Successful treatment of CMDs relies on the ability of therapeutic agents to act in cellular compartments where damage and inflammatory processes are occurring. CMDs are characterized by loss of ECM proteins that provide support and protection from the shearing forces of muscle contraction, thus resulting in chronic inflammation, fibrotic changes and a progressive loss of muscle function. Current treatments focusing on relieving the immediate symptoms of the inflammatory processes do little to stem the tide of disease progression and alternatives that slow or stop damage to muscles through replacement of critical extracellular matrix proteins are needed.
The primary challenge in the therapeutic application of stem cells for hereditary muscle disorders, particularly CMD, is the inefficient recruitment of therapeutic cells to the damaged muscles. Directed cell migration is a tightly regulated process, critical for numerous biological processes including proper tissue development, wound healing, and protection against invading pathogens. The precise mechanisms responsible for stem cell migration to skeletal muscles in normal and pathological conditions are still largely unknown but recent studies suggested that chemokines are important actors in skeletal muscle regeneration . It was shown that experimentally injured skeletal muscles and dystrophic muscles of mdx mice are characterized by an inflammatory “molecular signature” in which CC and CXC chemokines are prominent .
The understanding of the mechanisms regulating recruitment of systemically transplanted stem cells is crucial to the success of any clinical strategy. To date no sufficient data is available regarding chemokine expression in CMD-affected muscles. To broaden our knowledge, here we reported a detailed study of the chemotactic signatures in muscle tissues of CMD-affected patients and CMD animal models by employing proteome profile screens.
Chronic inflammation plays an important role in the pathogenesis of CMD. One of the critical features of inflamed muscle tissue is continuous infiltration of muscles with immune cells under the guidance of chemokines. High levels of chemokines secreted around muscle vasculature within the lesions determine which types of cells migrate to the lesion. Our proteome data indicate that multiple chemokines are induced in muscles affected with CMD. Comparative analysis of muscle biopsies from COL6RM and MDC1A patients showed consistently elevated levels of CCL5, CXCL6, CXCL7, CCL2 and GRO, suggesting that only a small subset of chemokines is available to provide the appropriate chemotactic gradient to patient’s muscle tissue. All identified molecules are prominent chemotactic factors that activate and attract various inflammatory cells, including lymphocytes, macrophages, neutrophils and granulocytes. CCL5, also known as RANTES (Regulated upon activation, normal T-cell expressed, and secreted), belongs to the CC chemokine family whose members include monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, I-309, macrophage inhibitory protein-1α and macrophage inhibitory protein-1β. CCL5 plays an essential role in inflammation by recruiting T cells, macrophages, dendritic cells, eosinophils, natural killer (NK) cells, mast cells, and basophils to the sites of inflammation. In collaboration with certain cytokines that are released by T cells such as IL-2 and IFN-γ, CCL5 also induces the activation and proliferation of NK cells to generate CC chemokine-activated killer cells. The activities of CCL5 are mediated through its binding to CCR1, CCR3 and CCR5. There are several ongoing clinical trials targeting the CCL5 receptors, but fewer studies specifically targeting the chemokine itself, and clinical studies with anti-CCL5 antibodies are still to be carried out. However, targeting CCL5 could result in novel therapies to decrease inflammatory responses and fibrosis, striking features of CMD muscles. Although our study demonstrated that CCL5 levels are associated with CMD severity, lowest in BM samples and highest in UCMD and LAMA2 biopsies, prognostic value of this molecule will require further statistical analysis in a larger cohort of patients with careful phenotypic evaluation. CXCL7, also known as NAP-2 (Neutrophil-activating peptide 2), is involved in neutrophil chemotaxis and activation. The functions described for CXCL7 suggest that in CMD muscle this chemokine could not only exacerbate muscle inflammation but also promote its chronicity by attracting monocytes to the inflamed tissue and activating them following recruitment to the muscles. CCL2, also called MCP-1, is a ligand of CCR2. CCL2/CCR2 signaling is critical for tissue recruitment of monocytes/macrophages upon inflammation and infection . It plays a significant pathogenic role in chronic inflammatory diseases, including multiple sclerosis, atherosclerosis, rheumatoid arthritis , and bronchitis obliterans syndrome . Conversely, CCL2/CCR2-mediated inflammatory response is essential to repair acute skeletal muscle injury. Ccr2-/- and Ccl2-/- mice show markedly reduced macrophage infiltration in response to acute muscle injuries induced by ischemia or myotoxic agents , and the diminished inflammatory response is accompanied by poor muscle regeneration. The GRO proteins are members of the chemokine superfamily, including the GROa, GROb, GROg, and all have been implicated in inflammatory signaling and shown to be chemotactic for neutrophils. These chemokines