Characterization of macrophages, giant cells and granulomas during muscle regeneration after irradiation
Introduction
Muscle injury in healthy adults induces a series of well-coordinated events involved in the repair of damaged tissue. The development of strategies to improve muscle regeneration after damage created by pathological events such as irradiation is of major scientific and clinical importance [1]. Indeed, after irradiation, several deleterious pathways are activated such as oxidative stress and cell death by apoptosis, necrosis and mitotic death. These events lead to uncontrolled and unpredictable inflammation waves, which may become chronic and resistant to classical anti-inflammatory treatment and thus are a major challenge for the treatment of radiation injury [2]. It has been shown that adipose tissue derived stem cell (ASC) injections can reduce inflammation and improve muscle regeneration after irradiation, but the mechanisms are not fully understood [3], [4], [5].
Mesenchymal stem cells derived from bone marrow (BM-MSC) have been shown to improve wound healing by recruiting macrophages [6]. Inflammation and tissue regeneration are linked and synchronized in the coordination of different biological and cellular processes. The inflammatory response within a tissue lesion begins with the infiltration of neutrophils followed by an infiltration of macrophages of the M1 phenotype [7], [8]. M1 macrophages produce pro-inflammatory cytokines that are involved in tissue clearing in order to remove the material which otherwise would maintain inflammation [9]. Subsequent differentiation/invasion by M2 macrophages attenuates M1 populations. Each type of macrophage subpopulation is involved in a specific biological process and expresses different cytokines (Schema 1A) [10], [11], [12], [13].
This understanding of the role of macrophages as derived from in vitro experiments does not necessarily reflect their precise role in vivo. The M1-like and M2-like macrophages (or resolving) are the usually preferred nomenclature for in vivo macrophages. Macrophages resolving inflammation exhibit shared M2 features but carry functions specific to M1 [14], [15], [16], [17], [18]. Data in the literature suggest that macrophages activated during inflammation resolution are more akin to M2b cells, but the real phenotype of these cells during host defense and tissue regeneration remains enigmatic [19]. Tissue-resident macrophages have also been thought to improve tissue repair. However, the actual function of these macrophages is still being debated (Schema 1A) [1].
The kinetics of the appearance of M1 and M2 macrophages are critical for tissue regeneration, because cytokines produced by these cells modulate the extracellular matrix [20], [21]. An imbalance of M1 and M2 macrophages induces an alteration of the function of the macrophages and interferes with tissue regeneration. Such an alteration leads to an accumulation of pro-fibrotic factors and induces fibrosis [22]. Recent studies have further indicated that the switch between proper regeneration and development of fibrosis is controlled by various factors, among them the transforming growth factor beta (TGF-βs) family. TGF-β is able to inhibit myoblast proliferation but also able to promote fibrosis [23]. Thus, specific inhibition of the TGF-β1 signaling pathway can significantly improve muscle repair [24].
Acute inflammation is an indispensable process of the innate immune response which can clean-up the tissue, and normally, resolves after one to two weeks [25]. In specific situations such as irradiation, inflammation can become prolonged and may result in chronic inflammation [8]. In contrast to acute inflammation, chronic inflammation lasts weeks, months or even indefinitely and causes tissue damage. In the context of chronic inflammation, the persistence of neutrophils alters the differentiation of M1 macrophages into M2 macrophages [8], [26]. Chronic inflammation is very dangerous for skeletal muscle healing because: i, the increased level of pro-inflammatory factors can block the shift in macrophage polarization from M1 to M2 [27]. The continuous presence of pro-inflammatory cytokines changes the regenerative capacity of satellite cells, which can hamper muscle healing [28]. ii, IL-6 changes the function of satellite cells and promotes muscle loss [29]. Chronic inflammation can transform into granulomatous inflammation. The reason for this conversion remains incompletely understood. iii, neutrophils usually remove molecules that promote acute inflammation. If the material is indigestible, it can induce several cycles of an acute inflammatory response. iiii, chronic inflammation causes tissue injury. The tissue can respond to this damage by forming granulomas. Granulomas can limit the inflamed area but the antigen presentation properties are restricted [30]. In granulomas the main cells are macrophages and lymphocytes. Macrophages undergo structural changes, and become epithelioid cells (large cytoplasm and morphologically similar to epithelial cells). These epithelioid cells can fuse together and form giant cells, their role is not clear (Schema 1B) [31], [32], [33].
The molecular and cellular mechanisms of tissue regeneration are not fully understood, but cytokines are thought to play a fundamental role. These molecules seem to be produced by many cell populations, mainly macrophages and helper T cells. The aim of this study was therefore to characterize macrophages and giant cells during chronic inflammation, to analyze the expression of their cytokines in order to better understand tissue regeneration after irradiation, as well as the role-played by stem cells during this process.
Section snippets
Methods
This study was approved by the French Army Animal Ethics Committee (N°2011/22.1). All minipigs were treated in compliance with the European legislation (dir 2010/63/EU) implemented into French low (decree 2013-118) regulating animal experimentation.
Results and discussion
Muscle lesions are characterized by drastic changes in muscle cells and in their extracellular matrices [37]. During regeneration, if the M1 pro-inflammatory response lasts for an extended period of time, excessive development of fibrosis may occur and cause tissue dysfunction [24]. Previously, we demonstrated that ASC treatment can enhance skin and muscle regeneration after irradiation [3], [4], [34], [38]. To gain further insight into the mechanisms of muscle regeneration we conducted a new
Conclusion
Tissue regeneration techniques are often ineffective after irradiation, because irradiation can cause incalculable spatio-temporal inflammatory waves within tissues. Stem cell-based therapies are novel therapeutic strategies that may key for developing new treatments for disease conditions with very few or no cures. Although there has been an increase in the number of clinical trials involving stem cell-based therapies in the last few years, the long-term risks and benefits of these therapies
Credit authorship contribution statement
Krisztina Nikovics: conceptualization, data curation, formal analysis, writing - original draft. Anne-Laure Favier: formal analysis, writing - original draft, supervision. Laure Barbier: data curation, methodology. Michel Drouet: Visualization. Diane Riccobono: methodology, supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We are very grateful to Robert Drillien for his helpful advice and critically reviewing our manuscript. We are thankful to Halima Morin and Abdelhafid Bendahmane for their invaluable help on the HIS technique. We wish to thank Xavier Butigieg and Muriel Rosset-Berthelot for technical support and Zsolt Kelemen for help in preparation of the manuscript. We thank the Imagery platform of INRA-Versailles for technical assistance on confocal microscopy. This work was supported by the Service de Santé
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