Research
Abstract
T cell receptor (TCR) stimulation leads to expression of the transcription factor TOX. Prolonged TCR signaling, such as encountered during chronic infections or in tumors, leads to sustained TOX expression, which is required for the induction of a state of exhaustion or dysfunction. While CD8 memory T cells (Tmem) in mice typically do not express TOX at steady state, some human Tmem express TOX, but appear fully functional. This seeming discrepancy between mouse and human T cells has led to the speculation that TOX is differentially regulated between these species, which could complicate the interpretation of pre-clinical mouse model studies. We report here that similarly to TCR-mediated signals, inflammatory cytokines are also sufficient to increase TOX expression in human and mouse Tmem. Thus, TOX expression is controlled by the environment, which provides an explanation for the different TOX expression patterns encountered in T cells isolated from specific pathogen free laboratory mice versus humans. Finally, we report that TOX is not necessary for cytokine-driven expression of PD-1. Overall, our data highlight that the mechanisms regulating TOX expression are conserved across species and indicate that TOX expression reflects a T cell’s activation state, and does not necessarily correlate with T cell dysfunction.
Authors
Nicholas J. Maurice, Jacqueline Berner, Alexis K. Taber, Dietmar Zehn, Martin Prlic
Abstract
Alloimmune responses driven by donor-specific antibodies (DSAs) can lead to antibody-mediated rejection (ABMR) in organ transplantation. Yet, the cellular states underlying alloreactive B cell responses and the molecular components controlling them remain unclear. Using high dimensional profiling of B cells in a cohort of 96 kidney transplant recipients, we identified expanded numbers of CD27+CD21- activated memory (AM) B cells that expressed the transcription factor T-bet in patients who developed DSAs and progressed to ABMR. Notably, AM cells were less frequent in DSA+ABMR- patients and at baseline levels in DSA- patients. RNA-seq analysis of AM cells in patients undergoing ABMR revealed these cells to be poised for plasma cell differentiation and to express restricted IGHV sequences reflective of clonal expansion. In addition to T-bet, AM cells manifested elevated expression of IRF4 and Blimp1, and upon co-culture with autologous T follicular helper cells, differentiated into DSA-producing plasma cells in an IL-21 dependent manner. The frequency of AM cells was correlated with the timing and severity of ABMR manifestations. Importantly, T-bet+ AM cells were detected within kidney allografts along with their restricted IGHV sequences. This study delineates a pivotal role for AM cells in promoting humoral responses and ABMR in organ transplantation and highlights them as important therapeutic targets.
Authors
Kevin Louis, Elodie Bailly, Camila Macedo, Louis Lau, Bala Ramaswami, Alexander Chang, Uma Chandran, Douglas Landsittel, Xinyan Gu, Geetha Chalasani, Adriana Zeevi, Parmjeet Randhawa, Harinder Singh, Carmen Lefaucheur, Diana Metes
Abstract
Cigarette smoke (CS) is the main etiological factor in the pathogenesis of emphysema/Chronic Obstructive Pulmonary Disease (COPD), which is associated with abnormal epithelial-mesenchymal-transition (EMT). Previously, we have shown an association between circadian rhythms and CS-induced lung inflammation, and nuclear-heme-receptor α (REV-ERBα) acting as an anti-inflammatory target in both pulmonary epithelial cells and fibroblasts. We hypothesized that molecular clock REV-ERBα plays an important role in CS-induced circadian dysfunction and EMT alteration. C57BL/6J wild type (WT) and REV-ERBα heterozygous (Het) and knockout (KO) mice were exposed to CS for 30 days (sub-chronic) and 4 months (chronic), and WT mice were exposed to CS for 10 days with or without REV-ERBα agonist (SR9009) administration. Sub-chronic/chronic CS exposure caused circadian disruption and dysregulated EMT in the lungs of WT and REV-ERBα KO mice, both circadian and EMT dysregulation were exaggerated in REV-ERBα KO condition. REV-ERBα agonist, SR9009 treatment reduced acute CS-induced inflammatory response and abnormal EMT in the lungs. Further, REV-ERBα agonist (GSK4112) inhibited TGFβ/CS-induced fibroblast differentiation in human fetal lung fibroblast 1 (HFL-1).Thus, CS-induced circadian gene alterations and EMT activation are mediated through a Rev-erbα-dependent mechanism, which suggests activation of REV-ERBα as a novel therapeutic approach for smoking-induced chronic inflammatory lung diseases.
