HSP90ß controls NLRP3 autoactivation.

Gain-of-function mutations in NLRP3 are linked to cryopyrin-associated periodic syndromes (CAPS). Although NLRP3 autoinflammasome assembly triggers inflammatory cytokine release, its activation mechanisms are not fully understood. Our study used a functional genetic approach to identify regulators of NLRP3 inflammasome formation. We identified the HSP90ß-SGT1 chaperone complex as crucial for autoinflammasome activation in CAPS. A deficiency in HSP90ß, but not in HSP90α, impaired the formation of ASC specks without affecting the priming and expression of inflammasome components. Conversely, activating NLRP3 with stimuli such as nigericin or alum bypassed the need for SGT1 and HSP90ß, suggesting the existence of alternative inflammasome assembly pathways. The role of HSP90ß was further demonstrated in PBMCs derived from CAPS patients. In these samples, the pathological constitutive secretion of IL-1ß could be suppressed using a pharmacological inhibitor of HSP90ß. This finding underscores the potential of SGT1-HSP90ß modulation as a therapeutic strategy in CAPS while preserving NLRP3's physiological functions.

Receptor interacting protein kinase-3 mediates both myopathy and cardiomyopathy in preclinical animal models of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle degenerative disorder, culminating in a complete loss of ambulation, hypertrophic cardiomyopathy and a fatal cardiorespiratory failure. Necroptosis is the form of necrosis that is dependent upon the receptor-interacting protein kinase (RIPK) 3; it is involved in several inflammatory and neurodegenerative conditions. We previously identified RIPK3 as a key player in the acute myonecrosis affecting the hindlimb muscles of the mdx dystrophic mouse model. Whether necroptosis also mediates respiratory and heart disorders in DMD is currently unknown. METHODS: Evidence of activation of the necroptotic axis was examined in dystrophic tissues from Golden retriever muscular dystrophy (GRMD) dogs and R-DMDdel52 rats. A functional assessment of the involvement of necroptosis in dystrophic animals was performed on mdx mice that were genetically depleted for RIPK3. Dystrophic mice aged from 12 to 18 months were analysed by histology and molecular biology to compare the phenotype of muscles from mdxRipk3+/+ and mdxRipk3-/- mice. Heart function was also examined by echocardiography in 40-week-old mice. RESULTS: RIPK3 expression in sartorius and biceps femoris muscles from GRMD dogs positively correlated to myonecrosis levels (r = 0.81; P = 0.0076). RIPK3 was also found elevated in the diaphragm (P ≤ 0.05). In the slow-progressing heart phenotype of GRMD dogs, the phosphorylated form of RIPK1 at the Serine 161 site was dramatically increased in cardiomyocytes. A similar p-RIPK1 upregulation characterized the cardiomyocytes of the severe DMDdel52 rat model, associated with a marked overexpression of Ripk1 (P = 0.007) and Ripk3 (P = 0.008), indicating primed activation of the necroptotic pathway in the dystrophic heart. MdxRipk3-/- mice displayed decreased compensatory hypertrophy of the heart (P = 0.014), and echocardiography showed a 19% increase in the relative wall thickness (P < 0.05) and 29% reduction in the left ventricle mass (P = 0.0144). Besides, mdxRipk3-/- mice presented no evidence of a regenerative default or sarcopenia in skeletal muscles, moreover around 50% less affected by fibrosis (P < 0.05). CONCLUSIONS: Our data highlight molecular and histological evidence that the necroptotic pathway is activated in degenerative tissues from dystrophic animal models, including the diaphragm and the heart. We also provide the genetic proof of concept that selective inhibition of necroptosis in dystrophic condition improves both histological features of muscles and cardiac function, suggesting that prevention of necroptosis is susceptible to providing multiorgan beneficial effects for DMD.

Thyroid-stimulating hormone receptor signaling restores skeletal muscle stem cell regeneration in rats with muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive myopathy leading to motor and cardiorespiratory impairment. We analyzed samples from patients with DMD and a preclinical rat model of severe DMD and determined that compromised repair capacity of muscle stem cells in DMD is associated with early and progressive muscle stem cell senescence. We also found that extraocular muscles (EOMs), which are spared by the disease in patients, contain muscle stem cells with long-lasting regenerative potential. Using single-cell transcriptomics analysis of muscles from a rat model of DMD, we identified the gene encoding thyroid-stimulating hormone receptor (Tshr) as highly expressed in EOM stem cells. Further, TSHR activity was involved in preventing senescence. Forskolin, which activates signaling downstream of TSHR, was found to reduce senescence of skeletal muscle stem cells, increase stem cell regenerative potential, and promote myogenesis, thereby improving muscle function in DMD rats. These findings indicate that stimulation of adenylyl cyclase leads to muscle repair in DMD, potentially providing a therapeutic approach for patients with the disease.

