Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGFβ plays a pivotal role in the pathobiology of MF by promoting bone marrow fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF MKs elaborated significantly greater levels of TGFβ1 than TGFβ2 and TGFβ3 to a varying degree, and evaluated the ability of AVID200 a potent TGFβ1/3 protein trap, to block the excessive TGFβ signaling. Treatment of human mesenchymal stromal cells (MSCs) with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGFβ1 to induce collagen expression. Moreover, treatment of MF mononuclear cells (MNCs) with AVID200 led to increased numbers of progenitor cells (PC) with wild type JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PC was attributed to its ability to block TGFβ1-induced p57Kip2 expression and SMAD2 activation thereby allowing normal rather than MF PCs to preferentially proliferate, and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1low mice, a murine model of MF, were treated with AVID200 resulting in the reduction in bone marrow (BM) fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short and long term HSCs. Collectively, these data provide the rationale for TGFβ1 blockade with AVID200 as a therapeutic strategy for MF patients.
Lilian Varricchio, Camelia Iancu-Rubin, Bhaskar Upadhyaya, Maria Zingariello, Fabrizio Martelli, Paola Verachi, Cara Clementelli, Jean-Francois Denis, Adeeb H. Rahman, Gilles Tremblay, John Mascarenhas, Ruben A. Mesa, Maureen O'Connor-McCourt, Anna Rita Migliaccio, Ronald Hoffman
We identified a microRNA (miRNA) profile characterizing HIV lipodystrophy and explored the downstream mechanistic implications with respect to adipocyte biology and the associated clinical phenotype. miRNA profiles were extracted from small extracellular vesicles (sEV) of HIV-infected individuals with and without lipodystrophic changes and individuals without HIV, among whom we previously showed significant reductions in adipose Dicer expression related to HIV. miR-20a-3p was increased and miR-324-5p and miR-186 reduced in sEV from HIV lipodystrophic individuals. Changes in these miRNAs correlated with adipose Dicer expression and clinical markers of lipodystrophy, including fat redistribution, insulin resistance, and hypertriglyceridemia. Human preadipocytes transfected with mimic miR-20a-3p, anti-miR-324-5p or anti-miR-186 induced consistent changes in Ltbp2, Wisp2, and Nebl expression. Knockdown of Ltbp2 (Latent-transforming growth factor beta-binding protein 2) downregulated markers of adipocyte differentiation (Fabp4, Pparg, C/ebpa, Fasn, adiponectin, Glut4, CD36), and Lamin C, and increased expression of genes involved in inflammation (IL1β, IL6, and Ccl20). Our studies suggest a unique sEV miRNA signature related to dysregulation of Dicer in adipose in HIV. Enhanced miR-20a-3p or depletion of miR-186 and miR-324-5p may downregulate Ltbp2 in HIV leading to dysregulation in adipose differentiation and inflammation, which could contribute to acquired HIV lipodystrophy and associated metabolic and inflammatory perturbations.
