Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome

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Abstract

Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy, characterized by a pathognomonic hindbrain malformation. All known JBTS-genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we use the recently identified JBTS-associated protein ARMC9 in tandem-affinity purification and yeast two-hybrid screens to identify a novel ciliary module whose dysfunction underlies JBTS. In addition to known JBTS-associated proteins CEP104 and CSPP1, we identify CCDC66 and TOGARAM1 as ARMC9 interaction partners. We show that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses and characterization of patient-derived fibroblasts, CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrate that dysfunction of ARMC9 or TOGARAM1 results in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant cold- and serum-induced ciliary loss in both ARMC9 and TOGARAM1 patient cell lines suggests a role for this new JBTS-associated protein module in ciliary stability.

Authors

Brooke L. Latour, Julie C. Van De Weghe, Tamara D.S. Rusterholz, Stef J.F. Letteboer, Arianna Gomez, Ranad Shaheen, Matthias Gesemann, Arezou Karamzade, Mostafa Asadollahi, Miguel Barroso-Gil, Manali Chitre, Megan E. Grout, Jeroen van Reeuwijk, Sylvia E.C. van Beersum, Caitlin V. Miller, Jennifer C. Dempsey, Heba Morsy, Michael J. Bamshad, Deborah A. Nickerson, Stephan C.F. Neuhauss, Karsten Boldt, Marius Ueffing, Mohammad Keramatipour, John A. Sayer, Fowzan S. Alkuraya, Ruxandra Bachmann-Gagescu, Ronald Roepman, Dan Doherty

Rathke’s cleft-like cysts arise from Isl1 deletion in murine pituitary progenitors

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Abstract

The transcription factor ISL1 is expressed in pituitary gland stem cells and the thyrotrope and gonadotrope lineages. Pituitary-specific Isl1 deletion causes hypopituitarism with increased stem cell apoptosis, reduced differentiation of thyrotropes and gonadotropes, and reduced body size. Conditional Isl1 deletion causes development of multiple Rathke’s cleft-like cysts, with 100% penetrance. Foxa1 and Foxj1 are abnormally expressed in the pituitary gland and associated with a ciliogenic gene expression program in the cysts. We confirmed expression of FOXA1, FOXJ1 and stem cell markers in human Rathke's cleft cyst tissue, but not craniopharyngiomas, which suggests these transcription factors are useful, pathological markers for diagnosis of Rathke's cleft cysts. These studies support a model whereby expression of ISL1 in pituitary progenitors drives differentiation into thyrotropes and gonadotropes, and without it, activation of FOXA1 and FOXJ1 permits development of an oral epithelial cell fate with mucinous cysts. This pituitary specific Isl1 mouse knockout sheds light on the etiology of Rathke's cleft cysts and the role of ISL1 in normal pituitary development.

Authors

Michelle L. Brinkmeier, Hironori Bando, Adriana C. Camarano, Shingo Fujio, Koji Yoshimoto, Flávio S. J. de Souza, Sally A. Camper

Donor monocyte-derived macrophages promote human acute graft versus host disease

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Abstract

Myelopoiesis is invariably present, and contributes to pathology, in animal models of graft versus host disease (GVHD). In humans, a rich inflammatory infiltrate bearing macrophage markers has also been described in histological studies. In order to determine the origin, functional properties and role in pathogenesis of these cells, we isolated single cell suspensions from acute cutaneous GVHD and subjected them to genotype, transcriptome and in vitro functional analysis. A donor-derived population of CD11c+CD14+ cells was the dominant population of all leukocytes in GVHD. Surface phenotype and nanostring gene expression profiling indicated the closest steady-state counterpart of these cells to be monocyte-derived macrophages. In GVHD, however, there was upregulation of monocyte antigens SIRPα and S100A8/9, and transcripts associated with leukocyte trafficking, pattern recognition, antigen presentation, and co-stimulation. Isolated GVHD macrophages stimulated greater proliferation and activation of allogeneic T cells, and secreted higher levels of inflammatory cytokines than their steady-state counterparts. In HLA-matched mixed leukocyte reactions, we also observed differentiation of activated macrophages with a similar phenotype. These exhibited cytopathicity to a cell line and mediated pathological damage to skin explants, independently of T cells. Together, these results define the origin, functional properties and potential pathogenic roles of human GVHD macrophages.

