Regulation of muscle growth and regeneration by the immune system

Abstract

Diseases of muscle that are caused by pathological interactions between muscle and the immune system are devastating, but rare. However, muscle injuries that involve trauma and regeneration are fairly common, and inflammation is a clear feature of the regenerative process. Investigations of the inflammatory response to muscle injury have now revealed that the apparently nonspecific inflammatory response to trauma is actually a complex and coordinated interaction between muscle and the immune system that determines the success or failure of tissue regeneration.

Figure 1. Myogenic precursor cells proceed through a tightly regulated sequence of myogenic-regulatory gene expression during muscle regeneration

Satellite cells are myogenic precursor cells (MPCs) that reside in a quiescent state on the surface of fully differentiated muscle fibres, and they can be identified by their location and by their expression of myogenic-regulatory genes (they express PAX7, lack expression of myoblast determination protein 1 (MYOD) and myogenin (MYOG), and may or may not express myogenic factor 5 (MYF5)). Following their activation by injury, exercise or other perturbations, they can enter the cell cycle to give rise to two daughter cells that have the same developmental destiny (symmetrical cleavage) or cells that have non-identical developmental paths (asymmetrical cleavage). Many of the proliferative daughter cells will begin to express MYOD and continue to differentiate. Other daughter cells will return to the quiescent state and remain PAX7⁺MYOD⁻MYOG⁻. If the cells continue to differentiate and lose the capacity to self-renew, they permanently downregulate PAX7 expression and begin to express the transcription factor MYOG, which is required for further differentiation and survival^,. Those PAX7⁻MYOD⁺MYOG⁺MYF5^+/− cells then have the capacity to fuse with neighbouring MPCs to form multinucleated myotubes. Myotubes then undergo rapid growth and begin to express genes required for terminal differentiation, many of which are under the control of MYOG, and become nascent muscle fibres. Eventually, nuclei derived from the originally activated satellite cell population become myonuclei that reside within the muscle fibre, in which myogenic-regulatory genes are permanently silenced (PAX7⁻MYOD⁻MYOG⁻MYF5⁻).

Figure 2. Myogenesis during muscle regeneration is temporally linked with a transition from the initial inflammatory response to a type 2 immune response

Within hours of trauma, muscles experience rapid activation of resident myeloid cells, and invasion by neutrophils and by macrophages that are biased towards an M1 phenotype. During the first 24 hours, the immune response is characterized by high levels of expression of tumour necrosis factor (TNF), which promotes the initial inflammatory response, and CC-chemokine ligand 2 (CCL2), which drives the recruitment of both myeloid and lymphoid populations. However, elevated expression of interleukin-10 (IL-10) coincides with the attenuation of the initial inflammatory response and with the reduced proliferation of myogenic precursor cells (MPCs) as they begin to exit the cell cycle and differentiate, which corresponds to a change in the macrophage population from an M1-biased phenotype to an M2-biased phenotype. The shift towards a pro-regenerative environment is also coupled with an increase in regulatory T (T_reg) cell numbers in the regenerative tissue. The time courses are compiled from aggregate data from numerous publications (REFS 20,24,25,37,42,54,56,125). ECM, extracellular matrix; IGF1, insulin-like growth factor 1; iNOS, inducible nitric oxide synthase.

Figure 3. Pro-inflammatory cytokines link inflammation with muscle regeneration

a | Interferon-γ (IFNγ)-mediated induction of macrophage activation coordinates the initial inflammatory response and the regulation of muscle differentiation. In the well-characterized signalling pathway through which IFNγ activates the M1 phenotype, the cytokine binds its receptor, leading to the recruitment of the tyrosine kinases Janus kinase 1 (JAK1) and JAK2, which in turn phosphorylate and activate signal transducer and activator of transcription 1 (STAT1) and IFN-regulatory factor 1 (IRF1). Nuclear targeting of dimerized, phosphorylated STAT1 enables it to bind γ-activated sequences (GASs) of target genes, leading to the activation of a range of genes that can promote the pro-inflammatory M1 phenotype. Simultaneously, IFNγ-activated signalling in muscle cells through the JAK–STAT1 pathway increases the expression of MHC class II transactivator (CIITA). During early stages of myogenesis, CIITA binds directly to myogenin (MYOG) and inactivates it, preventing MYOG-mediated induction of myogenic genes that are required for muscle differentiation and function (for example, TNNI2 (which encodes fast skeletal muscle troponin I2 and LMOD2 encoding leiomodin 2)). This helps to retain myogenic precursor cells (MPCs) in an undifferentiated, proliferative state. In later stages of differentiation, increased levels of CIITA lead to its recruitment of EZH2 to the Polycomb-response element (PRE) of muscle-specific genes, which leads to their silencing and prevents the expression of genes that are required to reach the fully mature muscle phenotype. b | Increased release of tumour necrosis factor (TNF) coordinates early myogenic events with the initial inflammatory response during early stages of muscle injury and repair. Although numerous cells, including those of muscle itself, can express TNF, inflammatory cells are the primary sources at early stages post injury. TNF binding to its receptor (TNFR) on macrophages activates signalling through TNFR-associated factor 2 (TRAF2), leading to activation of the inhibitor of nuclear factor-κB kinase (IKK), and the subsequent phosphorylation of inhibitor of nuclear factor-κB (IκB) and activation of nuclear factor-κB (NF-κB). Activated NF-κB then translocates to the cell nucleus to bind to the NF-κB-response element (NRE) of target genes to activate their expression. Expression of those target genes is essential to maintain the initial inflammatory response. Alternatively, the binding of molecules containing damage-associated molecular patterns (DAMPs), especially high mobility group protein B1 (HMGB1) can also activate NF-κB. HMGB1 binds to Toll-like receptor 4 (TLR4) on macrophages to activate NF-κB via myeloid differentiation primary response gene 88 (MYD88) and TRAF6. At the same time, TNF can bind its receptor on the MPC surface to activate mitogen-activated protein kinase (MAPK) kinase 3 (MKK3) or MKK6, leading to the activation of the MAPK p38α. Phosphorylation of EZH2 by p38α causes EZH2 recruitment to the Polycomb repressive complex 2 (PRC2), which leads to binding at the PRE of target genes. Activated EZH2 then trimethylates histone 3 lysine 27 (H3K27) at the target genes, leading to their repression. Two functionally important target genes in activated satellite cells are PAX7 and NOTCH1, and their silencing prepares the satellite cells to transition to the early stages of differentiation. CCL2, CC-chemokine ligand 2; CXCL9, CXC-chemokine ligand 9; IFNγR, IFNγ receptor; IL, interleukin; IL-6R, IL-6 receptor; IL-15R, IL-15 receptor; iNOS, inducible nitric oxide synthase; PCG2, Polycomb group 2; SOCS, suppressor of cytokine signalling.

