Hif-1alpha stabilisation is protective against infection in zebrafish comorbid models

Abstract

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 factor-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 and it remains unclear 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 a comorbid setting by combining two well-characterised in vivo zebrafish models - TB infection (Mycobacterium marinum infection) and sterile injury (tailfin transection). Using a local Mm infection near to the tailfin wound site caused neutrophil migration between the two sites that was reduced during Hif-1α stabilisation. During systemic Mm infection, wounding leads to increased infection burden, but the protective effect of Hif-1α stabilisation remains. Our data indicate that Hif-1α stabilisation alters neutrophil migration dynamics between comorbid sites, and 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.

Abbreviations

CFU

colony forming unit

CHT

caudal hematopoietic tissue

COPD

chronic obstructive pulmonary disease

DA Hif-1α

dominant active Hif-1α

DAMPS

damage associated molecular patterns

DMOG

dimethyloxaloylglycine

DMSO

dimethyl sulfoxide

dpf

days post fertilisation

dpi

days post infection

GPCRs

G protein coupled receptors

Hif-1α

hypoxia inducible factor-1alpha

HIV

human immunodeficiency virus

HK

heatkilled

hpf

hours post infection

hpw

hours post wound

Il-1β

Interleukin 1 beta

Mm

Mycobacterium marinum

mpc

minutes post (photo)conversion

Mtb

Mycobacterium tuberculosis

NI

not injected

PAMPS

pathogen associated molecular patterns

PR

phenol red

PVP

polyvinylpyrrolidone

TB

tuberculosis

WHO

World Health Organization

Introduction

Multi-drug resistance is an increasing problem worldwide and in 2017 WHO estimated that there were 490,000 cases of multi-drug resistant Mycobacterium tuberculosis infections (the bacterial pathogen that causes tuberculosis), alongside 600,000 new cases with resistance to the front-line drug rifampicin [1]. There is an urgent and unmet need for host-derived therapeutic targets that would circumvent the problems of emerging drug-resistance and could work in combination with current antimicrobials to completely clear patients of TB burden more rapidly [2].

Neutrophil activation can be viewed as a double-edged sword during disease, with pathogen elimination being beneficial to the host while surrounding tissue damage caused by excessive neutrophil inflammation bringing negative outcomes [3]. Neutrophils must distinguish between sterile and infected tissue injuries to determine an appropriate response [4], one that strikes a balance between infection control and tissue damage, but the mechanisms behind this are not well understood in complex in vivo tissue environments, partially due to a lack of appropriate models. Damage associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs) share some receptor repertoires and downstream signalling components, but there is evidence to suggest that neutrophils can differentiate between these signals [5].

Neutrophils are involved early in TB infection with influx associated with killing of bacteria in a number of cellular and animal models [3,6–8], but their function during mycobacterial infection is not well characterised. Neutrophils are important in infection control, however, they are also the drivers of many chronic inflammatory diseases such as chronic obstructive pulmonary disease (COPD) [9]. Neutrophils are one of the first immune cell types to respond to tissue injury and migrate to the wound to clear fragments of dead cells and protect against pathogen invasion [10]. However, in order for wounds to heal, neutrophilic inflammation must resolve, either by programmed cell death (apoptosis), or by movement away from the wound in a process called reverse migration [11,12]. If neutrophils persist, then excessive degranulation, alongside more recently described neutrophil extracellular traps (NETs), lead to build-up of toxic components and tissue damage, causing further neutrophil recruitment; a vicious cycle of chronic inflammation that underpins many inflammatory diseases like COPD [13,14].

Chronic diseases, such as TB and COPD, often do not occur individually but exist together in patients, a situation called comorbidity. This is true of TB, as one-third of the world’s population live healthily with latent TB infection for decades before a “second-hit” comorbidity leads to progression to active TB [15]. Some of the best characterised comorbidities are co-infections with other communicable diseases, most notably HIV which causes immune deficiency and allows TB to breakout of granulomas leading to active disease [1]. However, at the same time as anti-retroviral therapy is bringing HIV under greater control, there is an alarming rise in non-communicable diseases, such as diabetes and COPD, in the same populations that have been linked to TB activation [15,16]. Many of these non-communicable diseases have an inflammatory component, yet treatment of these diseases, and indeed TB itself, is currently tailored towards the single condition rather than considering the holistic outcome of the comorbidity [17]. This is reflected in animal models used to investigate cellular and molecular mechanisms of disease often being based on a single condition rather than considering comorbidities, and there is a pressing need for comorbid models to understand the complex interactions of cells in these contexts in vivo.

Neutrophils are exquisitely sensitive to low levels of oxygen (hypoxia), which pro-longs their lifespan and increases their bactericidal mechanisms [12,18,19]. The cellular response to hypoxia involves the activation and stabilisation of hypoxia inducible factor-1α (HIF-1α) transcription factor [20,21]. We have previously demonstrated that activating neutrophils, via stabilisation of Hif-1α, is host protective during in vivo mycobacterial infection - a good therapeutic outcome [22]. However, hypoxia and Hif-1α have also been shown to delay neutrophil apoptosis and reverse migration of neutrophils away from wounds in chronic inflammation models - a bad therapeutic outcome [12,23]. Therefore, the beneficial effects of Hif-1α stabilisation on a holistic-scale during infection remains unclear, due to the potential for neutrophil damage and chronic inflammation.

