In-Press Preview
Articles in this category appear as authors submitted them for publication, prior to copyediting and publication layout.
Abstract
Glioblastoma is a highly malignant brain tumor with no curative treatment options, and immune checkpoint blockade has not yet shown major impact. We hypothesized that drugs targeting mitosis might impact the tumor microenvironment and sensitize cancer cells to immunotherapy. We used two glioblastoma mouse models with different immunogenicity profiles, GL261 and SB28, to test the efficacy of antineoplastic and immunotherapy combinations. The spindle assembly checkpoint activator BAL101553 (lisavanbulin), agonistic anti-CD40 antibody, and double immune checkpoint blockade (anti-PD-1 and anti-CTLA-4) were evaluated individually or in combination for treating orthotopic GL261 and SB28 tumors. Genomic and immunological analyses were used to predict and interpret therapy responsiveness. BAL101553 monotherapy increased survival in immune checkpoint blockade resistant SB28 glioblastoma tumors and synergized with anti-CD40 antibody, in a T-cell independent manner. In contrast, the more immunogenic and highly mutated GL261 model responded best to anti-PD-1 and anti-CTLA-4 therapy and more modestly to BAL101553 and anti-CD40 combination. Our results show that BAL101553 is a promising therapeutic agent for glioblastoma and could synergize with innate immune stimulation. Overall, these data strongly support immune profiling of glioblastoma patients and preclinical testing of combination therapies with appropriate models for particular patient groups.
Authors
Vassilis Genoud, Felipe I. Espinoza, Eliana Marinari, Viviane Rochemont, Pierre-Yves Dietrich, Paul McSheehy, Felix Bachmann, Heidi A. Lane, Paul R. Walker
Abstract
Kawasaki disease (KD) is the leading cause of acquired heart disease among children. Murine and human data suggest that the NLRP3-IL-1β pathway is the main driver of KD pathophysiology. NLRP3 can be activated during defective autophagy/mitophagy. We used the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis, to examine the role of autophagy/mitophagy on cardiovascular lesion development. LCWE-injected mice had impaired autophagy/mitophagy and increased levels of ROS in cardiovascular lesions, together with increased systemic 8-OHdG release. Enhanced autophagic flux significantly reduced cardiovascular lesions in LCWE-injected mice, whereas autophagy blockade increased inflammation. Vascular smooth muscle cell specific deletion of Atg16l1 and global Parkin-/- significantly increased disease formation, supporting the importance of autophagy/mitophagy in this model. Ogg1-/- mice had significantly increased lesions with increased NLRP3 activity, whereas treatment with MitoQ, reduced vascular tissue inflammation, ROS production and systemic 8-OHdG release. Treatment with MN58b or Metformin (increasing AMPK and reducing ROS), resulted in decreased disease formation. Our results demonstrate that impaired autophagy/mitophagy and ROS-dependent damage exacerbate the development of murine KD vasculitis. This pathway can be efficiently targeted to reduce disease severity. These findings enhance our understanding of KD pathogenesis and identify novel therapeutic avenues for KD treatment.
Authors
Stefanie Marek-Iannucci, A. Beyza Ozdemir, Debbie Moreira, Angela C. Gomez, Malcolm Lane, Rebecca A Porritt, Youngho Lee, Kenichi Shimada, Masanori Abe, Aleksandr Stotland, David Zemmour, Sarah Parker, Elsa Sanchez-Lopez, Jennifer Van Eyk, Roberta A. Gottlieb, Michael Fishbein, Michael Karin, Timothy R Crother, Magali Noval Rivas, Moshe Arditi
Abstract
Oligoarticular juvenile idiopathic arthritis (oligo JIA) is the most common form of chronic inflammatory arthritis in children; yet, the cause of this disease remains unknown. To understand immune responses in oligo JIA, we immunophenotyped synovial fluid T cells with flow cytometry, bulk and single-cell RNA sequencing, DNA methylation studies, and Treg suppression assays. In synovial fluid, CD4+, CD8+, and γδ T cells expressed Th1-related markers, while Th17 cells were not enriched. Th1 skewing was prominent in CD4+ T cells, including Tregs, and was associated with severe disease. Transcriptomic studies confirmed a Th1 signature in CD4+ T cells from synovial fluid. The regulatory gene expression signature was preserved in Tregs, even those exhibiting Th1 polarization. These Th1-like Tregs maintained Treg specific methylation patterns and suppressive function, supporting the stability of this Treg population in the joint. While synovial fluid CD4+ T cells displayed an overall Th1 phenotype, scRNA-seq uncovered heterogeneous effector and regulatory sub-populations, including interferon-induced Tregs, peripheral helper T cells, and cytotoxic CD4+ T cells. In conclusion, oligo JIA is characterized by Th1 polarization that encompasses Tregs but does not compromise their regulatory identity. Targeting Th1-driven inflammation and augmenting Treg function may represent important therapeutic approaches in oligo JIA.
