Dysfunctional polycomb transcriptional repression contributes to Lamin A/C dependent muscular dystrophy

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Abstract

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. Polycomb group of proteins (PcG) 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 showed here that Lamin A loss deregulated PcG positioning in muscle satellite stem cells leading to de-repression of non-muscle specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional programme caused impairment in self-renewal, loss of cell identity and premature exhaustion of quiescent satellite cell pool. Genetic ablation of Cdkn2a locus restored muscle stem cell properties in Lamin A/C null dystrophic mice. Our findings established a direct link between Lamin A and PcG epigenetic silencing and indicated that Lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.

Authors

Andrea Bianchi, Chiara Mozzetta, Gloria Pegoli, Federica Lucini, Sara Valsoni, Valentina Rosti, Cristiano Petrini, Alice Cortesi, Francesco Gregoretti, Laura Antonelli, Gennaro Oliva, Marco De Bardi, Roberto Rizzi, Beatrice Bodega, Diego Pasini, Francesco Ferrari, Claudia Bearzi, Chiara Lanzuolo

TRPV4 channel opening mediates pressure-induced pancreatitis initiated by Piezo1 activation

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Abstract

Elevated pressure in the pancreatic gland is the central cause of pancreatitis following abdominal trauma, surgery, endoscopic retrograde cholangiopancreatography (ERCP), and gallstones. In the pancreas excessive intracellular calcium causes mitochondrial dysfunction, premature zymogen activation, and necrosis ultimately leading to pancreatitis. Although stimulation of the mechanically activated, calcium-permeable ion channel, Piezo1, in the pancreatic acinar cell is the initial step in pressure-induced pancreatitis, activation of Piezo1 produces only transient elevation in intracellular calcium that is insufficient to cause pancreatitis. Therefore, how pressure produces a prolonged calcium elevation necessary to induce pancreatitis is unknown. We demonstrate that Piezo1 activation in pancreatic acinar cells caused a prolonged elevation in intracellular calcium levels, mitochondrial depolarization, intracellular trypsin activation, and cell death. Notably, these effects were dependent on the degree and duration of force applied to the cell. Low or transient force were insufficient to activate these pathological changes whereas higher and prolonged application of force triggered sustained elevation in intracellular calcium leading to enzyme activation and cell death. All of these pathological events were rescued in acinar cells treated with a Piezo1 antagonist and in acinar cells from mice with genetic deletion of Piezo1. We discovered that Piezo1 stimulation triggered TRPV4 channel opening which was responsible for the sustained elevation in intracellular calcium that caused intracellular organelle dysfunction. Moreover, TRPV4 gene knockout mice were protected from Piezo1 agonist- and pressure-induced pancreatitis. These studies unveil a calcium signaling pathway in which Piezo1-induced TRPV4 channel opening causes pancreatitis.

Authors

Sandip M. Swain, Joelle M.J. Romac, Rafiq A. Shahid, Stephen J. Pandol, Wolfgang Liedtke, Steven R. Vigna, Rodger A. Liddle

Marked and rapid effects of pharmacological HIF-2α antagonism on hypoxic ventilatory control

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Abstract

Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer and there is great interest in applying inhibitors of HIF as anti-cancer therapeutics. The most advanced of these are small molecules that target the HIF-2 isoform through binding the PAS-B domain of HIF-2α. These molecules are undergoing clinical trials with promising results in renal and other cancers where HIF-2 is considered to be driving growth. Nevertheless, a central question remains as to whether such inhibitors impact on physiological responses to hypoxia at relevant doses. Here we show that pharmacological HIF-2α inhibition with PT2385, at doses similar to those reported to inhibit tumour growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatisation and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerisation with HIF-1β, did not respond to PT2385 indicating that these effects are on target. Furthermore, the finding of a hypomorphic ventilatory phenotype in untreated HIF-2α S305M mutant mice suggests a function for the HIF-2α PAS-B domain beyond heterodimerisation with HIF-1β. Although PT2385 was well-tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.

