Origins and functions of eosinophils in two non-mucosal tissues.

Eosinophils are a type of granulocyte named after the presence of their eosin-stained granules. Traditionally, eosinophils have been best known to play prominent roles in anti-parasitic responses and mediating allergic reactions. Knowledge of their behaviour has expanded with time, and they are now recognized to play integral parts in the homeostasis of gastrointestinal, respiratory, skeletal muscle, adipose, and connective tissue systems. As such, they are implicated in a myriad of pathologies, and have been the target of several medical therapies. This review focuses on the lifespan of eosinophils, from their origins in the bone marrow, to their tissue-resident role. In particular, we wish to highlight the functions of eosinophils in non-mucosal tissues with skeletal muscle and the adipose tissues as examples, and to discuss the current understanding of their participation in diseased states in these tissues.

Rebuilding and rebooting immunity with stem cells.

Advances in modern medicine have enabled a rapid increase in lifespan and, consequently, have highlighted the immune system as a key driver of age-related disease. Immune regeneration therapies present exciting strategies to address age-related diseases by rebooting the host's primary lymphoid tissues or rebuilding the immune system directly via biomaterials or artificial tissue. Here, we identify important, unanswered questions regarding the safety and feasibility of these therapies. Further, we identify key design parameters that should be primary considerations guiding technology design, including timing of application, interaction with the host immune system, and functional characterization of the target patient population.

Fibroblast and myofibroblast activation in normal tissue repair and fibrosis.

The term 'fibroblast' often serves as a catch-all for a diverse array of mesenchymal cells, including perivascular cells, stromal progenitor cells and bona fide fibroblasts. Although phenotypically similar, these subpopulations are functionally distinct, maintaining tissue integrity and serving as local progenitor reservoirs. In response to tissue injury, these cells undergo a dynamic fibroblast-myofibroblast transition, marked by extracellular matrix secretion and contraction of actomyosin-based stress fibres. Importantly, whereas transient activation into myofibroblasts aids in tissue repair, persistent activation triggers pathological fibrosis. In this Review, we discuss the roles of mechanical cues, such as tissue stiffness and strain, alongside cell signalling pathways and extracellular matrix ligands in modulating myofibroblast activation and survival. We also highlight the role of epigenetic modifications and myofibroblast memory in physiological and pathological processes. Finally, we discuss potential strategies for therapeutically interfering with these factors and the associated signal transduction pathways to improve the outcome of dysregulated healing.

Cellular activation patterns of CD10+ fibro-adipogenic progenitors across acquired disease states in human skeletal muscle biopsies.

Background: Fibro-adipogenic progenitors (FAP) are muscle resident mesenchymal stem cells pivotal for regulation of myofiber repair. Experimental results show in addition involvement in a range of other pathological conditions and potential for pharmacological intervention. FAP histopathology in human muscle biopsies is largely unknown, but has potential to inform translational research. Methods: CD10+ FAPs in 32 archival muscle biopsies from 8 groups (normal, dermatomyositis, inclusion body myositis (IBM), anti-synthetase syndrome, immune-mediated necrotizing myopathy (IMNM), denervation, type 2 atrophy, rhabdomyolysis) were visualized by CD10 immunohistochemistry and their histology compared. Groups are compared by semi-quantitative scoring. Results: Histological activation of endomysial CD10+ FAPs includes prominent expansion of a network of cell processes surrounding muscle fibers, as well as endomysial cell clusters evidencing proliferation. Prominence of periarteriolar processes is a notable feature in some pathologies. FAP activation is often associated with fiber degeneration/regeneration, foci of inflammation, and denervation in keeping with experimental results. Type 2 atrophy shows no evidence of FAP activation. Dermatomyositis and anti-synthetase syndrome associated myositis demonstrate diffuse activation. Conclusion: Assessment of CD10+ FAP activation is routinely possible using CD10 immunohistochemistry and demonstrates several patterns in keeping with preclinical results. Prominent expansion of FAP processes surrounding myofibers suggests enhanced interaction between myofiber/basement membranes and FAPs during activation. The presence of diffuse FAP activation in dermatomyositis biopsies unrelated to fiber repair raises the possibility of FAP activation as part of the autoimmune process. Future diagnostic applications, clinical significance and therapeutic potential remain to be elucidated.

DPPIV+ fibro-adipogenic progenitors form the niche of adult skeletal muscle self-renewing resident macrophages.