The zebrafish has become an invaluable animal model for TB and inflammatory disease over the last fifteen years [24]. Infection of zebrafish larvae with Mycobacterium marinum (Mm), a closely related strain to human Mtb and a natural fish pathogen, has been used to identify important molecular mechanisms involved in TB pathogenesis and granuloma formation [25]. Zebrafish embryos are transparent and innate immune cell transgenic lines have been used over the last decade in tailfin transection models to better understand the molecular mechanisms involved in both neutrophil recruitment to, and reverse migration from, a site of inflammation [12,26,27]. Here, we investigated the effects of Hif-1α stabilisation on neutrophil dynamics in comorbid models of infection and wounding by combining well-characterised zebrafish Mm infection and tailfin transection models [12,22]. During systemic infection, neutrophil inflammation dynamics at the tailfin wound occur as normal while presence of a wound exacerbates infection burden. By switching to a localised infection we show that interaction between tailfin inflammation neutrophils and the site of infection occurs and that infection can attract neutrophils away from the tailfin wound prematurely. Our data show that, on a local scale, stabilisation of Hif-1α can alter neutrophil migration dynamics, but that, on an entire organism level, the host-protective effect of Hif-1α stabilisation against infection remains. These findings demonstrate that comorbidities may have multiscale effects ranging from the local tissue to holistic levels and that Hif-1α is a promising drug target against TB, even in the presence of an inflammatory comorbidity.

Results

Mm infection induced neutrophil emergency haematopoeisis and was increased by a comorbid wound

Infection and inflammation commonly occur in the same individual during disease, yet many in vivo experimental systems investigate immune responses to these processes independently of each other. We set out to develop in vivo zebrafish models of infection and inflammation, that we have termed “comorbid models”. Initially we combined two well-defined models; a Mycobacterium marinum (Mm) model of systemic infection with injection of bacteria into the caudal vein at 30-32 hours post fertilisation (hpf) that allows assessment of bacterial burden at 4 days post infection (dpi), and a tailfin wound model of neutrophilic inflammation (transection of the tailfin at 2 days post fertilisation (dpf) with neutrophil inflammation resolving at 24 hours post wound (hpw)) [28,29] (Figure 1A).

Figure 1. Mm infection induced neutrophil emergency haematopoeisis and was increased by a comorbid wound.

(A) Schematic of experiment for B, C and E.

(B) Neutrophil numbers at the wound at 6 and 24 hours post wound (hpw) of mpx:GFP embryos. Groups are not injected (NI), control injection with PVP (PVP) and Mm injection (Mm). Data shown are mean ± SEM, n=75-85 accumulated from 3 independent experiments. Statistics were determined using one-way ANOVA (with Bonferonni’s multiple comparisons test). P values shown are: *P <.05, **P <.01, and ***P <.001.

(C) Percentage resolution of neutrophilic inflammation (percentage change in neutrophil number at the tailfin wound between 6 and 24 hpw). Groups are not injected (NI), control injection with PVP (PVP) and Mm injection (Mm). Data shown are mean ± SEM, n=75-85 from 3 independent experiments. Statistics were determined using one-way ANOVA (with Bonferonni’s multiple comparisons test). P values shown are: *P <.05, **P <.01, and ***P <.001.

(D) Total, whole-body neutrophil numbers at 2dpf, after 18 hours post infection (hpi) with PVP or Mm. Data shown are mean ± SEM, n=69-74 accumulated from 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

(E) Bacterial burden of larvae with or without a tailfin wound. Data shown are mean ± SEM, n=58 accumulated from 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

We first assessed whether injury at the caudal vein (the site of Mm infection) caused by the microinjection process itself would affect neutrophil behaviour at the tailfin wound. Injection of PVP into the caudal vein (mock infection control) caused no difference to the number of neutrophils at the peak of recruitment to the tailfin wound (6hpw), nor after neutrophil inflammation resolution at 24hpw (not injected, NI, compared to PVP injected) (Figure 1B).

The presence of systemic Mm infection increased neutrophil number at the wound at both the 6hpw and 24hpw timepoints compared to NI and PVP controls (Figure 1B).

Although overall neutrophil numbers were increased by infection at 6hpw and 24hpw (Figure 1B), the percentage change in neutrophil number between 6-24hpw remained the same across groups (Figure 1C). We assessed whole body neutrophil counts after Mm infection without a tailfin injury and confirmed that total neutrophil number was increased after Mm infection (Figure 1D) consistent with emergency haematopoeisis, likely contributing to the higher neutrophil numbers observed at the wound after infection [30]. Infection levels were measured in the comorbid model using fluorescent Mm and assessing bacterial burden at 4dpi. Levels of Mm infection were significantly increased in the presence of neutrophilic inflammation at the wound site compared to non-wounded controls (Figure 1E) indicating that the presence of localised tailfin inflammation is detrimental to infection control.