Authors
Qixin Wang, Isaac K. Sundar, Joseph H. Lucas, Thivanka Muthumalage, Irfan Rahman
Abstract
While antiretroviral therapy suppresses HIV replication, it does not eliminate viral reservoirs or restore damaged lymphoid tissue, posing obstacles to HIV eradication. Using the simian immunodeficiency virus (SIV) model of AIDS, we investigated the effect of mesenchymal stem/stromal cell (MSC) infusions on gut mucosal recovery, anti-viral immunity, viral suppression and determined associated molecular/metabolic signatures. MSC administration to SIV-infected macaques resulted in viral reduction and heightened virus-specific responses. Marked clearance of SIV-positive cells from gut mucosal effector sites was correlated with robust regeneration of germinal centers (GC), restoration of follicular B cells, T follicular helper (Tfh) cells, and enhanced antigen presentation by viral trapping within follicular dendritic cell network. Gut transcriptomic analyses showed increased antiviral response mediated by unique pathways of type-I/II IFN signaling, viral restriction factors, innate immunity and B cell proliferation and provided the molecular signature underlying enhanced host immunity. Metabolic analysis revealed strong correlations between B and Tfh cell activation, anti-SIV antibodies, and IL-7 expression with enriched retinol metabolism, which facilitates gut homing of antigen-activated lymphocytes. Our findings discover new MSC functions in modulating anti-viral immunity for enhanced viral clearance predominantly through type-I/II IFN signaling and B cell signature and provide a roadmap for multi-pronged HIV eradication strategies.
Authors
Mariana G. Weber, Chara J. Walters-Laird, Amir Kol, Clarissa Santos Rocha, Lauren A. Hirao, Abigail Mende, Bipin Balan, Juan Arredondo, Sonny R. Elizaldi, Smita S. Iyer, Alice Tarantal, Satya Dandekar
Abstract
Several studies have associated the presence of residual insulin secretion capability (also referred to as being C-peptide positive) with lower risk of insulin-induced hypoglycemia in patients with type 1 diabetes (T1D), although the reason is unclear. We tested the hypothesis that C-peptide infusion would enhance glucagon secretion in response to hyperinsulinemia during euglycemic and hypoglycemic conditions in dogs (5m/4f). After a 2 hr basal period, an IV-infusion of insulin was started, and dextrose was infused to maintain euglycemia for 2 hrs. At the same time, an IV-infusion of either saline (SAL) or C-peptide (CPEP) was started. After this euglycemic period, the insulin and SAL/CPEP infusions were continued for another 2 hrs, but the glucose was allowed to fall to ~50 mg/dL. In response to euglycemic-hyperinsulinemia, glucagon secretion decreased in SAL, but remained unchanged from basal in CPEP. During hypoglycemia, glucagon secretion in CPEP was two times higher than SAL, which increased net hepatic glucose output and reduced the amount of exogenous glucose required to maintain glycemia. These data suggest that the presence of C-peptide during IV insulin infusion can preserve glucagon secretion during euglycemia, and enhance it during hypoglycemia, which could explain why T1D patients with residual insulin secretion are less susceptible to hypoglycemia.
