Non-myogenic mesenchymal cells contribute to muscle degeneration in facioscapulohumeral muscular dystrophy patients.

Muscle-resident non-myogenic mesenchymal cells play key roles that drive successful tissue regeneration within the skeletal muscle stem cell niche. These cells have recently emerged as remarkable therapeutic targets for neuromuscular disorders, although to date they have been poorly investigated in facioscapulohumeral muscular dystrophy (FSHD). In this study, we characterised the non-myogenic mesenchymal stromal cell population in FSHD patients' muscles with signs of disease activity, identified by muscle magnetic resonance imaging (MRI), and compared them with those obtained from apparently normal muscles of FSHD patients and from muscles of healthy, age-matched controls. Our results showed that patient-derived cells displayed a distinctive expression pattern of mesenchymal markers, along with an impaired capacity to differentiate towards mature adipocytes in vitro, compared with control cells. We also demonstrated a significant expansion of non-myogenic mesenchymal cells (identified as CD201- or PDGFRA-expressing cells) in FSHD muscles with signs of disease activity, which correlated with the extent of intramuscular fibrosis. In addition, the accumulation of non-myogenic mesenchymal cells was higher in FSHD muscles that deteriorate more rapidly. Our results prompt a direct association between an accumulation, as well as an altered differentiation, of non-myogenic mesenchymal cells with muscle degeneration in FSHD patients. Elucidating the mechanisms and cellular interactions that are altered in the affected muscles of FSHD patients could be instrumental to clarify disease pathogenesis and identifying reliable novel therapeutic targets.

Vitamin C Deficiency in Patients With Acute Myeloid Leukemia.

Vitamin C has been shown to play a significant role in suppressing progression of leukemia through epigenetic mechanisms. We aimed to study the role of vitamin C in acute myeloid leukemia (AML) biology and clinical course. To this purpose, the plasma levels of vitamin C at diagnosis in 62 patients with AML (including 5 cases with acute promyelocytic leukemia, APL),7 with myelodysplastic syndrome (MDS), and in 15 healthy donors (HDs) were studied. As controls, vitamins A and E levels were analysed. Expression of the main vitamin C transporters and of the TET2 enzyme were investigated by a specific RQ-PCR while cytoplasmic vitamin C concentration and its uptake were studied in mononuclear cells (MNCs), lymphocytes and blast cells purified from AML samples, and MNCs isolated from HDs. There were no significant differences in vitamin A and E serum levels between patients and HDs. Conversely, vitamin C concentration was significantly lower in AML as compared to HDs (p<0.0001), inversely correlated with peripheral blast-counts (p=0.029), significantly increased at the time of complete remission (CR) (p=0.04) and further decreased in resistant disease (p=0.002). Expression of the main vitamin C transporters SLC23A2, SLC2A1 and SLC2A3 was also significantly reduced in AML compared to HDs. In this line, cytoplasmic vitamin C levels were also significantly lower in AML-MNCs versus HDs, and in sorted blasts compared to normal lymphocytes in individual patients. No association was found between vitamin C plasma levels and the mutation profile of AML patients, as well as when considering cytogenetics or 2017 ELN risk stratification groups. Finally, vitamin C levels did not play a predictive role for overall or relapse-free survival. In conclusion, our study shows that vitamin C levels are significantly decreased in patients with AML at the time of initial diagnosis, further decrease during disease progression and return to normal upon achievement of CR. Correspondingly, low intracellular levels may mirror increased vitamin C metabolic consumption in proliferating AML cells.

Human Conventional and Plasmacytoid Dendritic Cells Differ in Their Ability to Respond to Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a commensal yeast colonizer of mucosal surfaces and an emerging opportunistic pathogen in the mucosa and bloodstream. The role of S. cerevisiae has been largely characterized in peripheral blood mononuclear cells and monocyte-derived dendritic cells, where yeast cells induce the production of inflammatory cytokines through the interaction with mannose receptors, chitin receptors, DC SIGN, and dectin1. However, the response of blood-circulating dendritic cells (DCs) to S. cerevisiae has never been investigated. Among blood DCs, conventional DCs (cDCs) are producers of inflammatory cytokines, while plasmacytoid DCs (pDCs) are a specialized population producing a large amount of interferon (IFN)-α, which is involved in the antiviral immune response. Here we report that both human DC subsets are able to sense S. cerevisiae. In particular, cDCs produce interleukin (IL)-6, express activation markers, and promotes T helper 17 cell polarization in response to yeasts, behaving similarly to monocyte-derived DCs as previously described. Interestingly, pDCs, not cDCs, sense fungal nucleic acids, leading to the generation of P1-pDCs (PD-L1+CD80-), a pDC subset characterized by the production of IFN-α and the induction of a Th profile producing IL-10. These results highlight a novel role of pDCs in response to S. cerevisiae that could be important for the regulation of the host microbiota-immune system balance and of anti-fungal immune response.