CDC42 regulates PYRIN inflammasome assembly.

The PYRIN inflammasome pathway is part of the innate immune response against invading pathogens. Unprovoked continuous activation of the PYRIN inflammasome drives autoinflammation and underlies several autoinflammatory diseases, including familial Mediterranean fever (FMF) syndrome. PYRIN inflammasome formation requires PYRIN dephosphorylation and oligomerization by molecular mechanisms that are poorly understood. Here, we use a functional genetics approach to find regulators of PYRIN inflammasome function. We identify the small Rho GTPase CDC42 to be essential for PYRIN activity and oligomerization of the inflammasome complex. While CDC42 catalytic activity enhances PYRIN phosphorylation, thereby inhibiting it, the inflammasome-supportive role of CDC42 is independent of its GDP/GTP binding or hydrolysis capacity and does not affect PYRIN dephosphorylation. These findings identify the dual role of CDC42 as a regulator of PYRIN and as a critical player required for PYRIN inflammasome assembly in health and disease.

From Diagnosis to Prognosis: Revisiting the Meaning of Muscle ISG15 Overexpression in Juvenile Inflammatory Myopathies.

OBJECTIVE: Juvenile idiopathic inflammatory/immune myopathies (IIMs) constitute a highly heterogeneous group of disorders with diagnostic difficulties and prognostic uncertainties. Circulating myositis-specific autoantibodies (MSAs) have been recognized as reliable tools for patient substratification. Considering the key role of type I interferon (IFN) up-regulation in juvenile IIM, we undertook the present study to investigate whether IFN-induced 15-kd protein (ISG-15) could be a reliable biomarker for stratification and diagnosis and to better elucidate its role in juvenile IIM pathophysiology. METHODS: The study included 56 patients: 24 with juvenile dermatomyositis (DM), 12 with juvenile overlap myositis (OM), 10 with Duchenne muscular dystrophy, and 10 with congenital myopathies. Muscle biopsy samples were assessed by immunohistochemistry, immunoblotting, and real-time quantitative polymerase chain reaction. Negative regulators of type I IFN (ISG15 and USP18) and positive regulators of type I IFN (DDX58 and IFIH1) were analyzed. RESULTS: ISG15 expression discriminated patients with juvenile IIM from those with nonimmune myopathies and, among patients with juvenile IIM, discriminated those with DM from those with OM. Among patients with juvenile DM, up-regulation of the type I IFN positive regulators DDX58 and IFIH1 was similar regardless of MSA status. In contrast, the highest levels of the type I IFN negative regulator ISG15 were observed in patients who were positive for melanoma differentiation-associated gene 5 (MDA-5). Finally, ISG15 levels were inversely correlated with the severity of muscle histologic abnormalities and positively correlated with motor performance as evaluated by the Childhood Myositis Assessment Scale and by manual muscle strength testing. CONCLUSION: Muscle ISG15 expression is strongly associated with juvenile DM, with patients exhibiting a different ISG-15 muscle signature according to their MSA class. Patients with juvenile DM who are positive for MDA-5 have higher expression of ISG15 in both gene form and protein form compared to the other subgroups. Moreover, our data show that negative regulation of type I IFN correlates with milder muscle involvement.

Distinct interferon signatures stratify inflammatory and dysimmune myopathies.

Objective: The role of interferons (IFN) in the pathophysiology of primary inflammatory and dysimmune myopathies (IDM) is increasingly investigated, notably because specific neutralisation approaches may constitute promising therapeutic tracks. In present work we analysed the muscular expression of specific IFNα/ß and IFNγ-stimulated genes in patients with various types of IDM. Methods: 39 patients with IDM with inclusion body myositis (IBM, n=9), dermatomyositis (DM, n=10), necrotising autoimmune myopathies (NAM, n=10) and antisynthetase myositis (ASM, n=10), and 10 controls were included. Quantification of expression levels of IFNγ, ISG15, an IFNα/ß-inducible gene and of six IFNγ-inducible genes (GBP2, HLA-DOB, HLA-DPB, CIITA, HLA-DRB and HLA-DMB) was performed on muscle biopsy samples. Results: DM usually associated with strong type I IFNα/ß signature, IBM and ASM with prominent type II IFNγ signature and NAM with neither type I nor type II IFN signature. Immunofluorescence study in ASM and IBM showed myofibre expression of major histocompatibility class 2 (MHC-2) and CIITA, confirming the induction of the IFNγ pathway. Furthermore, MHC-2-positive myofibres were observed in close proximity to CD8+ T cells which produce high levels of IFNγ. Conclusion: Distinct IFN signatures allow a more distinct segregation of IDMs and myofibre MHC-2 expression is a reliable biomarker of type II IFN signature.

