Fibro–Adipogenic Progenitors Cross-Talk in Skeletal Muscle: The Social Network

1Skeletal muscle is the most abundant tissue in healthy humans, accounting for 40% of body weight. It is composed of multinucleated contractile cells called myofibers, which are formed during development by fusion of differentiated mononuclear muscle cells, and their number remains constant during post-natal growth. The regenerative potential of skeletal muscle relies primarily on satellite cells (SCs), the prototypical muscle stem cells. Upon muscle injury SCs enter the cell cycle, proliferate, and differentiate to repair damaged myofibers, while self-renewing to repopulate the reserve pool.

2Recently, several studies have indicated that the establishment of functional cross-talk between SCs and other cell types within the muscle niche, including motor neurons, endothelial cells, immune cells, fibrogenic cells, and adipogenic precursors, is crucial for muscle repair and homeostasis. Indeed, different stem/progenitor cell types are recruited to the regenerative niche and influence muscle regeneration either by directly differentiating into muscle cells or by releasing paracrine factors (i.e., growth factors, cytokines) that control the regenerative response of SCs. Among the non-cellular components of the SCs niche the extra-cellular matrix (ECM) plays a crucial role by undergoing a transient remodeling during acute injury, followed by a prompt termination to avoid pathological fibrosis and tissue degeneration. Although recent findings have shown that myogenic cells can produce ECM components, and a recently identified population of interstitial tenocytes has been implicated in ECM deposition in vivo, the main cellular sources of ECM proteins are fibroblasts, myo-fibroblast, and fibro–adipogenic progenitors (FAPs).

3Since their discovery FAPs have attracted a considerable attention, in particular, their phenotypical plasticity, which appears critical for efficient muscle repair. FAPs have been defined as multi-potent progenitors, having the ability to differentiate into fibroblasts, adipocytes, and possibly into osteoblasts and chondrocytes, although not into myoblasts. They share the expression of cell surface markers, such as Sca-1 and PDGFRα with mesenchymal stem cells and can therefore be broadly defined as mesenchymal precursors.

4Under quiescent conditions FAPs frequently localize close to blood vessels but unlike pericytes FAPs reside outside the capillary basement membrane and do not express NG2.

5However, upon muscle injury, FAPs become activated, proliferate and expand, and provide a transient favorable environment to promote SCs-mediated regeneration. FAPs expansion is critical during regeneration in order to sustain SCs differentiation in a paracrine manner and to maintain the SCs pool. Indeed, in vivo depletion of FAPs clearly established their absolute requirement for regeneration and long-term maintenance of skeletal muscle. However, as regeneration proceeds, FAPs are cleared from the regenerative niche by apoptosis and failure in doing so has been associated with their pathological accumulation and with a number of muscle dysfunctions. In fact, beyond their supportive role in muscle regeneration, FAPs have been identified as the major source of infiltrating fibroblasts and adipocytes in degenerating dystrophic muscles. Similarly, in chronic atrophic conditions, caused by moto-neurons deficits, increased fibrosis is associated with accumulation of FAPs in the interstitium of denervated muscles. Likewise, intra-muscular fatty infiltration and obesity-associated muscle dysfunctions have been also linked to FAPs accumulation and fibro–adipogenic differentiation.

6These findings emphasize that the FAPs lineage decisions are dramatically influenced by signals released in their microenvironment, whose pathological alteration might culminate in excessive ECM accumulation, acquisition of altered cell fates, as in the case of heterotypic ossification, and impaired myogenesis. In physiological conditions, FAPs’ cross-talk with other cell populations is emerging as an important and finely orchestrated process crucial for a successful muscle regeneration. While it is now well established that a cross-talk between SCs and fibrogenic cells is necessary for efficient SCs expansion in response to injury, and to prevent interstitial fibrosis accumulation, increasing evidence indicates that FAPs also actively interact with immune cells in a finely tuned manner.

7Taken together, these observations demonstrate that FAPs orchestrate a plethora of processes involved in regenerative myogenesis, which have been recently reviewed elsewhere. In this mini-review, we will instead specifically focus on the secreted signals, cytokines, and paracrine factors controlling FAPs function and those released by FAPs monitoring the different cell types involved in muscle repair. We will first describe the signals secreted by the various cell populations present in the regenerative niche known to directly influence FAPs activity and then discuss the signals released by FAPs themselves, highlighting their cellular targets and functions (Table 1).