Characterization of FLT3-ITDmut acute myeloid leukemia: molecular profiling of leukemic precursor cells.

Acute myeloid leukemia (AML) with FLT3-ITD mutations (FLT3-ITDmut) remains a therapeutic challenge, with a still high relapse rate, despite targeted treatment with tyrosine kinase inhibitors. In this disease, the CD34/CD123/CD25/CD99+ leukemic precursor cells (LPCs) phenotype predicts for FLT3-ITD-positivity. The aim of this study was to characterize the distribution of FLT3-ITD mutation in different progenitor cell subsets to shed light on the subclonal architecture of FLT3-ITDmut AML. Using high-speed cell sorting, we sequentially purified LPCs and CD34+ progenitors in samples from patients with FLT3-ITDmut AML (n = 12). A higher FLT3-ITDmut load was observed within CD34/CD123/CD25/CD99+ LPCs, as compared to CD34+ progenitors (CD123+/-,CD25-,CD99low/-) (p = 0.0005) and mononuclear cells (MNCs) (p < 0.0001). This was associated with significantly increased CD99 mean fluorescence intensity in LPCs. Significantly higher FLT3-ITDmut burden was also observed in LPCs of AML patients with a small FLT3-ITDmut clones at diagnosis. On the contrary, the mutation burden of other myeloid genes was similar in MNCs, highly purified LPCs and/or CD34+ progenitors. Treatment with an anti-CD99 mAb was cytotoxic on LPCs in two patients, whereas there was no effect on CD34+ cells from healthy donors. Our study shows that FLT3-ITD mutations occur early in LPCs, which represent the leukemic reservoir. CD99 may represent a new therapeutic target in FLT3-ITDmut AML.

Lack of PKCθ Promotes Regenerative Ability of Muscle Stem Cells in Chronic Muscle Injury.

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by muscle wasting and chronic inflammation, leading to impaired satellite cells (SCs) function and exhaustion of their regenerative capacity. We previously showed that lack of PKCθ in mdx mice, a mouse model of DMD, reduces muscle wasting and inflammation, and improves muscle regeneration and performance at early stages of the disease. In this study, we show that muscle regeneration is boosted, and fibrosis reduced in mdxθ-/- mice, even at advanced stages of the disease. This phenotype was associated with a higher number of Pax7 positive cells in mdxθ-/- muscle compared with mdx muscle, during the progression of the disease. Moreover, the expression level of Pax7 and Notch1, the pivotal regulators of SCs self-renewal, were upregulated in SCs isolated from mdxθ-/- muscle compared with mdx derived SCs. Likewise, the expression of the Notch ligands Delta1 and Jagged1 was higher in mdxθ-/- muscle compared with mdx. The expression level of Delta1 and Jagged1 was also higher in PKCθ-/- muscle compared with WT muscle following acute injury. In addition, lack of PKCθ prolonged the survival and sustained the differentiation of transplanted myogenic progenitors. Overall, our results suggest that lack of PKCθ promotes muscle repair in dystrophic mice, supporting stem cells survival and maintenance through increased Delta-Notch signaling.

Dysfunctional polycomb transcriptional repression contributes to lamin A/C-dependent muscular dystrophy.

Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the lamin A/C gene cause several diseases belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of lamin A-dependent dystrophies are still largely unknown. The polycomb group (PcG) of proteins are epigenetic repressors and lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss muscular dystrophy (EDMD), we show here that lamin A loss deregulated PcG positioning in muscle satellite stem cells, leading to derepression of non-muscle-specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional program caused impairment in self-renewal, loss of cell identity, and premature exhaustion of the quiescent satellite cell pool. Genetic ablation of the Cdkn2a locus restored muscle stem cell properties in lamin A/C-null dystrophic mice. Our findings establish a direct link between lamin A and PcG epigenetic silencing and indicate that lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.

Macrophages fine tune satellite cell fate in dystrophic skeletal muscle of mdx mice.

