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

Context and background

Tuberculosis (Tb) is one of the leading causes of death worldwide with 1.5 million deaths in 2018 (1). Alarmingly, multi-drug resistant Tb is rising with 484,000 new cases of Tb with resistance to rifampicin (the most effective first-line drug).

During Tb infection, one of the major immune cells to respond are the neutrophils. Neutrophils are innate immune cells that primarily clear bacteria through phagocytosis and the release of cytotoxic granules (2). Through these mechanisms, neutrophils can help to remove the Tb infection. However, neutrophils can behave as “double agents” with increasing numbers of neutrophils aiding in bacterial survival and evasion from the immune system (3).

It is increasingly important to find new drugs or new treatment strategies for Tb. Powerful microscopic techniques and the transparent nature of zebrafish embryos can be utilised to visualise and track the immune response in real time, in vivo. Although Tb cannot infect zebrafish, a close relative, Mycobacterium marinum (Mm) can, providing a useful system for modelling tuberculosis infection and studying the immune response in vivo in zebrafish. In this preprint, the authors utilise a comorbidity model (that is the presence of additional conditions co-occurring with the primary condition) with Mm infection and tail-fin wounding. The authors wished to investigate whether the stabilisation of HIF-1α is beneficial in a disease (Tb) when there is both infection and inflammation present.

Key findings

1. Neutrophils prioritise the response to infection over wounding responses

Utilising a neutrophil reporter line (GFP or Kaede), the authors investigate the role of neutrophils in Tb comorbidity. The authors injected labelled Mm bacteria and later induced tail-fin wounding to recreate comorbidity. They found that in the presence of prior infection, more neutrophils were located at the wound than in non-infected controls. This resulted in a higher bacterial burden. However, when the wound and infection were induced at the same time, the authors observed higher numbers of neutrophils at the infection site compared to the wound. This suggests that neutrophils may prioritise a new infection over a wound. The authors then induced a wound prior to the Mm infection. Experiments in which they photoconverted and tracked the labelled neutrophils at the wound site confirmed that neutrophils indeed preferentially migrate to the infection rather than patrol the wound site.

2. HIF-1α stabilisation is beneficial for Mm control during comorbidity

During Mm infection, stabilisation of HIF-1α (and therefore functioning HIF signalling) has been shown to reduce bacterial burden (4). However, in a tail-fin wounding model, HIF-1α stabilisation delayed inflammation resolution (5). The authors therefore investigated the role of stabilising HIF-1α in a comorbidity model. Using pharmacological or genetic methods to stabilise HIF-1α, the authors found that stabilisation retained neutrophils at the site of infection, at the detriment of migration towards a wound. To further explore this, the authors stabilised HIF-1α, then investigated wound experienced vs naïve neutrophils.  Wound experienced neutrophils that are HIF-1α stabilised migrated away from wounds, and towards the site of infection more slowly, confirming that HIF-1α activation promotes an inflammatory phenotype. However, neutrophils were still capable of controlling the Mm bacterial burden when HIF-1α was stabilised as these fish demonstrated a significantly lower bacterial count then wounded fish without HIF-1α stabilisation. This suggests that the positive clinical benefit of HIF-1α stabilisation in neutrophils is retained in comorbidity.

Why I chose this paper

I always enjoy reading microscopy papers, especially where zebrafish or Drosophila models are used. This paper is a good example of how useful non-mouse models can be for clinically relevant studies. This is particularly important for advancing our understanding of Tb, as this represents one of the top 10 causes of death worldwide.

 

Open questions

 

1. The authors show an increased bacterial burden in fish that have also been wounded. Do the neutrophils reduce this bacterial burden if given long enough (i.e. looking at later time points)?

 

2. Are the wound experienced neutrophils “primed” to better respond to infection than the wound naïve neutrophils? In essence, if you first induce a wound then infect at a later time point do you see more neutrophils at the site of infection compared to no-wound?

 

3. It appear that neutrophils more readily respond to infection than a wound (Fig 2, panel B and figure 3). Is this due to spatial restrictions at the chosen wound site? Or are these cells “primed” to better respond to infection? Or is it that an infection represents a stronger stimulus or higher priority for a neutrophil? Alternatively, are the neutrophils simply responding to the newest stimuli, as in figure 1?

 

4. Do neutrophils that phagocytose the Mm bacteria preferentially remain at the site of infection? Is there a correlation between the number of bacteria phagocytosed and tracking to/from a wound?

 

5. Are there any plans to investigate the transcriptional profile of the neutrophils to compare wound experience to naïve or HIF-stabilised vs wild type?

 

6. Could the authors speculate on what they believe the active HIF-1α is doing functionally to retain neutrophils at the site of infection? Is this a trafficking issue (considering the data in Fig 5)?

 

7. What role are other immune cells, such as macrophages, playing in the comorbidity situation? Are they aiding in wound healing, thereby enabling the neutrophils to return to/prioritise the infection?

 

8. What was the bacterial burden in fish where the authors first induced a wound or induced a wound and infection at the same time? In this situation the authors found more neutrophils at the site of infection so did that mean less bacterial cells?

 

9. In HIF-1α stabilised neutrophils, there is a greater retainment of neutrophils at the site of infection and better control of Mm bacterial burden. However, there is also a reduction in the resolution of inflammation. What happens to the infection over a longer period in comorbidity? For example, failure to resolve inflammation and the neutrophil response has been implicated in promoting Tb progression (3). Is this something the authors would expect to occur here?

 

References

1. World Health Organisation. Tuberculosis (TB) [Internet]. [cited 2019 Oct 23]. Available from: https://www.who.int/news-room/fact-sheets/detail/tuberculosis
2. Mayadas TN, Cullere X, Lowell CA. The Multifaceted Functions of Neutrophils. Annu Rev Pathol. 2014;9:181–218.
3. Kroon EE, Coussens AK, Kinnear C, Orlova M, Möller M, Seeger A, et al. Neutrophils: Innate Effectors of TB Resistance? Front Immunol [Internet]. 2018 Nov 14 [cited 2019 Oct 23];9. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6246713/
4. Elks PM, Brizee S, van der Vaart M, Walmsley SR, van Eeden FJ, Renshaw SA, et al. Hypoxia inducible factor signaling modulates susceptibility to mycobacterial infection via a nitric oxide dependent mechanism. PLoS Pathog. 2013;9(12):e1003789.
5. Elks PM, van Eeden FJ, Dixon G, Wang X, Reyes-Aldasoro CC, Ingham PW, et al. Activation of hypoxia-inducible factor-1α (Hif-1α) delays inflammation resolution by reducing neutrophil apoptosis and reverse migration in a zebrafish inflammation model. Blood. 2011 Jul 21;118(3):712–22.

Tags: comorbidity, hif-1a, imaging, mm, tb, tuberculosis, zebrafish

doi: https://doi.org/10.1242/prelights.14820

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