Local somite Mm infection lowers neutrophil numbers at the tailfin wound

To investigate neutrophil migration to infection and wound stimuli in a comorbid model we switched from a systemic infection to a local infection challenge in order to create a single focus of infection. We challenged 3dpf zebrafish larvae with a tailfin wound immediately followed by a local somite infection into the 26-27^th somite (Mm or PVP mock infection control) and counted neutrophils at each site over time. Unlike systemic infection (Figure 1D), local somite infection did not increase wholebody neutrophil number over the 4 hours post infection/wound time period of this assay (Figure 2A-B). When challenged with Mm infection alone or tailfin wound alone, migrated to each respective site and peaked at 4-6hpw/i (Figure 2C-E). Of note, some neutrophils were present at the site of infection before challenge (on average 10 neutrophils) due to the natural distribution of neutrophils at this stage, with very few present at the end of the tail (the wound site, <5 neutrophils) (Figure 2C-E). When tailfin wounding was followed by PVP injection (as a mock infection control), neutrophils migrated to both the somite PVP site and the tailfin wound site, indicating that a wound in the somite was sufficient to attract neutrophils (Figure 2D-E). When tailfin wounding was followed by somite Mm infection, neutrophils migrated to the somite infection site at the expense of tailfin wound neutrophils (Figure 2C-E).

Figure 2. Local somite Mm infection lowers neutrophil numbers at the tailfin wound.

(A) Schematic of experiment for B-E.

(B) Total, whole-body neutrophil numbers in mpx:GFP embryos at 4hpw after local somite injection with PVP or Mm. Data shown are mean ± SEM, n=30-35 accumulated from 3 independent experiments.

(C) Number of neutrophils at site of infection and tailfin wound at 4hpi/w in mpx:GFP embryos. The groups are Wound (tailfin wound alone), Mm infection (Mm infection alone), Mock infection (PVP)/wound (injection of PVP into the somite alongside a tailfin wound) and Mm infection/wound (injection of Mm into the somite alongside a tailfin wound). Individual embryos are represented as lines joining their respective infection and tailfin wound neutrophil numbers. Data shown are mean ± SEM, n=9-13 representative of 3 independent experiments.

(D) Neutrophil numbers at the site of infection at 4hpi. Data shown are mean ± SEM, n=9-13 representative of 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

(E) Neutrophil numbers at the site of tailfin wound at 4hpi. Data shown are mean ± SEM, n=9-13 representative of 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

We next investigated whether neutrophils migrate from a somite wound to the tailfin wound. Photoconversion of Tg(mpx:Gal4/UAS:Kaede) neutrophils allows fate-tracking of specific populations of neutrophils. We photoconverted neutrophils in the caudal vein region, anterior to the somite wound, and tracked their migration over 4 hours post somite infection/tailfin transection (Figure 3A). We observed that in PVP somite injections, red neutrophils that were photoconverted in the CHT, primarily migrate to the site of somite injection, but are not retained at this injection wound, instead migrate towards the tailfin transection wound (Figure 3B). Conversely, when Mm was injected into the somite, the majority of photoconverted neutrophils were recruited to the infection and were retained at the site instead of migrating towards the tailfin transection (Figure 3B-D). Over the 4 hour timelapse, few neutrophils migrated to the tailfin wound in Mm infected larva (Figure 3B-D), and of those that did they took a direct route to the tailfin wound, bypassing the infection (Figure 3B, red track). Together, these data indicate that the signal gradient caused by Mm infection is additive to that of the somite injury alone and that neutrophils preferentially migrate to Mm and are retained at infection rather than travelling further along the trunk to the tailfin wound.

Figure 3. Mm retains neutrophils at the somite infection site at the expense of migration to the tailfin wound.

(A) Schematic of experiment for B-D, showing photoconversion of neutrophils in the caudal hematopoietic tissue (CHT) of mpx:Kaede embryos.

(B) Example fate-tracks of red neutrophils that were photoconverted in the CHT and migrated to either PVP or Mm infection in the somite over 4hpi/w. Data shown are examples from 2 independent experiments with 10 fish per group. Scale bar = 100 μM

(C) Stereo-fluorescence micrographs of the location of red photoconverted neutrophils that originated in the CHT in PVP and Mm infected larvae. Data shown are examples from 2 independent experiments with 10 fish per group. Scale bar = 100 μM

(D) Heatmap of location of red photoconverted neutrophils that originated in the CHT over time. Data shown are n=5 embryos per group accumulated from 2 independent experiments.

Finally, we examined whether the attractant effect of Mm on neutrophils was dependent on live bacteria. We used heatkilled bacteria and found that there was no statistical difference between the neutrophil number at 4hpi/w at either the somite infection or the tailfin wound compared to live Mm (Figure 4A-D). HK Mm caused a significant increase in neutrophil numbers at infection compared to PVP injected controls, similar to live Mm (Figure 4C). However, the neutrophil localisation pattern of the heatkilled Mm infected larva appeared to be intermediate between PVP injection and live Mm (Figure 4B), and indeed HK Mm did not significantly decrease neutrophil numbers at the tailfin wound compared to PVP injected controls, while live Mm did (Figure 4D). Therefore, these data indicate that signals from HK Mm are sufficient to attract neutrophils to the site of infection, but suggest a minor contribution of the bacteria being alive to neutrophil migration behaviours in this assay.

Figure 4. Heatkilled Mm are sufficient for increased neutrophil recruitment to the infection site.

(A) Schematic of experiment for B-D.

(B) Number of neutrophils at site of infection and tailfin wound at 4hpi/w in mpx:GFP embryos. The groups are Wound (tailfin wound alone), Mock infection (PVP)/wound (injection of PVP into the somite alongside a tailfin wound), Mm infection/wound (injection of Mm into the somite alongside a tailfin wound) and heatkilled (HK) Mm infection/wound (injection of HK Mm into the somite alongside a tailfin wound). Individual embryos are represented as lines joining their respective infection and tailfin wound neutrophil numbers. Data shown are mean ± SEM, n=21 representative of 3 independent experiments.