Authors
Mary Courtney Moore, Shana O. Warner, Yufei Dai, Nicole Sheanon, Marta Smith, Ben Farmer, Rebecca L. Cason, Alan D. Cherrington, Jason J. Winnick
Abstract
Epigenetic modifications of the genome, including DNA methylation, histone methylation/acetylation and noncoding RNAs, have been reported to play a fundamental role in regulating immune response during the progression of atherosclerosis. SETDB2 is a member of the KMT1 family of lysine methyltransferases and members of this family typically methylate histone H3 Lys9 (H3K9), an epigenetic mark associated with gene silencing and previous studies have shown SETDB2 is involved in innate and adaptive immunity, the pro-inflammatory response and hepatic lipid metabolism. Here we report that the expression of SETDB2 is markedly upregulated in human and murine atherosclerotic lesions. The upregulation of SETDB2 is observed in pro-inflammatory M1, but not anti-inflammatory M2 macrophages (MΦ). Notably, we found that genetic deletion of SETDB2 in hematopoietic cells promotes vascular inflammation and enhances the progression of atherosclerosis in bone marrow transfer studies in LDLR knockout mice. Single cell RNA-Seq analysis in isolated CD45+ cells from atherosclerotic plaques from mice with SETDB2 deficient bone marrow revealed a significant increase in inflammatory macrophage population and enhanced expression of genes involved in inflammation, myeloid cell recruitment and lipid metabolism. Additionally, we found that loss of SETDB2 in hematopoietic cells is associated with macrophage accumulation in atherosclerotic lesions, macrophage proliferation and attenuated efferocytosis. Overall, these studies identify SETDB2 as an important inflammatory cell regulator that controls macrophage activation in atherosclerotic plaques.
Authors
Xinbo Zhang, Jonathan Sun, Alberto Canfrán-Duque, Binod Aryal, George Tellides, Ying Ju Chang, Yajaira Suárez, Timothy F. Osborne, Carlos Fernández-Hernando
Abstract
Hepatocellular carcinoma (HCC) is the 6th-most common and the 4th-most deadly cancer worldwide. The development cost of new therapeutics is a major limitation in patient outcomes. Importantly, there is a paucity of preclinical HCC models in which to test new small molecules. Herein, we implemented novel patient-derived organoid (PDO) and patient-derived xenografts (PDX) strategies for high-throughput drug screening. Omacetaxine, an FDA-approved drug for chronic myelogenous leukemia (CML), was found to be a top effective small molecule in HCC PDOs. Next, omacetaxine was tested against a larger cohort of 40 human HCC PDOs. Serial dilution experiments demonstrated that omacetaxine is effective at low (nanomolar) concentrations. Mechanistic studies established that omacetaxine inhibits global protein synthesis, with a disproportionate effect on short-half-life proteins. High-throughput expression screening newly identified molecular targets for omacetaxine, including key oncogenes, such as PLK1. In conclusion, by using an innovative strategy, we report, for the first time, the effectiveness of omacetaxine in HCC. In addition, we newly elucidate key mechanisms of omacetaxine action. Finally, we provide a proof-of-principle basis for future studies applying drug screening PDOs sequenced with candidate validation in PDX models. Clinical trials could be considered to evaluate omacetaxine in patients with HCC.
Authors
Ling Li, Gilad Halpert, Michael G. Lerner, Haijie Hu, Peter Dimitrion, Matthew J. Weiss, Jin He, Benjamin Philosophe, Richard Burkhart, William R. Burns, Russell N. Wesson, Andrew MacGregor Cameron, Christopher L. Wolfgang, Christos Georgiades, Satomi Kawamoto, Nilofer S. Azad, Mark Yarchoan, Stephen J. Meltzer, Kiyoko Oshima, Laura M. Ensign, Joel S. Bader, Florin M. Selaru
Abstract
Haploidentical hematopoietic stem cell transplantation (h-HSCT) represents an efficient curative approach for patients affected by hematologic malignancies in which the reduced intensity conditioning induces a state of immunologic tolerance between donor and recipient. However, opportunistic viral infections greatly affect h-HSCT clinical outcomes. Natural Killer (NK) cells are the first lymphocytes recovering after transplant and provide a prompt defense against human Cytomegalovirus (HCMV) infection/reactivation. By undertaking a longitudinal single cell computational profiling of multiparametric flow cytometry, we show that HCMV accelerates NK cell immune-reconstitution together with the expansion of CD158b1b2jpos/NKG2Aneg/NKG2Cpos/NKp30low NK cells. The frequency of this subset correlates with HCMV viremia, further increases in recipients experiencing multiple episodes of viral reactivations and persists for months after the infection. The transcriptional profile of FACS-sorted CD158b1b2jpos NK cells confirmed the ability of HCMV to de-regulate NKG2C, NKG2A and NKp30 gene expression, thus inducing the expansion of NK cells with adaptive traits. These NK cells are characterized by the down-modulation of several gene pathways associated with cell migration, cell-cycle, effector-functions and by a state of metabolic/cellular exhaustion. This profile reflects the functional impairments of adaptive NK cells to produce IFN-γ, a phenomenon also due to the viral-induced expression of LAG-3 and PD-1 checkpoint-inhibitors.
