiogenic therapy increases HIF1alpha activation in mycobacterial 250

granulomas in vivo [38]. 251

252

Another recently appreciated anti-mycobacterial killing mechanism is activation of the Warburg 253

effect in mycobacteria-infected macrophages resulting in a switch to glycolysis at the expense of 254

oxidative phosphorylation [72]. This metabolic shift produces sufficient ATP to fuel antibacterial 255

effector activity, glucose-derived metabolites that are toxic to M. tuberculosis under hypoxic 256

conditions, and, in a positive feedback loop, stabilises HIF1alpha to further enhance cellular 257

immunity [73]. 258

259

Together, this potentiation of anti-mycobacterial immunity by hypoxia and HIF1alpha signalling 260

demonstrate a molecular pathway linking anti-angiogenic therapy to reduced mycobacterial burden. 261

262

7. Host-detrimental effects of hypoxia 263

While HIF1alpha activation leads to an enhancement of anti-mycobacterial immunity, it also 264

drastically induces the production of tissue-destructive matrix metalloproteinases (MMPs) [20, 74]. 265

MMPs are produced as part of inflammatory responses and are central to the pathogenesis of many 266

inflammatory diseases when over produced, and their production in mycobacterial infection aids 267

granuloma formation by remodelling infected tissue [21, 75]. The collagenase activity of MMPs 268

aids the growth and dissemination of mycobacteria, facilitating the escape of mycobacteria away 269

from areas of growth-limiting hypoxia. This effect could be analogous to the pro-metastatic effects 270

of hypoxia on tumours (Figure 1). 271

272

The switch to glycolysis is a double-edged sword as prolonged engagement of glycolysis leads to 273

the accumulation of intracellular lipid droplets and the transformation of macrophages into foam 274

cells laden with triacylglycerols (Figure 1). Foam cells are deficient in anti-bacterial function and 275

are thought to provide a ready carbon source for mycobacterial growth in the granuloma core [76]. 276

277

Hypoxia may inhibit the function of T cells and exclude T cells from mycobacterial granulomas. It 278

is evident from the cancer literature that hypoxia-induced cytokine profiles create a 279

microenvironment conducive to Treg retention at the expense of anti-tumour effector T cell types 280

[77]. It is unknown if a similar mechanism functions in TB granulomas. 281

282

It has also been shown that inhibition of lymphangiogenesis by VEGFC reduces systemic T cell 283

responses to infection [78]. If a VEGF family-targeting therapeutic is used to induce hypoxia in TB 284

granulomas, it is conceivable that it could affect inflammatory lymphangiogenesis and the priming 285

of adaptive immune responses. An important distinction between lung collapse or high altitude 286

environmental hypoxia and anti-angiogenic therapy is that the former methods of inducing hypoxia 287

do not prevent blood flow to the granuloma. Hypoxia induced by anti-angiogenic therapy may thus 288

have compounding effects on protective adaptive immunity. 289

290

Mycobacterial granulomas are naturally resistant to penetration by antibiotics, and this is a major 291

therapeutic hurdle for antibiotic delivery [79]. There is thus a valid concern that anti-angiogenesis 292

therapy will create avascular granulomas that could be impenetrable to antibiotics. Vascular 293

normalisation experiments provide a contrasting intervention that decreases granuloma hypoxia by 294

facilitating circulation to the granuloma neovascular network. Work in the rabbit TB model by 295

Datta et al. utilised a short duration of treatment with the monoclonal antibody bevacizumab to 296

neutralise infection-induced VEGF and normalise the granuloma vasculature resulting in better 297

delivery of a small molecule dye to the granuloma core [34]. Xu et al. next demonstrated that 298

inhibiting matrix metalloprotease activity normalised the vasculature and improved antibiotic 299

delivery to mouse granulomas [80]. These two studies highlight the importance of vascular 300

perfusion to delivering antibiotics into granulomas and suggest anti-angiogenic therapies could 301

hinder antibiotic delivery. 302

303

8. Necessary elements of a preclinical model to test the hypothesis 304

TB is a disease that exacerbates economic inequity at personal and international levels. Despite the 305

efficacious application of anti-VEGF biologics to small numbers of ocular TB patients, I believe 306

small molecule VEGFR inhibitors are the most feasible intervention to advance. From an 307

economics standpoint, small molecules can be chemically synthesised as generic drugs as patents 308

expire. Importantly, small molecule VEGFR inhibitors have been formulated for delivery by tablets 309

and capsules extending their delivery range into areas lacking sufficient infrastructure to handle 310

biologics. 311

312

Mouse models of TB infection are the gold standard small animal models for testing new therapies. 313

The ideal mouse TB model would replicate: 314

1. Angiogenic and hypoxic granulomas 315

2. A natural ability to control or sterilise infection 316

3. The pharmacokinetics of antibiotic delivery into granulomas 317

318

Most mouse strains do not develop hypoxic granulomas following mycobacterial infection like that 319

seen in the zebrafish-M. marinum model and in human TB infection [37, 81]. This has recently 320

changed with the characterisation of the C3HeB/FeJ mouse strain that recapitulates necrotic 321

granulomas with areas of hypoxia following pulmonary infection with M. tuberculosis [82-84]. The 322

reproduction of hypoxic granulomas is essential to determine if hypoxia-inducing anti-angiogenic 323

therapies are effective in treating M. tuberculosis infection in mammals [81]. Although granuloma 324

angiogenesis has not been directly described in this strain, it is likely to recapitulate this phenotype 325

as mycobacterial infection-induced angiogenesis is highly conserved across species pairings. 326