Suman Srinivasa, Ruben Garcia-Martin, Martin Torriani, Kathleen V. Fitch, Anna R. Carlson, C. Ronald Kahn, Steven K. Grinspoon
BACKGROUND. Immune checkpoint inhibitors (ICIs), which have transformed the care of multiple malignancies, fail to demonstrate efficacy in pancreatic cancer. Recently, genomic biomarkers have been associated with response to ICIs: microsatellite instability high (MSI-H) and tumor mutation burden (TMB) ≥10 mutations/Mb. Some investigations suggest that alterations in Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling genes may predispose to improved outcomes with immunotherapy. The current study examined a possible role for SWI/SNF complex abnormalities in pancreatic cancer responsiveness to ICIs. METHODS. We interrogated a database of 6,831 cancer patients that had undergone next generation sequencing (NGS) in order to evaluate those with advanced pancreatic cancer, SWI/SNF alterations, and outcomes depending on immunotherapy treatment. RESULTS. Of 6,831 cancer patients, nine had metastatic pancreatic adenocarcinoma harboring SWI/SNF chromatin remodeling gene alterations and had received ICIs: seven had an ARID1A alteration (77%); two, ARID1B (22%); three, SMARCA4 (33%); one, SMARCB1 (11%); and one, PBRM1 (11%). Three patients possessed more than one SWI/SNF complex alteration. Only three tumors were microsatellite unstable. Eight of 9 patients (89%) achieved an objective response, including a complete remission, with the two longest responses ongoing at 33+ and 36+ months. Median progression-free and overall survival was 9 and 15 months, respectively. Responses occurred even in the presence of microsatellite stability, low TMB and/or low PD-L1 expression. CONCLUSION. A small subset of patients with pancreatic cancer have genomic alterations in the SWI/SNF chromatin remodeling components and these patients appear to be responsive to ICIs, suggesting the need for prospective trials. TRIAL REGISTRATION. ClinicalTrials.gov NCT02478931 FUNDING. Joan and Irwin Jacobs Fund and by National Cancer Institute at the National Institutes of Health [Grant No. NIH P30 CA023100 (RK) and LRP KYGF9753 (GPB), as well as the Gershenson Family, the Duarte Family, and anonymous patient donors (GPB).
Gregory P. Botta, Shumei Kato, Hitendra Patel, Paul Fanta, Suzanna Lee, Ryosuke Okamura, Razelle Kurzrock
Nox2 is a ROS-generating enzyme, deficiency of which increases suppression by Tregs in vitro and in an in vivo model of cardiac remodelling. Since Tregs have emerged as a candidate therapy in autoimmunity and transplantation, we hypothesised that Nox2 deficiency in Tregs in recipient mice may improve outcomes in a heart transplant model. A novel B6129 mouse model with Treg-targeted Nox2 deletion (Nox2fl/flFoxP3Cre+) was generated and transplanted with hearts from CB6F1 donors. As compared to littermate controls, Nox2fl/flFoxP3Cre+ mice had lower plasma levels of alloantibodies and troponin-I, reduced levels of IFN-γ in heart allograft homogenates and diminished cardiomyocyte necrosis and allograft fibrosis. Single cell analyses of allografts revealed higher absolute numbers of Tregs and lower CD8+ T cell infiltration in Nox2-deficient recipients compared to Nox2-replete mice. Mechanistically, in addition to a greater suppression of CD8+CD25- T effector cell proliferation and IFN-γ production, Nox2-deficient Tregs expressed higher levels of CCR4 and CCR8, driving cell migration to allografts; this was associated with increased expression of miR214-3p. These data indicate that Nox2 deletion in Tregs enhances their suppressive ability and migration to heart allografts. Therefore, Nox2 inhibition in Tregs may be a useful approach to improve their therapeutic efficacy.
Silvia C. Trevelin, Anna Zampetaki, Greta Sawyer, Aleksandar Ivetic, Alison C. Brewer, Lesley Ann Smyth, Federica Marelli-Berg, Robert Köchl, Robert I. Lechler, Ajay M. Shah, Giovanna Lombardi
Development of primary liver cancer is a multi-stage process. Detailed understanding of sequential epigenetic alterations is largely missing. Here, we performed Infinium Human Methylation 450k BeadChips and RNA sequencing analyses for genome-wide methylome and transcriptome profiling of cirrhotic liver (n=7), low- (n=4) and high-grade (n=9) dysplastic lesions, early (n=5) and progressed (n=3) hepatocellular carcinomas (HCC) synchronously detected in eight HCC patients with chronic hepatitis B infection. Integrative analyses of epigenetically driven molecular changes were identified and validated in two independent cohorts comprising 887 HCC. Mitochondrial DNA sequencing was further employed for clonality analyses and indicates multi-clonal origin in the majority of investigated HCC. Alterations in DNA methylation progressively increased from CL to dysplastic lesions and reached a maximum in early HCC. Associated early alterations identified by IPA pathway analyses involved apoptosis, immune regulation and stemness pathways, while late changes centered on cell survival, proliferation and invasion. We further validated putative 23 epi-drivers with concomitant expression changes and associated with overall survival. Functionally, Striatin 4 (STRN4) was demonstrated to be epigenetically regulated and inhibition of STRN4 significantly suppressed tumorigenicity of HCC cell lines. Overall, application of integrative genomic analyses defines epigenetic driver alterations and provides promising targets for novel therapeutic approaches.