(C) Neutrophil numbers at the site of infection at 4hpi. Data shown are mean ± SEM, n=42 representative of 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

(D) Neutrophil numbers at the site of tailfin wound at 4hpi. Data shown are mean ± SEM, n=42 representative of 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

Neutrophils preferentially migrated to a new infection stimulus rather than patrol the wound site

In a single model of tailfin wound, once neutrophils have migrated to a wound site (between 1-6hpw), they are retained at the wound, patrolling until the resolution phase of inflammation (6-12hpw) [12,27]. We have previously demonstrated that neutrophils migrate away from the wound by a diffusion process at around 8-12hpw when neutrophils become desensitised to signals that retains them at the wound [31]. We hypothesised that infection can overcome this retention signal at the wound site and attract neutrophils prematurely away from the wound. To determine whether infection can overcome retention signals and attract neutrophils away from a tailfin wound, we changed the localised infection model so that the infection challenge was performed at a time when neutrophils are present at the tailfin wound and are still being recruited. At 4hpw, a localised Mm infection was introduced into the 26-27^th somite (Figure 5A). 4hpw is a timepoint at which neutrophils would not have started to reverse migrate away in a single wound model, a process that normally occurs after 6-12hpw [10,12].

Figure 5. Neutrophils preferentially migrated to a new infection stimulus rather than patrol the wound site.

(A) Schematic of experiment for B-F.

(B) Stereo-fluorescence micrographs of a tailfin transected mpx:Kaede embryo after either 30^th somite, 26/27^th somite, or 23-24 somite infection with Mm at 0hpi and 2hpi. The local infection site is shown by a yellow ring and photoconversion (PC) of wound neutrophils is shown by the box. With 30 somite injection red (wound-experienced) neutrophil have started migration to the infection site by the time the timelapse has been started and are almost all at the infection site by 2hpi (white ring). The 26^th-27^th somite infection does not start recruiting wound-experienced neutrophil by the time of the timelapse, but has done so after 2hpi (white arrowheads). Infection into the 23^rd-24^th somite does not recruit any wound-experienced neutrophils over a 2 hour time-course. Representative example from n=9 embryos per group from 3 independent experiments. Scale bar = 150 μM

(C) Stereo-fluorescence micrographs of a tailfin transected mpx:Kaede embryo after 26/27^th somite infection with Mm. Wound-naïve neutrophils are green only and those photoconverted at the wound at timepoint zero (wound-experienced) begin as red-only and regain GFP (therefore giving a yellow overlay) over the course of the timelapse as nascent Kaede fluorescent protein is made. Both wound-naïve (white arrowhead) and wound-experienced (yellow arrowhead) are recruited to the localised site of Mm infection before 110 minutes post conversion (mpc), even though the timelapse is begun at 5hpw, a timepoint when neutrophils would normally still be recruited to the tailfin transection. Representative example from data shown in D-F, n=12 embryos from 3 independent experiments.Scale bar = 100 μM

(C) Number of green, wound-naïve neutrophils at infection site over 1.5hpi. Data shown are mean ± SEM, n=12 embryos accumulated from 3 independent experiments.

(D) Number of red, wound-experienced neutrophils at infection site over 1.5hpi. Data shown are mean ± SEM, n=12 embryos accumulated from 3 independent experiments.

(E) Number of red, wound-experienced neutrophils at wound site over 1.5hpi. Data shown are mean ± SEM, n=12 embryos accumulated from 3 independent experiments.

Photoconversion of Tg(mpx:Gal4/UAS:Kaede) neutrophils at the tailfin wound at 4hpw allowed identification of neutrophils that had visited the wound (“wound experienced” red neutrophils), compared to those that had not (“wound naïve” green neutrophils). We demonstrated that injection of Mm into the 26-27^th somite was sufficient to attract neutrophils away from the wound (wound experienced neutrophils) between 4hpw-6hpw (Figure 5B). The movement of neutrophils between the two sites did not occur to the same extent when Mm was injected further away from the tailfin wound (23-24^th somite) and injection closer to the wound site (30^th somite) caused early neutrophil movement to the infection site before the embryos could be mounted for microscopy, therefore injection into the 26^th-27^th somite was chosen as being optimal (Figure 5B). Wound naïve neutrophils were attracted to the infection site, but numbers remained steady between 0-110mpc (minutes post conversion) (Figure 5B). By 100mpc almost all wound-experienced neutrophils had been attracted away from the tailfin wound towards the infection site (Figure 5C-F). These data demonstrate that the “second hit” of infection was sufficient to overcome signalling that retains neutrophils at the initial tailfin wound site.