8Interleukin-4 (IL-4) and IL-13 are Th2 cytokines, which have been implicated as mediators in the cross-talk between the immune system and FAPs. The innate immune system is activated rapidly upon muscle injury and triggers the recruitment of Th2 lymphocytes, macrophages, mast cells, and eosinophils to the injured sites.

9Interleukin-4/IL-13 signaling is crucial for skeletal muscle repair, as demonstrated by studies showing a complete absence of regenerated myofibers, persistence of cellular debris, and an inflammatory infiltrate, in the muscles of IL-4/IL-13–/– mice following cardiotoxin-induced injury. Although activation of type 2 immune responses has been classically associated with alternatively activated (M2) macrophages, eosinophils have been recently identified as the dominant cell source of IL-4 and IL-13 during skeletal muscle regeneration. Specifically, it has been shown that eosinophils secrete IL-4 to activate the regenerative actions of FAPs. Indeed, identified FAPs as the cells specifically expressing the IL-4Rα, demonstrating, both in vitro and in vivo, that FAPs are the cellular targets of IL-4/IL-13 signaling during muscle regeneration. Intriguingly, they also unveiled a previously unrecognized function of FAPs: their capacity to phagocytoze necrotic debris, a crucial process for successful completion of muscle repair. In addition, IL-4/IL-13 signaling, via activation of STAT6, promotes FAPs proliferation to support myogenesis, while inhibiting their differentiation into adipocytes. Accordingly, in the injured muscles of IL-4-knockout mice, the levels of adipocytes are increased, while in vitro and in vivo administration of IL-4 inhibits FAPs adipogenesis. In agreement with these observations, glucocorticoids (GCs)-induced repression of IL-4 leads to intramuscular adipogenic accumulation by promoting FAPs proliferation and differentiation into adipocytes. Since GCs are known to suppress eosinophils, it is likely that they inhibit IL-4 signaling by reducing the number of infiltrating eosinophils upon muscle injury. Yet, IL-4-polarized, anti-inflammatory macrophages have been shown to induce adipogenesis of human FAPs isolated from dystrophic muscles, suggesting that IL-4 signaling might govern more complex cellular interactions than previously expected.

10Interleukin-15 is expressed in human skeletal muscle and it has been identified as an anabolic factor involved in muscle growth. Indeed, IL-15 can decrease protein degradation in muscle and modulate muscle–adipose tissues interactions. A recent work identified IL-15 as a myokine able to prevent intramuscular fatty infiltration, likely affecting FAPs differentiation capacities. In this work, the authors showed that IL-15 stimulates FAPs proliferation and it directly inhibits their adipogenic differentiation, both in vitro and in vivo, ultimately facilitating myofibers regeneration. Moreover, intramuscular administration of a recombinant IL-15 prevented fat accumulation in the murine model of glycerol-induced fatty degeneration. Accordingly, in vitro treatment of FAPs with IL-15 impaired their capacity to differentiate into adipocytes, likely through the induction of desert Hedgehog (DHH) signaling, a known repressor of FAPs adipogenesis. Although these results suggest a positive role for IL-15 in muscle regeneration, the evidence that IL-15 administration, and expression, correlates with increased collagen deposition in vivo after muscle damage, poses several unresolved issues that warrant further investigation. Indeed, whether IL-15 directly promotes FAPs differentiation into fibroblasts has not been tested yet. Furthermore, even though FAPs expansion and regenerative fibrogenesis have a positive impact on acute muscle regeneration, the evidence that IL-15 expression is positively correlated with the number of FAPs and collagen deposition in subjects with rotator cuff tear indicates that IL-15 might serve, instead, as a signal to sustain FAPs pathogenic fibrogenesis in chronically fibrotic muscles.

11Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine secreted rapidly upon muscle damage by infiltrating inflammatory cells and its impact on muscle regeneration is still under debate. Indeed, while pharmacological blockade of TNF-α has been associated with reduced muscle necrosis and amelioration of the histological profile of dystrophic muscles. More recently, TNF-α has been implicated in preventing FAPs aberrant accumulation, suggesting that anti-TNF therapies might instead exert a pro-fibrotic effect.

