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.
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
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.
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
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.
Yee-Shiuan Chen, Iuan-Bor Chen, Giang Pham, Tzu-Yu Shao, Hansraj Bangar, Sing Sing Way, David B. Haslam
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..
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
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.
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
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.
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 (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.
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
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.
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
After trauma, regeneration of adult CNS axons is abortive causing devastating neurologic deficits. Despite progress in rehabilitative care, there is no effective treatment stimulating axon growth following injury. Using models with different regenerative capacities, followed by gain- and loss-of-function analysis, we identified profilin1 (Pfn1) as a coordinator of actin and microtubules (MTs), powering axon growth and regeneration. In growth cones, Pfn1 increased actin retrograde flow, MT growth speed and invasion of filopodia by MTs, orchestrating cytoskeleton dynamics towards axon growth. In vitro, active Pfn1 promoted MT growth in a formin-dependent manner, whereas localization of MTs to growth cone filopodia was facilitated by direct MT binding and interaction with formins. In vivo, Pfn1 ablation limited regeneration of growth-competent axons after sciatic nerve and spinal cord injury. Adeno-associated viral (AAV) delivery of constitutively active Pfn1 to rodents promoted axon regeneration, neuromuscular junction maturation and functional recovery of injured sciatic nerves, and increased the ability of regenerating axons to penetrate the inhibitory spinal cord glial scar. Thus, we identify Pfn1 as an important regulator of axon regeneration and suggest that AAV-mediated delivery of constitutively active Pfn1, together with the identification of modulators of Pfn1 activity, should be considered to treat the injured nervous system.
Rita Pinto-Costa, Sara Castro Sousa, Sérgio C. Leite, Joana Nogueira-Rodrigues, Tiago Ferreira da Silva, Diana Machado, Joana Beatriz Moreira Marques, Ana Catarina Costa, Márcia A. Liz, Francesca Bartolini, Pedro Brites, Mercedes Costell, Reinhard Fässler, Monica M. Sousa
Severe alcoholic hepatitis (SAH) is a deadly liver disease without an effective medical therapy. Although SAH mortality is known to correlate with hepatic accumulation of immature liver cells, why this occurs, and how it causes death is unclear. Here, we demonstrated that expression of epithelial splicing regulatory protein-2 (ESRP2), an RNA splicing factor that maintains the non-proliferative, mature phenotype of adult hepatocytes, was suppressed in both human SAH and various mouse models of SAH in parallel with the severity of alcohol consumption and liver damage. Inflammatory cytokines released by excessive alcohol ingestion reprogrammed adult hepatocytes into proliferative, fetal-like cells by suppressing ESRP2. Sustained loss of ESRP2 permitted re-emergence of a fetal RNA splicing program that attenuates the Hippo signaling pathway and thus, allows fetal transcriptional regulators to accumulate in adult liver. We further showed that depleting ESRP2 in mice exacerbated alcohol-induced steatohepatitis, enabling surviving hepatocytes to shed adult hepatocyte functions and become more regenerative but threatens overall survival by populating the liver with functionally-immature hepatocytes. Our findings revealed a novel mechanism that explains why liver failure develops in patients with the clinical syndrome of SAH, suggesting that recovery from SAH might be improved by limiting adult-to-fetal reprogramming in hepatocytes.
Jeongeun Hyun, Zhaoli Sun, Ali Reza Ahmadi, Sushant Bangru, Ullas V. Chembazhi, Kuo Du, Tianyi Chen, Hidekazu Tsukamoto, Ivan Rusyn, Auinash Kalsotra, Anna Mae Diehl
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