Hif-1α stabilisation retained neutrophils at infection at the expense of migration to a tailfin wound

Hypoxia signalling, via stabilisation of Hif-1α, has profound effects on neutrophil behaviours and antimicrobial activity [12,22,23]. We set out to understand whether Hif-1α stabilisation affected neutrophil behaviour in our comorbid models of infection and inflammation. We first stabilised Hif-1α in our simultaneous local infection and tailfin wound comorbid model to determine neutrophil migrations towards the two sites. Endogenous Hif-1α was stabilised pharmacologically using the hydroxylase inhibitors FG4592 and DMOG [12] 4 hours before infection with Mm into the 26-27th muscle somite. This was followed by immediate tailfin wound and neutrophil numbers were counted at each site at 6pw/I (Figure 6A). The solvent control for both hydroxylase inhibitors (DMSO), caused no difference in neutrophil migration to infection and wound at 6hpw/i compared to untreated larvae (Figure 6B-F). Treatment with either FG5492 or DMOG caused significantly increased neutrophil migration to the infection site with fewer neutrophils migrating to the tailfin wound compared to DMSO controls (Figure 6B-F). These findings were confirmed by genetic stabilisation of Hif-1α using dominant active Hif-1α (Figure 6G-I). These data suggest that neutrophils primed with Hif-1α are more sensitive to the local infection chemokine gradient at the expense of the more distant gradient emanating from the wound.

Figure 6. Hif-1α stabilisation increased neutrophil numbers at infection at the expense of those at the tailfin wound.

(A) Schematic of experiment for B-F.

(B) Number of neutrophils at site of infection and tailfin wound of mpx:GFP embryos at 4hpi/w after Hif-1α stabilisation with FG4592 or DMOG with no treatment and DMSO controls. Data shown are mean ± SEM, n=9-15 representative of 3 independent experiments.

(C) Neutrophil numbers at the infection site at 4hpi with DMSO and FG4592 treatment.

Data shown are mean ± SEM, n=10-11 representative of 3 independent experiments. Statistics were determined using one-way ANOVA (with Bonferonni’s multiple comparisons test). P values shown are: *P <.05, **P <.01, and ***P <.001.

(D) Neutrophil numbers at the wound site at 4hpi with DMSO and FG4592 treatment.

Data shown are mean ± SEM, n=10-11 representative of 3 independent experiments. Statistics were determined using one-way ANOVA (with Bonferonni’s multiple comparisons test). P values shown are: *P <.05, **P <.01, and ***P <.001.

(E) Neutrophil numbers at the infection site at 4hpi with DMSO and DMOG treatment.

Data shown are mean ± SEM, n=19 representative of 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

(F) Neutrophil numbers at the wound site at 4hpi with DMSO and DMOG treatment.

Data shown are mean ± SEM, n=19 representative of 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

(G) Number of neutrophils at site of infection and tailfin wound at 4hpi/w after Hif-1α stabilisation with dominant active Hif-1α (DA1) or phenol red (PR) controls. Data shown are mean ± SEM, n=20-22 representative of 3 independent experiments.

(H) Neutrophil numbers at the infection site at 4hpi with PR and DA1. Data shown are mean ± SEM, n=19 representative of 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

(I) Neutrophil numbers at the wound site at 4hpi with PR and DA1 treatment. Data shown are mean ± SEM, n=36-41 accumulated from 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

Hif-1α stabilisation delayed wound-experienced neutrophil migration to Mm infection

We have previously demonstrated, in a single tailfin wound model, that stabilisation of Hif-1α delays neutrophil reverse migration away from the wound [12]. However, here we show that a local Mm infection is able to attract neutrophils away from the tailfin wound prematurely (Figure 5). We therefore hypothesised that Hif-1α would prevent wound-experienced neutrophils from exiting the injury site prematurely to migrate to a localised infection site. We tested this in our comorbid model where local infection was performed 4 hours post tailfin wound. Wound-naïve neutrophil attraction to the site of Mm infection was not altered by DA Hif-1α compared to phenol red (PR) controls (Figure 7A-B). Infection was sufficient to attract wound-experienced neutrophils away from the wound prematurely, but neutrophils in DA Hif-1α embryos were significantly delayed in their migration towards localised Mm infection compared to PR controls (Figure 7B-C). The migration speed of wound-experienced neutrophils was lower in the DA Hif-1α group compared to the PR group, largely due to their tighter association to the wound edge and less migration away (Figure 7D). This decrease in migration speed was more marked in wound-experienced neutrophils that were successful in migrating away from the wound edge towards the Mm infection site (Figure 7E). These neutrophils migrated to the infection site at two-thirds of the speed in DA Hif-1α embryos compared to the PR controls (Figure 7E). Furthermore, they took a less direct route to the infection, with the meandering index of these neutrophils significantly lower in the DA Hif-1α group compared to PR controls (Figure 7F). Neutrophils in Hif-1α stabilised embryos therefore remain more sensitive to the wound signalling gradient, even if successful in escaping the wound to a second hit of infection.

Figure 7. Stabilisation of Hif-1α delayed the migration of wound-experienced neutrophils to a local site of Mm infection.

(A) Schematic of experiment for B-F.

(B) Number of green, wound-naïve neutrophils at infection site over 1 hour post wound (hpw) in mpx:Kaede embryos. Groups shown are DA Hif-1α (DA, red points) and phenol red controls (PR, black points). Data shown are mean ± SEM, n=7-9 embryos accumulated from 3 independent experiments. Line of best fit shown is calculated by linear regression. P value shown is for the difference between the 2 slopes. P values shown are: *P <.05, **P <.01, and ***P <.001.

(C) Number of red, wound-experienced neutrophils at infection site over 1 hpw.

Groups shown are DA Hif-1α (DA, red points) and phenol red controls (PR, black points). Data shown are mean ± SEM, n=7-9 embryos accumulated from 3 independent experiments. Line of best fit shown is calculated by linear regression. P value shown is for the difference between the 2 slopes. P values shown are: *P <.05, **P <.01, and ***P <.001.