Satellite cells (SCs) are muscle stem cells that remain quiescent during homeostasis and are activated in response to acute muscle damage or in chronic degenerative conditions such as Duchenne Muscular Dystrophy. The activity of SCs is supported by specialized cells which either reside in the muscle or are recruited in regenerating skeletal muscles, such as for instance macrophages (MΦs). By using a dystrophic mouse model of transient MΦ depletion, we describe a shift in identity of muscle stem cells dependent on the crosstalk between MΦs and SCs. Indeed MΦ depletion determines adipogenic conversion of SCs and exhaustion of the SC pool leading to an exacerbated dystrophic phenotype. The reported data could also provide new insights into therapeutic approaches targeting inflammation in dystrophic muscles.

Distinct Expression of Inflammatory Features in T Helper 17 Cells from Multiple Sclerosis Patients.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). T helper (Th) 17 lymphocytes play a role in the pathogenesis of MS. Indeed, Th17 cells are abundant in the cerebrospinal fluid and peripheral blood of MS patients and promote pathogenesis in the mouse model of MS. To gain insight into the function of Th17 cells in MS, we tested whether Th17 cells polarized from naïve CD4 T cells of healthy donors and MS patients display different features. To this end, we analysed several parameters that typify the Th17 profile during the differentiation process of naïve CD4 T cells obtained from relapsing-remitting (RR)-MS patients (n = 31) and healthy donors (HD) (n = 28). Analysis of an array of cytokines produced by Th17 cells revealed that expression of interleukin (IL)-21, tumour necrosis factor (TNF)-ß, IL-2 and IL-1R1 is significantly increased in Th17 cells derived from MS patients compared to healthy donor-derived cells. Interestingly, IL-1R1 expression is also increased in Th17 cells circulating in the blood of MS patients compared to healthy donors. Since IL-2, IL-21, TNF-ß, and IL-1R1 play a crucial role in the activation of immune cells, our data indicate that high expression of these molecules in Th17 cells from MS patients could be related to their high inflammatory status.

AMBRA1 Controls Regulatory T-Cell Differentiation and Homeostasis Upstream of the FOXO3-FOXP3 Axis.

Regulatory T cells (Treg) are necessary to maintain immunological tolerance and are key players in the control of autoimmune disease susceptibility. Expression of the transcription factor FOXP3 is essential for differentiation of Treg cells and indispensable for their suppressive function. However, there is still a lack of knowledge about the mechanisms underlying its regulation. Here, we demonstrate that pro-autophagy protein AMBRA1 is also a key modulator of T cells, regulating the complex network that leads to human Treg differentiation and maintenance. Indeed, through its ability to interact with the phosphatase PP2A, AMBRA1 promotes the stability of the transcriptional activator FOXO3, which, in turn, triggers FOXP3 transcription. Furthermore, we found that AMBRA1 plays a significant role in vivo by regulating Treg cell induction in mouse models of both tumor growth and multiple sclerosis, thus highlighting the role of AMBRA1 in the control of immune homeostasis.

Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy.

Fibro-adipogenic progenitors (FAPs) are currently defined by their anatomical position, expression of non-specific membrane-associated proteins, and ability to adopt multiple lineages in vitro. Gene expression analysis at single-cell level reveals that FAPs undergo dynamic transitions through a spectrum of cell states that can be identified by differential expression levels of Tie2 and Vcam1. Different patterns of Vcam1-negative Tie2high or Tie2low and Tie2low/Vcam1-expressing FAPs are detected during neonatal myogenesis, response to acute injury and Duchenne Muscular Dystrophy (DMD). RNA sequencing analysis identified cell state-specific transcriptional profiles that predict functional interactions with satellite and inflammatory cells. In particular, Vcam1-expressing FAPs, which exhibit a pro-fibrotic expression profile, are transiently activated by acute injury in concomitance with the inflammatory response. Aberrant persistence of Vcam1-expressing FAPs is detected in DMD muscles or upon macrophage depletion, and is associated with muscle fibrosis, thereby revealing how disruption of inflammation-regulated FAPs dynamics leads to a pathogenic outcome.

Denervation-activated STAT3-IL-6 signalling in fibro-adipogenic progenitors promotes myofibres atrophy and fibrosis.