Authors

Xiaotong Cheng, Maria Prange-Barczynska, James W. Fielding, Minghao Zhang, Alana L. Burrell, Joanna D.C.C. Lima, Luise Eckardt, Isobel L.A. Argles, Christopher W. Pugh, Keith J. Buckler, Peter A. Robbins, Emma J. Hodson, Richard K. Bruick, Lucy M. Collinson, Fraydoon Rastinejad, Tammie Bishop, Peter J. Ratcliffe

TGFβ-induced epigenetic deregulation of SOCS3 facilitates STAT3-signaling to promote fibrosis

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Abstract

Fibroblasts are key-effector cells in tissue remodeling. They remain persistently activated in fibrotic diseases, resulting in progressive deposition of extracellular matrix. Although fibroblast activation maybe initiated by external factors, prolonged activation can induce an “autonomous”, self-maintaining pro-fibrotic phenotype in fibroblasts. Accumulating evidence suggests that epigenetic alterations play a central role to establish this persistently activated pathologic phenotype of fibroblasts. We demonstrated that in fibrotic skin of patients with systemic sclerosis (SSc), a prototypical idiopathic fibrotic disease, transforming growth factor-β (TGFβ) induced the expression of DNA-methyltransferase 3A (DNMT3A) and DNMT1 in fibroblasts in a SMAD-dependent manner to silence the expression of suppressor of cytokine signaling 3 (SOCS3) by promoter hypermethylation. Downregulation of SOCS3 facilitated activation of signal transducers and activators of transcription 3 (STAT3) to promote fibroblast-to–myofibroblast transition, collagen release and fibrosis in vitro and in vivo. Re-establishment of the epigenetic control of STAT3 signaling by genetic or pharmacological inactivation of DNMT3A reversed the activated phenotype of SSc fibroblasts in tissue culture, inhibited TGFβ-dependent fibroblast activation and ameliorated experimental fibrosis in murine models. These findings identify a novel pathway of epigenetic imprinting of fibroblasts in fibrotic disease with translational implications for the development of new targeted therapies in fibrotic diseases.

Authors

Clara Dees, Sebastian Pötter, Yun Zhang, Christina Bergmann, Xiang Zhou, Markus Luber, Thomas Wohlfahrt, Emmanuel Karouzakis, Andreas Ramming, Kolja Gelse, Akihiko Yoshimura, Rudolf Jaenisch, Oliver Distler, Georg Schett, Jörg H.W. Distler

IL-17 producing γδ T cells protect against Clostridium difficile infection

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Abstract

Colitis caused by C. difficile infection is an increasing cause of human morbidity and mortality, especially after antibiotic use in healthcare settings. The natural immunity of newborn infants and protective host immune mediators against C. difficile infection are not fully understood, with data suggesting that inflammation can be either protective or pathogenic. Here we show an essential role for IL-17A produced by γδ T cells in host defense against C. difficile infection. Fecal extracts of children with C. difficile infection showed increased IL-17A and T cell receptor γ-chain expression, and IL-17 production by intestinal γδ T cells was efficiently induced after infection in mice. C. difficile induced tissue inflammation and mortality were each significantly increased in mice deficient in IL-17A or γδ T cells. neonatal mice, with naturally expanded ROR-γ+ γδ T cells poised for IL-17 production were resistant to C. difficile infection, whereas eliminating γδ T cells or IL-17A each efficiently overturned neonatal resistance against infection. These results reveal an expanded role for IL-17 producing γδ T cells in neonatal host defense against infection and provide a mechanistic explanation for the clinically observed resistance of infants to C. difficile colitis.

Authors

Yee-Shiuan Chen, Iuan-Bor Chen, Giang Pham, Tzu-Yu Shao, Hansraj Bangar, Sing Sing Way, David B. Haslam

Cytotoxic CD4+ T lymphocytes may induce endothelial cell apoptosis in systemic sclerosis

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Abstract

Systemic sclerosis (SSc) is an autoimmune fibrotic disease whose pathogenesis is poorly understood and lacks effective therapies. We undertook quantitative analyses of T cell infiltrates in the skin of thirty-five untreated patients with early diffuse SSc and here show that CD4+ cytotoxic T cells and CD8+ T cells contribute prominently to these infiltrates. We also observed an accumulation of apoptotic cells in SSc tissues, suggesting that recurring cell death may contribute to tissue damage and remodeling in this fibrotic disease. HLA-DR expressing endothelial cells were frequent targets of apoptosis in SSc, consistent with the prominent vasculopathy seen in patients with this disease. A circulating effector population of cytotoxic CD4+ T cells, which exhibited signatures of enhanced metabolic activity, was clonally expanded in systemic sclerosis patients. These data suggest that cytotoxic T cells may induce the apoptotic death of endothelial and other cells in systemic sclerosis. Cell loss driven by immune cells may be followed by overly exuberant tissue repair processes that lead to fibrosis and tissue dysfunction..