(D) Speed of red, wound-experienced neutrophil movement at the wound site. Groups shown are DA Hif-1α (DA) and phenol red controls (PR). Data shown are mean ± SEM, n=5-6 embryos accumulated from 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

(E) Speed of red, wound-experienced neutrophils migrating from the wound site to the infection site. Groups shown are DA Hif-1α (DA) and phenol red controls (PR). Data shown are mean ± SEM, n=5-6 embryos accumulated from 3 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001.

(F) Meandering index of red, wound-experienced neutrophils migrating from the wound site to the infection site. Groups shown are DA Hif-1α (DA) and phenol red controls (PR). Data shown are mean ± SEM, n=15-18 embryos accumulated from 2 independent experiments. Statistics were determined using an unpaired T-test. P values shown are: *P <.05, **P <.01, and ***P <.001. Scale bars = 500 μM

Taken together, these data indicate that wound-experienced neutrophils in Hif-1α stabilised larvae remain more sensitive to the wound gradient and are less likely to migrate to the second hit infection site compared to normal controls.

Mm burden was decreased by Hif-1α stabilisation, despite delayed resolution of neutrophilic inflammation

In the single model of Mm infection we have previously shown that Hif-1α stabilisation reduced bacterial burden; a good therapeutic outcome [22]. However, in the single tailfin model, Hif-1α delayed neutrophil inflammation resolution away from the wound; a bad therapeutic outcome in diseases of chronic inflammation [12]. As infection and chronic inflammation are common attributes of comorbidities, we investigated whether the beneficial therapeutic outcome of Hif-1α stabilisation in systemic infection would be maintained in the presence of chronic inflammation.

We observed an increase in neutrophil recruitment to the tailfin wound after Mm infection (at 6hpw) in PR controls (Figure 8A-B), in keeping with the emergency hematopoietic effect of infection observed earlier (Figure 1D). No effect of DA Hif-1α was observed on neutrophil recruitment compared to PR controls (Figure 8B), consistent with previous observations in the single tailfin transection model [12]. Neutrophil numbers at the wound after resolution, at 24hpw were increased by DA Hif-1α compared to PR controls in the presence (Mm) or absence (PVP) of Mm infection (Figure 8C) and the percentage resolution (6-24hpw) was reduced by Hif-1α stabilisation compared to PR controls (Figure 8D), indicating that Hif-1α stabilisation delays neutrophil inflammation resolution in the presence of systemic infection.

Figure 8. Mm burden was decreased by Hif-1α stabilisation, despite delayed resolution of neutrophilic inflammation.

(A) Schematic of experiment for B-D.

(B) Neutrophil numbers recruited to the tailfin wound at 6hpw in mpx:GFP embryos.

Groups are phenol red (PR) and DA Hif-1α (DA) injected at 30hpf with PVP or Mm. Data shown are mean ± SEM, n=62-111 accumulated from 3 independent experiments. Statistics were determined using one-way ANOVA (with Bonferonni’s multiple comparisons test). P values shown are: *P <.05, **P <.01, and ***P <.001.

(C) Neutrophil numbers at the tailfin wound at 24hpw. Groups are phenol red (PR) and DA Hif-1α (DA) injected at 30hpf with PVP or Mm. Data shown are mean ± SEM, n=62-111 accumulated from 3 independent experiments. Statistics were determined using one-way ANOVA (with Bonferonni’s multiple comparisons test). P values shown are: *P <.05, **P <.01, and ***P <.001.

(D) Percentage resolution of neutrophil inflammation (between 6 to 24 hpw). Groups are phenol red (PR) and DA Hif-1α (DA) injected at 30hpf with PVP or Mm. Data shown are mean ± SEM, n=62-111 accumulated from 3 independent experiments. Statistics were determined using one-way ANOVA (with Bonferonni’s multiple comparisons test). P values shown are: *P <.05, **P <.01, and ***P <.001.

(E) Schematic of experiment for G-F.

(F) Stereo-fluorescence micrographs of Mm mCherry infected 4dpi larvae after injection with DA Hif-1α (DA1) and phenol red (PR) as a negative control and either wounded at 48hpf or non-wounded in mpx:GFP embryos. Representative images from data shown in G, with n=58 accumulated from 3 independent experiments.

(G) Bacterial burden of larvae shown in (F). Data shown are mean ± SEM, n=58 accumulated from 3 independent experiments. Statistics were determined using one-way ANOVA (with Bonferonni’s multiple comparisons test). P values shown are: *P <.05, **P <.01, and ***P <.001.

DA Hif-1α larvae had decreased bacterial burden compared to PR controls indicating that the protective effects of Hif-1α stabilisation remained, even in the presence of tailfin inflammation (Figure 8E-F). This is despite our finding that an inflammatory process (tailfin wound) during systemic infection caused a marked increase in infection levels in the absence of Hif-1α stabilisation (Figure 8E-F). These results indicate that Hif-1α remains protective against Mm even when neutrophil inflammation resolution is delayed at the tailfin.