12During acute injury, TNF-α has been reported to promote muscle repair by activating promyogenic p38 signaling, thus inducing SCs differentiation. Recently, it has been suggested that TNF-α regulates matrix production during acute damage, thus unveiling a crucial function for TNF-α in mediating FAPs apoptosis and clearance. Specifically, TNF-α was found to be predominantly expressed and produced by infiltrating monocytes that rapidly differentiate into pro-inflammatory macrophages (M1) that accumulate in close proximity to expanding FAPs. By using a mouse model unable to recruit circulating monocytes to damaged muscles [the C–C chemokine receptor type 2 (Ccr2)–/– mouse strain], elegantly demonstrated that in the absence of infiltrating TNF-a-producing macrophages, FAPs accumulate in the sites of damage and aberrantly differentiate into fibrogenic cells. Inflammatory cell-derived TNF-α production is therefore required for FAPs clearance to prevent pathological ECM accumulation. Of note, this physiological role is altered in chronically damaged muscles, such as those of dystrophic mice, where the abundance of transforming growth factor beta 1 (TGF-β1) signaling impairs the pro-apoptotic effects of TNF-α on FAPs. These data might offer a possible explanation for the apparent controversial results reporting a positive effect of anti-TNF-α therapies on dystrophic mice. Indeed, when TGF-β1 is abundant, as in chronic degenerating muscles, the anti-fibrotic role of TNF-α is irrelevant and pharmacological approaches aimed at inhibiting its activity most likely exert their beneficial effects through targeting of the pro-myogenic, SC-mediated function of TNF-α.

13The transforming growth factor beta (TGF-β) superfamily comprises pleiotropic and multifunctional secreted peptides implicated in a wide range of cell functions, including tissue homeostasis and repair, immune and inflammatory responses, ECM deposition, cell differentiation, and growth. Studies in a wide range of experimental models have firmly established TGF-β1 as a crucial mediator of fibrinogenesis and inhibition of its activity has consistently been associated with reduced fibrosis.

14In the context of skeletal muscle, inhibition of TGF-β1 has been linked to improvement in muscle regeneration and decreased fibrosis, consistent with the importance of the TGF-β1 signaling in regulating both regeneration and matrix production. Several works have elucidated the detrimental, cell-autonomous, impact of TGF-β signaling on muscle stem cells by inhibiting their activation and terminal differentiation while promoting a fibrogenic switch in chronically degenerating muscles.

15Nonetheless, recent findings point toward a prominent role for inflammatory cell-derived TGF-β signaling in the survival and fibrotic differentiation of FAPs. Specifically, during chronic muscle damage, macrophages express and secrete high levels of TGF-β1, antagonizing the TNF-mediated apoptosis of FAPs, and instead induce their fibrogenic differentiation and consequent ECM deposition. Thus, in conditions of chronic muscle damage, TGF-β1 acts as a dominant, pro-survival signal that overrides the beneficial effect of the pro-inflammatory cell-derived, and anti-fibrotic cytokine, TNF-α. Thus, treatment with nilotinib, via specific inhibition of TGF-β1-induced p38 signaling, restores FAPs apoptosis and prevents fibrotic accumulation in dystrophic mice.

16Of note, FAPs from chronic fibrotic dystrophic muscles have been identified as the main source of TGF-β-activating enzymes. Indeed, once released, latent TGF-β1 must be activated, either via enzymatic or mechanical mechanisms, to exert its properties and to bind to its receptors. FAPs exhibit high expression of a series of latent TGF-β1 activators, among which matrix metallo proteinase 14 (MMP14) and bone morphogenetic protein 1 (BMP1) are able to activate the latent TGF-β1 released by pro-inflammatory macrophages. Notably, pharmacological inhibition of BMP1 or MMP14 reduced muscle fibrosis in dystrophic mice resulting in increased muscle fiber size and reduced necrosis.

17In summary, these data support a model through which chronic inflammation and fibrosis reciprocally sustain themselves in degenerating dystrophic muscles, by reinforcing a regulatory cross-talk between inflammatory cells and FAPs.

18In skeletal muscle, IL-6 is classified as a myokine produced and released by muscle fibers in response to contraction. It promotes lipolytic and anti-inflammatory beneficial effects during exercise, working as an energy sensor and exerting both local and endocrine metabolic effects.