Carolin Czauderna, Alicia Poplawski, Colm J. O´Rourke, Darko Castven, Benjamín Pérez-Aguilar, Diana Becker, Stefanie Heilmann-Heimbach, Margarete Odenthal, Wafa Amer, Marcel Schmiel, Uta Drebber, Harald Binder, Dirk A. Ridder, Mario Schindeldecker, Beate K. Straub, Peter R. Galle, Jesper B. Andersen, Snorri S. Thorgeirsson, Young Nyun Park, Jens U. Marquardt
Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here we use clinical information, neuroimaging, and large-scale exome sequencing data to uncover the occurrence and phenotypic spectrum of a novel DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PDZ-binding motif of human (h)DAT. We identified the rare, but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset, neurodegenerative parkinsonism and comorbid neuropsychiatric disease. We show that hDAT-K619N displays reduced uptake capacity, decreased surface expression, and accelerated turnover in cell cultures. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDAT-WT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila leads to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.
Freja Herborg, Kathrine L. Jensen, Sasha Tolstoy, Natascha V. Arends, Leonie P. Posselt, Aparna Shekar, Jenny I Aguilar, Viktor K. Lund, Kevin Erreger, Mattias Rickhag, Matthew D. Lycas, Markus N. Lonsdale, Troels Rahbek-Clemmensen, Andreas T. Sørensen, Amy H. Newman, Annemette Løkkegaard, Ole Kjaerulff, Thomas Werge, Lisbeth B. Møller, Heinrich J.G. Matthies, Aurelio Galli, Lena E. Hjermind, Ulrik Gether
Chronic obstructive pulmonary disease (COPD) is mainly caused by cigarette smoking, characterized by chronic inflammation in vulnerable individuals. However, it is unknown how genetic factors may shape chronic inflammation in COPD. To understand how hedgehog interacting protein, encoded by HHIP gene identified in the genome-wide association study in COPD, plays a role in inflammation, we utilized Hhip+/- mice that resemble persistent inflammation and emphysema observed in human COPD upon aging. By performing single cell RNA sequencing of the whole lung from mice at different ages, we found that Hhip+/- mice develop a cytotoxic immune response with a specific increase in Klrg1+CD8+T cells with upregulated Ifnγ expression recapitulating human COPD. Hhip expression was restricted to a lung fibroblast subpopulation that has increased interaction with CD8+T lymphocytes in Hhip+/- compared to Hhip+/+ during aging. Hhip-expressing lung fibroblasts have upregulated IL-18 pathway genes in Hhip+/- lung fibroblasts, which was sufficient to drive increased levels of IFNγ in CD8+T cells ex vivo. Our finding provides insight into how a common genetic variation contributes to the amplified lymphocytic inflammation in COPD.
Jeong H. Yun, ChangHee Lee, Tao Liu, Siqi Liu, Edy Y. Kim, Shuang Xu, Jeffrey L. Curtis, Luca Pinello, Russell P. Bowler, Edwin K. Silverman, Craig P. Hersh, Xiaobo Zhou
Cell lines are the mainstay in understanding the biology of COVID-19 infection, but do not recapitulate many of the complexities of human infection. The use of human lung tissue is one solution for the study of such novel respiratory pathogens. We hypothesized that a cryopreserved bank of human lung tissue allows for the ex vivo study of the inter-individual heterogeneity of host response to SARS-CoV-2 infection, thus providing a bridge between studies with cell lines and studies in animal models. We generated a cryobank of tissues from 21 donors, many of whom had clinical risk factors for severe COVID-19. Cryopreserved tissues preserved 90% cell viability and contained heterogenous populations of metabolically active epithelial, endothelial, and immune cell subsets of the human lung. Samples were readily infectable with HCoV-OC43 and SARS-CoV-2 coronaviruses, and demonstrated comparable susceptibility to infection. In contrast, we observed a marked donor-dependent heterogeneity in the expression of IL6, CXCL8 and IFNB1 in response to SARS-CoV2 infection. Treatment of tissues with dexamethasone and the experimental drug, n-hydroxycytidine, suppressed viral growth in all samples, whereas chloroquine and remdesivir had no detectable effect. Metformin and sirolimus, molecules with predicted but unproven antiviral activity, each suppressed viral replication in tissues from a subset of donors. In summary, we developed a novel system for the ex vivo study of human SARS-CoV- 2 infection using primary human lung tissue from a library of donor tissues. This model may be useful for drug screening and for understanding basic mechanisms of COVID-19 pathogenesis.