Discussion

With the emergence of antibiotic resistance, there is increasing interest to find host-derived factors that could act as therapeutic targets [2]. We have previously identified in zebrafish in vivo models of tuberculosis infection that targeting neutrophils is a potential mechanism to decrease infection burden via Hif-1α stabilisation [22]. Physiological hypoxia and Hif-1α stabilisation have been demonstrated to have activating effects on neutrophils in a growing number of models, increasing their antimicrobial capabilities in vitro, ex vivo and in vivo [18,19]. These findings have been tempered by clinical observations that activated neutrophils are associated with chronic disease, leading to excess tissue damage and poor disease outcomes [11,23]. Patient studies address neutrophil behaviour at chronic stages of disease by which time there is a cycle of neutrophil overactivation, degranulation, tissue damage and further recruitment. Targeting neutrophils at earlier disease stages has the potential to be highly beneficial before this chronic cycle begins, but potential effects on patients with TB and comorbid inflammatory conditions, such as COPD, remain unclear.

By combining zebrafish Mm infection and a tailfin wound to make a comorbid model we have shown that Hif-1α stabilisation remains protective against Mm infection in the presence of comorbid inflammation. This was despite infection burden being increased by the presence of a tailfin wound in the wildtype situation. While the increase in infection alongside a wound may be due to neutrophils migrating to the tailfin wound, it may also be due to differences in neutrophil activation by wounding and Hif-1α. After both wounding and Hif-1α stabilisation there is robust upregulation of pro-inflammatory signalling, for example increased Il-1β in neutrophils [32,33], but with wounding neutrophil activation appears detrimental to infection control while with Hif-1α stabilisation this is host protective. Signs of Hif-1α stabilisation being detrimental to neutrophilic inflammation resolution were observed at the tailfin wound in the comorbid model where resolution of neutrophil inflammation was delayed. However no other adverse effects were observed, consistent with findings from the single tailfin wound model [12]. These data indicate that stimulation of neutrophils by inflammation (wounding) and Hif-1α stabilisation are different, and that if neutrophils are appropriately activated by Hif-1α they could be highly beneficial to host infection control without damaging holistic effects.

We developed a local infection and tailfin wound comorbid model to investigate the effects of Hif-1α stabilisation on neutrophil migration to wound and infection sites simultaneously. We found that neutrophils dispersed between infection and wound sites, but when Hif-1α was stabilised, neutrophils seldom migrated beyond the local infection on to the tailfin wound. Conversely, Hif-1α stabilisation retained neutrophils at the tailfin wound when a second hit of infection was introduced, while in wildtype larvae infection caused premature migration away from the wound to the infection site. These data indicate that Hif-1α stabilisation causes increased sensitivity to wound or infection gradients, leading to retention of neutrophils and reduced ability of these cells to respond to competing signals. Our previous findings have shown that Hif-1α stabilisation caused no effect on neutrophil recruitment to the tailfin wound in the single inflammation model, therefore Hif-1α is unlikely to have effects on neutrophil recruitment signalling [12]. Taken together, these data indicate that recognition of “retention signals” by neutrophils is sensitised by stabilised Hif-1α, keeping neutrophils at the wound or infection site, and that there is an as yet unidentified molecular change in neutrophils in Hif-1α stabilised embryos that alters their sensitivity to these tissue gradients. Likely candidates for Hif-1α targets include G protein coupled receptors (GPCRs) that are involved in neutrophil migration (many chemokine receptors are GPCRs) and are regulated by Hif-1α in immune cells (eg CXCR1, CXCR2 or CXCR4) [34–38]. Cxcr1/2 have been implicated in retention of neutrophils at wounds and we have recently demonstrated that decreasing Cxcr4 signalling causes premature reverse migration away from the tailfin wound [39].

Previous findings in a zebrafish model of tailfin infection followed by localised infection demonstrated that, during the reverse migration phase of neutrophil inflammation (>12hpw), wound-experienced neutrophils that are migrating away from the wound towards infection stimuli (Staphylococcus aureus and zymosan) display unaltered migration behaviour compared to nearest-neighbour-wound-naive neutrophils [40]. In the absence of Hif-1α stabilisation, this appears to be the case in our Mm/wound comorbid model, with both wound-naïve and wound-experienced neutrophils equally able to respond to the secondary local infection. When Hif-1α is stabilised differences in neutrophil migration behaviour become evident - wound-experienced neutrophils change behaviour and are slower to migrate to the second hit, while wound-naïve neutrophils migrate as normal. This indicates that neutrophils that have visited the wound may differ from those that have not in certain contexts (eg, when Hif-1α is stabilised), however the mechanisms behind these differences have yet to be determined.

Here, we kept as many aspects of each individual model as close as possible to those previously characterised in order to avoid setting up comorbid models with undefined individual characteristics that could potentially complicate interpretation [12,22]. Comorbidities can be incredibly complex and further in vivo comorbid models are required to understand the full range of cellular and molecular mechanisms involved. Here, we only considered neutrophil migration towards infection and injury, however macrophages also play important roles in the outcomes of wound and infection, as previously demonstrated in single-model zebrafish studies [10,29]. Future investigations into the role of macrophages in comorbid models may uncover novel mechanisms for macrophage migration and activity in wounds and infections when they occur together.

We used infection and sterile wounds as a comorbidity in this study, however comorbidities can be more complex and further underlying conditions can affect both processes. A good example of this is diabetes, which suppresses the immune system, decreases wound healing and causes complex HIF dysregulation. Diabetes and hyperglycaemia can lead to localised tissue hypoxia due to links to obesity and changes in immunometabolism, however, hyperglycaemia also destabilises HIF-1α, which in part is responsible for the defective wound healing and infection responses [41,42]. Indeed, diabetes triples the chance of developing TB (WHO). The effects of HIF stabilisation in comorbidities such as diabetes and TB remain unclear.