elicit their effects by signaling through the chemokine receptors CXCR1 and CXCR2. In models of chronic muscle disease, such as muscular dystrophies and other myopathies, the presence of inflammatory cells and/or their mediators within the muscle has been associated with an aggravation of muscle pathology . Both neutrophils and macrophages also have the capacity to kill muscle cells . On the other hand, macrophages recruited to areas of acutely damaged muscle have been shown to promote more effective muscle repair . This dichotomy is probably related to the presence of different subpopulations of macrophages, which express specialized and polarized functions under the influence of different environmental cues . Chemokines are excellent candidate molecules for playing a central role in regulating the proportions of different subpopulations of macrophages and other leukocytes within damaged muscles, as well as the chronicity of inflammation and efficacy of the subsequent muscle remodeling response. In principle, the chemokines released from damaged muscles under these conditions could originate from multiple sources including non-muscle cell types (e.g. resident macrophages, endothelial cells, etc.) and infiltrating leukocytes, as well as the muscle cells themselves. Our data are in a good agreement with previous studies. In fact, increased expression levels of several chemokine ligands and their cognate receptors have been found in muscle biopsies obtained from animal models and human patients suffering from muscular dystrophy or inflammatory myopathies. In particular, a predominant up-regulation of the CC chemokines, including CCL2 (MCP-1), CCL3 (MIP-1α) and CCL4 (MIP-1β), has been reported . More direct evidence for the importance of CC chemokines in muscle regeneration has been provided by the demonstration that recovery of normal muscle structure and force production after acute injury in vivo is significantly impaired in mice receiving antibodies against CCL2 or lacking its primary receptor, CCR2 . In experimental models of skeletal muscle injury, major leukocyte accumulation also occurs at sites of muscle regeneration, consisting initially of neutrophils and then primarily of macrophages [33, 34]. It has been shown that interference with macrophage influx delays subsequent muscle repair . It is increasingly evident that chemokines can exert multiple functions that extend well beyond their more established effects on leukocyte activation and trafficking. For example, chemokines have been shown to have important effects on non-myeloid cell types as diverse as endothelial cells, synoviocytes, neural cells, and smooth muscle cells . More recently, interference with CXCL12 (SDF-1) signaling through its cognate receptor, CXCR4, was found to be associated with impaired migration and increased apoptosis of skeletal muscle progenitor cells during embryogenesis . Overall, our data suggest that a distinct subset of chemokines is available to provide the appropriate chemotactic gradient for systemic transplantation of stem cells to CMD-affected muscle tissue.
Interestingly, proteome analysis of muscle biopsies from dyw and Col6a1-/- mice revealed the significant induction of many chemokines, in contrast to the CMD patients. These findings in the dyw model are consistent with observations that the dyw mouse model presents with a severe phenotype correlating to the pathology of human MDC1A. This is in contrast to the Col6a1-/- mouse model, which produces no detectable COL6 protein, but displays a mild phenotypic severity when compared to human UCMD and more similar to the milder BM. Given the relatively mild phenotype of Col6a1-/- animals under physiological conditions, induction of the inflammatory process in the Col6a1-/- mouse was accomplished through administration of a myonecrotic agent, the Naja mossambica mossambica venom CTX, which causes selective damage by targeting myofibrils and inducing inflammation, as previously shown in the same model (22). Treatment with CTX revealed that once damage to muscle was initiated many of chemokines identified in LAMA2-deficient muscles were also present in CTX-damaged muscle tissue of Col6a1-/- mice, suggesting that both diseases share similar chemotactic signals in mice. Temporal patterns of identified chemokines showed early induction and maintained expression, indicating that rapid induction and sustained expression of identified chemotactic molecules may provide a selective mechanism for inflammatory cell recruitment. The biological functions of identified chemokines suggest potential inflammatory infiltrate in muscle tissues. For example, CCL6 (identified only in rodents) is a potent chemoattractant of macrophages, as well as of B cells, CD4+ lymphocytes and eosinophils. In mice, CCL6 is expressed in cells from neutrophil and macrophage lineages, and can be induced under conditions suitable for myeloid cell differentiation. The cell surface receptor for CCL6 is believed to be the chemokine receptor CCR1. Our mouse data are in good agreement with a recently published study, showing that muscles of UCMD patients are significantly infiltrated with M2 macrophages . Also, it was demonstrated that COL6 has a key role for macrophages and its deficiency affects macrophage recruitment and polarization . The same work showed that nerve injury triggers a strong increase of some inflammatory chemokines, such as IL-1beta and MCP1, in wild-type mice but not in COL6 KO mice.