Authors

Laura Jardine, Urszula Cytlak, Merry Gunawan, Gary Reynolds, Kile Green, Xiao-nong Wang, Sarah Pagan, Maharani Paramitha, Christopher A. Lamb, Anna Long, Erin Hurst, Smeera Nair, Graham H. Jackson, Amy Publicover, Venetia Bigley, Muzlifah Haniffa, AJ Simpson, Matthew Collin

Human CD83-targeted chimeric antigen receptor T cells prevent and treat graft-versus-host disease

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Abstract

Graft-versus-host disease (GVHD) remains an important cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). For decades, GVHD prophylaxis has included calcineurin-inhibitors, despite their incomplete efficacy and impairment of graft-versus-leukemia (GVL). Distinct from pharmacologic immune suppression, we have developed a novel, human CD83-targeted chimeric antigen receptor (CAR) T cell for GVHD prevention. CD83 is expressed on allo-activated, conventional CD4+ T cells (Tconv) and proinflammatory dendritic cells (DC); which are both implicated in GVHD pathogenesis. Human CD83 CAR T cells eradicate pathogenic CD83+ target cells, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide durable prevention of xenogeneic GVHD. CD83 CAR T cells are also capable of treating xenogeneic GVHD. We show human, acute myeloid leukemia (AML) expresses CD83 and myeloid leukemia cell lines are readily killed by CD83 CAR T cells. Human CD83 CAR T cells are a promising cell-based approach to prevent two critical complications of allo-HCT; GVHD and relapse. Thus, human CD83 CAR T cells warrant clinical investigation in GVHD prevention and treatment, as well as targeting CD83+ AML.

Authors

Bishwas Shrestha, Kelly Walton, Jordan Reff, Elizabeth M. Sagatys, Nhan Tu, Justin C. Boucher, Gongbo Li, Tayyeb Ghafoor, Martin Felices, Jeffrey Miller, Joseph Pidala, Bruce R. Blazar, Claudio Anasetti, Brian C. Betts, Marco L. Davila

Residual endotoxin induces primary graft dysfunction through ischemia-reperfusion-primed alveolar macrophages

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Abstract

Despite the widespread use of antibiotics, bacterial pneumonias in donors strongly predispose to the fatal syndrome of primary graft dysfunction (PGD) following lung transplantation. We report that bacterial endotoxin persists in human donor lungs after pathogen is cleared with antibiotics and is associated with neutrophil infiltration and PGD. In mouse models, depletion of tissue-resident alveolar macrophages (TRAM) attenuated neutrophil recruitment in response to endotoxin as shown by compartmental staining and intravital imaging. Bone marrow chimeric mice revealed that neutrophils were recruited by TRAM through activation of TLR4 in a MyD88-dependent manner. Intriguingly, low levels of endotoxin, insufficient to cause donor lung injury, promoted TRAM-dependent production of CXCL2, increased neutrophil recruitment, and led to PGD, which was independent of donor non-classical monocytes. Reactive oxygen species (ROS) increased in human donor lungs starting from the warm-ischemia phase and were associated with increased transcription and translocation to the plasma membrane of TLR4 in donor TRAM. Consistently, scavenging ROS or inhibiting their production to prevent TLR4 transcription/translocation or blockade of TLR4 or co-receptor CD14 on donor TRAM prevented neutrophil recruitment in response to endotoxin and ameliorated PGD. Our studies demonstrate that residual endotoxin after successful treatment of donor bacterial pneumonia promotes PGD through ischemia-reperfusion-primed donor TRAM..