Adult tissue-resident macrophages (RMs) are either maintained by blood monocytes or through self-renewal. While the presence of a nurturing niche is likely crucial to support the survival and function of self-renewing RMs, evidence regarding its nature is limited. Here, we identify fibro-adipogenic progenitors (FAPs) as the main source of colony-stimulating factor 1 (CSF1) in resting skeletal muscle. Using parabiosis in combination with FAP-deficient transgenic mice (PdgfrαCreERT2 × DTA) or mice lacking FAP-derived CSF1 (PdgfrαCreERT2 × Csf1flox/null), we show that local CSF1 from FAPs is required for the survival of both TIM4- monocyte-derived and TIM4+ self-renewing RMs in adult skeletal muscle. The spatial distribution and number of TIM4+ RMs coincide with those of dipeptidyl peptidase IV (DPPIV)+ FAPs, suggesting their role as CSF1-producing niche cells for self-renewing RMs. This finding identifies opportunities to precisely manipulate the function of self-renewing RMs in situ to further unravel their role in health and disease.

Protocol for generation of a time-resolved cellular interactome during tissue remodeling in adult mice.

Efficient skeletal muscle regeneration necessitates fine-tuned coordination among multiple cell types through an intricate network of intercellular communication. We present a protocol for generation of a time-resolved cellular interactome during tissue remodeling. We describe steps for isolating distinct cell populations from skeletal muscle of adult mice after acute damage and extracting RNA from purified cells prior to the generation of RNA sequencing data. We then detail procedures for generating and deciphering a time- and lineage-resolved model of intercellular crosstalk. For complete details on the use and execution of this protocol, please refer to Groppa et al. (2023).1.

Activation of ß-catenin in mesenchymal progenitors leads to muscle mass loss.

Loss of muscle mass is a common manifestation of chronic disease. We find the canonical Wnt pathway to be activated in mesenchymal progenitors (MPs) from cancer-induced cachectic mouse muscle. Next, we induce ß-catenin transcriptional activity in murine MPs. As a result, we observe expansion of MPs in the absence of tissue damage, as well as rapid loss of muscle mass. Because MPs are present throughout the organism, we use spatially restricted CRE activation and show that the induction of tissue-resident MP activation is sufficient to induce muscle atrophy. We further identify increased expression of stromal NOGGIN and ACTIVIN-A as key drivers of atrophic processes in myofibers, and we verify their expression by MPs in cachectic muscle. Finally, we show that blocking ACTIVIN-A rescues the mass loss phenotype triggered by ß-catenin activation in MPs, confirming its key functional role and strengthening the rationale for targeting this pathway in chronic disease.

Spatial compartmentalization of signaling imparts source-specific functions on secreted factors.

Efficient regeneration requires multiple cell types acting in coordination. To better understand the intercellular networks involved and how they change when regeneration fails, we profile the transcriptome of hematopoietic, stromal, myogenic, and endothelial cells over 14 days following acute muscle damage. We generate a time-resolved computational model of interactions and identify VEGFA-driven endothelial engagement as a key differentiating feature in models of successful and failed regeneration. In addition, the analysis highlights that the majority of secreted signals, including VEGFA, are simultaneously produced by multiple cell types. To test whether the cellular source of a factor determines its function, we delete VEGFA from two cell types residing in close proximity: stromal and myogenic progenitors. By comparing responses to different types of damage, we find that myogenic and stromal VEGFA have distinct functions in regeneration. This suggests that spatial compartmentalization of signaling plays a key role in intercellular communication networks.

Methylation of the Hippo effector YAP by the methyltransferase SETD7 drives myocardial ischaemic injury: a translational study.

AIMS: Methylation of non-histone proteins is emerging as a central regulatory mechanism in health and disease. The methyltransferase SETD7 has shown to methylate and alter the function of a variety of proteins in vitro; however, its function in the heart is poorly understood. The present study investigates the role of SETD7 in myocardial ischaemic injury. METHODS AND RESULTS: Experiments were performed in neonatal rat ventricular myocytes (NRVMs), SETD7 knockout mice (SETD7-/-) undergoing myocardial ischaemia/reperfusion (I/R) injury, left ventricular (LV) myocardial samples from patients with ischaemic cardiomyopathy (ICM), and peripheral blood mononuclear cells (PBMCs) from patients with ST-elevation MI (STEMI). We show that SETD7 is activated upon energy deprivation in cultured NRVMs and methylates the Hippo pathway effector YAP, leading to its cytosolic retention and impaired transcription of antioxidant genes manganese superoxide dismutase (MnSOD) and catalase (CAT). Such impairment of antioxidant defence was associated with mitochondrial reactive oxygen species (mtROS), organelle swelling, and apoptosis. Selective pharmacological inhibition of SETD7 by (R)-PFI-2 restored YAP nuclear localization, thus preventing mtROS, mitochondrial damage, and apoptosis in NRVMs. In mice, genetic deletion of SETD7 attenuated myocardial I/R injury, mtROS, and LV dysfunction by restoring YAP-dependent transcription of MnSOD and CAT. Moreover, in cardiomyocytes isolated from I/R mice and ICM patients, (R)-PFI-2 prevented mtROS accumulation, while improving Ca2+-activated tension. Finally, SETD7 was up-regulated in PBMCs from STEMI patients and negatively correlated with MnSOD and CAT. CONCLUSION: We show a methylation-dependent checkpoint regulating oxidative stress during myocardial ischaemia. SETD7 inhibition may represent a valid therapeutic strategy in this setting.

