Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle
2020; Springer Nature; Volume: 21; Issue: 4 Linguagem: Inglês
10.15252/embr.201949075
ISSN1469-3178
AutoresFrancescopaolo Iavarone, Ombretta Guardiola, Alessandra Scagliola, Gennaro Andolfi, Federica Esposito, Antonio L. Serrano, Eusebio Perdiguero, Silvia Brunelli, Pura Muñoz‐Cánoves, Gabriella Minchiotti,
Tópico(s)Muscle Physiology and Disorders
ResumoArticle27 February 2020Open Access Transparent process Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle Francescopaolo Iavarone Stem Cell Fate Laboratory, CNR, Institute of Genetics and Biophysics "A. Buzzati-Traverso", Naples, Italy Search for more papers by this author Ombretta Guardiola Stem Cell Fate Laboratory, CNR, Institute of Genetics and Biophysics "A. Buzzati-Traverso", Naples, Italy Search for more papers by this author Alessandra Scagliola School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy Search for more papers by this author Gennaro Andolfi Stem Cell Fate Laboratory, CNR, Institute of Genetics and Biophysics "A. Buzzati-Traverso", Naples, Italy Search for more papers by this author Federica Esposito Stem Cell Fate Laboratory, CNR, Institute of Genetics and Biophysics "A. Buzzati-Traverso", Naples, Italy Search for more papers by this author Antonio Serrano Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBER on Neurodegenerative Diseases (CIBERNED), Barcelona, Spain Search for more papers by this author Eusebio Perdiguero Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBER on Neurodegenerative Diseases (CIBERNED), Barcelona, Spain Search for more papers by this author Silvia Brunelli School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy Search for more papers by this author Pura Muñoz-Cánoves Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBER on Neurodegenerative Diseases (CIBERNED), Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Search for more papers by this author Gabriella Minchiotti Corresponding Author [email protected] orcid.org/0000-0003-0225-9043 Stem Cell Fate Laboratory, CNR, Institute of Genetics and Biophysics "A. Buzzati-Traverso", Naples, Italy Search for more papers by this author Francescopaolo Iavarone Stem Cell Fate Laboratory, CNR, Institute of Genetics and Biophysics "A. Buzzati-Traverso", Naples, Italy Search for more papers by this author Ombretta Guardiola Stem Cell Fate Laboratory, CNR, Institute of Genetics and Biophysics "A. Buzzati-Traverso", Naples, Italy Search for more papers by this author Alessandra Scagliola School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy Search for more papers by this author Gennaro Andolfi Stem Cell Fate Laboratory, CNR, Institute of Genetics and Biophysics "A. Buzzati-Traverso", Naples, Italy Search for more papers by this author Federica Esposito Stem Cell Fate Laboratory, CNR, Institute of Genetics and Biophysics "A. Buzzati-Traverso", Naples, Italy Search for more papers by this author Antonio Serrano Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBER on Neurodegenerative Diseases (CIBERNED), Barcelona, Spain Search for more papers by this author Eusebio Perdiguero Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBER on Neurodegenerative Diseases (CIBERNED), Barcelona, Spain Search for more papers by this author Silvia Brunelli School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy Search for more papers by this author Pura Muñoz-Cánoves Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBER on Neurodegenerative Diseases (CIBERNED), Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Search for more papers by this author Gabriella Minchiotti Corresponding Author [email protected] orcid.org/0000-0003-0225-9043 Stem Cell Fate Laboratory, CNR, Institute of Genetics and Biophysics "A. Buzzati-Traverso", Naples, Italy Search for more papers by this author Author Information Francescopaolo Iavarone1, Ombretta Guardiola1, Alessandra Scagliola2, Gennaro Andolfi1, Federica Esposito1, Antonio Serrano3, Eusebio Perdiguero3, Silvia Brunelli2, Pura Muñoz-Cánoves3,4,5 and Gabriella Minchiotti *,1 1Stem Cell Fate Laboratory, CNR, Institute