Fibro-adipogenic progenitors (FAPs) are typically activated in response to muscle injury, and establish functional interactions with inflammatory and muscle stem cells (MuSCs) to promote muscle repair. We found that denervation causes progressive accumulation of FAPs, without concomitant infiltration of macrophages and MuSC-mediated regeneration. Denervation-activated FAPs exhibited persistent STAT3 activation and secreted elevated levels of IL-6, which promoted muscle atrophy and fibrosis. FAPs with aberrant activation of STAT3-IL-6 signalling were also found in mouse models of spinal cord injury, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS) and in muscles of ALS patients. Inactivation of STAT3-IL-6 signalling in FAPs effectively countered muscle atrophy and fibrosis in mouse models of acute denervation and ALS (SODG93A mice). Activation of pathogenic FAPs following loss of integrity of neuromuscular junctions further illustrates the functional versatility of FAPs in response to homeostatic perturbations and suggests their potential contribution to the pathogenesis of neuromuscular diseases.

Modulation of monocytes by bioactive lipid anandamide in multiple sclerosis involves distinct Toll-like receptors.

Monocytes are believed to be involved in the immunopathogenesis of multiple sclerosis (MS). The aim of this study was to investigate their role in MS and their immunomodulation by the endocannabinoid system (ECS), a novel target for the treatment of this disease. We compared the level of cytokine production from monocytes in healthy subjects and MS patients upon stimulation with viral or bacterial Toll-like receptors (TLR) and we evaluated the ECS immunomodulatory role in these cells. Here we show that MS monocytes produced more TNF-α, IL-12 and IL-6 following activation of TLR2/4 with LPS or of TLR5 with flagellin, as opposed to TLR7/8 stimulation with R848. Furthermore AEA, the main endocannabinoid, suppressed cytokine production and release from healthy monocytes upon stimulation with both bacterial and viral TLR receptors but not in cells from MS patients, where its immunosuppressive activity was TLR7/8-dependent. Altered expression levels of key ECS members in MS monocytes paralleled these data. Our data disclose a distinct immunomodulatory effect of AEA and an alteration of AEA-related members of the ECS in monocytes from MS patients that involves viral but not bacterial TLR. These findings not only may help to better understand the role of monocytes in MS immunopathogenesis but also could be of help to exploit new endocannabinoid-based drugs that target innate immune cells.

Altered cerebellum development and impaired motor coordination in mice lacking the Btg1 gene: Involvement of cyclin D1.

Cerebellar granule neurons develop postnatally from cerebellar granule precursors (GCPs), which are located in the external granule layer (EGL) where they massively proliferate. Thereafter, GCPs become postmitotic, migrate inward to form the internal granule layer (IGL), further differentiate and form synapses with Purkinje cell dendrites. We previously showed that the Btg family gene, Tis21/Btg2, is required for normal GCP migration. Here we investigated the role in cerebellar development of the related gene, Btg1, which regulates stem cell quiescence in adult neurogenic niches, and is expressed in the cerebellum. Knockout of Btg1 in mice caused a major increase of the proliferation of the GCPs in the EGL, whose thickness increased, remaining hyperplastic even after postnatal day 14, when the EGL is normally reduced to a few GCP layers. This was accompanied by a slight decrease of differentiation and migration of the GCPs and increase of apoptosis. The GCPs of double Btg1/Tis21-null mice presented combined major defects of proliferation and migration outside the EGL, indicating that each gene plays unique and crucial roles in cerebellar development. Remarkably, these developmental defects lead to a permanent increase of the adult cerebellar volume in Btg1-null and double mutant mice, and to impairment in all mutants, including Tis21-null, of the cerebellum-dependent motor coordination. Gain- and loss-of-function strategies in a GCP cell line revealed that Btg1 regulates the proliferation of GCPs selectively through cyclin D1. Thus, Btg1 plays a critical role for cerebellar maturation and function.

The p38 mitogen-activated protein kinase cascade modulates T helper type 17 differentiation and functionality in multiple sclerosis.

The p38 mitogen-activated protein kinase cascade is required for the induction of a T helper type 17 (Th17) -mediated autoimmune response, which underlies the development and progression of several autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis (MS). However, the contribution of p38 phosphorylation to human Th cell differentiation has not been clarified. Here we demonstrate that the p38 signalling pathway is implicated in the generation of Th17 lymphocytes from human CD4(+)  CD27(+)  CD45RA(+) naive T cells, both in healthy donors and in patients affected by the relapsing-remitting form of MS. Our data also indicate that p38 activation is essential for interleukin-17 release from central memory lymphocytes and committed Th17 cell clones. Furthermore, CD4(+) T cells isolated from individuals with relapsing-remitting MS display an altered responsiveness of the p38 cascade, resulting in increased p38 phosphorylation upon stimulation. These findings suggest that the p38 signalling pathway, by modulating the Th17 differentiation and response, is involved in the pathogenesis of MS, and open new perspectives for the use of p38 inhibitors in the treatment of Th17-mediated autoimmune diseases.

