Hif-1alpha stabilisation is protective against infection in a zebrafish model of comorbidity

Multi-drug resistant tuberculosis is a worldwide problem and there is an urgent need for host-derived therapeutic targets, circumventing emerging drug resistance. We have previously shown that hypoxia inducible-1α (Hif-1α) stabilisation helps the host to clear mycobacterial infection via neutrophil activation. However, Hif-1α stabilisation has also been implicated in chronic inflammatory diseases caused by prolonged neutrophilic inflammation. Comorbid infection and inflammation can be found together in disease settings, so it is unclear as to whether Hif-1α stabilisation would be beneficial in a holistic disease setting. Here, we set out to understand the effects of Hif-1α on neutrophil behaviour in disease-relevant settings by combining two well-characterised in vivo zebrafish models: TB infection (Mycobacterium marinum infection) and wounding (tailfin transection). We demonstrate during systemic infection, that wounding leads to increased infection burden, but the protective effect of Hif-1α stabilisation remains. A local Mm infection near to the tailfin wound site caused neutrophil migration between sites that was reduced by Hif-1α stabilisation. Our data indicate that the protective effect of Hif-1α against Mm is maintained in the presence of inflammation, highlighting its potential as a host-derived target against TB infection in a disease relevant setting.


Introduction 45
Multi-drug resistance is an increasing problem worldwide and in 2017 WHO estimated 46 that there were 490,000 cases of multi-drug resistant Mycobacterium tuberculosis 47 infections (the cause of tuberculosis), alongside 600,000 new cases with resistance to 48 the front-line drug rifampicin [1]. There is an urgent and unmet need for host-derived 49 therapeutic targets that would circumvent the problems of emerging drug-resistance 50 and could work in combination with current antimicrobials to completely clear patients 51 of TB burden more rapidly [2]. 52 Neutrophil activation is often viewed as a double-edged sword in terms of disease 53 control [3]. Neutrophils must distinguish between sterile and infected tissue injuries to 54 determine an appropriate response [4], one that strikes a balance between infection 55 control and tissue damage, but the mechanisms behind this are not well understood 56 in complex in vivo tissue environments, partially due to a lack of appropriate models. 57 Damage associated molecular patterns (DAMPs) and pathogen associated molecular 58 patterns (PAMPs) share some receptor repertoires and downstream signalling 59 components, but there is evidence to suggest that neutrophils can differentiate 60 between these signals [5]. Neutrophils are involved early in TB infection with influx 61 associated with killing of bacteria in a number of cellular and animal models [3,6-8], 62 but their function during mycobacterial infection is not well characterised. Neutrophils 63 are important in infection control, however, they are also the drivers of many chronic 64 inflammatory diseases such as chronic obstructive pulmonary disease (COPD) [9]. 65 Neutrophils are one of the first immune cell types to respond to tissue injury and 66 migrate to the wound to clear up fragments of cells and protect against pathogen 67 invasion [10]. However, in order for wounds to heal, neutrophilic inflammation must 68 resolve, either by programmed cell death (apoptosis), or by movement away from the 69 wound in a process called reverse migration [11,12]. If neutrophils persist, then 70 degranulation occurs leading to release of toxic components, further tissue damage, 71 and consequent neutrophil recruitment; a vicious cycle of chronic inflammation that 72 underpins many inflammatory diseases like COPD. 73 Chronic diseases, such as TB and COPD, often do not occur individually but exist 74 together in patients, a situation called a comorbidity. This is especially true of TB, as 75

