Arrhythmogenic cardiomyopathy (AC) is a heart disease often caused by mutations in genes coding for desmosomal proteins including desmoglein-2 (DSG2), plakoglobin (PG), and desmoplakin (DP). Therapy is symptomatic to limit arrhythmia since the mechanisms by which desmosomal components control cardiomyocyte function are largely unknown. A new paradigm would be to stabilize desmosomal cardiomyocyte adhesion and hyper-adhesion, which renders desmosomal adhesion independent from Ca2+. Here, we further characterized the mechanisms behind enhanced cardiomyocyte adhesion and hyper-adhesion. Dissociation assays performed in HL-1 cells and murine ventricular cardiac slice cultures allowed us to define a set of signaling pathways regulating cardiomyocyte adhesion under basal and hyper-adhesive conditions. Adrenergic signaling, activation of PKC and inhibition of p38MAPK enhanced cardiomyocyte adhesion, referred to as positive adhesiotropy, and induced hyper-adhesion. Activation of ERK1/2 paralleled positive adhesiotropy, whereas adrenergic signaling induced Pg phosphorylation at S665 under both basal and hyper-adhesive conditions. Adrenergic signaling and p38MAPK inhibition recruited DSG2 to cell junctions. In PG-deficient mice with an AC phenotype, only PKC activation and p38MAPK inhibition enhanced cardiomyocyte adhesion. Our results demonstrate that cardiomyocyte adhesion can be stabilized by different signaling mechanisms, which are in part off-set in PG-deficient AC.
Maria Shoykhet, Sebastian Trenz, Ellen Kempf, Tatjana Williams, Brenda Gerull, Camilla Schinner, Sunil Yeruva, Jens Waschke
ETV6 is an ETS family transcription factor which plays a key role in hematopoiesis and megakaryocyte development. Our group and others have identified germline mutations in ETV6 resulting in autosomal dominant thrombocytopenia and predisposition to malignancy; however, molecular mechanisms defining the role of ETV6 in megakaryocyte development have not been well established. Using a combination of molecular, biochemical, and sequencing approaches in patient-derived PBMCs, we demonstrate abnormal cytoplasmic localization of ETV6 and the HDAC3/NCOR2 repressor complex that leads to overexpression of HDAC3-regulated interferon response genes. This transcriptional dysregulation is also reflected in patient-derived platelet transcripts, and drives aberrant proplatelet formation in megakaryocytes. Our results suggest that aberrant transcription may predispose patients with ETV6 mutations to bone marrow inflammation, dysplasia, and megakaryocyte dysfunction.
Marlie H. Fisher, Gregory D. Kirkpatrick, Brett M. Stevens, Courtney L. Jones, Michael U. Callaghan, Madhvi Rajpurkar, Joy Fulbright, Megan A. Cooper, Jesse Rowley, Christopher C. Porter, Arthur Gutierrez-Hartmann, Kenneth Jones, Craig T. Jordan, Eric M. Pietras, Jorge Di Paola
Background: Baseline expression of FCRL5, a marker of naïve and memory B cells, was shown to predict response to rituximab (RTX) in rheumatoid arthritis. This study investigated baseline expression of FCRL5 as a potential biomarker of clinical response to RTX in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). Methods: A previously validated RT-qPCR-based platform was used to assess FCRL5 expression in patients with GPA/MPA (RAVE trial, NCT00104299). Results: Baseline FCRL5 expression was significantly higher in patients achieving complete response (CR) at 6, 12, and 18 months, independent of other clinical and serological variables, among those randomized to RTX but not CYC/AZA. Patients with baseline FCRL5 expression ≥ 0.01 expression units (termed FCRL5hi) exhibited significantly higher CR rates at 6, 12, and 18 months as compared to FCRL5low subjects (84% vs 57% p=0.016, 68% vs 40% p=0.02 and 68% vs 29% p=0.0009, respectively). Conclusion: Our data taken together suggest that FCRL5 is a biomarker of B cell lineage associated with increased achievement and maintenance of complete remission among patients treated with RTX and warrant further investigation in a prospective manner.
Kasia Owczarczyk, Matthew D. Cascino, Cecile Holweg, Gaik W. Tew, Ward Ortmann, Timothy W. Behrens, Thomas Schindler, Carol A. Langford, E. William St. Clair, Peter A. Merkel, Robert Spiera, Philip Seo, Cees G.M. Kallenberg, Ulrich Specks, Noha Lim, John H. Stone, Paul Brunetta, Marco Prunotto
The emergence of SARS-CoV-2 has created an international health crisis. Small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection due to low affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promotor. In contrast to non-transgenic mice, intranasal exposure of K18-hACE2 animals to two different doses of SARS-CoV-2 resulted in acute disease including weight loss, lung injury, brain infection and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals revealed increases in transcripts involved in lung injury and inflammatory cytokines. In the lower dose challenge groups, there was a survival advantage in the female mice with 60% surviving infection whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared to female mice. This is the first highly lethal murine infection model for SARS-CoV-2. The K18-hACE2 murine model will be valuable for the study of SARS-CoV-2 pathogenesis and the assessment of MCMs.