19Interleukin-6 regulates both muscle hypertrophy and regeneration. Indeed, IL-6 knockout mice show a reduced hypertrophic response to overloading, ascribed to impaired myonuclei incorporation as a consequence of the defective proliferation and migration of SCs. Treatment with IL-6 promotes murine SCs proliferation, via regulation of cell-cycle associated genes, Cyclin D1 and c-Myc, while during regeneration the IL-6/STAT3 axis controls SCs fate. Interestingly, FAPs were identified as one of the main source of IL-6 during muscle regeneration. Upon muscle injury, IL-6 expression remains constant in myogenic progenitors but increases nearly 10-fold in FAPs and in vitro co-culture experiments have shown that IL-6 mediates the pro-myogenic activity of FAPs.

20The positive effect of IL-6, and others myokines, is normally associated with transient production and short-term action. By contrast, persistent inflammatory conditions, denervation, and some types of cancer and other chronic diseases have been associated with long-lasting elevated IL-6 levels. In agreement with this notion, IL-6 has been shown to promote skeletal muscle atrophy. Accordingly, in denervated muscles, FAPs show a persistent activation of IL-6, thus promoting muscle atrophy without other systemic effects. Notably, in vivo pharmacological inhibition of IL-6 effectively counteracts denervation-mediated muscle atrophy and accumulation of FAPs with hyper-activation of IL-6 signaling has also been found in mouse models of amyotrophic lateral sclerosis (ALS).

21Taken together, these observations suggest two apparently opposing effects of FAPs-derived IL-6 during muscle regeneration or in denervation-induced muscle wasting, and further studies are needed to shed light on the molecular mechanisms behind these apparently contradictory roles.

22Interleukin-33 is a nuclear chromatin-associated cytokine, belonging to the IL-1 family, and constitutively expressed in the nucleus of a wide variety of cell types, including fibroblasts, epithelial cells, and endothelial cells. IL-33 appears to function as an alarmin (alarm signal) that is rapidly released upon cellular damage and stress and mediates a potent effect on the activation of regulatory T cell lymphocytes (Treg).

23In skeletal muscle, the major IL-33-producing cell type has been identified within the FAP cell population. FAPs start to express IL-33 within 6–12 h after acute injury, inducing proliferation of muscle resident Treg. As previously demonstrated, Treg cells promote muscle repair, accumulating in both acutely and chronically injured skeletal muscles. Interestingly, a severe decline in Treg accumulation, caused by an impairment in IL-33-producing FAPs, has been linked to regeneration defects in aged muscles. On the other hand, in vivo treatment with IL-33 restored the Treg population in injured muscles of old mice, enhancing tissue regeneration.

24Intriguingly, IL-33-expressing FAPs have been found in close association with muscle spindles, which are stretch-sensitive mechanoreceptors that lie within the skeletal muscle and comprise both sensory and motor neurons. This finding raises the possibility that FAPs might function as mechano-cellular sensors that modulate the cross-talk between neural and immune cells to facilitate proper homeostatic reorganization of skeletal muscle and neural circuits upon injury. In agreement with this possibility, IL-33 expression is increased in fibroblasts upon mechanical stress and PDGFRα+ mesenchymal precursors, found within the endoneurium of peripheral nerves, have been recently implicated in tissue repair and regeneration.

25Wnt family member 1 (WNT1) inducible signaling pathway protein 1 (WISP1) is encoded by the cellular communication network factor 4 (CCN4) gene, a member of the CCN family of matricellular proteins that are involved in diverse biological processes, such as ECM remodeling, tissue repair, and tumor growth. CCN4/WISP1 is important in the musculoskeletal system, where it regulates osteogenesis and chondrogenesis, as well as skin repair.

26A recent study showed that in young mice CCN4/WISP1 is upregulated in FAPs following muscle injury, but this induction is lost in FAPs of old muscles. The FAP-secreted WISP1 plays an important role in SCs expansion and asymmetric commitment to myogenic differentiation. Indeed, similar to aging, the loss of WISP1 in knockout mice affects SCs function and impairs myogenesis. In agreement with this, the transplantation of young, but not aged or WISP–/– FAPs, rescues the myogenic dysfunction of aged SCs and their regeneration ability. Even better, systemic treatment with recombinant WISP1 mimics rejuvenation beneficial effects, opening new prospects in the use of this approach as a strategy to counteract aging and associated muscular diseases.