Authors
Elisa Zaghi, Michela Calvi, Simone Puccio, Gianmarco Spata, Sara Terzoli, Clelia Peano, Alessandra Roberto, Federica De Paoli, Jasper J.P. van Beek, Jacopo Mariotti, Chiara De Philippis, Barbara Sarina, Rossana Mineri, Stefania Bramanti, Armando Santoro, Vu Thuy Khanh Le-Trilling, Mirko Trilling, Emanuela Marcenaro, Luca Castagna, Clara Di Vito, Enrico Lugli, Domenico Mavilio
Abstract
Background: Dietary sodium intake mismatches urinary sodium excretion over prolonged periods. Our aims were to localize and quantify electrostatically bound sodium within human skin using triple quantum filtered (TQF) protocols for magnetic resonance imaging (MRI) and spectroscopy (MRS), and explore dermal sodium in Type 2 Diabetes Mellitus (T2D). Methods: We recruited adult participants with T2D (n=9) and euglycemic participants with no history of Diabetes Mellitus (n=8). All had undergone lower limb amputations or abdominal skin reduction surgery for clinical purposes. We used 20μm in-plane resolution 1H MRI to visualise anatomical skin regions ex vivo from skin biopsies taken intra-operatively, 23Na TQF MRI/MRS to explore distribution and quantification of freely dissolved and bound sodium and Inductively Coupled Plasma Mass Spectrometry to quantify sodium in selected skin samples. Results: Human dermis has a preponderance (>90%) of bound sodium that co-localizes with the glycosaminoglycan (GAG) scaffold. Bound and free sodium have similar anatomical locations. T2D associates with a severely reduced dermal bound sodium capacity. Conclusion: We provide the first evidence for high levels of bound sodium within human dermis, co-locating to the GAG scaffold, consistent with a dermal ‘third space repository’ for sodium. T2D associates with diminished dermal electrostatic binding capacity for sodium.
Authors
Petra Hanson, Christopher J. Philp, Harpal S. Randeva, Sean James, J Paul O'Hare, Thomas Meersmann, Galina E. Pavlovskaya, Thomas M. Barber
Abstract
Metastases cause 90% of human cancer deaths. The metastatic cascade involves local invasion, intravasation, extravasation, metastatic site colonization, and proliferation. While individual mediators of these processes have been investigated, interactions between these mediators remain less well defined. We previously identified a complex between receptor tyrosine kinase c-Met and β1 integrin in metastases. Using novel cell culture and in vivo assays, we found that c-Met/β1 complex induction promotes intravasation and vessel wall adhesion in triple-negative breast cancer cells, but does not increase extravasation. These effects may be driven by the ability of the c-Met/β1 complex to increase mesenchymal and stem cell characteristics. Multiplex transcriptomic analysis revealed upregulated Wnt and hedgehog pathways after c-Met/β1 complex induction. A β1 integrin point mutation that prevented binding to c-Met reduced intravasation. OS2966, a therapeutic antibody disrupting c-Met/β1 binding, decreased breast cancer cell invasion and mesenchymal gene expression. Bone-seeking breast cancer cells exhibited higher c-Met/β1 complex levels than parental controls and preferentially adhered to tissue-specific matrix. Patient bone metastases demonstrated higher c-Met/β1 complex than brain metastases. Thus, the c-Met/β1 complex drives intravasation of triple-negative breast cancer cells and preferential affinity for bone-specific matrix. Pharmacological targeting of the complex may prevent metastases, particularly osseous metastases.
Authors
Darryl Lau, Harsh Wadhwa, Sweta Sudhir, Alexander Chih-Chieh Chang, Saket Jain, Ankush Chandra, Alan T. Nguyen, Jordan M. Spatz, Ananya Pappu, Sumedh S. Shah, Justin Cheng, Michael M. Safaee, Garima Yagnik, Arman Jahangiri, Manish K. Aghi
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