Authors

Mahzad Akbarpour, Emilia Lecuona, Stephen Chiu, Qiang Wu, Melissa Querrey, Ramiro Fernandez, Felix Luis Nunez-Santana, Haiying Sun, Sowmya Ravi, Chitaru Kurihara, James M. Walter, Nikita Joshi, Ziyou Ren, Scott C. Roberts, Alan R. Hauser, Daniel Kreisel, Wenjun Li, Navdeep Chandel, Alexander V. Misharin, Thalachallour Mohanakumar, G.R. Scott Budinger, Ankit Bharat

Targeting the innate immunoreceptor RIG-I overcomes melanoma-intrinsic resistance to T cell immunotherapy

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Abstract

Understanding tumor resistance to T cell immunotherapies is critical to improve patient outcomes. Our study revealed a role for transcriptional suppression of the tumor-intrinsic HLA class I (HLA-I) antigen processing and presentation machinery (APM) in therapy resistance. Low HLA-I APM mRNA levels in melanoma metastases prior to immune checkpoint blockade (ICB) correlated with non-responsiveness to therapy and poor clinical outcome. Patient-derived melanoma cells with silenced HLA-I APM escaped recognition by autologous CD8+ T cells. However, targeted activation of the innate immunoreceptor RIG-I initiated de novo HLA-I APM transcription thereby overcoming T cell resistance. Antigen presentation was restored in interferon (IFN)-sensitive but also immunoedited IFN-resistant melanoma models through RIG-I-dependent stimulation of an IFN-independent salvage pathway involving IRF1 and IRF3. Likewise, enhanced HLA-I APM expression was detected in RIG-I (DDX58)-high melanoma biopsies, correlating with improved patient survival. Induction of HLA-I APM by RIG-I synergized with antibodies blocking PD-1 and TIGIT inhibitory checkpoints in boosting the anti-tumor T cell activity of ICB non-responders. Overall, the herein identified IFN-independent effect of RIG-I on tumor antigen presentation and T cell recognition proposes innate immunoreceptor targeting as a strategy to overcome intrinsic T cell resistance of IFN-sensitive and IFN-resistant melanomas and improve clinical outcomes in immunotherapy.

Authors

Lina Such, Fang Zhao, Derek Liu, Beatrice Thier, Vu Thuy Khanh Le-Trilling, Antje Sucker, Christoph Coch, Natalia Pieper, Sebastian Howe, Hilal Bhat, Halime Kalkavan, Cathrin Ritter, Robin Brinkhaus, Selma Ugurel, Johannes Köster, Ulrike Seifert, Ulf Dittmer, Martin Schuler, Karl Sebastian Lang, Thomas A Kufer, Gunther Hartmann, Jürgen Christian Becker, Susanne Horn, Soldano Ferrone, David Liu, Eliezer M. Van Allen, Dirk Schadendorf, Klaus Griewank, Mirko Trilling, Annette Paschen

Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy

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Abstract

Dominant mutations in the HSP70 co-chaperone DNAJB6 cause a late onset muscle disease termed limb girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70, and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of HSP70’s diffusion in LGMDD1 mouse muscle was diminished likely because it has an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small molecule inhibitor of the DNAJ-HSP70 complex re-mobilized HSP70, improved strength and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counter-intuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70:DNAJB6 may be effective in treating this inherited muscular dystrophy.

Authors

Rocio Bengoechea, Andrew R. Findlay, Ankan K. Bhadra, Hao Shao, Kevin C. Stein, Sara K. Pittman, Jill Daw, Jason E. Gestwicki, Heather L. True, Conrad C. Weihl

Selective role of Nck1 in atherogenic inflammation and plaque formation

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Abstract

While Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS) established the role of treating inflammation in atherosclerosis, our understanding of endothelial activation at atherosclerosis-prone sites remains limited. Disturbed flow at atheroprone regions primes plaque inflammation by enhancing endothelial NF-κB signaling. Herein, we demonstrate a role for the Nck adaptor proteins in disturbed flow-induced endothelial activation. Although highly similar, only Nck1 deletion, but not Nck2 deletion, limited flow-induced NF-κB activation and proinflammatory gene expression. Nck1 knockout mice showed reduced endothelial activation and inflammation in both models of disturbed flow and high fat diet-induced atherosclerosis, whereas Nck2 deletion did not. Bone marrow chimeras confirmed that vascular Nck1, but not hematopoietic Nck1, mediated this effect. Domain swap experiments and point mutations identified the Nck1 SH2 domain and the first SH3 domain as critical for flow-induced endothelial activation. We further characterized Nck1’s proinflammatory role by identifying interleukin-1 type I receptor kinase-1 (IRAK-1) as a Nck1-selective binding partner, demonstrating IRAK-1 activation by disturbed flow required Nck1 in vitro and in vivo, showing endothelial Nck1 and IRAK-1 staining in early human atherosclerosis, and demonstrating that disturbed flow-induced endothelial activation required IRAK-1. Taken together, our data reveal a hitherto unknown link between Nck1 and IRAK-1 in atherogenic inflammation.

