Visualising and manipulating macrophage phenotypes during mycobacterial infection in vivo

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a global health emergency,
heightened by rising antimicrobial resistance. Host-directed therapies (HDTs) are promising strategies
by enhancing immune responses. Macrophages, key innate immune cells, polarise along a spectrum
from pro- to anti-inflammatory states, yet how these phenotypes arise in vivo during infection remains
poorly understood. As macrophages serve as the principal intracellular niche for Mtb survival, they
represent powerful HDT targets. A central unresolved question is how anti-inflammatory macrophages
arise in infected tissues: through repolarisation of pro-inflammatory populations or recruitment in
subsequent waves.

Using Mycobacterium marinum-infected zebrafish larvae, I evaluated the TgBAC(arg2:GFP) line
as an in vivo reporter of anti-inflammatory macrophages. Macrophage arg2:GFP expression required
virulent bacteria and Toll-like receptor signalling. Timelapse imaging revealed that arg2:GFP+
macrophages were smaller, rounder and faster than arg2:GFP- macrophages, mirroring antiinflammatory
behaviours described in mammalian macrophages. To test whether arg2:GFP+
macrophages arise through repolarisation, I combined arg2:GFP with a novel pro-inflammatory
reporter, Tg(tnfa:mCherry-F). I identified a distinct tnfa+arg2+ population with characteristics of
arg2:GFP+ macrophages. A subpopulation of tnfa:mCherry+ macrophages upregulated arg2:GFP over
time, accompanied by this phenotypic switch, providing the first in vivo visualisation of proinflammatory
macrophages transitioning to an anti-inflammatory-like state.

To manipulate repolarisation, I modulated hypoxia inducible factors (HIFs), key regulators of
macrophage function. Genetic Hif-2α stabilisation induced arg2:GFP in resting leukocytes, and Hif-2α
was partially required for tnfa:mCherry+ cells to become arg2:GFP+ during infection, aligning with
mammalian studies identifying arginase as a HIF-2α target. Genetic and pharmacological disruption of
macrophage arginase impaired bacterial control, indicating that this phenotypic switch is hostprotective.

Collectively, this work demonstrates that pro-inflammatory macrophages can repolarise,
supporting macrophage plasticity rather than distinct inflammatory subsets. It further
establishes arg2:GFP as a powerful platform for in vivo assessment of macrophage plasticity andbacterial burden, providing an early-stage screening tool for macrophage-targeted HDTs.