Authors
Amélie M. Julé, Kacie J. Hoyt, Kevin Wei, Maria Gutierrez-Arcelus, Maria L. Taylor, Julie Ng, James A. Lederer, Siobhan M. Case, Margaret H. Chang, Ezra M. Cohen, Fatma Dedeoglu, Melissa M. Hazen, Jonathan S. Hausmann, Olha Halyabar, Erin Janssen, Jeffrey Lo, Mindy S. Lo, Esra Meidan, Jordan E. Roberts, Mary Beth F. Son, Robert P. Sundel, Pui Y. Lee, Talal Chatila, Peter A. Nigrovic, Lauren A. Henderson
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the pandemic Coronavirus Disease 2019 (COVID-19) and now many face the burden of prolonged symptoms—long-lasting COVID-19 symptoms or “long-COVID”. Long-COVID is thought to be linked to immune dysregulation due to harmful inflammation, with the exact causes being unknown. Given the role of the microbiome in mediating inflammation, we aimed to examine the relationship between the oral microbiome and the duration of long-COVID symptoms. Tongue swabs were collected from patients presenting with symptoms concerning for COVID-19. Confirmed infections were followed until resolution of all symptoms. Bacterial composition was determined by metagenomic sequencing. We used random forest modeling to identify microbiota and clinical covariates that associated with long-COVID symptoms. Of the patients followed, 63% (17/27) developed ongoing symptomatic COVID-19 and 37% (10/27) went on to long-COVID. Patients with prolonged symptoms had significantly higher abundances of microbiota that induce inflammation, such as members of the genera Prevotella and Veillonella. Of note are species that produce lipopolysaccharides and the similarity of long-COVID patients’ oral microbiome to those of patients with chronic fatigue syndrome. All together, we our findings suggest an association with the oral microbiome and long-COVID revealing the possibility that dysfunction of the oral microbiome may contribute to this draining disease.
Authors
John P. Haran, Evan Bradley, Abigail L. Zeamer, Lindsey Cincotta, Marie-Claire Salive, Protiva Dutta, Shafik Mutaawe, Otuwe Anya, Mario Meza-Segura, Ann M. Moormann, Doyle V. Ward, Beth A. McCormick, Vanni Bucci
Abstract
Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and main indication for heart transplantation in children. Therapies specific to pediatric DCM remains limited due to lack of a disease model. Our previous study showed that treatment of neonatal rat ventricular myocytes (NRVMs) with non-failing or DCM pediatric patient serum activates the fetal gene program (FGP). Here we show that serum treatment with Proteinase K prevents activation of the FGP, whereas RNase treatment exacerbates it, suggesting that circulating proteins, but not circulating microRNAs, promote these pathological changes. Evaluation of the protein secretome showed that midkine (MDK) is up-regulated in DCM serum, and NRVM treatment with MDK activates the FGP. Changes in gene expression in serum-treated NRVMs, evaluated by next-generation RNA sequencing (RNA-Seq), indicates extracellular matrix remodeling and focal adhesion pathways are upregulated in pediatric DCM serum and serum-treated NRVMs, suggesting alterations in cellular stiffness. Cellular stiffness was evaluated by Atomic Force Microscopy, which showed an increase in stiffness in DCM serum-treated NRVMs. Of the proteins increased in DCM sera, secreted frizzled related protein 1 (sFRP1) was a potential candidate for the increase in cellular stiffness, and sFRP1 treatment of NRVMs recapitulated the increase in cellular stiffness observed in response to DCM-serum treatment. Our results show that serum circulating proteins promote pathological changes in gene expression and cellular stiffness, and circulating miRNAs are protective against pathological changes.
Authors
Danielle A. Jeffrey, Julie Pires Da Silva, Anastacia M. Garcia, Xuan Jiang, Anis Karimpour-Fard, Lee S. Toni, Thomas Lanzicher, Brisa Peña, Carissa A. Miyano, Karin Nunley, Armin Korst, Orfeo Sbaizero, Matthew R.G. Taylor, Shelley D. Miyamoto, Brian L. Stauffer, Carmen C. Sucharov
Abstract
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.
Authors
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
Abstract
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.
Authors
Suman Srinivasa, Ruben Garcia-Martin, Martin Torriani, Kathleen V. Fitch, Anna R. Carlson, C. Ronald Kahn, Steven K. Grinspoon
Abstract
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).