Authors

Takashi Maehara, Naoki Kaneko, Cory Adam Perugino, Hamid Mattoo, Jesper Kers, Hugues Allard-Chamard, Vinay S. Mahajan, Hang Liu, Samuel J.H. Murphy, Musie Ghebremichael, David A. Fox, Aimee S. Payne, Robert Lafyatis, John H. Stone, Dinesh Khanna, Shiv Pillai

Local microvascular leakage promotes trafficking of activated neutrophils to remote organs

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Abstract

Increased microvascular permeability to plasma proteins and neutrophil emigration are hallmarks of innate immunity and key features of numerous inflammatory disorders. Whilst neutrophils can promote microvascular leakage, the impact of vascular permeability on neutrophil trafficking is unknown. Here, through the application of confocal intravital microscopy, we reported that vascular permeability enhancing stimuli caused a significant frequency of neutrophil reverse transendothelial cell migration (rTEM). Furthermore, mice with a selective defect in microvascular permeability enhancement (VEC-Y685F-ki) showed reduced incidence of rTEM. Mechanistically, elevated vascular leakage promoted movement of interstitial chemokines into the blood stream, a response that supported abluminal-to-luminal neutrophil TEM. Through development of an in vivo cell labelling method we provided direct evidence for the systemic dissemination of rTEM neutrophils, showed them to exhibit an activated phenotype and capable of trafficking to the lungs where their presence was aligned with regions of vascular injury. Collectively, we demonstrated that increased microvascular leakage reverses the localisation of directional cues across venular walls, thus causing neutrophils engaged in diapedesis to re-enter the systemic circulation. This cascade of events offers a mechanism to explain how local tissue inflammation and vascular permeability can induce downstream pathological effects in remote organs, most notably in the lungs.

Authors

Charlotte Owen-Woods, Régis Joulia, Anna Barkaway, Loïc Rolas, Bin Ma, Astrid Fee Nottebaum, Kenton P. Arkill, Monja Stein, Tamara Girbl, Matthew Golding, David O. Bates, Dietmar Vestweber, Mathieu-Benoit Voisin, Sussan Nourshargh

Steroid-resistance in Diamond Blackfan anemia associates with p57^Kip2 dysregulation in erythroid progenitors

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Abstract

Despite the effective clinical use of steroids for the treatment of Diamond Blackfan anemia (DBA), the mechanistic bases via which glucocorticoids regulate human erythropoiesis remain poorly understood. Here, we report that the sensitivity of erythroid differentiation to dexamethasone (Dex) is dependent on the developmental origin of human CD34+ progenitor cells, specifically increasing the expansion of CD34+ progenitors from peripheral blood (PB) but not cord blood (CB). Dexamethasone treatment of erythroid-differentiated PB, but not CB, CD34+ progenitors resulted in the expansion of a novel CD34+CD36+CD71hiCD105med immature colony-forming unit-erythroid (CFU-E) population. Furthermore, proteomics analyses revealed the induction of distinct proteins in dexamethasone-treated PB and CB erythroid progenitors. Dexamethasone treatment of PB progenitors resulted in the specific upregulation of p57Kip2, a Cip/Kip cyclin-dependent kinase inhibitor, and we identified this induction as critical; shRNA-mediated downregulation of p57Kip2, but not the related p27Kip1, significantly attenuated the impact of dexamethasone on erythroid differentiation and inhibited the expansion of the immature CFU-E subset. Notably, in the context of DBA, we found that steroid resistance was associated with a dysregulated p57Kip2 expression. Altogether, these data identify a novel glucocorticoid-responsive human erythroid progenitor and provide new insights into glucocorticoid-based therapeutic strategies for the treatment of patients with DBA.

