Full spectrum cytometry improves the resolution of highly autofluorescent biological samples: Identification of myeloid cells in regenerating skeletal muscles.

Autofluorescence (AF) is an intrinsic characteristic of cells caused by the presence of fluorescent biological compounds within the cell; these can include structural proteins (e.g., collagen and elastin), cellular organelles, and metabolites (e.g., aromatic amino acids). In flow cytometric studies, the presence of AF can lead to reduced antigen and population resolution, as well as the presence of artifacts due to false positive events. Here, we describe a methodology that uses the inherent ability of full spectrum cytometry to treat AF as a fluorochrome and to thereby separate it from the other fluorochromes of the assay. This method can be applied to complex inflamed tissues; for instance, in regenerating skeletal muscle we have developed a 16-color panel targeting highly autofluorescent myeloid cells. This represents a first step toward overcoming technological limitations in flow cytometry due to AF.

High-Dimensional Immunophenotyping with 37-Color Panel Using Full-Spectrum Cytometry.

A comprehensive study of the cellular components of the immune system demands both deep and broad immunophenotyping of numerous cell subsets in an effective and practical way. Novel full-spectrum technology reveals the complete emission spectrum of each dye maximizing the amount of information that can be obtained on a single sample regarding conventional flow cytometry and provide an expanded knowledge of biological processes. In this chapter, we describe a 37-color protocol that allows to identify more than 45 different cell populations on whole blood samples of SARS-CoV-2-infected patients.

Genome-wide analysis of TIAR RNA ligands in mouse macrophages before and after LPS stimulation.

TIA-1 related protein (TIAR) is a RNA-binding protein involved in several steps of gene expression such as RNA splicing Aznarez et al. (2008) [1] and translation Piecyk et al. (2000) [2]. TIAR contains three RNA recognition motifs (RRMs) allowing its interaction with specific sequences localized in the untranslated regions (UTRs) of several mRNAs. In myeloid cells, TIAR has been shown to bind and regulate the translation and stability of various mRNA-encoding proteins important for the inflammatory response, such as TNFα Piecyk et al. (2000), Gueydan et al. (1999) [2], [3], Cox-2 Cok et al. (2003) [4] or IL-8 Suswam et al. (2005) [5]. Here, we generated two macrophage-like RAW 264.7 cell lines expressing either a tagged full-length TIAR protein or a RRM2-truncated mutant unable to bind RNA with high affinity Dember et al. (1996), Kim et al. (2013) . By a combination of RNA-IP and microarray analysis (RIP-chip), we identified mRNAs specifically bound by the full-length protein both in basal conditions and in response to LPS (GSE77577).

Fibrogenic Cell Plasticity Blunts Tissue Regeneration and Aggravates Muscular Dystrophy.

Preservation of cell identity is necessary for homeostasis of most adult tissues. This process is challenged every time a tissue undergoes regeneration after stress or injury. In the lethal Duchenne muscular dystrophy (DMD), skeletal muscle regenerative capacity declines gradually as fibrosis increases. Using genetically engineered tracing mice, we demonstrate that, in dystrophic muscle, specialized cells of muscular, endothelial, and hematopoietic origins gain plasticity toward a fibrogenic fate via a TGFß-mediated pathway. This results in loss of cellular identity and normal function, with deleterious consequences for regeneration. Furthermore, this fibrogenic process involves acquisition of a mesenchymal progenitor multipotent status, illustrating a link between fibrogenesis and gain of progenitor cell functions. As this plasticity also was observed in DMD patients, we propose that mesenchymal transitions impair regeneration and worsen diseases with a fibrotic component.

Understanding the process of fibrosis in Duchenne muscular dystrophy.

Fibrosis is the aberrant deposition of extracellular matrix (ECM) components during tissue healing leading to loss of its architecture and function. Fibrotic diseases are often associated with chronic pathologies and occur in a large variety of vital organs and tissues, including skeletal muscle. In human muscle, fibrosis is most readily associated with the severe muscle wasting disorder Duchenne muscular dystrophy (DMD), caused by loss of dystrophin gene function. In DMD, skeletal muscle degenerates and is infiltrated by inflammatory cells and the functions of the muscle stem cells (satellite cells) become impeded and fibrogenic cells hyperproliferate and are overactivated, leading to the substitution of skeletal muscle with nonfunctional fibrotic tissue. Here, we review new developments in our understanding of the mechanisms leading to fibrosis in DMD and several recent advances towards reverting it, as potential treatments to attenuate disease progression.

