Virus-specific CD8+ T cells play a central role in HIV-1 natural controllers to maintain suppressed viremia in the absence of antiretroviral therapy. These cells display a memory program that confers them stemness properties, high survival, polyfunctionality, proliferative capacity, metabolic plasticity, and antiviral potential. The development and maintenance of such qualities by memory CD8+ T cells appear crucial to achieving natural HIV-1 control. Here we show that targeting the signaling pathways Wnt/TCF-1 and mTORC through GSK3 inhibition to reprogram HIV-specific CD8+ T cells from non-controllers promoted functional capacities associated with natural control of infection. Features of such reprogrammed cells included the enrichment in TCF-1+ less-differentiated subsets, superior response to antigen, enhanced survival, polyfunctionality, metabolic plasticity, less mTORC1-dependency, improved response to γ-chain cytokines and stronger HIV suppressive capacity. Thus, such CD8+ T cell reprogramming, combined with other available immunomodulators, might represent a promising strategy for adoptive cell therapy in the search for an HIV-1 cure.
Federico Perdomo-Celis, Caroline Passaes, Valérie Monceaux, Stevenn Volant, Faroudy Boufassa, Pierre de Truchis, Morgane Marcou, Katia Bourdic, Laurence Weiss, Corinne Jung, Christine Bourgeois, Cécile Goujard, Laurence Meyer, Michaela Müller-Trutwin, Olivier Lambotte, Asier Sáez-Cirión
Ischemic stroke prompts a strong inflammatory response which is associated with exacerbated outcomes. In this study, we investigated mechanistic regulators of neutrophil extracellular trap (NET) formation in stroke and if they contribute to stroke outcomes. NET forming neutrophils were found throughout brain tissue of ischemic stroke patients and elevated plasma NET biomarkers correlated with worse stroke outcomes. Additionally, we observed increased plasma and platelet surface expressed high mobility group box 1 (HMGB1) in stroke patients. Mechanistically, platelets were identified as the critical source for HMGB1 causing NETs in the acute phase of stroke. Depleting platelets or platelet-specific knockout of HMGB1 significantly reduced plasma HMGB1 and NET levels after stroke, and greatly improved stroke outcomes. We subsequently investigated the therapeutic potential of neonatal NET inhibitory factor (nNIF) in stroke. Mice treated with nNIF had smaller brain infarcts, improved long-term neurological and motor function, and enhanced survival after stroke. nNIF specifically blocked NET formation without affecting neutrophil recruitment after stroke. Importantly, nNIF also improved stroke outcomes in diabetic and aged mice and was still effective when given 1 hour after stroke onset. These results support a pathological role for NETs in ischemic stroke and warrant further investigation of nNIF for stroke therapy.
Frederik Denorme, Irina Portier, John L. Rustad, Mark J. Cody, Claudia V. de Araujo, Chieko Hoki, Matthew D. Alexander, Ramesh Grandhi, Mitchell R. Dyer, Matthew D. Neal, Jennifer J. Majersik, Christian C. Yost, Robert A. Campbell
BACKGROUND. It is unclear whether the level of serum hepatitis B virus (HBV) DNA at baseline impacts the on-treatment risk of hepatocellular carcinoma (HCC) in HBeAg positive, non-cirrhotic patients with chronic hepatitis B (CHB). METHODS. We conducted a multicenter cohort study including 2,073 entecavir- or tenofovir-treated, HBeAg-positive, non-cirrhotic, adult CHB patients with baseline HBV DNA levels ≥5.00 log10 IU/mL at three centers in Korea between January 2007 and December 2016. We evaluated the on-treatment incidence rate of HCC by baseline HBV DNA levels. RESULTS. During a median 5.7 years of continuous antiviral treatment, 47 patients developed HCC (0.39 per 100 person-years). By Kaplan–Meier analysis, HCC risk was the lowest in those with baseline HBV DNA levels ≥8.00 log10 IU/mL, increased incrementally with decreasing viral load, and the highest with HBV DNA levels 5.00–5.99 log10 IU/mL (P<0.001). By multivariable analysis, baseline HBV DNA level was an independent factor that was inversely associated with HCC risk. Compared with HBV DNA ≥8.00 log10 IU/mL, the adjusted hazard ratios for HCC risk with HBV DNA 7.00–7.99 log10 IU/mL, 6.00–6.99 log10 IU/mL, and 5.00–5.99 log10 IU/mL were 2.48 (P=0.03), 3.69 (P=0.002), and 6.10 (P<0.001), respectively. CONCLUSION. On-treatment HCC risk increased incrementally with decreasing baseline HBV DNA levels in the range of ≥5.00 log10 IU/mL in HBeAg-positive, non-cirrhotic, adult patients with CHB. Early initiation of antiviral treatment with a high viral load (≥8.00 log10 IU/mL) may maintain the lowest risk of HCC in those patients. FUNDING. Korean Government.
