Abstract
Macrophages are central drivers of chronic inflammation, yet how a sustained inflammatory state alters their function remains unclear. Using GFP knock-in zebrafish targeting irg1/acod1 that marks macrophage activation, we track the dynamic transitions of macrophage states during acute muscle injury under homeostatic and chronically inflamed conditions, induced by genetic mutation of nlrc3l. In the chronic inflammation model, muscle repair is impaired and expression of the mannose receptor mrc1b/cd206 is severely downregulated in a myd88-dependent manner. Two reparative macrophage subtypes, defined by their cellular behavior and single-cell transcriptomics profile, clustering and muscle-encasing, are lost. A chronic infection model recapitulates these defects, under…
Abstract
Macrophages are central drivers of chronic inflammation, yet how a sustained inflammatory state alters their function remains unclear. Using GFP knock-in zebrafish targeting irg1/acod1 that marks macrophage activation, we track the dynamic transitions of macrophage states during acute muscle injury under homeostatic and chronically inflamed conditions, induced by genetic mutation of nlrc3l. In the chronic inflammation model, muscle repair is impaired and expression of the mannose receptor mrc1b/cd206 is severely downregulated in a myd88-dependent manner. Two reparative macrophage subtypes, defined by their cellular behavior and single-cell transcriptomics profile, clustering and muscle-encasing, are lost. A chronic infection model recapitulates these defects, underscoring the link to macrophage mrc1b repression. Depleting either mrc1b or macrophages impairs muscle repair. Reinstating normal macrophage states by restoring macrophage nlrc3l expression or ablating myd88-mediated inflammatory pathways rescues muscle repair in nlrc3l mutants. Contrary to conventional discrete states, we identify hybrid M1/M2 macrophage states post-injury. While transient during normal injury response, a pro-inflammatory hybrid state persists during chronic activation, which restricts macrophage heterogeneity, represses mrc1b, and inhibits intracellular cathepsin K accumulation, a hallmark of reparative subtypes. Thus, our study provides mechanistic insight into the dynamics of macrophage activation during muscle injury and repair, and how these processes are modulated under chronic inflammation.
Data availability
Data generated in this study are provided in the publication, Supplementary Information and Source Data File (Supplementary Data 2). RNA sequencing data is freely available at the GEO repository as GSE283438 for bulk RNA-seq, and GSE301079 for scRNA-seq.
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Acknowledgements
We thank Maura McGrail and Jeff Essner for the generous sharing of GeneWeld plasmids and protocols, Michel Bagnat for the tnfa:GFP line, Brian Conlon and Anika Rueppell for the E. coli MG1655 support, and Peng Huang for reagent support. We are grateful to Mike Vernon and staff of UNC High-Throughput Sequencing Facility (supported by P30-CA016086 and P30-ES010126) for library preparations and RNA-sequencing, Gabrielle Cannon of UNC Advanced Analytics Core (UNC CGIBD, P30 DK034987) for scRNA-seq library preparations, researchers of UNC Bioinformatics and Analytics Research Collabrative (BARC): Tara Brennan, Matt Niederhuber, Ismael Gomez, and Austin Hepperla for processing and analzying scRNA-seq data, and Michelle Altemara and staff of UNC Zebrafish Aquaculture Core Facility for zebrafish housing and care. This work was supported by NIH NIGMS grant R35GM124719 to C.E.S.
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Author notes
These authors contributed equally: Caroline G. Spencer, Matthew Hamilton.
Authors and Affiliations
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Caroline G. Spencer, Matthew Hamilton, Ethan Bedsole, Yingshan N. Wei, Alison M. Rojas, Andrew Burciu, John Zhu, Keith Z. Sabin & Celia E. Shiau 1.
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Celia E. Shiau
Authors
- Caroline G. Spencer
- Matthew Hamilton
- Ethan Bedsole
- Yingshan N. Wei
- Alison M. Rojas
- Andrew Burciu
- John Zhu
- Keith Z. Sabin
- Celia E. Shiau
Contributions
C.G.S.: data analysis and visualization, experimentation (injury model, time course analysis, epistasis experiments, multiple mutant phenotyping, timelapse and static injury imaging, CRISPR), writing; M.H.: data analysis and visualization, experimentation (bulk and single-cell RNA-seq analysis, time course analysis, imaging, injury analysis, cloning, qPCR, HCR, scRNA-seq, IHC), writing; E.B.: data analysis and visualization, experimentation (KI versus TOL2 imaging, CRISPR, injury analysis, scRNA-seq, infection); Y.N.W.: data analysis and visualization, experimentation (subtype and muscle imaging, genetic experiments, infection); A.M.R.: experimentation (generation of GFP knock-in and TOL2 lines, germ-free experimentation); A.B.: experimentation (FACS sorting and protocol development, bulk RNA-seq preparations); JZ: experimentation (KI versus TOL2 analysis); K.S.: experimentation (initial IHC analysis of injury); C.E.S.: conceptualization and experimental designs, data analysis and visualization, supervision, funding acquisition, writing- original draft and all revisions. All authors reviewed and edited the manuscript.
Corresponding author
Correspondence to Celia E. Shiau.
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Spencer, C.G., Hamilton, M., Bedsole, E. et al. Chronic macrophage activation derails muscle repair by disrupting mannose-receptor-linked plasticity revealed by endogenous irg1/acod1 tracking. Nat Commun (2026). https://doi.org/10.1038/s41467-025-68204-3
Received: 19 November 2024
Accepted: 19 December 2025
Published: 07 January 2026
DOI: https://doi.org/10.1038/s41467-025-68204-3