27Interestingly, these findings together with the reported impaired interplay between FAPs and Treg during aging, the sensitivity of FAPs to muscle denervation, and the recently reported atrophic phenotype of FAPs-depleted skeletal muscles clearly point toward FAPs as promising new cellular targets for the treatment of muscle defects associated with sarcopenia.

28Follistatin is a potent natural antagonist of myostatin and activin A, two TGF-β superfamily cytokines implicated in muscle growth inhibition, and it is therefore a potent pro-myogenic factor.

29In the context of skeletal muscle regeneration, follistatin expression is induced 12 h after muscle injury and remains elevated for 5 days, concurrent with SCs activation. Of note, FAPs have been described as the major source of follistatin, displaying 10-fold higher expression levels than SCs. Follistatin is considered the central mediator of the fusogenic effects exerted by histone deacetylase inhibitors (HDACi) on skeletal muscles. Indeed, HDACi treatment in dystrophic mice induces the upregulation of follistatin in muscle progenitor cells, promoting the formation of multinucleated myotubes. In agreement with its pro-myogenic activity, follistatin knock-down in FAPs reduced the ability of HDACi to stimulate SCs-mediated formation of myotubes, suggesting a crucial role of FAP-derived follistatin as a mediator of the pro-differentiative activity of FAPs.

30A proper balance between follistatin and its antagonists is crucial to preserve reciprocal functional interactions between FAPs and SCs and to preserve muscle homeostasis. Indeed, pharmacological inhibition of the activin receptor type-2B pathway (AcvR2B), which blocks both myostatin and activin A activity, reverses muscle atrophy in SC-depleted skeletal muscles, while also restoring stem cells regenerative potential. Of note, beneficial effects of AcvR2B targeting in SCs-depleted muscles are accompanied by an increased number of FAPs, an observation that further supports the notion that restoration of a proper balance of regulatory factors between the different cells within the regenerative niche is key for muscle repair.

31Once again, a role of FAPs emerges not only in regulating muscle regeneration but also in mediating signaling pathways associated with maintaining muscle mass. Future experiments should elucidate the possible use of FAPs as a source of trophic factors.

32Interleukin is a broadly expressed anti-inflammatory cytokine that inhibits the activation of the innate immune system and Th1 activation, preventing inflammatory and autoimmune pathologies. Like IL-6, IL-10 is also considered a myokine expressed in skeletal muscle in a wide range of conditions. It influences different aspects of muscle biology, such as regeneration, exercise, metabolism, and aging. Its anti-inflammatory activity has been investigated in different muscle-related disorders. The main source of IL-10 in regenerating skeletal muscle is macrophages and Tregs although it has also been demonstrated that FAPs increase IL-10 expression upon muscle damage. This evidence is in line with the crucial pro-myogenic activity of FAPs, which likely contribute to muscle repair also through the secretion of an anti-inflammatory cytokine, such as IL-10, to counteract the anti-myogenic activity of TNF-α. Although the function of IL-10 released by FAPs has not yet been demonstrated in vivo, this work underscores the complexity of the interplay between inflammatory cells and the other players in muscle regeneration. Future studies are needed to better understand this mechanism.

33In conclusion, the available evidence reviewed above clearly indicates that FAPs act as crucial regulators of skeletal muscle homeostasis (Figure 1). However, several critical issues need to be addressed before defining them as the co-star of skeletal muscle repair. First, their molecular heterogeneity makes it difficult to target them genetically, to uniquely assess their requirement, and to define the function of FAP-specific expression of the different factors described above. Future single-cell transcriptomic approaches will help identify sub-populations differently altered during the diverse stages of muscle regeneration and, more importantly, in pathological situations.

34Finally, the evidence of the association of FAPs with nerve structures, and the ability of FAPs to respond to nerve lesions, suggest a mechano-sensitivity of FAPs and emphasize the urgency to improve our understanding of the molecular regulation governing FAPs activity during muscle adaptation.

35LM and CM wrote and edited the manuscript. BB and DP wrote the manuscript.

36The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

37The handling Editor declared a shared affiliation, though no other collaboration, with several of the authors, BB, CM, and DP, at the time of review.