TNFα and IFNγ cooperate for efficient pro- to anti-inflammatory transition of macrophages during muscle regeneration.

IFNγ is traditionally known as a proinflammatory cytokine with diverse roles in antimicrobial and antitumor immunity. Yet, findings regarding its sources and functions during the regeneration process following a sterile injury are conflicting. Here, we show that natural killer (NK) cells are the main source of IFNγ in regenerating muscle. Beyond this cell population, IFNγ production is limited to a small population of T cells. We further show that NK cells do not play a major role in muscle regeneration following an acute injury or in dystrophic mice. Surprisingly, the absence of IFNγ per se also has no effect on muscle regeneration following an acute injury. However, the role of IFNγ is partially unmasked when TNFα is also neutralized, suggesting a compensatory mechanism. Using transgenic mice, we showed that conditional inhibition of IFNGR1 signaling in muscle stem cells or fibro-adipogenic progenitors does not play a major role in muscle regeneration. In contrast to common belief, we found that IFNγ is not present in the early inflammatory phase of the regeneration process but rather peaks when macrophages are acquiring an anti-inflammatory phenotype. Further transcriptomic analysis suggests that IFNγ cooperates with TNFα to regulate the transition of macrophages from pro- to anti-inflammatory states. The absence of the cooperative effect of these cytokines on macrophages, however, does not result in significant regeneration impairment likely due to the presence of other compensatory mechanisms. Our findings support the arising view of IFNγ as a pleiotropic inflammatory regulator rather than an inducer of the inflammatory response.

Thermoneutral Housing and a Western Diet Combination Exacerbates Dysferlin-Deficient Muscular Dystrophy.

INTRODUCTION/AIMS: Most mouse models of muscular dystrophy (MD) show mild phenotypes, which limits the translatability of experimental therapies to patients. A growing body of evidence suggests that MD is accompanied by metabolic abnormalities that could potentially exacerbate the primary muscle wasting process. Since thermoneutral (TN) housing of mice (~30°C) has been shown to affect many metabolic parameters, particularly when combined with a Western diet (WD), our aim was to determine whether the combination of TN and WD exacerbates muscle wasting in dysferlin-deficient BLAJ mice, a common model of limb-girdle MD type 2b (LGMD2b). METHODS: The 2-mo-old wild-type (WT) and BLAJ mice were housed at TN or room temperature (RT) and fed a WD or regular chow for 9 mo. Ambulatory function, muscle histology, and protein immunoblots of skeletal muscle were assessed. RESULTS: BLAJ mice at RT and fed a chow diet showed normal ambulation function similar to WT mice, whereas 90% of BLAJ mice under WD and TN combination showed ambulatory dysfunction (p < 0.001), and an up to 4.1-fold increase in quadriceps and gastrocnemius fat infiltration. Western blotting revealed decreased autophagy marker microtubules-associated protein 1 light chain 3-B (LC3BII/LC3BI) ratio and up-regulation of protein kinase B/AKT and ribosomal protein S6 phosphorylation, suggesting inefficient cellular debris and protein clearance in TN BLAJ mice fed a WD. Male and female BLAJ mice under TN and WD combination showed heterogenous fibro-fatty infiltrate composition. DISCUSSION: TN and WD combination exacerbates rodent LGMD2b without affecting WT mice. This improves rodent modeling of human MD and helps elucidate how metabolic abnormalities may play a causal role in muscle wasting.

Pleiotropic activation of endothelial function by angiotensin II receptor blockers is crucial to their protective anti-vascular remodeling effects.