Won-Mook Choi, Gi-Ae Kim, Jonggi Choi, Seungbong Han, Young-Suk Lim
BACKGROUND. Tuberous Sclerosis Complex (TSC) is a neurogenetic syndrome due to loss-of-function mutations in TSC2 or TSC1, characterized by tumors at multiple body sites, including facial angiofibroma (FAF). Here, an ultrasensitive assessment of the extent and range of UV-induced mutations in TSC facial skin was performed. METHODS. A Multiplex High-sensitivity PCR Assay (MHPA) was developed, enabling mutation detection at extremely low (<0.1%) variant allele frequencies (VAF). RESULTS. MHPA assays were developed for both TSC2 and TP53, and applied to 81 samples, including 66 skin biopsies. UV-induced second hit mutation causing inactivation of TSC2 was pervasive in TSC facial skin with an average of 4.8 mutations per 2 mm biopsy at median VAF 0.08%, generating >150,000 incipient facial tumors (subclinical ‘micro-FAFs’) in the average TSC subject. The MHPA analysis also led to the identification of a refined UV-related indel signature and a recurrent complex mutation pattern, consisting of both a single or dinucleotide variant, and a 1-9 nt deletion, in cis. CONCLUSION. TSC facial skin can be viewed as harboring a patchwork of clonal fibroblast proliferations (micro-FAF) with indolent growth, a small proportion of which develop into clinically observable FAF. Our observations also expand the spectrum of UV-related mutation signatures. FUNDING. This work was supported by the TSC Alliance, Engles Family Fund for Research in TSC and LAM, and National Institutes of Health, National Heart, Lung, and Blood Institute [U01HL131022-04; Intramural Research Program].
Katarzyna Klonowska, Joannes M. Grevelink, Krinio Giannikou, Barbara A. Ogorek, Zachary T. Herbert, Aaron R. Thorner, Thomas N. Darling, Joel Moss, David J. Kwiatkowski
Pericytes (PC) are abundant yet remain the most enigmatic and ill-defined cell population in the heart. Here, we investigated if PC can be reprogrammed to aid neovascularization. Primary PC from human and mouse hearts acquired cytoskeleton proteins typical of vascular smooth muscle cells (VSMC) upon exclusion of EGF/bFGF, which signal through ERK1/2, or exposure to the MEK-inhibitor PD0325901. Differentiated PC became more proangiogenic, more responsive to vasoactive agents, and insensitive to chemoattractants. RNA-Sequencing revealed transcripts marking the PD0325901-induced transition into proangiogenic, stationary VSMC-like cells, including the unique expression of two angiogenesis-related markers, aquaporin 1 (AQP1) and cellular retinoic acid-binding protein 2 (CRABP2), which were further verified at the protein level. This enabled us to trace PC during in vivo studies. In mice, implantation of Matrigel plugs containing human PC+PD0325901 promoted the formation of α-SMApos neovessels compared with PC only. Two-week oral administration of PD0325901 to mice increased the heart arteriolar density, total vascular area, arteriole coverage by PDGFRβposAQP1posCRABP2pos PC, and myocardial perfusion. Short-duration PD0325901 treatment of mice after myocardial infarction enhanced the peri-infarct vascularization, reduced the scar, and improved systolic function. In conclusion, myocardial PC have intrinsic plasticity that can be pharmacologically modulated to promote reparative vascularization of the ischemic heart.