of Genetics and Biophysics "A. Buzzati-Traverso", Naples, Italy 2School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy 3Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBER on Neurodegenerative Diseases (CIBERNED), Barcelona, Spain 4Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain 5Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain *Corresponding author. Tel: +39 0816 132357; E-mail: [email protected] EMBO Rep (2020)21:e49075https://doi.org/10.15252/embr.201949075 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Macrophages are characterized by a high plasticity in response to changes in tissue microenvironment, which allows them to acquire different phenotypes and to exert essential functions in complex processes, such as tissue regeneration. Here, we report that the membrane protein Cripto plays a key role in shaping macrophage plasticity in skeletal muscle during regeneration and disease. Conditional deletion of Cripto in the myeloid lineage (CriptoMy-LOF) perturbs MP plasticity in acutely injured muscle and in mouse models of Duchenne muscular dystrophy (mdx). Specifically, CriptoMy-LOF macrophages infiltrate the muscle, but fail to properly expand as anti-inflammatory CD206+ macrophages, which is due, at least in part, to aberrant activation of TGFβ/Smad signaling. This reduction in macrophage plasticity disturbs vascular remodeling by increasing Endothelial-to-Mesenchymal Transition (EndMT), reduces muscle regenerative potential, and leads to an exacerbation of the dystrophic phenotype. Thus, in muscle-infiltrating macrophages, Cripto is required to promote the expansion of the CD206+ anti-inflammatory macrophage type and to restrict the EndMT process, providing a direct functional link between this macrophage population and endothelial cells. Synopsis The membrane protein Cripto is an extrinsic determinant of macrophage plasticity in skeletal muscle regeneration and disease. Cripto-dependent modulation of macrophage phenotypes controls endothelial plasticity and contributes to proper muscle repair. Cripto is predominantly expressed in expanding anti-inflammatory macrophages in the regenerating tissue. Cripto promotes the proper accumulation of CD206+ anti-inflammatory MPs, by modulating TGF-β/Smad signaling. Lack of Cripto in the myeloid lineage affects vascular remodeling by increasing EndMT and collagen deposition. Introduction Skeletal muscle regeneration relies on highly coordinated sequential events, which involve a complex network of interaction between tissue-resident and recruited cells, including muscle stem cells, inflammatory cells, endothelial-derived progenitors, and fibro/adipogenic progenitors (FAPs) 1. Progressive impairment of the interplay between the inflammatory cells, mainly the infiltrated macrophages, and the different muscle cellular components is emerging as a key event in switching regeneration from compensatory to pathogenic regeneration 2. Macrophages exhibit substantial plasticity and can quickly change their phenotype in response to different stimuli in the microenvironment. This unique feature makes them serve different functions during the process of skeletal muscle regeneration, including resolution of the pro-inflammatory phase and transition to the regeneration phase. Soon after an acute injury, invading macrophages adopt a pro-inflammatory phenotype, phagocyte necrotic tissue, and secrete growth factors, promoting an environment that favors satellite cell activation and proliferation 3-5. Subsequently, a second wave of macrophages with anti-inflammatory phenotype suppresses the process of inflammation and supports restorative function. These anti-inflammatory macrophages arise from resident macrophages and/or circulating pro-inflammatory macrophages that switch into an anti-inflammatory phenotype 6. Besides supporting myoblast differentiation, anti-inflammatory macrophages produce angiogenic factors promoting vascular remodeling, which is necessary for a proper regenerative process 7. Furthermore, macrophages promote the differentiation of endothelial-derived progenitors by