Authors

Mabruka Alfaidi, Christina H. Acosta, Dongdong Wang, James G. Traylor, A. Wayne Orr

Noggin regulates foregut progenitor cell programming and mis-expression leads to esophageal atresia

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Abstract

Esophageal atresia (EA/TEF) are common congenital abnormalities of the gastrointestinal tract. The etiology of EA/TEF is not well understood. We hypothesized that EA/TEF may be the direct consequence of abnormal expression of Noggin (NOG) signaling cascade. Here we showed that, in neonates with EA/TEF, NOG was missing from the atretic esophagus, resulting in immature esophagus that contains respiratory glands, and cilia. When using mouse esophageal organoid units (EOUs) or tracheal organoid units (TOU) as a model of foregut development in vitro, NOG determined the fate of foregut progenitors by allowing expression of esophageal epithelium proteins. When NOG was present in the culture of mTOU, it altered the cell morphology of the organoid unit epithelium, allowing expression of squamous cell proteins normally found in esophagus. On the other hand, when NOG was inhibited in mEOU, the organoid epithelium began to express respiratory markers mimicking the phenotype seen in pathology samples of human EA/TEF. Moreover, human EOU derived from EA/TEF patients were small, fibrotic and lack esophageal epithelium, but when NOG was added, the EOU grew larger, healthier and express esophageal proteins. These results indicate that Noggin is a critical regulator of cell fate decisions between esophageal and pulmonary morphogenesis.

Authors

Carolina Pinzon-Guzman, Sreedhara Sangadala, Katherine M. Riera, Evgenya Y. Popova, Elizabeth Manning, Won Jae Huh, Matthew S. Alexander, Julia S. Shelton, Scott D. Boden, James R. Goldenring

Blocking endothelial apoptosis revascularises the retina in a model of ischemic retinopathy

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Abstract

Aberrant, neovascular retinal blood vessel growth is a vision-threatening complication in ischemic retinal diseases. It is driven by retinal hypoxia frequently caused by capillary non-perfusion and endothelial cell (EC) loss. We investigated the role of EC apoptosis in this process using a mouse model of ischemic retinopathy, in which vessel closure and EC apoptosis cause capillary regression and retinal ischemia followed by neovascularisation. Protecting ECs from apoptosis in this model did not prevent capillary closure or retinal ischemia. Nonetheless, it prevented the clearance of ECs from closed capillaries, delaying vessel regression and allowing ECs to persist in clusters throughout the ischemic zone. In response to hypoxia, these preserved ECs underwent a vessel sprouting response and rapidly reassembled into a functional vascular network. This alleviated retinal hypoxia, preventing subsequent pathogenic neovascularisation. Vessel reassembly was not limited by VEGFA neutralisation, suggesting it was not dependent on the excess VEGFA produced by the ischemic retina. Neutralisation of ANG2 did not prevent vessel reassembly, but did impair subsequent angiogenic expansion of the reassembled vessels. Blockade of EC apoptosis may promote ischemic tissue re-vascularisation by preserving ECs within ischemic tissue that retain the capacity to reassemble a functional network and rapidly restore blood supply.

Authors

Zoe L. Grant, Lachlan Whitehead, Vickie H. Y. Wong, Zheng He, Richard Y. Yan, Abigail R. Miles, Andrew V. Benest, David O. Bates, Claudia Prahst, Katie Bentley, Bang V. Bui, Robert C.A. Symons, Leigh Coultas