CCL2 exhibits a chemotactic activity for monocytes and basophils, but does not attract neutrophils or eosinophils. RARRES2 (retinoic acid receptor responder protein 2), also known as chemerin, is a chemoattractantprotein that acts as a ligand for the G protein-coupled receptorCMKLR1, also known as ChemR23. Chemerin was found to stimulate chemotaxis of dendritic cells and macrophages to the site of inflammation. CCL8, also known as monocyte chemoattractant protein 2 (MCP-2), is chemotactic for and activates many different immune cells, including mast cells, eosinophils and basophils as well as monocytes, T cells and NK cells, which are involved in the inflammatory response. CCL8 elicits its effects by binding to several different cell surface receptors, including CCR1, CCR2B and CCR5. CCL9/CCL10, also called macrophage inflammatory protein-1 gamma (MIP-1γ), macrophage inflammatory protein-related protein-2 (MRP-2) and CCF18, in rodents attracts dendritic cells that possess the cell surface molecule CD11b and the chemokine receptorCCR1. CCL9/10 (found exclusively in rodents) is constitutively expressed in macrophages and myeloid cells. IL-16 has been characterized as a chemoattractant and activator for many immune cells expressing the cell surface molecule CD4, including monocytes, eosinophils, and dendritic cells. CCL12, also known as monocyte chemotactic protein 5 (MCP-5) or MCP-1-related chemokine, specifically attracts eosinophils, monocytes and lymphocytes. Its expression can be hugely induced in macrophages. CCL12 is a ligand for CCR2. CXCL1, also known as growth-regulated oncogene (GRO), can bind with high affinity to the IL-8 receptor type B and is very potent neutrophil attractant and activator. CXCL12 is well known to have chemotactic and activating functions on T-lymphocytes, monocytes, but not neutrophils, mainly during acute inflammatory responses. CXCL12 acts as a positive regulator of monocyte migration and a negative regulator of monocyte adhesion. It also stimulates migration of monocytes and T-lymphocytes through its receptors, CXCR4 and ACKR3, and decreases monocyte adherence to surfaces. The CXCL12-CXCR4 axis was shown to play a significant role in regulating migration of both proliferating and terminally differentiated muscle stem cells.
The capacity of systemically administered ADSC to participate in regeneration of skeletal muscle was evaluated using the CTX-induced myonecrosis model with actively ongoing regeneration and remodeling of muscle tissue. Our data clearly demonstrate that the specific populations of ADSC selected with an affinity for the chemokines being released at the site of muscle damage are able to migrate from systemic compartment and efficiently colonize in muscle, sustaining their long-term maintenance and the continuous replenishment of COL6. CTX-treated Col6a1-/- muscles receiving the Ccr2- and Cxcr2-positive ADSC transplants showed significant increase in the number of COL6-positive myofibers as compared to muscle without CTX injury. Moreover, COL6-producing ADSC were able to migrate intramuscularly and repopulated significant area of the damaged tissue and adjacent myofibers. This directional migration was likely induced in response to the presence of Ccl2, Cxcl1/2 and Cxcl7 ligands released in the course of CTX injury and regeneration elicited by various mechanisms. Also, it is possible that pro-inflammatory cytokines, chemokines and growth factors that typically result in homing of immune cells to damaged site are released from muscle resident cells, stimulating directional migration of ADSC within muscle tissue. In fact, a prominent feature of CTX-injured muscle is a striking inflammatory infiltrate of immune cells, such as macrophages and neutrophils. Also, it is well known that CCR2 and CXCR2 are major regulators of induced macrophage and neutrophils trafficking in vivo [40, 41]. In addition, CXCR4-CXCL12 chemotactic axis was shown to play a significant role in regulating migration of both proliferating and terminally differentiated muscle cells . It is plausible that Ccr2- and Cxcr2-positive ADSC were recruited to the CTX-damaged site by chemotactic mechanisms of inflammatory and satellite cells, respectively. Together, these observations suggest that mobilization of ADSC from blood stream in damaged muscle may highly depend on the local inflammatory state rather than other factors. The ability of ADSC to donate the therapeutic COL6 protein to the injured muscle strongly supports the possibility of stem cell therapy for COL6RM, which exhibits substantially more severe myopathology than the mouse model.