inhibiting a process known as the Endothelial-to-Mesenchymal Transition (EndMT) process, in which endothelial cells acquire myofibroblastic traits while losing endothelial-specific gene expression 8-10. While in acute muscle injury, macrophages adopt well-coordinated sequential waves of pro- and anti-inflammatory phenotypes, this balance is skewed in chronic muscle diseases like muscular dystrophies, where muscles fail to regenerate and the tissue is progressively substituted by adipocytes and collagen fibers 11, 12. Emerging evidence indicates that different macrophage populations with mixed phenotypes coexist in dystrophic muscles, but their precise function is only recently starting to be characterized 13. Research efforts are currently focused on understanding how macrophage plasticity is controlled and how macrophages crosstalk with other muscle populations in injured and dystrophic muscles. Recent findings from our laboratory and others place the extracellular membrane protein Cripto within this complex regulatory network. Cripto is a glycosylphosphatidylinositol (GPI)-anchored protein and acts as coreceptor for different members of the TGF-β superfamily 14. Of note, depending on the context it also exists as a soluble protein either secreted or shed from the membrane 15, 16. Cripto is a developmental gene known to regulate early embryonic development 17, 18 and it is usually not expressed in normal adult tissues including resting skeletal muscles. However, Cripto becomes rapidly and transiently re-expressed upon acute injury both in activated/proliferating satellite cells and in a subpopulation of infiltrating macrophages 19. Emerging evidence indicates that Cripto is a key regulator of the myogenic program during skeletal muscle regeneration 16, 19, 20. However, no studies have been reported so far on the inflammatory cell/macrophage-specific role of Cripto in skeletal muscle regeneration. Here, we show in vivo evidence that Cripto is preferentially expressed by anti-inflammatory macrophages and is a key regulator of macrophage plasticity in injured and in dystrophic skeletal muscles. Moreover, we suggest that Cripto mediates the crosstalk between macrophages and endothelial cells promoting vascular remodeling, at least in part, by restricting TGF-β-induced EndMT and preventing excessive fibrosis in dystrophic muscles. Results Expression profile of cell-surface Cripto in subpopulations of macrophages during acute muscle injury The timely attraction of macrophages (MPs), the ordered transition between the pro- and anti-inflammatory phenotypes, and the precise termination of their activity are prerequisites for a successful regeneration process 4. Cripto is expressed in the MPs that infiltrate the injured muscle 19; yet, the dynamics of Cripto expression in the pro- and anti-inflammatory MPs was not investigated so far. To address this issue directly, hind limb muscles of wild-type mice were injected with cardiotoxin (CTX) and the expression of Cripto in the different inflammatory cell populations was analyzed at days 2, 3, and 5 after injury (Fig 1A). To this end, injured muscles were dissociated by enzymatic digestion and the bulk cells were stained for Cripto and the MP markers CD11b, F4/80, and Ly6C. We first assessed the expression of Cripto in the CD11b+ immune cells at the different time points after injury; the number of CD11b+ that expressed Cripto progressively increased from days 2 to 5 up to 40% (6.5 ± 0.7% at day 2 vs. 40.7 ± 1.3% at day 5; n = 5; P = 1.14E-08; Figs 1B and EV1A), consistent with previous data of Cripto expression in the infiltrating MPs 19. We then examined the distribution of pro-inflammatory (F4/80+/Ly6CHigh) and anti-inflammatory (F4/80+/Ly6CLow) MPs in the CD11b+ cells throughout the time course (Fig 1C). As expected, while the F4/80+/Ly6CLow MPs (blue) and F4/80+/Ly6CHigh MPs (red) were almost equally distributed in the CD11b+ cells at day 2, from day 3 onward a dramatic increase in F4/80+/Ly6CLow MPs was observed, at the expense of the F4/80+/Ly6CHigh MPs (Fig 1C and D). Finally, to directly examine the nature of the