Authors
Gregory P. Botta, Shumei Kato, Hitendra Patel, Paul Fanta, Suzanna Lee, Ryosuke Okamura, Razelle Kurzrock
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
Ryosuke Hiwa, Hailyn V. Nielsen, James L. Mueller, Ravi Mandla, Julie Zikherman
Abstract
G protein-coupled receptors (GPCRs) are highly desirable drug targets for human disease. Although GPCR dysfunction drives development and progression of many tumors, including breast cancer (BC), targeting individual GPCRs has limited efficacy as a cancer therapy because numerous GPCRs are activated. Here, we sought a new way of blocking GPCR activation in HER2+-BC by targeting a subgroup of GPCRs that couple to Gi/o proteins (Gi/o-GPCRs). In mammary epithelial cells of transgenic mouse models, and BC cell lines, HER2 hyperactivation altered GPCR expression, particularly, Gi/o-GPCRs. Gi/o-GPCR stimulation transactivated EGFR and HER2 and activated the PI3K/AKT and Src pathways. If we uncoupled Gi/o-GPCRs from their cognate Gi/o proteins by pertussis toxin (PTx), then BC cell proliferation and migration was inhibited in vitro and HER2-driven tumor formation and metastasis suppressed in vivo. Moreover, targeting Gi/o-GPCR signaling via PTx, PI3K, or Src inhibitors enhanced HER2-targeted therapy. These results indicate that, in BC cells, HER2 hyperactivation drives aberrant Gi/o-GPCR signaling, and Gi/o-GPCR signals converge on PI3K/AKT and Src signaling pathways to promote cancer progression and resistance to HER2-targeted therapy. Our findings point to a new way to pharmacologically deactivate GPCR signaling to block tumor growth and enhance therapeutic efficacy.
Authors
Cancan Lyu, Yuanchao Ye, Maddison M. Lensing, Kay-Uwe Wagner, Ronald J. Weigel, Songhai Chen
Abstract
BACKGROUND. A previous Phase I study showed that the infusion of autologous Treg cells expanded ex-vivo into recent onset Type 1 Diabetes (T1D) patients had an excellent safety profile, however, the majority of the infused Tregs could no longer be detected in the peripheral blood three months post-infusion (NCT01210664-Treg-T1D Trial). Interleukin-2 (IL-2) has been shown to enhance human Treg cell survival and expansion at low doses in vivo. Therefore, we conducted a Phase 1 study (NCT02772679-TILT trial) combining polyclonal Treg and low-dose IL-2 and assessed the impact over time on Tregs populations and other immune cells. METHODS. T1D Patients were treated with a single infusion of autologous polyclonal Tregs, expanded ex vivo, followed by one or two 5-day courses of recombinant human low dose IL-2 (ld-IL-2) at 0.3 x 106 to 1 x 106 units/dose. Fluorescence-based flow cytometry, Cytometry by Time Of Flight (CyTOF) and 10X Genomics single cell RNAseq were used to follow the distinct immune cell populations phenotypes over time following immunotherapy. RESULTS. Multiparametric analysis revealed that the combination therapy led to an increase in the number of infused and endogenous Tregs but also resulted in a significant increase from baseline in a subset of activated NK, Mucosal-Associated Invariant T (MAIT) cells and clonal CD8+ T populations. CONCLUSIONS. These data support the hypothesis that Id-IL-2 expands exogenously administered Tregs but also can expand cytotoxic cells. These results have important implications for the use of a combination of ld-IL-2 and Tregs for the treatment of autoimmune diseases with pre-existing active immunity. TRIAL REGISTRATION. ClinicalTrials.gov NCT01210664 (Treg-T1D Trial), NCT02772679 (TILTtrial). FUNDING. Sean N. Parker Autoimmunity Research Laboratory Fund, National Center for Research Resources.