Matthew A. Schaller, Yamini Sharma, Zadia Dupee, Duy T. Nguyen, Juan M. Urueña, Ryan A. Smolchek, Julia C. Loeb, Tiago N. Machuca, John A. Lednicky, David J. Odde, Robert F. Campbell, W. Gregory Sawyer, Borna Mehrad
The inflammatory and IFN pathways of innate immunity play a key role in both resistance and pathogenesis of Coronavirus Disease 2019 (COVID-19). Innate sensors and SARS-CoV-2-Associated Molecular Patterns (SAMPs) remain to be completely defined. Here we identify single-stranded RNA (ssRNA) fragments from SARS-CoV-2 genome as direct activators of endosomal TLR7/8 and MyD88 pathway. The same sequences induced human DC activation in terms of phenotype and functions, such as IFN and cytokine production and Th1 polarization. A bioinformatic scan of the viral genome identified several hundreds of fragments potentially activating TLR7/8, suggesting that products of virus endosomal processing potently activate the IFN and inflammatory responses downstream these receptors. In vivo, SAMPs induced MyD88-dependent lung inflammation characterized by accumulation of proinflammatory and cytotoxic mediators and immune cell infiltration, as well as splenic DC phenotypical maturation. These results identify TLR7/8 as crucial cellular sensors of ssRNAs encoded by SARS-CoV-2 involved in host resistance and disease pathogenesis of COVID-19.
Valentina Salvi, Hoang Oanh Nguyen, Francesca Sozio, Tiziana Schioppa, Carolina Gaudenzi, Mattia Laffranchi, Patrizia Scapini, Mauro Passari, Ilaria Barbazza, Laura Tiberio, Nicola Tamassia, Cecilia Garlanda, Annalisa Del Prete, Marco Antonio Cassatella, Alberto Mantovani, Silvano Sozzani, Daniela Bosisio
The NR4A family of orphan nuclear receptors (Nr4a1-3) plays redundant roles to establish and maintain Treg identity; deletion of multiple family members in the thymus results in Treg deficiency and a severe inflammatory disease. Consequently, it has been challenging to unmask redundant functions of the NR4A family in other immune cells. Here we use a competitive bone marrow chimera strategy, coupled with conditional genetic tools, to rescue Treg homeostasis and unmask such functions. Unexpectedly, chimeras harboring Nr4a1–/– Nr4a3–/– (DKO) bone marrow develop autoantibodies and a systemic inflammatory disease despite a replete Treg compartment of largely wild-type origin. This disease differs qualitatively from that seen with Treg-deficiency and is B cell-extrinsic. Negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO T cells with functional, phenotypic, and transcriptional features of anergy accumulate in chimeric mice. Nevertheless, we observe upregulation of genes encoding inflammatory mediators in anergic DKO T cells, and DKO T cells exhibit enhanced capacity for IL-2 production. These studies reveal cell-intrinsic roles for the NR4A family in both central and peripheral T cell tolerance, and demonstrate that each is essential to preserve immune homeostasis.
Ryosuke Hiwa, Hailyn V. Nielsen, James L. Mueller, Ravi Mandla, Julie Zikherman
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