Cripto+ immune cells, we evaluated the distribution of F4/80+/Ly6CHigh and F4/80+/Ly6CLow MPs in the CD11b+/Cripto+ population. Of note, the F4/80+/Ly6CLow MPs were the vast majority at all the time points analyzed (Fig 1C and D). Figure 1. Time-course expression of surface Cripto in pro- and anti-inflammatory macrophages in acute skeletal muscle injury A. Experimental scheme of Cripto expression analysis in macrophage (MP) subpopulations from wild-type skeletal muscles at different time points after cardiotoxin (CTX) injection. B. Representative flow cytometry dot plots of CD11b+ and CD11b+/Cripto+ cells at days 2, 3, and 5 after injury. PE (Phycoerythrin/free channel). Data are mean ± SEM (n = 5 biological replicates; P ≤ 0.00008, Student's t-test). C. Representative flow cytometry dot plots of CD11b+/F4/80+/Ly6CHigh/Low and CD11b+/Cripto+/F4/80+/Ly6CHigh/Low cells at days 2, 3, and 5 after injury. D. Quantification of F4/80+/Ly6CHigh/Low cells in the CD11b+ and CD11b+/Cripto+ cell population from injured muscles at the indicated time points. Data represent mean ± SEM (n = 5 biological replicates; ***P < 0.001, Student's t-test). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Cripto isotype control and Cripto expression by qRT–PCR A. Representative flow cytometry dot plots of Cripto isotype control in CD11b+ cells at days 2, 3, and 5 after injury. PE (Phycoerythrin/free channel). B. Quantitative real-time PCR analysis of Cripto expression profile in Cd11b+/Ly6CLow MPs at days 2, 3, and 5 after injury. Relative mRNA levels were normalized to Gapdh. Data are mean ± SEM (n = 6 biological replicates; **P < 0.01; ***P < 0.00, Student's t-test). Download figure Download PowerPoint The gradual increase in Cripto expression levels in the CD11b+/F4/80+/Ly6CLow MP population was detected by qRT–PCR (Fig EV1B). Collectively, these results indicate that Cripto progressively increased in the immune cells that infiltrate the injured muscle and predominantly accumulated at the surface of anti-inflammatory MPs, concomitantly with their accumulation in the regenerating tissue. Cripto controls MP plasticity To investigate the physiological role of Cripto in the infiltrating MPs without interfering with its activity in the myogenic compartment 19, we generated myeloid-specific Cripto knockout (KO) mice. Tg:Criptofl/fl conditional mice 21 were crossed with the lysozyme M (LysM) Cre line (Tg:LysMCre) in which the Cre recombinase is expressed in the myeloid lineage 22. Homozygous conditional Tg:LysMCre::Cripto−/− mice (CriptoMy-LOF) were born in Mendelian ratio and developed normally. Acute injury was thus induced in tibialis anterior (TA) muscles of CriptoMy-LOF and Control mice by CTX injection and injured muscles were analyzed at days 2 and 5 after injury. Deletion of Cripto in the infiltrated MPs was verified by PCR (Appendix Fig S1A and B). Immunofluorescence analysis of injured muscle sections stained with F4/80 showed no significant difference in the F4/80+ area between CriptoMy-LOF and Control mice at both days 2 and 5 after injury (Fig 2A and B), suggesting that Cripto genetic ablation did not affect MP accumulation. Given the expression of Cripto in the anti-inflammatory MPs, we analyzed this MP population at days 2 and 5 after injury by staining muscle sections for the anti-inflammatory marker CD206 (Fig 2C). While the frequency distribution of CD206+ MPs was comparable in CriptoMy-LOF and Control muscles at day 2 (Fig 2C top panels and D), a significant decrease in CD206+ MPs was observed in CriptoMy-LOF at day 5 (Fig 2C bottom panels and E). In order to specifically identify and characterize Cripto KO MPs, we combined conditional lineage-tracing and genetic ablation of Cripto. CriptoMy-LOF mice were thus crossed with the Tg:R26mTmG transgenic line 23 to obtain the Tg:LysMCre::R26mTmG::Criptofl/fl (GFP-CriptoMy-LOF; Fig EV2A and B) and the Tg:LysMCre::R26mTmG (GFP-Control) as control. We first assessed the efficiency of LysMCre-mediated Cripto deletion by genomic qPCR analysis on Florescence-Activated Cell Sorting (FACS)-sorted F4/80+ MPs (CriptoMy-LOF) or GFP+ monocytes/MPs (GFP-CriptoMy-LOF). Genomic DNA from heterozygous Cripto KO (Cripto+/−) mice was used as reference for 50% of gene deletion. The efficiency of Cripto deletion was ~ 50% in the overall MP population, while it increased up to ~ 80% in the GFP+ population (Fig EV2C). Accordingly, ~ 50% of the MP population expressed the reporter GFP both in GFP-CriptoMy-LOF and GFP-Control mice (Fig EV2D). Finally, we measured Cripto protein by ELISA and found that it was significantly reduced in Cripto KO MPs compared to control (Fig EV2E). Figure 2. Myeloid-specific Cripto deletion perturbs MP plasticity without affecting monocyte/macrophage accumulation A. Representative pictures of immunostaining for F4/80 (green) in Control and CriptoMy-LOF TA sections at days 2 (top panel) and 5 (bottom panel) after injury. B. Quantification of F4/80 staining/damaged area (μm2) at days 2 (top graph) and 5 (bottom graph) after injury. C. Representative pictures of immunostaining for CD206 in Control and CriptoMy-LOF TA sections at days 2 (top panel) and 5 (bottom panel) after injury, respectively. D, E. Quantification of CD206+ MPs per area (mm2) at days 2 (D) and 5 (E) after injury. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5×. Data are expressed as box plots displaying minimum, first quartile, median, third quartile, and maximum (n ≥ 5 biological replicates; **P < 0.01, Student's t-test). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Efficiency of Cripto deletion A. Schematic representation of Tg:LysMCre::R26mTmG::Criptofl/fl (GFP-CriptoMy-LOF) transgenic mice. Red arrows indicate forward and reverse primers used for PCR genotyping. Green arrows indicate forward and reverse primers used to amplify a DNA region encompassing exon 4. Yellow arrows indicate forward and reverse primers used to amplify exon 2, as reference PCR. B. PCR genotyping on DNA extracted from FACS-isolated GFP+ cells from GFP-CriptoMy-LOF and GFP-Control mice at day 2 after injury. Gapdh was used as control. C. Genomic quantitative real-time PCR (qRT–PCR) analysis of wild-type Cripto allele on FACS-isolated F4/80+ (blue bar) or GFP+ (green bar) MPs from CriptoMy-LOF and GFP-CriptoMy-LOF muscles, respectively, at day 2 after injury. Genomic DNA extracted from Cripto KO heterozygous mice (Cripto+/−; black bar) was used as reference of 50% Cripto deletion. Data are expressed as percentage of wild-type allele over the reference PCR and are mean ± SEM (n = 6 biological replicates; ***P < 0.000004; Student's t-test). D. Representative flow cytometry dot plots showing the percentage of F4/80+ cell population and GFP+ cells expressing F4/80 in GFP-Control and GFP-CriptoMy-LOF muscles at day 2 after injury. Data are mean ± SEM (n = 4 biological replicates; P = ns; Student's t-test). E. ELISA-based assay of Cripto protein from total protein extracts of FACS-sorted GFP+ MPs from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data are expressed as box plots displaying minimum, first quartile, median, third quartile, and maximum (n = 5 biological replicates; **P < 0.01; Student's t-test). Download figure Download PowerPoint Given that the vast majority (> 90%) of GFP+ cells expressed F4/80 in both GFP-CriptoMy-LOF and GFP-Control muscles (Fig 3A), we quantified the percentage of GFP+ cells/MPs at both days 2 and 5 after injury and found that it was comparable in the two groups (Fig 3A), thus providing further evidence that CriptoMy-LOF did not affect MP accumulation in the injured muscle. To further investigate the nature of these MPs, CriptoMy-LOF and Control MPs were FACS-isolated by GFP expression, and the expression profile of genes associated with either pro- or anti-inflammatory phenotype analyzed by qRT–PCR at days 2 and 5 after injury. Cripto KO MPs showed a transient increase in the pro-inflammatory marker Nos2 at day 2, while other pro-inflammatory genes (Mcp1, Tnfα) were not affected at both time points (Fig 3B). Of note, at day 2, Arg1 and Fizz1, which identify the CD206+ MPs 24, were similarly expressed in the two groups (Fig 3B), consistent with the equal distribution of the CD206+ MPs at this time point (Fig 2D). Conversely, later on at day 5 both genes were significantly downregulated in Cripto KO cells, whereas other anti-inflammatory markers (Il10, Il4rα, and Tgfβ) were not affected (Fig 3B). These findings were consistent with the reduction of the CD206+ MPs in CriptoMy-LOF (Fig 2E) and provided molecular support for the idea that modulation of MP phenotypic plasticity is perturbed in the absence of Cripto. Figure 3. Cripto controls the proper expansion of CD206+ MPs by modulating TGFβ signaling A. Representative flow cytometry dot plots showing the percentage of GFP+ cell population in GFP-Control and GFP-CriptoMy-LOF muscles at days 2 (top panel) and 5 (bottom panel) after injury. Percentage of GFP+ cells expressing F4/80 in GFP-Control and GFP-CriptoMy-LOF muscles is shown at day 2 after injury (middle panel). Data are mean ± SEM (n = 4 biological replicates; P = ns; Student's t-test). B. qRT–PCR analysis of pro-inflammatory markers (Nos2, Mcp1, and Tnfα) and anti-inflammatory markers (Arg1, Fizz1, Il10, Il4rα, and Tgfβ) in GFP-CriptoMy-LOF and GFP-Control MPs at days 2 and 5 after injury. Data represent mean ± SEM of relative mRNA level normalized with Gapdh (n ≥ 4 biological replicates; ***P < 0.001, Student's t-test). C. Representative pictures of immunostaining for GFP (green), CD206 (red), and pSMAD3 (white) in GFP-Control and GFP-CriptoMy-LOF TA sections at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5×. D. Quantification of GFP+/CD206±/pSMAD3± cell distribution in TA sections from GFP-Control and GFP-CriptoMy-LOF at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Data are mean ± SEM (n = 5 biological replicates; **P < 0.01, Student's t-test). E. Representative flow cytometry dot plots of F4/80+/Ly6CLow cell population gated on GFP+ cells from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data are mean ± SEM (n = 3 biological replicates; P = ns; Student's t-test). F. Smear plot of RNA-seq data from GFP+/F4/80+/Ly6CLow cells FACS sorted from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data show the expression level as log2 fold change (FC) [log2(GFP-Control/GFP-CriptoMy-LOF)], against log2(expression) [log2(average of gene expression across all samples], for each individual gene. The blue lines correspond to LogFC of 1 and −1 (n = 3 biological replicates). Download figure Download PowerPoint Cripto modulates TGFβ signaling in the infiltrating MPs Fine-tuning of the TGFβ signaling plays a key role in shaping MP identity 13, 25 and Cripto is a well-known modulator of TGFβ signaling through interaction with TGF-β family ligands 14. For instance, it is known that Cripto attenuates TGFβ-1 signaling by interfering with its binding to the Act-R II receptors 26. This raised the possibility that Cripto-dependent modulation of TGFβ signaling might play a role in shaping MP plasticity. We therefore investigated the status of TGFβ signaling in Cripto KO infiltrating MPs by looking at the phosphorylation of the TGFβ effector SMAD3. Immunofluorescence analysis of pSMAD3 and CD206 within the GFP+ cell population revealed a significant increase in pSMAD3+ cells in the GFP+/CD206− population of CriptoMy-LOF mice, which inversely correlated with the decrease in GFP/CD206 double-positive cells (Fig 3C and D). Of note, the expression of both Tgfβ and the latent TGFβ binding protein 4 (LTBP4), which binds the latent TGFβ promoting its secretion 13, did not increase in Cripto KO MPs (Fig 3B and Appendix Fig S2), indicating that neither expression nor secretion of TGFβ was affected and suggesting that loss of Cripto induced aberrant activation of the signaling. To further explore this phenotype and based on the observation that Cripto was preferentially expressed in the anti-inflammatory Ly6CLow MPs, concomitantly with their accumulation in the regenerating tissue (Fig 1C), we compared the molecular signature of wild-type and Cripto KO Ly6CLow MPs. Of note, FACS analysis of GFP+ cells showed that the percentage of F4/80+/Ly6CLow MPs was comparable in CriptoMy-LOF and Control at day 5 after injury (75.9 ± 2.2% in GFP-Control