There are no therapeutics that directly enhance chronic endothelial nitric oxide (NO) release, which is typically associated with vascular homeostasis. In contrast, angiotensin II (AngII) receptor type 1 (AT1R) blockers (ARBs) can attenuate AngII-mediated oxidative stress, which often leads to increased endothelial NO bioavailability. Herein, we investigate the potential presence of direct, AngII/AT1R-independent ARB class effects on endothelial NO release and how this may result in enhanced aortic wall homeostasis and endothelial NO-specific transcriptome changes. Treatment of mice with four different ARBs induced sustained, long-term inhibition of vascular contractility by up to 82% at 16 weeks and 63% at 2 weeks, an effect reversed by L-NAME and absent in endothelial NO synthase (eNOS) KO mice or angiotensin converting enzyme inhibitor captopril-treated animals. In absence of AngII or in tissues with blunted AT1R expression or incubated with an AT2R blocker, telmisartan reduced vascular tone, supporting AngII/AT1R-independent pleiotropism. Finally, telmisartan was able to inhibit aging- and Marfan syndrome (MFS)-associated aortic root widening in NO-sensitive, BP-independent fashions, and correct aberrant TGF-ß signaling. RNAseq analyses of aortic tissues identified early eNOS-specific transcriptome reprogramming of the aortic wall in response to telmisartan. This study suggests that ARBs are capable of major class effects on vasodilatory NO release in fashions that may not involve blockade of the AngII/AT1R pathway. Broader prophylactic use of ARBs along with identification of non-AngII/AT1R pathways activated by telmisartan should be investigated.

Metabolic reprogramming of skeletal muscle by resident macrophages points to CSF1R inhibitors as muscular dystrophy therapeutics.

The role of tissue-resident macrophages during tissue regeneration or fibrosis is not well understood, mainly due to the lack of a specific marker for their identification. Here, we identified three populations of skeletal muscle-resident myelomonocytic cells: a population of macrophages positive for lymphatic vessel endothelial receptor 1 (LYVE1) and T cell membrane protein 4 (TIM4 or TIMD4), a population of LYVE1-TIM4- macrophages, and a population of cells likely representing dendritic cells that were positive for CD11C and major histocompatibility complex class II (MHCII). Using a combination of parabiosis and lineage-tracing experiments, we found that, at steady state, TIM4- macrophages were replenished from the blood, whereas TIM4+ macrophages locally self-renewed [self-renewing resident macrophages (SRRMs)]. We further showed that Timd4 could be reliably used to distinguish SRRMs from damage-induced infiltrating macrophages. Using a colony-stimulating factor 1 receptor (CSF1R) inhibition/withdrawal approach to specifically deplete SRRMs, we found that SRRMs provided a nonredundant function in clearing damage-induced apoptotic cells early after extensive acute injury. In contrast, in chronic mild injury as seen in a mouse model of Duchenne muscular dystrophy, depletion of both TIM4-- and TIM4+-resident macrophage populations through long-term CSF1R inhibition changed muscle fiber composition from damage-sensitive glycolytic fibers toward damage-resistant glycolytic-oxidative fibers, thereby protecting muscle against contraction-induced injury both ex vivo and in vivo. This work reveals a previously unidentified role for resident macrophages in modulating tissue metabolism and may have therapeutic potential given the ongoing clinical testing of CSF1R inhibitors.

Macrophages in Skeletal Muscle Dystrophies, An Entangled Partner.

While skeletal muscle remodeling happens throughout life, diseases that result in its dysfunction are accountable for many deaths. Indeed, skeletal muscle is exceptionally capable to respond to stimuli modifying its homeostasis, such as in atrophy, hypertrophy, regeneration and repair. In particular conditions such as genetic diseases (muscular dystrophies), skeletal muscle's capacity to remodel is strongly affected and undergoes continuous cycles of chronic damage. This induces scarring, fatty infiltration, as well as loss of contractibility and of the ability to generate force. In this context, inflammation, primarily mediated by macrophages, plays a central pathogenic role. Macrophages contribute as the primary regulators of inflammation during skeletal muscle regeneration, affecting tissue-resident cells such as myogenic cells and endothelial cells, but also fibro-adipogenic progenitors, which are the main source of the fibro fatty scar. During skeletal muscle regeneration their function is tightly orchestrated, while in dystrophies their fate is strongly disturbed, resulting in chronic inflammation. In this review, we will discuss the latest findings on the role of macrophages in skeletal muscle diseases, and how they are regulated.

