Myeloid cell diversification during regenerative inflammation: Lessons from skeletal muscle

Abstract

Understanding the mechanisms of tissue and organ regeneration in adult animals and humans is of great interest from a basic biology as well as a medical, therapeutical point of view. It is increasingly clear that the relatively limited ability to regenerate tissues and organs in mammals as oppose to lower vertebrates is the consequence of evolutionary trade-offs and changes during development and aging. Thus, the coordinated interaction of the immune system, particularly the innate part of it, and the injured, degenerated parenchymal tissues such as skeletal muscle, liver, lung, or kidney shape physiological and also pathological processes. In this review, we provide an overview of how morphologically and functionally complete (ad integrum) regeneration is achieved using skeletal muscle as a model. We will review recent advances about the differentiation, activation, and subtype specification of circulating monocyte to resolution or repair-type macrophages during the process we term regenerative inflammation, resulting in complete restoration of skeletal muscle in murine models of toxin-induced injury.

Keywords: Acute; Macrophage; Macrophage subtype specification; Monocytes; Muscle Regeneration; Myeloid cells; Regenerative inflammation; Sterile injury; Tissue repair.

Fig. 1.

Regenerative inflammation. Circulating neutrophils and Ly6C^high monocytes infiltrate early following acute sterile injury and give rise to Ly6C^high inflammatory macrophages to promote the clearance of necrotic debris. These cells stimulate MuSC proliferation and induce apoptosis of fibro-adipo-progenitors (FAPs) and fibroblasts. At the same time, muscle stem cells (MuSCs) exit quiescence and start to proliferate. Neutrophils typically undergo apoptosis shortly after. Integration of inflammatory lipid mediators (SPMs; specialized pro-resolving mediators) together with efferocytosis initiates the resolution of inflammation. The resolution phase is further supported by metabolic reprogramming of the Ly6C^high inflammatory macrophages. These events promote the phenotypic switch to a resolution/reparative macrophage phenotype. These Ly6C^low repair macrophages resolve inflammation while stimulating myoblasts, fibroblasts, and endothelial cells through the secretion of growth factors to promote cell cycle exit, fusion and myotube formation, re-vascularization, and ECM remodeling, respectively. The coordinated action and cellular interactions of macrophages with the injured cell milieu during regenerative inflammation are essential for efficient regeneration and the return to homeostasis.

Fig. 2.

Macrophage activation via Pattern Recognition Receptors (PRR) and autoantigens during regenerative inflammation. Schematic drawing depicting distinct ligand recognition and cellular location of pattern recognition receptors (PRRs) sensing various pathogen-associated (PAMPs) and damage-associated molecular patterns (DAMPs) during macrophage activation. Pathways involved in controlling macrophage phenotype and function during regenerative inflammation are summarized (AnxA1, AMPKa1, PPARg-GDF3, IGF-1, C/EBPb-IL-10, MKP-1, SRB1, BACH1-HMOX1, FPN, SPMs-Resolvin D2, OPN). Abbreviations: HMGB1, high-mobility group box-1; AKT, Akt serine/threonine kinase family; IRAK3, Interleukin 1 Receptor Associated Kinase 3; P2X7, P2X purinoceptor 7; NLRP3, NLR family, pyrin domain containing 3; TLR, toll-like receptor; HA, Hyaluronic acid; ATP, Adenosine triphosphate; SRB1, Scavenger receptor class B type 1; OPN, osteopontin; MKP-1, Mitogen-Activated Protein Kinase Phosphatase 1; p38, p38 Mitogen-Activated Protein Kinase; IFNGR, Interferon-gamma receptor; CD44, Cluster of Differentiation 44; FPR2/AXL, Formyl peptide receptor 2; GDF3, Growth differentiation factor 3; IGF-1, insulin-like growth factor 1; IL-10, Interleukin-10; FPN, ferroportin; AnxA1, Annexin A1; AMPKa1, AMP-activated protein kinase; BACH1, BTB Domain And CNC Homolog 1; HMOX1, Heme Oxygenase 1; NF-kB, Nuclear factor-kappa B; GPR18, G Protein-Coupled Receptor 18; C/EBPβ, CCAAT Enhancer Binding Protein Beta.

Fig. 3.

Key inflammatory pathways are dispensable for proper muscle regeneration. A. Schematic depiction of differentially expressed genes (p < 0.05, FC >= 1.5) during circulating monocyte to muscle-infiltrating inflammatory (Ly6C^high) and repair macrophage (Ly6C^low) transition. The number of genes changing is indicated per transition stage. Data are available under accession numbers GSE114291 and GSE164722. B. Heatmap representing the mRNA expression dynamics of key inflammation-associated genes in sorted blood monocyte, Ly6C^high or Ly6C^low muscle-infiltrating macrophages at indicated time points following cardiotoxin injury. Clustered RNA-seq expression values are visualized as Expression Z-score (calculated using the DEseq method). C. Average fiber CSA of regenerating tibialis anterior (TA) muscle in indicated mouse strains (8–10 weeks-old males) at day 8 post cardiotoxin (CTX) injury (n = at least 4 mice per group). Bars and lines represent mean ± SEM. D. Representative images of H&E-stained skeletal muscle (TA) from WT-control, Akt1 KO, Akt2 KO, and Irak3 KO animals at day 8 post CTX-induced injury. Scale bars in the upper left corner represent 100 μm. E. Representative images of H&E-stained skeletal muscle (TA) from Akt1^fl/fl control, Akt1^fl/fl LysM-Cre animals at day 8 post CTX-induced injury. Scale bars in the upper left corner represent 100 μm. F. Representative images of H&E-stained skeletal muscle (TA) from WT-control and Nlrp3 KO animals at day 8 post CTX-induced injury. Nlrp3 −/− mice were acquired through JAX (Stock #: 021302). Scale bars in the upper left corner represent 150 μm.

Fig. 4.

Macrophage diversification and cellular interactions during regenerative inflammation. Schematic depiction of participating muscle-infiltrating macrophage subtypes and their cellular interactions during acute muscle injury and regeneration. During an acute sterile muscle injury, Ly6C^high monocytes extravasate from the circulation and start to phagocytose myofiber debris. These cells transition into Ly6C^low repair macrophages to promote wound healing through growth factor production. Recent advances in single-cell technologies propose the presence of multiple functionally distinct subtypes or states of repair macrophages that could preferentially influence the cellular interactions within the regenerating cell milieu. Possible subtypes of repair macrophages include Growth Factor Expressing, Resolution-related, and Antigen-Presenting macrophages.