A Leukemia-Associated CD34/CD123/CD25/CD99+ Immunophenotype Identifies FLT3-Mutated Clones in Acute Myeloid Leukemia.

PURPOSE: We evaluated leukemia-associated immunophenotypes (LAIP) and their correlation with fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) gene mutational status in order to contribute a better identification of patients at highest risk of relapse in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: Bone marrow samples from 132 patients with AML were analyzed by nine-color multiparametric flow cytometry. We confirmed the presence of the mutation in diagnostic samples and in sorted cells by conventional RT-PCR and by patient-specific RQ-PCR. RESULTS: Within the CD34(+) cell fraction, we identified a discrete population expressing high levels of the IL3 receptor α-chain (CD123) and MIC-2 (CD99) in combination with the IL2 receptor α-chain (CD25). The presence of this population positively correlated with the internal tandem duplications (ITD) mutation in the FLT3 gene (r = 0.71). Receiver operating characteristics showed that, within the CD34(+) cell fraction a percentage of CD123/CD99/CD25(+) cells ≥11.7% predicted FLT3-ITD mutations with a specificity and sensitivity of >90%. CD34/CD123/CD99/CD25(+) clones were also detectable at presentation in 3 patients with FLT3 wild-type/NPM1(+) AML who relapsed with FLT3-ITD/NPM1(+) AML. Quantitative real-time PCR designed at relapse for each FLT3-ITD in these three cases confirmed the presence of low copy numbers of the mutation in diagnostic samples. CONCLUSIONS: Our results suggest that the CD34/CD25/CD123/CD99(+) LAIP is strictly associated with FLT3-ITD-positive cells.

The differential characterization of GPR55 receptor in human peripheral blood reveals a distinctive expression in monocytes and NK cells and a proinflammatory role in these innate cells.

G protein-coupled receptor 55 (GPR55) is activated by endogenous, plant-derived and synthetic cannabinoids. Recent studies reported a broad tissue distribution for GPR55 and found prominent roles for this receptor in inflammatory pain, gut and bone physiology, as well as cancer. However, little is known about the expression and function of GPR55 in immune cells. To address this question, we performed a detailed characterization of GPR55 in different human innate and adaptive immune populations using polychromatic flow cytometry and we found that monocytes and NK cells expressed remarkable levels of this receptor compared to several cells of adaptive immunity. GPR55 activation by the specific agonist O-1602 boosted IL-12 and TNF-α production, and decreased endocytic activity, in LPS-activated monocytes. In addition, it increased CD69 activation marker expression, granzyme B and CD107a-dependent cytotoxicity and IFN-γ and TNF-α production in NK cells activated by both IL-2 and IL-12. These over-stimulatory effects of GPR55 were antagonized by its selective antagonist cannabidiol. Altogether, our data thus unveil a proinflammatory role for GPR55 in innate immunity that may be important for the design of new immune therapeutic strategies.

Tis21 knock-out enhances the frequency of medulloblastoma in Patched1 heterozygous mice by inhibiting the Cxcl3-dependent migration of cerebellar neurons.

A failure in the control of proliferation of cerebellar granule neuron precursor cells (GCPs), located in the external granular layer (EGL) of the cerebellum, gives rise to medulloblastoma. To investigate the process of neoplastic transformation of GCPs, we generated a new medulloblastoma model by crossing Patched1 heterozygous mice, which develop medulloblastomas with low frequency, with mice lacking the Tis21 gene. Overexpression of Tis21 is known to inhibit proliferation and trigger differentiation of GCPs; its expression decreases in human medulloblastomas. Double-knock-out mice show a striking increase in the frequency of medulloblastomas and hyperplastic EGL lesions, formed by preneoplastic GCPs. Tis21 deletion does not affect the proliferation of GCPs but inhibits their differentiation and, chiefly, their intrinsic ability to migrate outside the EGL. This defect of migration may represent an important step in medulloblastoma formation, as GCPs, remaining longer in the EGL proliferative niche, may become more prone to transformation. By genome-wide analysis, we identified the chemokine Cxcl3 as a target of Tis21. Cxcl3 is downregulated in Tis21-null GCPs of EGL and lesions; addition of Cxcl3 to cerebellar slices rescues the defective migration of Tis21-null GCPs and, remarkably, reduces the area of hyperplastic lesions. As Tis21 activates Cxcl3 transcription, our results suggest that Tis21 induces migration of GCPs through Cxcl3, which may represent a novel target for medulloblastoma therapy.