Authors

Ryan J. Ashley, Hongxia Yan, Nan Wang, John Hale, Brian M Dulmovits, Julien Papoin, Meagan E. Olive, Namrata D Udeshi, Steven A. Carr, Adrianna Vlachos, Jeffrey M. Lipton, Lydie Da Costa, Christopher D. Hillyer, Sandrina Kinet, Naomi Taylor, Narla Mohandas, Anupama Narla, Lionel Blanc

Cystic fibrosis transmembrane conductance regulator dysfunction in platelets drives lung hyperinflammation

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Abstract

Cystic fibrosis (CF) lung disease is characterized by an inflammatory response that can lead to terminal respiratory failure. The cystic fibrosis transmembrane regulator (CFTR) is mutated in CF and we hypothesized that dysfunctional CFTR in platelets, which are key participants in immune responses, is a central determinant of CF inflammation. We found that deletion of CFTR in platelets produced exaggerated acute lung inflammation and platelet activation after intratracheal LPS or Pseudomonas aeruginosa challenge. CFTR loss of function in mouse or human platelets resulted in agonist-induced hyperactivation and increased calcium entry into platelets. Inhibition of the transient receptor potential cation channel 6 (TRPC6) reduced platelet activation and calcium flux, and reduced lung injury in CF mice after intratracheal LPS or Pseudomonas aeruginosa challenge. CF subjects receiving CFTR modulator therapy showed partial restoration of CFTR function in platelets, which may be a convenient approach to monitoring biological responses to CFTR modulators. We conclude that CFTR dysfunction in platelets produces aberrant TRPC6-dependent platelet activation, which is a major driver of CF lung inflammation and impaired bacterial clearance. Platelets, and TRPC6, are what we believe to be novel therapeutic targets in the treatment of CF lung disease.

Authors

Guadalupe Ortiz-Munoz, Michelle A. Yu, Emma Lefrançais, Benat Mallavia, Colin Valet, Jennifer J. Tian, Serena Ranucci, Kristin M. Wang, Zhe Liu, Nicholas Kwaan, Diana Dawson, Mary Ellen Kleinhenz, Fadi T. Khasawneh, Peter M. Haggie, Alan S. Verkman, Mark R. Looney

Gut epithelial TSC1/mTOR controls RIPK3-dependent necroptosis in intestinal inflammation and cancer

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Abstract

While the Western-diet and dysbiosis are the most prominent environmental factors associated with inflammatory bowel diseases (IBDs), the corresponding host factors and cellular mechanisms remain poorly defined. Here we report that the TSC1-mTOR pathway in the gut epithelium represents a metabolic and innate immune checkpoint for intestinal dysfunction and inflammation. mTOR hyperactivation triggered by the Western-diet or Tsc1-ablation led to epithelium necroptosis, barrier disruption, and predisposition to DSS (dextran sulfate sodium)-induced colitis and inflammation-associated colon cancer. Mechanistically, our results uncovered a critical role for TSC1-mTOR in restraining the expression and activation of RIPK3 in the gut epithelium through Trim11-mediated ubiquitination and autophagy-dependent degradation. Notably, microbiota-depletion by antibiotics or gnotobiotics attenuated RIPK3 expression and activation, thereby alleviating epithelial necroptosis and colitis driven by mTOR hyperactivation. mTOR primarily impinged on RIPK3 to potentiate TNF- and microbial PAMP-induced necroptosis, and hyperactive mTOR and aberrant necroptosis were intertwined in human IBDs. Together, our data reveal a previously unsuspected link between the Western-diet, microbiota and necroptosis, and identify the mTOR-RIPK3-necroptosis axis as a driving force for intestinal inflammation and cancer.

Authors

Yadong Xie, Yifan Zhao, Lei Shi, Wei Li, Kun Chen, Min Li, Xia Chen, Haiwei Zhang, Tiantian Li, Matsuzawa-Ishimoto Yu, Xiaomin Yao, Dianhui Shao, Zunfu Ke, Jian Li, Yan Chen, Xiaoming Zhang, Jun Cui, Shuzhong Cui, Qibin Leng, Ken Cadwell, Xiaoxia Li, Hong Wei, Haibing Zhang, Huabin Li, Hui Xiao