Image and Statistical Analysis 189
Microscopy data was analysed using Leica LASX (Leica Microsystems, Germany) and 190 Image J software. All data were analysed (Prism 7.0, GraphPad Software) using t-191 tests for comparisons between two groups and one-way ANOVA (with Bonferonni 192 post-test adjustment) for other data. P values shown are: *P < .05, **P < .01, and ***P identification of neutrophils that had visited the wound ("wound experienced" red 262 neutrophils), compared to those that had not ("wound naïve" green neutrophils) 263 ( Figure 3B). We demonstrated that injection of Mm into the 26-27th somite was 264 sufficient to attract neutrophils away from the wound (wound experienced neutrophils) 265 between 4hpw-6hpw ( Figure S1). By 100mpc (minutes post conversion) almost all 266 wound-experienced neutrophils had been attracted away from the tailfin wound by 267 infection ( Figure 3D). These data demonstrate that the "second hit" of infection was 268 sufficient to overcome signalling that retains neutrophils at the initial tailfin wound site. We have previously demonstrated, in a single tailfin wound model, that stabilisation of 291 Hif-1 delays neutrophil reverse migration away from the wound [12]. However, here 292 we show that a local Mm infection is able to attract neutrophils away from the tailfin 293 wound prematurely (Figure 3). We therefore hypothesised that Hif-1 would prevent 294 wound-experienced neutrophils from exiting the injury site prematurely to migrate to a 295 localised infection site. Wound-naïve neutrophil attraction to the site of Mm infection 296 was not altered by DA Hif-1 compared to phenol red (PR) controls ( Figure 5A-B). 297 Infection was sufficient to attract wound-experienced neutrophils away from the wound 298 prematurely, but DA Hif-1 neutrophils were significantly delayed in their migration 299 towards localised Mm infection compared to PR controls ( Figure 5B-C). The migration 300 speed of wound-experienced neutrophils was lower in the DA Hif-1 group compared 301 to the PR group, largely due to their tighter association to the wound edge and less 302 migration away ( Figure 5D). This decrease in migration speed was more marked in 303 wound-experienced neutrophils that were successful in migrating away from the 304 wound edge towards the Mm infection site ( Figure 5E). These neutrophils migrated to 305 the infection site at two-thirds of the speed in DA Hif-1 embryos compared to the PR 306 controls ( Figure 5E). Furthermore, they took a less direct route to the infection, with 307 the meandering index of these neutrophils significantly lower in the DA Hif-1 group 308 compared to PR controls ( Figure 5F). These data indicate that Hif-1 stabilised 309 neutrophils remain more sensitive to the wound signalling gradient, even if successful 310 in escaping the wound to a second hit of infection. It is interesting to note that, in many 311 cases, wound-experienced neutrophils migrating away from the wound in the DA Hif-312 1 group dithered between the wound and infection sites, with a shuttling movement 313 backwards and forwards, a behaviour not observed in PR controls (Movie S1). 314 Dithering between infection and wound sites was also not observed in DA Hif-1 315 wound-naïve neutrophils in the same individual larvae, suggesting a difference 316 between wound-experienced and wound-naïve neutrophils in their detection of the two 317

stimuli. 318
Taken together, these data indicate that wound-experienced neutrophils in Hif-1 319 stabilised larvae remain more sensitive to the wound gradient and are less likely to 320 migrate to the second hit infection site compared to normal controls.  We developed dual-infection/inflammation models to investigate the effects of Hif-1 370 on neutrophil migration to wound and infection sites simultaneously. Using localised 371

Mm infection and tailfin wound we found that neutrophils dispersed between infection 372
and wound sites, but that when Hif-1 was stabilised, neutrophils seldom migrated 373 past the local infection to the tailfin wound. Hif-1 stabilisation also retained 374 neutrophils at the tailfin wound when a second hit of infection was introduced, while in 375 wildtype larvae infection caused premature migration away from the wound to the 376 infection site. These data indicate that Hif-1 stabilisation causes increased sensitivity 377 to wound or infection gradients, leading to retention of neutrophils and reduced ability 378 of these cells to respond to competing signals. 379 Wound-naïve neutrophils were able to migrate to Mm at the same rate when Hif-1 is 380 stabilised, while wound-experienced neutrophils are slower to respond and remain at 381 the wound for longer. In some instances, when Hif-1 is stabilised the neutrophils 382 seem unable to decide which stimuli to migrate to, shuttling between the two sites. Hif-383 1 stabilisation caused no effect on neutrophil recruitment to the tailfin wound in the 384 single inflammation model, therefore is unlikely to have effects on recruitment 385 signalling [12]. Taken together, these data indicate that recognition of "retention 386 signals" by neutrophils is sensitised by stabilised Hif-1, keeping neutrophils at the 387 wound or infection site, and that there is an as yet unidentified molecular change in 388 Hif-1 stabilised neutrophils that alters their sensitivity to these tissue gradients. Likely 389 candidates for Hif-1 targets include G protein coupled receptors (GPCRs) that are 390 investigations of comorbidities increase we anticipate that dual-models will increase 420 in popularity, but with a plethora of possible combinations and timings of stimuli 421 available, care will be required to understand the relevance of these models to disease 422

situations. 423
Using dual-models of infection and wounding we have highlighted that comorbidity is 424 likely to have a range of effects on neutrophil behaviour during infection that differ on 425 the local tissue scale compared to the whole-organism, holistic level. Although Hif-1 426 stabilisation could be detrimental at local level inflammation, our dual-models suggest 427 that on a whole-organism level neutrophil activation by  stabilisation is not harmful 428 and could be a promising host-derived treatment strategy against TB.