vs. 71.7 ± 3.8 in GFP-CriptoMy-LOF; Fig 3E), indicating that Cripto deletion did not affect the proper downregulation of Ly6C expression. To further investigate this issue, GFP+/Ly6CLow cells were FACS-isolated from both groups and total RNA was extracted and analyzed by RNA-seq. Smear plot analysis of differentially expressed genes showed no main differences between Control and Cripto KO dataset (Fig 3F and Dataset EV1), indicating that Cripto KO MPs carried the overall expression signature of the Ly6CLow MPs and suggesting that Cripto was dispensable to induce/maintain the Ly6CLow phenotype. Altogether, these findings indicated that Cripto acts as an extrinsic modulator of MP plasticity and suggested that it is required for the proper expansion/maintenance of the CD206+ anti-inflammatory MP population. Cripto-dependent modulation of MP phenotypes promotes skeletal muscle regeneration in acute injury and disease We have recently shown that Cripto expression in the infiltrated MPs cannot compensate for the lack of Cripto in the myogenic compartment 19, suggesting that Cripto exerts a cell-type-specific role in the regeneration process. To investigate the effect of Cripto-dependent modulation of MP phenotypes in the repair process, we performed morphometric analysis of CriptoMy-LOF and Control TA muscles at days 5 and 30 after CTX-induced injury (CTX-I) (Fig 4A and B). Quantification of the minimal Feret's diameter of centrally nucleated fibers (CNF) showed no significant differences in the two groups at both time points (Fig 4C), indicating that regeneration was proceeding normally in CriptoMy-LOF mice following a single muscle injury. To investigate this phenotype further and given the incomplete deletion of Cripto in the overall MP population (Fig EV2C), we used a more challenging model of re-injury 27. To this end, CriptoMy-LOF and Control TA muscles were injected with CTX and allowed to recover for 30 days; regenerated muscles were then re-injured by a second CTX injection (CTX-II), and the minimal Feret's diameter of CNF was determined at days 5 and 30 after re-injury (Fig 4D and E). Unlike the absence of phenotype observed after a single muscle injury (CTX-I), a significant shift toward smaller regenerating myofibers was detected in CriptoMy-LOF muscles at both days 5 and 30 after re-injury (Fig 4F), which is a clear indication of altered maturation of regenerating myofibers. Furthermore, the CD206+ anti-inflammatory MPs significantly decreased in CriptoMy-LOF TA muscles at day 5 after re-injury (Fig EV3A), consistent with that observed in the single injury model (Figs 2E and 3D). Finally, to test whether myeloid Cripto deficiency may affect the satellite cell compartment, we analyzed the distribution of Pax7+ satellite cells. A transient reduction in Pax7+ cells was observed in CriptoMy-LOF at day 5 after single injury, which was recovered at late time points (Fig EV3B and C). Figure 4. Effect of myeloid-specific Cripto deletion on skeletal muscle regeneration process A. Schematic representation of the experimental design in the single injury model (CTX-I). B. Representative pictures of double immunostaining with laminin (green) and embryonic myosin heavy chain (eMHC; red) at days 5 (left panel) and 30 (right panel) after cardiotoxin (CTX) injection. C. Minimal Feret's diameter distribution of Control and CriptoMy-LOF centrally nucleated myofibers at days 5 (top panel) and 30 (bottom panel) after CTX injection. Data are mean ± SEM (n = 5 biological replicates; P = ns, Student's t-test). D. Schematic representation of the experimental design in the re-injury model (CTX-II). E. Representative pictures of double immunostaining with laminin (green) and eMHC (red) at days 5 (left panel) and 30 (right panel) after re-injury (CTX-II). F. Minimal Feret's diameter distribution of Control and CriptoMy-LOF centrally nucleated myofibers at days 5 (top panel) and 30 (bottom panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Data are mean ± SEM (n = 5 biological replicates; *P < 0.05, Student's t-test
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