Facts

  • Muscle regeneration involves a sequence of tissue repair mechanisms regulated by both pro- and anti-inflammatory immune cells.

  • Myositis pathomechanisms are not fully understood but may result from chronic exposure to immune cells and cytokines, leading to destruction and mis-repair of muscle, with fibrosis and vascular architecture disturbance.

  • Therapeutic options for inflammatory myopathies are predominantly based on immunosuppressive treatments, which are sometimes insufficient to regulate the different features of these complex pathologies.

  • Adipose tissue-derived stem cells and stromal vascular fraction have immunomodulatory, anti-fibrotic, proangiogenic and regenerative properties that could be exploited for the treatment of refractory or relapsed myositis.

  • However, the mechanisms behind these effects are still insufficiently understood and more preclinical studies are required before their clinical use.

Open questions

  • Does repeated acute muscular destruction trigger chronical autoimmune inflammation, or is auto-immunity responsible for muscle destruction in myositis?

  • In myositis, how does chronicle inflammation affect adipose-derived cells capacities?

  • Will adipose stem cells replace muscle stem cells and directly participate in tissue regeneration, or rather have a supporting role for immune and local stem cells?

Introduction

The mechanisms of muscle regeneration following injury are now well-known and involve both muscle and immune cells, through a regulated process [1]. A disruption of this process may be the cause of chronic inflammation and a failure of muscle regeneration, which can lead to autoimmune diseases such as myositis. Myositis, also called idiopathic inflammatory myopathies, represents a group of immune-mediated diseases, including Polymyositis (PM), Dermatomyositis (DM), Immune-Mediated Necrotising Myositis (IMNM), Inclusion Body Myositis (IBM) and overlap myositis [2]. They are clinically characterised by muscle weakness, and histologically by the presence of varying levels of myofibre necrosis and leucocyte infiltrates in muscles [3]. Predominantly muscular, some forms of myositis can also be associated with other manifestations, such as Interstitial Lung Disease (ILD), skin ulcers or Raynaud’s phenomenon. Corticosteroids and immunosuppressive drugs are commonly used but may be ineffective in some patients or even aggravating due to their possible side effects [4,5,6].

For these patients, cell therapies have sometimes shown long-term beneficial effects. Autologous hematopoietic stem cell transplants, which have been used to replace auto-reactive immune systems, have improved patients’ conditions, and even allowed some of them to enter into remission. These therapies generally seem to be safe, but can be complicated by severe or even life-threatening iatrogenic infections due to myeloablative conditioning regimens [7,8,9,10]. More recently, the discovery of the immunomodulatory effects of Mesenchymal Stem Cells (MSC) from Bone Marrow (BM-MSC) or Umbilical Cord Blood (UC-MSC), in addition to their well-known regenerative effect, has led to their use for the treatment of patients with refractory autoimmune diseases, including myositis [11]. If these types of stem cells seem safer, the invasiveness of the harvesting and the low rate of stem cells recovered from these sources remain important limitations to their use.

Adipose tissue is another source of MSC. They can be extracted safely and in large quantities from a lipoaspirate by enzymatic digestion or mechanical isolation for Stromal Vascular Fraction (SVF), followed by replicative cultures of adherent cells for Adipose-Derived Stem Cells (ADSC) [12]. If ADSC are known to possess immunomodulatory, proangiogenic, anti-fibrotic and regenerative capacities like MSC from other tissues [13], SVF share similar properties and is easier to prepare [14]. However, the therapeutically active components within this heterogeneous cellular product are not well defined, and the mechanisms responsible for its activity remain insufficiently understood.

In order to evaluate the potential of SVF as a treatment for refractory myositis, we first summarise here the physiological mechanisms of muscle regeneration and the pathological mechanisms involved in myositis. Next, we address the main features of these diseases through the known mechanisms of action of this cell therapy. Last, we discuss its clinical relevance by analysing results from various clinical trials.

Physiological muscle regeneration

Muscle regeneration after trauma is a process that involves both immune and muscular cells in order to restore normal muscle function. At first, satellite cell activation and proliferation accompanied by inflammation, followed by a progressive decrease of inflammation under the influence of anti-inflammatory cells, which stimulate muscle progenitor cell differentiation and tissue remodelling [15].

At the earliest stage of regeneration after injury, muscle damaged cells release Damage-Associated Molecular Patterns (DAMP) [16], which lead through Toll Like Receptor (TLR) to the activation and infiltration of immune cells, mostly mast cells [17] and neutrophils [18]. These cells start to clear the damaged myofibres and secrete pro-inflammatory cytokines (mostly IL-1, IL-6, IL-8 and TNFα). The pro-inflammatory signal spreads and after 24 h, macrophages can be observed at the lesion site [19]. They are mostly involved in the elimination of damaged muscular cells by the production of Reactive Oxygen Species (ROS), through the increased expression of Inducible Nitric Oxide Synthase (iNOS), and phagocytosis. Like neutrophils, they secrete a large amount of cytokines (mostly TNFα, IL-6, and IL-1β) which triggers a positive feedback loop between neutrophil and macrophage recruitment and production of cytotoxic substances, but also T-cell recruitment [20].