Figure 4. Interactions between myeloid cells, lymphoid cells, fibro-adipogenic progenitor cells and myogenic precursor cells determine the course of muscle growth and regeneration

Trauma to muscle can cause the release of molecules containing damage-associated molecular patterns (DAMPs), such as high mobility group protein B1 (HMGB1), that activate and recruit immune cells to the site of trauma^,. This corresponds to the activation of satellite cells to enter the cell cycle and to proliferate. The release of cytokines and chemokines, especially CC-chemokine ligand 2 (CCL2) and CXC-chemokine ligand 1 (CXCL1), from neutrophils, resident macrophages and CD8⁺ T cells promotes further recruitment of immune cells to an inflammatory response driven by interferon-γ (IFNγ) and tumour necrosis factor (TNF). Elevations in those pro-inflammatory cytokines sustain the initial inflammatory response but also act directly on myogenic precursor cells (MPCs) through epigenetic mechanisms to regulate their expression of myogenic-regulatory genes that control MPC development. Macrophages biased towards the M1 phenotype also release insulin-like growth factor 1 (IGF1), which increases MPC proliferation and further expands the MPC population. M1 macrophage populations then undergo a shift to a population that is biased towards the M2 pro-regenerative phenotype. Several ligands have important roles in promoting this phenotypic shift, including fibrinogen, amphiregulin (AREG) and interleukin-10 (IL-10) produced by regulatory T (T_reg) cells, and IL-10 and IGF1 produced by macrophages. In addition, phagocytosis of cellular debris also contributes to the macrophage phenotypic switch. AREG release by T_reg cells increases the expression of myogenic-regulatory factors that drive the later stages of muscle differentiation. This influence is further enhanced by the recruitment of T_reg cells to regenerated muscle by IL-33 released by fibro-adipogenic progenitor (FAP) cells and possibly by macrophages. FAP cell proliferation and differentiation into cells with phagocytic or fibrogenic phenotypes are influenced by the release of transforming growth factor-β (TGFβ) from macrophages and by IL-4 released by eosinophils. Those macrophages that differentiate to become fibroblasts then contribute to the extracellular matrix production that supports muscle repair and the release of IGF1, which increases the growth of fully differentiated muscle.

Figure 5. Dysregulation of the immune response to muscle injury increases muscle damage and fibrosis

If muscle experiences chronic trauma, or if the inflammatory response to muscle injury is perturbed by disease or ageing, immune cells can exacerbate fibre damage or fibrosis. Under conditions that amplify or prolong the initial inflammatory response, free radicals produced by neutrophils or macrophages can promote muscle membrane lysis and fibre death. In particular, neutrophil production of superoxide (O₂⁻), hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl), nitric oxide (NO) and peroxynitrite (ONOO⁻) by NADPH oxidase, myeloperoxidase and inducible nitric oxide synthase can greatly increase muscle damage. In addition, in mdx mouse skeletal muscle, which experiences chronic inflammation, there are persistently higher numbers of eosinophils that can cause further damage of muscle fibres through the release of major basic protein (MBP). Chronic muscle damage also increases the occurrence of a transitional macrophage population that can lead to pathological increases in the number of fibro-adipogenic progenitor (FAP) cells and their profibrotic activities because transforming growth factor-β (TGFβ) production by transitional macrophages counters the negative regulation of their numbers by tumour necrosis factor (TNF) released by M1 macrophages. In addition, chronic muscle inflammation leads to an M2-biased macrophage phenotype in which the levels of expression and activity of arginase are highly elevated, leading to high levels of polyamines that can further expand fibroblast populations and the production of proline, which can increase connective tissue production. IL-10, interleukin-10.