As investigations of comorbidities continue to rise we anticipate that comorbid models will increase in popularity, but with a plethora of possible conditions, combinations and timings of stimuli available, care will be required to understand the relevance of these models to the research question asked.

Using comorbid models of infection and wounding we have highlighted that comorbidity has a range of effects on neutrophil behaviour during infection that differ on the local tissue scale compared to the whole-organism, holistic, level. In our comorbid model Hif-1α stabilisation remains host protective effect while causing a delay in neutrophil inflammation resolution at a wound, properties that are consistent with the individual models [12,22]. Using a localised infection comorbid model we show that Hif-1α stabilisation increases neutrophil retention at infection and tailfin wound sites in vivo. Our comorbid models suggest that, on a whole-organism level, neutrophil activation by Hif-1α stabilisation is able to reduce infection burden and remains a promising host-derived therapeutic strategy against multi-drug resistant TB.

Materials and methods

Zebrafish husbandry

All the zebrafish used in this project were raised in the University of Sheffield Home Office approved aquarium and were kept under standard protocols as previously outlined [43]. Adult zebrafish were kept in tanks of no more than 40 adult fish, and experience a 14-hour light and 10-hour dark cycle. A recirculating water supply is maintained and the temperature of the water is kept at 28°C. Embryos for this study were generated by in-crossing TgBAC(mpx:Gal4.VP16);Tg(UAS:Kaede)i222 (shortened to mpx:Kaede) or Tg(mpx:GFP)i114 (shortened to mpx:GFP) [27,44].

Ethics

All procedures over the course of this project were performed on embryos that were less than 5.2 days post fertilisation (dpf) and were therefore considered outside of the Animals (Scientific Procedures) Act. Procedures were carried out to standards set by the UK Home Office on the Project Licence P1A4A7A5E held by Professor Stephen Renshaw at the University of Sheffield.

Tailfin transection

For all experiments, larval tailfins were transected at 48 hours post fertilisation (hpf) as previously described [12]. Kaede-expressing wound neutrophils were photoconverted at 4 hours post wound (hpw) using a SOLA light engine white light LED (Lumencor, Beaverton, OR, USA) through DAPI filters on a Leica DMi8 inverted widefield microscope (Leica Microsystems (UK), Milton Keynes, United Kingdom). Timelapse microscopy was performed using a Leica DMi8 inverted widefield microscope (Leica Microsystems) using a HC FL PLAB 10x/0.40 lens and captured using a Hammamatsu ORCA-Flash 4.0 camera (Hammamatsu, Hamamatsu-City, Japan). Neutrophil counts were performed with the investigator blinded to the experimental group on a Leica MZ10 F Stereomicroscope with fluorescence, with changes in focus and magnification allowing optical resolution of individual cells (Leica Microsystems).

Mycobacterium marinum infection

Mm infection experiments were performed using M. marinum M (ATCC #BAA-535), containing a psMT3-mCherry or psMT3 mCrimson vector [45]. Injection inoculum was prepared from an overnight liquid culture in the log-phase of growth resuspended in 2% polyvinylpyrrolidone40 (PVP40) solution (CalBiochem/Merck KGaA, Darmstadt, Germany) as previously described [22]. For heatkilled experiments, Mm were incubated at 80°C for 30 minutes as previously described [46].

For systemic infection 150-200 colony forming units (CFU) were injected into the caudal vein at 28-30hpf, as previously described [47].

For localised somite infection, fish were anaesthetised in 0.168 mg/ml Tricaine (Sigma-Aldrich/Merck KGaA, Darmstadt, Germany) and were microinjected with 500CFU (colony forming units) of Mm in the 26^th-27^th somite [40].

Hif-1α stabilisation

Embryos were injected with dominant active hif-1αb (ZFIN: hif1ab) variant RNA at the one cell stage as previously described [12,48]. Phenol red (PR) (Sigma-Aldrich) was used as a vehicle control.

Hif-1α was stabilised pharmacologically using hydroxylase inhibitors FG4592, 5μM or DMOG, 100μM (dimethyloxaloylglycine), with DMSO (dimethyl sulfoxide) control.

Bacterial pixel count

Infected zebrafish larvae were imaged at 4 days post infection (dpi) on an inverted Leica DMi8 with a 2.5x objective lens. Brightfield and fluorescent images were captured using a Hammamatsu OrcaV4 camera. Bacterial burden was assessed using dedicated pixel counting software as previously described [22,49].

Image and Statistical Analysis

Microscopy data was analysed using Leica LASX (Leica Microsystems) and Image J software. Meandering index was calculated by dividing the shortest possible path from start to endpoint by the total distance travelled by the neutrophil over the time period. Therefore a neutrophil travelling in a straight line would have a meandering index of 1 and a neutrophil deviating from the shortest path a meandering index of <1. All data shown are mean with SEM (Prism 8.0, GraphPad Software, San Diego, CA, USA) with statistics determined using t-tests for comparisons between two groups and one-way ANOVA (with Bonferonni post-test adjustment) for other data. P values shown are: *P <.05, **P <.01, and ***P <.001.