Elisa Avolio, Rajesh Katare, Anita C. Thomas, Andrea Caporali, Daryl Schwenke, Michele Carrabba, Marco Meloni, Massimo Caputo, Paolo Madeddu
Nonalcoholic fatty liver disease (NAFLD), the most common liver disease has become a silent worldwide pandemic. The incidence of NAFLD correlates with the rise in obesity, type 2 diabetes and metabolic syndrome. A hallmark feature of NAFLD is excessive hepatic fat accumulation or steatosis, due to dysregulated hepatic fat metabolism which can progress to nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Currently, there are no approved pharmacotherapies to treat this disease. Here we have identified that activation of the kisspeptin receptor (KISS1R) signaling pathway has therapeutic effects in NAFLD. Using high fat diet-fed mice, we demonstrated that a deletion of hepatic Kiss1r exacerbated hepatic steatosis. In contrast, enhanced stimulation of KISS1R protected against steatosis in wild-type C57BL/6J mice and decreased fibrosis using a diet-induced mouse model of NASH. Mechanistically, we found that hepatic KISS1R signaling activates the master energy regulator, AMPK, to thereby decrease lipogenesis and progression to NASH. In NAFLD patients and in HFD-fed mice, hepatic KISS1/KISS1R expression and plasma kisspeptin levels were elevated, suggesting a compensatory mechanism to reduce triglyceride synthesis. These findings establish KISS1R as a therapeutic target to treat NASH.
Stephania Guzman, Magdalena Dragan, Hyokjoon Kwon, Vanessa de Oliveira, Shivani Rao, Vrushank Bhatt, Katarzyna M. Kalemba, Ankit Shah, Vinod K. Rustgi, He Wang, Paul R. Bech, Ali Abbara, Chioma Izzi-Engbeaya, Pinelopi Manousou, Jessie Yanxiang Guo, Grace L. Guo, Sally Radovick, Waljit S. Dhillo, Fredric E. Wondisford, Andy V. Babwah, Moshmi Bhattacharya
Food addiction is characterized by a loss of behavioral control over food intake and is associated with obesity and other eating disorders. The mechanisms underlying this behavioral disorder are largely unknown. We aim to investigate the changes in miRNAs expression promoted by food addiction in animals and humans and their involvement in the mechanisms underlying the behavioral hallmarks of this disorder. Sharp similitudes were found between the miRNAs signatures in the medial prefrontal cortex (mPFC) of our animal cohort and the miRNAs circulating levels in our human cohort allowing to identify several miRNAs of potential interest for the development of this disorder. TuD inhibition of miRNA-29c-3p in the mouse mPFC promotes persistence to response and enhances the vulnerability to develop food addiction, whereas miRNA-665-3p inhibition promotes compulsive-like behavior and also enhances food addiction vulnerability. In contrast, miRNA-137-3p inhibition in the mPFC does not affect the development of food addiction. Therefore, miRNA-29c-3p and miRNA-665-3p could be acting as protective factors towards food addiction. The elucidation of these novel epigenetic mechanisms provides advances toward innovative biomarkers and possible future interventions for food addiction and related disorders based on the strategies now available to modify miRNA activity and expression.
Alejandra García-Blanco, Laura Domingo-Rodriguez, Judit Cabana-Domínguez, Noèlia Fernàndez-Castillo, Laura Pineda-Cirera, Jordi Mayneris-Perxachs, Aurelijus Burokas, Jose Espinosa-Carrasco, Silvia Arboleya, Jessica Latorre, Catherine Stanton, Bru Cormand, Jose-Manuel Fernández-Real, Elena Martín-García, Rafael Maldonado
Understanding the regulatory programs enabling cancer stem cells (CSCs) to self-renew and drive tumorigenicity could identify new treatments. Through comparative chromatin state and gene expression analyses in ovarian CSCs vs. non-CSCs, we identified FOXK2 as a highly expressed stemness-specific transcription factor in ovarian cancer. Its genetic depletion diminished stemness features and reduced tumor initiation capacity. Our mechanistic studies highlight that FOXK2 directly regulated IRE1α (ERN1 gene) expression, a key sensor for the unfolded protein response (UPR). Chromatin immunoprecipitation-sequencing revealed that FOXK2 bound to an intronic regulatory element of ERN1. Blocking FOXK2 from binding to this enhancer by using a catalytically inactive CRISPR/Cas9 (dCas9) diminished IRE1α transcription. At the molecular level, FOXK2-driven upregulation of IRE1α led to alternative XBP1 splicing and activation of stemness pathways, while genetic or pharmacological blockade of this sensor of the UPR inhibited ovarian CSCs. Collectively, these data establish a new function for FOXK2 as a key transcriptional regulator of CSCs and a mediator of the UPR, providing insight into potentially targetable new pathways in CSCs.