[The effect of interferon-gamma on skeletal muscle cell biology]. / 15es JSFM : Prix Master 2017* - Effet de l'interféron-gamma sur la biologie des cellules musculaires squelettiques.

Dysimmune and inflammatory myopathies (DIMs) affect around 14/100,000 people worldwide. Based on immupour nopathological criteria, DIMs are divided in four groups: (1) polymyositis (PM)/inclusion body myositis (IBM), (2) dermatomyositis (DM), (3) immune-mediated necrotizing myopathies (IMNM) and (iv) overlapping myositis including anti-synthetase syndrome (ASS). ASS and PM/IBM are characterized by the activation of inflammation with lymphocytic infiltrations. Recently, we showed that an expression of the major histocompatibility complex class 2 (MHC2) was present in myofibers from ASS and IBM muscle biopsies. Interestingly, MHC2 expression is known to be stimulated by Interferon-gamma (IFNγ) in myogenic cells. LTCD8 cells, which are well-known producers of IFNγ, are commonly found in close vicinity to MHC2 positive myofibers. This inflammatory cytokine also inhibits myogenic differentiation in vitro by CIITA-myogenin interaction. The mechanisms involved in the lymphocyte-driven muscle toxicity in DIMs are unclear. The objectives of this project are to characterize IFNγ effects on the biology of human myogenic cells by morphological, molecular and cellular approaches. Then, we aim to investigate the role of IFNγ in these myopathies and its impact during muscular regeneration. In vitro preliminary studies have been performed using human and mouse myoblasts treated or not with IFNγ. Our results should lead to a better understanding of the role of IFNγ in the pathophysiology of DIMs, and would hopefully help identify new therapeutic targets.

Long non-coding RNAs in human early embryonic development and their potential in ART.

BACKGROUND: Human long non-coding RNAs (lncRNAs) are an emerging category of transcripts with increasingly documented functional roles during development. LncRNAs and roles during human early embryo development have recently begun to be unravelled. OBJECTIVE AND RATIONALE: This review summarizes the most recent knowledge on lncRNAs and focuses on their expression patterns and role during early human embryo development and in pluripotent stem cells (PSCs). Public mRNA sequencing (mRNA-seq) data were used to illustrate these expression signatures. SEARCH METHODS: The PubMed and EMBASE databases were first interrogated using specific terms, such as 'lncRNAs', to get an extensive overview on lncRNAs up to February 2016, and then using 'human lncRNAs' and 'embryo', 'development', or 'PSCs' to focus on lncRNAs involved in human embryo development or in PSC.Recently published RNA-seq data from human oocytes and pre-implantation embryos (including single-cell data), PSC and a panel of normal and malignant adult tissues were used to describe the specific expression patterns of some lncRNAs in early human embryos. OUTCOMES: The existence and the crucial role of lncRNAs in many important biological phenomena in each branch of the life tree are now well documented. The number of identified lncRNAs is rapidly increasing and has already outnumbered that of protein-coding genes. Unlike small non-coding RNAs, a variety of mechanisms of action have been proposed for lncRNAs. The functional role of lncRNAs has been demonstrated in many biological and developmental processes, including cell pluripotency induction, X-inactivation or gene imprinting. Analysis of RNA-seq data highlights that lncRNA abundance changes significantly during human early embryonic development. This suggests that lncRNAs could represent candidate biomarkers for developing non-invasive tests for oocyte or embryo quality. Finally, some of these lncRNAs are also expressed in human cancer tissues, suggesting that reactivation of an embryonic lncRNA program may contribute to human malignancies. WIDER IMPLICATIONS: LncRNAs are emerging potential key players in gene expression regulation. Analysis of RNA-seq data from human pre-implantation embryos identified lncRNA signatures that are specific to this critical step. We anticipate that further studies will show that these new transcripts are major regulators of embryo development. These findings might also be used to develop new tests/treatments for improving the pregnancy success rate in IVF procedures or for regenerative medicine applications involving PSC.