327

With respect to human-like immune control, the CC001/UncJ mouse strain was identified from a 328

screen of mouse strains as more able to reduce M. tuberculosis burden between 3 and 6 weeks post 329

infection [85]. The ability of a mouse model to restrain TB infection is unusual and suggests an 330

effective immune response against M. tuberculosis that could be leveraged to examine the effect of 331

hypoxia-inducing therapy in the background of successful immune-mediated control of infection. 332

333

As addressed in section 7, anti-angiogenesis therapy could reduce antibiotic penetration into 334

granulomas. Testing of anti-angiogenic therapy in conjunction with conventional antibiotics will 335

determine if anti-angiogenic therapy should be used to treat antibiotic-sensitive TB or only as a 336

“last line” therapy for antibiotic resistant infections. 337

338

9. Conclusion 339

While hypoxia in cancer has been suggested to ultimately lead to more aggressive malignancies, 340

hypoxia in mycobacterial infection may fit a converse paradigm where lack of oxygen slows 341

bacterial growth and improves anti-TB immunity. The observations and the findings discussed here 342

demonstrate potential adjunctive, host-directed therapies and may open new avenues to treating 343

recalcitrant TB infections, including multi-drug resistant disease. However concerns of the wider 344

effects of these treatments on antibiotic susceptibility call for further pre-clinical testing and 345

compassionate use of anti-angiogenic therapies in antibiotic resistant infections. 346

347

While induction of hypoxia appears to be effective in slowing the growth of mycobacteria in vivo, 348

this approach is probably not sterilising given the low “cure” rate (5-10%) compared with 349

“improved” status (~50%) measured for collapse therapy patients [12], and the highly variable 350

improvement rate of sanatoria from 90% to 5% declining with initial severity of disease and with an 351

average 9 year fitness rate of 60% [1]. These observations could be due to the conversion of active 352

lesions to more latent lesions with a reduced but not sterile load of mycobacteria. While this is 353

certainly preferable to the high mortality rate seen in the pre-antibiotic era it is unsatisfactory 354

compared to the 86% cure rate for drug susceptible TB recorded by the WHO 355

(http://www.who.int/gho/tb/epidemic/treatment/en/). Where hypoxia-inducing therapies may find a 356

niche is in the treatment of MDR TB (50% cure rate) and XDR TB (23% cure rate). 357

358

10. Acknowledgements 359

I would like to thanks Drs Mark Cronan and David Tobin, and all members of the Tobin lab and 360

Tuberculosis Research Program at the Centenary Institute for discussions that helped shape this 361

hypothesis. I believe Dr Cronan coined the phrase “bacterial tumour” that shaped so much of my 362

research into TB-associated angiogenesis. 363

364

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Figure legends 627 Figure 1: The “bacterial tumour”: similarities between the TB granuloma and solid tumours. 628 (Top left) Both lesions are hypoxic and drive the formation of a leaky and haemostatic vasculature 629 through the production of VEGF and ANG-2. The lack of sufficient perfusion prevents the 630 penetration of drugs into each lesion resulting in phenotypic insensitivity. Activation of haemostasis 631 is a common strategy to subvert the immune response. The hypoxic conditions activate a HIF1-632 mediated transcriptional program that also feed into metabolism and tissue destruction. 633 (Top right) There are shared immunological markers of disease prognosis. A robust type 1 immune 634 response with spatial penetration of lymphocytes is associated with lesion apoptosis and immune 635 containment. A skewing to a type 2 response with insufficient lymphocyte penetration is associated 636 with poor immune control and a worse prognosis. 637 (Bottom right) Matrix metalloprotease (MMP)-mediated remodelling and destruction of the 638 surrounding stromal tissue is essential to facilitate lesion spread from primary foci through 639 surrounding tissue and eventually facilitating haematogenous spread. MMP activity is increased by 640 hypoxia and the HIF1-mediated transcriptional program. 641 (Bottom left) Metabolic syndrome-associated diseases diabetes and dyslipidaemia are important 642 comorbidities for both diseases. At a molecular level, these comorbidities feed lesions with glucose 643 and lipid. In the TB granuloma this results in loss of effective immune control and fuels bacterial 644 growth, while in cancer the malignant cells use the nutrients. 645 646 Figure 2: VEGFR inhibition drives a granuloma maturation phenotype in zebrafish 647 (A) Representative image of an organised cellular zebrafish-M. marinum granuloma. 648 (B) Representative image of an organised necrotic zebrafish-M. marinum granuloma. 649 (C) Quantification of granuloma necrosis in 6 week-post infection zebrafish adults treated with 1 650 µM pazopanib as indicated. Each dot represents a single granuloma. 651

TB dissemination

Cancer metastasis

MMP-mediated matrix destruction

Vascular dysfunction: weak perfusion

Drug insensitivity

M1 Th1

M2 Th2

Good prognosis Lesion apoptosis

Poor prognosis Immune evasion

Hypoxia / HIF1alpha

O2

Vascular dysfunction: thrombosis

TNF, IFNgamma

IL-10, PD-L1

Aerobic glycolysis Diabetes Dyslipidemia Citrate / acetyl CoA

Lipid accumulation

Granuloma caseation

Tumour survival