Emerging skeletal muscle stromal cell diversity: Functional divergence in fibro/adipogenic progenitor and mural cell populations.

While the majority of healthy skeletal muscle consists of multinucleated syncytial repetitive contractile myofibers, repaired by skeletal muscle stem cells when damaged, the maintenance of muscle function also requires a range of tissue-resident stromal populations. In fact, the careful orchestration of damage response processes upon muscle injury relies heavily on stromal cell contribution for effective repair. The two main types of muscle-resident stromal cells are fibro/adipogenic progenitors and mural cells. The latter is comprised of pericytes and vascular smooth muscle cells. Recent publications identifying common markers for stromal cell populations have allowed investigating population dynamics throughout the regenerative process at a higher resolution. Mounting evidence now suggests that subpopulations with distinct roles may exist among stromal cells. In various degenerative muscle wasting conditions, critical cross-talk and spatial signalling amongst various cell populations become dysregulated. This can result in the failure to curb pathological fibro/adipogenic progenitor proliferation and propensity for laying down excessive extracellular matrix, which in turn leads to fibrotic infiltration, reduced contractile units and gradual decline in muscle function. Restoration of physiologically appropriate stromal cell function is therefore just as crucial for therapeutic targeting as the homeostatic maintenance of muscle function.

Human skeletal muscle CD90+ fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients.

Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)-the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by in vitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle.

Multipotent stromal cells: One name, multiple identities.

Multipotent stromal cells (MSCs) are vital for development, maintenance, function, and regeneration of most tissues. They can differentiate along multiple connective lineages, but unlike most other stem/progenitor cells, they carry out various other functions while maintaining their developmental potential. MSCs function as damage sensors, respond to injury by fostering regeneration through secretion of trophic factors as well as extracellular matrix (ECM) molecules, and contribute to fibrotic reparative processes when regeneration fails. Tissue-specific MSC identity, fate(s), and function(s) are being resolved through fate mapping coupled with single cell "omics," providing unparalleled insights into the secret lives of tissue-resident MSCs.

Origins, potency, and heterogeneity of skeletal muscle fibro-adipogenic progenitors-time for new definitions.

Striated muscle is a highly plastic and regenerative organ that regulates body movement, temperature, and metabolism-all the functions needed for an individual's health and well-being. The muscle connective tissue's main components are the extracellular matrix and its resident stromal cells, which continuously reshape it in embryonic development, homeostasis, and regeneration. Fibro-adipogenic progenitors are enigmatic and transformative muscle-resident interstitial cells with mesenchymal stem/stromal cell properties. They act as cellular sentinels and physiological hubs for adult muscle homeostasis and regeneration by shaping the microenvironment by secreting a complex cocktail of extracellular matrix components, diffusible cytokines, ligands, and immune-modulatory factors. Fibro-adipogenic progenitors are the lineage precursors of specialized cells, including activated fibroblasts, adipocytes, and osteogenic cells after injury. Here, we discuss current research gaps, potential druggable developments, and outstanding questions about fibro-adipogenic progenitor origins, potency, and heterogeneity. Finally, we took advantage of recent advances in single-cell technologies combined with lineage tracing to unify the diversity of stromal fibro-adipogenic progenitors. Thus, this compelling review provides new cellular and molecular insights in comprehending the origins, definitions, markers, fate, and plasticity of murine and human fibro-adipogenic progenitors in muscle development, homeostasis, regeneration, and repair.

Migration of Lung Resident Group 2 Innate Lymphoid Cells Link Allergic Lung Inflammation and Liver Immunity.

Group 2 innate lymphoid cells (ILC2s) are tissue resident in the lung and activated by inhaled allergens via epithelial-derived alarmins including IL-33. Activated ILC2s proliferate, produce IL-5 and IL-13, and induce eosinophilic inflammation. Here, we report that intranasal IL-33 or the protease allergen papain administration resulted in increased numbers of ILC2s not only in the lung but also in peripheral blood and liver. Analyses of IL-33 treated parabiosis mice showed that the increase in lung ILC2s was due to proliferation of lung resident ILC2s, whereas the increase in liver ILC2s was due to the migration of activated lung ILC2s. Lung-derived ILC2s induced eosinophilic hepatitis and expression of fibrosis-related genes. Intranasal IL-33 pre-treatment also attenuated concanavalin A-induced acute hepatitis and cirrhosis. These results suggest that activated lung resident ILC2s emigrate from the lung, circulate, settle in the liver and promote type 2 inflammation and attenuate type 1 inflammation.