Authors
Shen Dong, Kamir J. Hiam-Galvez, Cody T. Mowery, Kevan C. Herold, Stephen E. Gitelman, Jonathan H. Esensten, Weihong Liu, Angela P. Lares, Ashley S. Leinbach, Michael Lee, Vinh Nguyen, Stanley J. Tamaki, Whitney Tamaki, Courtney M. Tamaki, Morvarid Mehdizadeh, Amy L. Putnam, Matthew H. Spitzer, C. Jimmie Ye, Qizhi Tang, Jeffrey A. Bluestone
Abstract
cGMP-dependent protein kinase 1α (PKG1α) promotes left ventricle (LV) compensation to pressure overload. PKG1-activating drugs improve heart failure (HF) outcomes but are limited by vasodilation-induced hypotension. Signaling molecules which mediate PKG1α cardiac therapeutic effects but do not promote PKG1α-induced hypotension could therefore represent improved therapeutic targets. We investigated roles of mixed lineage kinase 3 (MLK3) in mediating PKG1α effects on LV function after pressure overload, and in regulating blood pressure (BP). In a transaortic constriction HF model PKG activation with sildenafil preserved LV function in MLK3+/+, but not MLK3-/- littermates. MLK3 co-immunoprecipitated with PKG1α. MLK3-PKG1α co-interaction decreased in failing LVs. PKG1a phosphorylated MLK3 on Thr-277/Ser-281 sites required for kinase activation. MLK3-/- mice displayed hypertension and increased arterial stiffness, though PKG stimulation with sildenafil or the sGC stimulator BAY41-2272 still reduced BP in MLK3-/- mice. MLK3 kinase inhibition with URMC-099 did not affect BP, but induced LV dysfunction in mice. These data reveal MLK3 as a PKG1α substrate mediating PKG1α preservation of LV function but not acute PKG1α BP effects. Mechanistically, MLK3 kinase-dependent effects preserve LV function, while MLK3 kinase-independent signaling regulates BP. These findings suggest augmenting MLK3 kinase activity could preserve LV function in HF but avoid hypotension from PKG1α activation.
Authors
Timothy D. Calamaras, Suchita Pande, Robert A.U. Baumgartner, Seung Kyum Kim, Joseph C. McCarthy, Gregory L. Martin, Kelly Tam, Angela L. McLaughlin, Guang-rong Wang, Mark J. Aronovitz, Weiyu Lin, Jonathan I. Aguirre, Paulina Baca, Peiwen Liu, Daniel A. Richards, Roger J. Davis, Richard H. Karas, Iris Z. Jaffe, Robert M. Blanton
Abstract
Cardiac inflammation and fibrosis contribute significantly to hypertension-related adverse cardiac remodeling. IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been demonstrated as a key molecular link between inflammation and cardiovascular disease. However, the cell-specific contribution of IKKβ signaling towards adverse cardiac remodeling remains elusive. Cardiac fibroblasts are one of the most populous non-myocyte cell types in the heart that play a key role in mediating cardiac fibrosis and remodeling. To investigate the function of fibroblast IKKβ, we generated inducible fibroblast-specific IKKβ-deficient mice. Here we report an important role of IKKβ in the regulation of fibroblast functions and cardiac remodeling. Fibroblast-specific IKKβ deficient male mice were protected from angiotensin II (Ang II)-induced cardiac hypertrophy, fibrosis, and macrophage infiltration. Ablation of fibroblast IKKβ inhibited Ang II-stimulated fibroblast proinflammatory and profibrogenic responses, leading to ameliorated cardiac remodeling and improved cardiac function in IKKβ-deficient mice. Findings from this study establish fibroblast IKKβ as a key factor regulating cardiac fibrosis and function in hypertension-related cardiac remodeling.
Authors
Weiwei Lu, Zhaojie Meng, Rebecca Hernandez, Changcheng Zhou
Abstract
Innate immunity and chronic inflammation are involved in atherosclerosis and atherothrombosis leading to target organ damage in essential hypertension (EH). However, the role of neutrophils in EH is still elusive. We investigated the association between angiotensin II (Ang II) and neutrophil extracellular traps (NETs) in pathogenesis of EH. Plasma samples, kidney biopsies and surgical specimens of abdominal aortic aneurysms (AAA) from EH patients were used. Cell-based assays, NETs/human aortic endothelial cells co-cultures and in situ studies were performed. Increased plasma levels of NETs and tissue factor (TF) activity were detected in untreated, newly-diagnosed, EH patients. Stimulation of control neutrophils with plasma from untreated EH patients generated TF-enriched NETs promoting endothelial collagen production. Ang II induced NETosis in vitro via a reactive oxygen species (ROS)/peptidylarginine deiminase type 4 and autophagy-dependent pathway. Circulating NETs and thrombin generation levels were reduced significantly in EH patients starting treatment with Ang II receptor blockers, whereas their plasma was unable to trigger procoagulant NETs. Moreover, TF-bearing NETotic neutrophils/remnants were accumulated in sites of interstitial renal fibrosis and in the subendothelial layer of AAA. These data reveal the important pathogenic role of Ang II/ROS/NETs/TF axis in EH, linking thromboinflammation with endothelial dysfunction and fibrosis.
Authors
Akrivi Chrysanthopoulou, Eugenia Gkaliagkousi, Antonios Lazaridis, Stella Arelaki, Panagiotis Pateinakis, Maria Ntinopoulou, Alexandros Mitsios, Christina Antoniadou, Christos Argyriou, George S. Georgiadis, Vasileios Papadopoulos, Alexandra Giatromanolaki, Konstantinos Ritis, Panagiotis Skendros