Yaqi Zhang, Yinu Wang, Guangyuan Zhao, Edward J. Tanner, Mazhar Adli, Daniela Matei
BACKGROUND. Responses to conventional donor lymphocyte infusion (DLI) for post-allogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell-based therapy is a promising modality to treat post-HCT relapse. METHODS. We initiated this ongoing phase I trial of adoptively transferred cytokine induced memory-like (CIML) NK cells in patients with myeloid malignancies relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5–10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High resolution profiling with mass cytometry and single cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion. RESULTS. In the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10 to 50-fold in vivo expansion that was sustained over months. The infusion was well-tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires. CONCLUSION. Given their rapid expansion and long-term persistence in an immune compatible environment, CIML NK cells serve as a promising platform for the treatment of post-transplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies. TRIAL REGISTRATION. NCT04024761 FUNDING. Supported by Dunkin Donuts Breakthrough Award, the NIH/National Cancer Institute R21 CA245413, the Leukemia and Lymphoma Society Scholar and TRP awards.
Roman M. Shapiro, Grace C. Birch, Guangan Hu, Juliana Vergara Cadavid, Sarah Nikiforow, Joanna Baginska, Alaa K. Ali, Mubin Tarannum, Michal Sheffer, Yasmin Z. Abdulhamid, Benedetta Rambaldi, Yohei Arihara, Carol Reynolds, Max S. Halpern, Scott J. Rodig, Nicole Cullen, Jacquelyn O. Wolff, Kathleen L. Pfaff, Andrew A. Lane, R. Coleman Lindsley, Corey S. Cutler, Joseph H. Antin, Vincent T. Ho, John Koreth, Mahasweta Gooptu, Haesook T. Kim, Karl-Johan Malmberg, Catherine J. Wu, Jianzhu Chen, Robert J. Soiffer, Jerome Ritz, Rizwan Romee
As blood transitions from steady laminar flow (S-flow) in healthy arteries to disturbed flow (D-flow) in aneurysmal arteries, platelets are subjected to external forces. Biomechanical platelet activation is incompletely understood and is a potential mechanism behind antiplatelet medication resistance. While it was demonstrated that anti-platelet drugs suppress growth of abdominal aortic aneurysms (AAA) in patients, we revealed a certain degree of platelet reactivity persisted in spite of aspirin therapy urging us to consider additional anti-platelet therapeutic targets. Transcriptomic profiling of platelets from patients with AAA revealed upregulation of a signal transduction pathway common to olfactory receptors (ORs), and this was explored as a mediator of AAA progression. Healthy platelets subjected to D-flow ex vivo, platelets from patients with AAA, and platelets in murine models of AAA demonstrated increased membrane olfactory receptor 2L13 (OR2L13) expression. A drug screen identified a molecule activating platelet OR2L13 which limited both biochemical and biomechanical platelet activation as well as AAA growth. This observation was further supported by selective deletion of the OR2L13 ortholog in a murine model of AAA that accelerated aortic aneurysm growth and rupture. These studies reveal that ORs regulate platelet activation in AAA and aneurysmal progression through platelet-derived mediators of aortic remodeling.
Craig N. Morrell, Doran Mix, Anu Aggarwal, Rohan Bhandari, Matthew Godwin, A. Phillip Owens III, Sean P. Lyden, Adam Doyle, Krystin Krauel, Matthew T. Rondina, Amy Mohan, Charles J. Lowenstein, Sharon Shim, Shaun Stauffer, Vara Prasad Josyula, Sara K. Ture, David I. Yule, Larry E. Wagner III, John M. Ashton, Ayman Elbadawi, Scott J. Cameron
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