Macrophage plasticity and the role of inflammation in skeletal muscle repair.

Effective repair of damaged tissues and organs requires the coordinated action of several cell types, including infiltrating inflammatory cells and resident cells. Recent findings have uncovered a central role for macrophages in the repair of skeletal muscle after acute damage. If damage persists, as in skeletal muscle pathologies such as Duchenne muscular dystrophy (DMD), macrophage infiltration perpetuates and leads to progressive fibrosis, thus exacerbating disease severity. Here we discuss how dynamic changes in macrophage populations and activation states in the damaged muscle tissue contribute to its efficient regeneration. We describe how ordered changes in macrophage polarization, from M1 to M2 subtypes, can differently affect muscle stem cell (satellite cell) functions. Finally, we also highlight some of the new mechanisms underlying macrophage plasticity and briefly discuss the emerging implications of lymphocytes and other inflammatory cell types in normal versus pathological muscle repair.

MKP-1 coordinates ordered macrophage-phenotype transitions essential for stem cell-dependent tissue repair.

Re-establishing tissue homoeostasis in response to injury requires infiltration of inflammatory cells and activation of resident stem cells. However, full tissue recovery also requires that the inflammation is resolved. While it is known that disturbing the interactions between inflammatory cells and tissue resident cells prevents successful healing, the molecular mechanisms underlying the paracrine interactions between these cell types are practically unknown. Here, and in a recent study, we provide mechanistic evidence that macrophages control stem cell-dependent tissue repair. In particular, we found that the temporal spacing of the pro- to anti-inflammatory macrophage polarization switch is controlled by the balance of p38 MAPK (termed here p38) and the MAPK phosphatase MKP-1 during the muscle healing process. Moreover, we demonstrate a new function for MKP-1-regulated p38 signaling in deactivating macrophages during inflammation resolution after injury. Specifically, at advanced stages of regeneration, MKP-1 loss caused an unscheduled "exhaustion-like" state in muscle macrophages, in which neither pro- nor anti-inflammatory cytokines are expressed despite persistent tissue damage, leading to dysregulated reparation by the tissue stem cells. Mechanistically, we demonstrate that p38 and MKP-1 control the AKT pathway through a miR-21-dependent PTEN regulation. Importantly, both genetic and pharmacological interference with the individual components of this pathway restored inflammation-dependent tissue homeostasis in MKP-1-deficient mice and delayed inflammation resolution and tissue repair dysregulation in wild-type mice. Because the process of tolerance to bacterial infection involves a progressive attenuation of pro-inflammatory gene expression, we discuss here the potential similarities between the mechanisms underlying inflammation resolution during tissue repair and those controlling endotoxin tolerance.

Aberrant repair and fibrosis development in skeletal muscle.

The repair process of damaged tissue involves the coordinated activities of several cell types in response to local and systemic signals. Following acute tissue injury, infiltrating inflammatory cells and resident stem cells orchestrate their activities to restore tissue homeostasis. However, during chronic tissue damage, such as in muscular dystrophies, the inflammatory-cell infiltration and fibroblast activation persists, while the reparative capacity of stem cells (satellite cells) is attenuated. Abnormal dystrophic muscle repair and its end stage, fibrosis, represent the final common pathway of virtually all chronic neurodegenerative muscular diseases. As our understanding of the pathogenesis of muscle fibrosis has progressed, it has become evident that the muscle provides a useful model for the regulation of tissue repair by the local microenvironment, showing interplay among muscle-specific stem cells, inflammatory cells, fibroblasts and extracellular matrix components of the mammalian wound-healing response. This article reviews the emerging findings of the mechanisms that underlie normal versus aberrant muscle-tissue repair.

Impaired embryonic development in mice overexpressing the RNA-binding protein TIAR.

BACKGROUND: TIA-1-related (TIAR) protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs). Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. METHODOLOGY/PRINCIPAL FINDINGS: To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR) allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2alpha that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. CONCLUSIONS/SIGNIFICANCE: This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming.