Modulating Macrophage Phenotypes During Infection In Zebrafish

Macrophages are important white blood cells that phagocytose invading bacteria and react to infection by upregulating antimicrobial killing mechanisms (termed proinflammatory macrophage polarisation). After the initial response, macrophage populations change behaviours to more anti-inflammatory phenotypes to participate in healing of damaged tissues in order that tissues return to normal (termed anti-inflammatory macrophage polarisation). Despite a deep understanding of macrophage phenotypes in vitro, how diverse macrophage behaviours manifest in infected tissues is not well understood and represents an opportunity for developing new medicines against infections where antimicrobial resistance is an increasing problem (for example in Mycobacterium tuberculosis infection).

Here we propose to develop a zebrafish larval model of anti-inflammatory macrophages that will allow examination of macrophage polarisation during tuberculosis infection in vivo. This will replace mammalian models of invasive infection and reduce the overall number of animals used.

A hallmark of macrophage polarisation is their antimicrobial nitric oxide (NO) output, with proinflammatory macrophages having upregulated inducible Nitric Oxide Synthase (iNOS) leading to high NO levels and anti-inflammatory macrophages having upregulated Arginase leading to decreased NO. We have made an arginase2:GFP transgenic line, that if developed and validated will be the first zebrafish transgenic model for anti-inflammatory macrophages. We have observed GFP expressing macrophages in granulomas, the hallmark structure of TB infection. We have shown that manipulation of Hypoxia Inducible Factor-1alpha (HIF-1alpha, a master transcriptional regulator of the cellular hypoxia response) and Tribbles signalling pathways, improve infection outcomes in a zebrafish Mycobacterium marinum (Mm) model of tuberculosis and have profound effects on macrophages.

We hypothesise that macrophage pro- and anti-inflammatory signalling is dysregulated during mycobacterial infection and can be modulated in vivo to improve infection outcomes.

Our specific objectives are:

1. Determine the macrophage anti-inflammatory response to Mm infection in vivo.

We will cross the arg2:GFP into existing macrophage:red marker lines and use confocal and spinning disk microscopy to define which cells upregulate arg2:GFP from early infection, 1 day post infection (dpi), to granuloma stages at 4dpi. We will quantify arg2:GFP positive macrophage behaviours, including migration, phagocytosis and bacterial killing. arg2:GFP positive macrophages will be FACS purified and RNAseq/qPCR performed to identify the profile of arg2:GFP+ve macrophages and compare them to murine/human datasets.

2. Characterise the dynamics of proinflammatory/anti-inflammatory macrophage polarisation during infection.

We will cross the arg2:GFP transgenic line with proinflammatory markers transgenic lines and use timelapse microscopy and expression studies to determine the dynamics of macrophage marker expression, addressing whether arg2:GFP macrophages originate from a proinflammatory population, or whether other macrophages migrate into infection sites and differentiate directly to an arg2:GFP expressing phenotype.

3. Therapeutically manipulate macrophage proinflammatory/anti-inflammatory polarisation to improve infection outcomes.

We will manipulate HIF and Tribbles signalling pathways in the double transgenics, known modulators of proinflammatory/anti-inflammatory macrophage phenotypes. Readouts will include proinflammatory/anti-inflammatory reporter lines and bacterial burden to assess the effect on infection outcome. In parallel, potential targets from the RNAseq in aim 1 will be investigated via a small CRISPR-Cas9 based genetic screen alongside siRNA knockdown in immortalised, murine bone marrow-derived macrophage lines.