Around three days after injury, both CD8+ and CD4+ T cells appear at the lesion site and can be detected for up to ten days [21]. CD8+ T cells pursue the task of macrophages and neutrophils, by releasing many cytokines which amplify the recruitment of leucocytes and by acting on extracellular matrix remodelling to speed up cellular debris elimination [Full size image

First, the immunomodulatory effect of SVF is supported mainly by three cell populations: AD-MSC, macrophages and Treg cells. AD-MSC immunomodulatory capacities are similar or higher to those of BM-MSC according to different studies [110, 111] and have already been tested in vitro [112] and in many in vivo models [113]. Even if the mechanisms involved are not fully understood for these cells, several studies support their effects on T-cell activation, proliferation and differentiation from Th1 cells into Th2 cells, through soluble factors like PGE2 and IDO [114,115,116] and direct interactions via CD54/CD2 and CD58/CD11a, which increase IL-10 production [117]. They also induce Treg proliferation, via TGFβ and IL-33 secretion [118, 121]. Recent studies have shown that the interactions between MSC and pro-inflammatory cells enhance the immunosuppressive capacities of MSC. Indeed, these authors observed that IFNγ and TNFα secreted by Th1 lymphocytes or CD54 expressed by pro-inflammatory macrophages increased IDO activities [122, 123]. SVF also contains macrophages which exhibit anti-inflammatory activities through the secretion of high levels of IL-10 and IL-1 decoy receptors [124] that attenuate TNFα inflammatory signals via activation of STAT3 [125, 126], and modulation of inflammatory gene transcription rates [127]. The modification of the balance between Arg-1 and iNOS activities, which both use L-arginine as a substrate, leads to decreased ROS production, and thus to reduced oxidative stress and destruction of myofibres.

ADSC and SVF can also act on fibrosis via their immunomodulatory effects. Indeed, by reprogramming immune cells into anti-inflammatory cells, they increase the expression of IL-10, which presents several anti-fibrotic properties: inhibition of neutrophil and macrophage invasion and ROS release [128], down-regulation of TGFβ1 expression [129], up-regulation of MMP and down-regulation of collagen expression [130]. Preclinical and clinical studies suggest that SVF anti-fibrotic effects are strongly related to the secretion of HGF by MSC during inflammatory responses, as evidenced by clinical and histological parameters [131, 132]. Indeed, through the paracrine effect of this factor, SVF and ADSC reduce the expression of TGFβ1 and thus the differentiation of collagen type I/III producing cells (fibroblasts) and alpha-Smooth Muscle Actin producing cells (myofibroblasts). ADSC also induce a significant increase in TGFβ3, which reduces the expression of these genes, and stimulates MMP-1, -2 and -3 expressions, which increase fibrotic molecule degradation. The change in the TGFβ1/TGFβ3 and MMP-2/TIMP-2 ratio tips the scales in favour of an anti-fibrotic effect [133, 134]. MMP expression is also stimulated by proangiogenic factors, like bFGF or VEGF, to degrade extracellular matrix and prepare neo-angiogenesis [135].

Indeed, SVF is known to express high levels of IGF1, IL-8, Platelet-Derived Growth Factor-beta (PDGFβ), bFGF and VEGF, and to have robust angiogenic and vasculogenic activities demonstrated both in vitro and in vivo in a hind limb ischaemia model [136]. These growth factors help to maintain a vascular-like micro-environment that supports MSC differentiation into endothelial cells, and thus participate in angiogenesis and vascular repair during muscular regeneration [137]. Traktuev et al. demonstrated that VEGF helps the migration of MSC and promotes the secretion of PDGFβ by EPC, which enable MSC to proliferate [138, 139]. PDGFβ is well-known for its action during vascular development [140] but also plays a role in the proangiogenic properties of SVF, by inducing the secretion of proangiogenic extracellular vesicles by both MSC and EPC [141, 142]. These extracellular vesicles contain proangiogenic molecules such as c-KIT and Stem cell factor, which participate in the recruitment of EPC and their differentiation into endothelial cells [143]. PDGFβ secretion by EPC also induces pericyte recruitment [144] which is known to play an essential role in angiogenesis regulation [145].

SVF, through its immunomodulatory properties, acts on both chronic inflammation and muscle repair via cytokine release (mainly IL-4 and IL-13). Moreover, growth factors secreted by stromal cells may have a positive effect on muscle regeneration, and some of them are currently under evaluation in the management of muscle disorders, such as sarcopenia [146]. But one advantage to using cell therapy, rather than hormones or cytokines, could be its ability to differentiate in situ depending on its cellular environment. This could strengthen and help satellite cells to replace defective cells. The conversion of ADSC or SVF to a myogenic phenotype has been obtained in vitro by addition of inductive media, containing horse serum and hydrocortisone. This leads to the expression of the myogenic transcription factors Myod1 and myogenin and then the fusion and formation of multinucleated cells expressing the myosin heavy chain [147,148,149]. Based on histological evidence, ADSC fuse to form multinucleated myotubes in vitro. In their study, Di Rocco et al. showed that ADSC and SVF cells were able to differentiate into skeletal muscle cells when cultured in the presence of differentiating primary myoblasts [150]. Furthermore, the conversion of SVF to a myogenic phenotype is enhanced by myogenic environment in the absence of cell-cell contacts (transwell culture) and even in absence of muscle cells but to a lesser extent. This myogenic conversion has also been demonstrated in vivo by several studies. In a lagomorphic model of muscular injury induced by cardiotoxin, the intramuscular injection of short-term cultured (3 days) SVF cells induced an increase in muscle mass and functional capacities [151]. The myogenic differentiation of SVF and fusion with muscular cells have been demonstrated using SVF genetically modified to express β-galactosidase or GFP, showing evidence of the contribution of SVF cells to muscular regeneration in vivo with 20% of GFP-positive fibres in the total area of sections from treated hind limbs [150, 151]. This contribution could be enhanced by pretreating SVF with anti-inflammatory cytokine IL-4 or SDF1 before use to increase the myogenic capacity of ADSC in vitro and in vivo [152]. To finish, it has also been reported that injection of human ADSC into immunocompetent mdx mice resulted in a substantial expression of human dystrophin in both injected and adjacent muscle, revealing the spread of cells to other muscles [153].