Joseph W. Golden, Curtis R. Cline, Xiankun Zeng, Aura R. Garrison, Brian D. Carey, Eric M. Mucker, Lauren E. White, Joshua D. Shamblin, Rebecca L. Brocato, Jun Liu, April M. Babka, Hypaitia B. Rauch, Jeffrey M. Smith, Bradley S. Hollidge, Collin Fitzpatrick, Catherine V. Badger, Jay W. Hooper
Atrial fibrillation (AF) is the most common cardiac arrhythmia, yet the molecular signature of the vulnerable atrial substrate is not well understood. Here, we delineated a distinct transcriptional signature in right versus left atrial cardiomyocytes (CMs) at baseline, and identified chamber-specific gene expression changes in patients with history of AF in the setting of end-stage heart failure (AF+HF) that are not present in heart failure alone (HF). We observed that human left atrial (LA) CMs exhibit Notch pathway activation and increased ploidy in AF+HF, but not in HF alone. Transient activation of Notch signaling within adult CMs in a murine genetic model is sufficient to increase ploidy in both atrial chambers. Notch activation within LA CMs generated a transcriptomic fingerprint resembling AF, with dysregulation of transcription factor and ion channel genes including Pitx2, Tbx5, Kcnh2, Kcnq1, and Kcnip2. Notch activation also produced distinct cellular electrophysiologic responses in LA versus RA CMs, prolonging the action potential duration (APD) without altering the upstroke velocity in the LA, and reducing the maximal upstroke velocity without altering the APD in the RA. Our results support a shared human/murine model of increased Notch pathway activity predisposing to AF.
Catherine E. Lipovsky, Jesus Jimenez, Qiusha Guo, Gang Li, Tiankai Yin, Stephanie Hicks, Somya Bhatnagar, Kentaro Takahashi, David M. Zhang, Brittany D. Brumback, Uri Goldsztejn, Rangarajan D. Nadadur, Carlos Perez-Cervantes, Ivan P. Moskowitz, Shaopeng Liu, Bo Zhang, Stacey L. Rentschler
Increased metabolism distinguishes myofibroblasts or fibrotic lung fibroblasts (fLfs) from the normal lung fibroblasts (nLfs). The mechanism of metabolic activation in fLfs has not been fully elucidated. Further, the anti-fibrogenic effects of caveolin-1 scaffolding domain peptide CSP/CSP7 involve metabolic reprogramming in fLfs is unclear. We therefore analyzed lactate and succinate levels, and the expression of glycolytic enzymes, and hypoxia inducible factor-1alpha (HIF-1α). Lactate and succinate levels as well as the basal expression of glycolytic enzymes and HIF-1α αwere increased in fLfs. These changes were reversed following restoration of p53 or its transcriptional target microRNA-34a (miR-34a) expression in fLfs. Conversely, inhibition of basal p53 or miR-34a increased glucose metabolism, glycolytic enzymes and HIF-1α in nLfs. Treatment of fLfs or mice having bleomycin- or TGF-beta1-induced lung fibrosis with CSP/CSP7, reduced the expression of glycolytic enzymes and HIF-1α. Further, inhibition of p53 or miR-34a abrogated CSP/CSP7-mediated restoration of glycolytic flux in fLfs in vitro and in mice with pulmonary fibrosis and lacking p53 or miR-34a expression in fibroblasts in vivo. Our data indicate that dysregulation of glucose metabolism in fLfs is causally linked to loss of basal expression of p53 and miR-34a. Treatment with CSP/CSP7 constrains aberrant glucose metabolism through restoration of p53 and miR-34a.
Venkadesaperumal Gopu, Liang Fan, Rashmi Shetty, MR Nagaraja, Sreerama Shetty
Hidradenitis suppurativa (HS) is a highly prevalent and morbid inflammatory skin disease with limited treatment options. The major cell types and inflammatory pathways in skin of HS patients are poorly understood. In addition, it is currently unknown which patients will respond to TNFα blockade. Herein, we comprehensively elucidate and functionally define the immune cell infiltrate and major inflammatory pathways in HS skin, before and after anti-TNFα therapy. We discovered that clinically and histologically healthy appearing skin (i.e., nonlesional skin) is dysfunctional in HS patients with a relative loss of immune regulatory pathways. At the cellular level, HS skin lesions were characterized by quantitative and qualitative dysfunction of type 2 dendritic cells (cDC2s), relatively reduced regulatory T cells (Tregs), an influx of memory B cells and a plasma cell/plasmablast infiltrate predominantly in end-stage fibrotic skin. At the molecular level, there was a relative bias towards the IL-1 pathway and type 1 T cell responses when compared to both healthy skin and skin from psoriasis patients. Anti-TNFα therapy significantly attenuated B cell activation with minimal effect on other inflammatory pathways. Finally, we identified an immune activation signature in skin prior to anti-TNFα treatment that correlated with subsequent lack of response to this modality. Taken together, our results reveal the fundamental immunopathogenesis of HS and provide a molecular foundation for future studies focused on stratifying patients based on likelihood of clinical response to TNFα blockade.
Margaret M. Lowe, Haley B. Naik, Sean Clancy, Mariela Pauli, Kathleen M. Smith, Yingtao Bi, Robert Dunstan, Johann Gudjonsson, Maia Paul, Hobart W. Harris, Esther A. Kim, Uk Sok Shin, Richard Ahn, Wilson Liao, Scott L. Hansen, Michael Rosenblum
Compromised muscle mitochondrial metabolism is a hallmark of peripheral arterial disease, especially in patients with the most severe clinical manifestation - critical limb ischemia (CLI). We asked whether inflexibility in metabolism is critical for the development of myopathy in ischemic limb muscles. Using Polg mtDNA mutator (D257A) mice, we reveal remarkable protection from hindlimb ischemia (HLI) due to a unique and beneficial adaptive enhancement of glycolytic metabolism and elevated ischemic muscle PFKFB3. Similar to the relationship between mitochondria from CLI and claudicating patient muscles, BALB/c muscle mitochondria are uniquely dysfunctional after HLI onset as compared to the BL6 parental strain. AAV-mediated over-expression of PFKFB3 in BALB/c limb muscles improved muscle contractile function and limb blood flow following HLI. Enrichment analysis of RNA sequencing data on muscle from CLI patients revealed a unique deficit in the Glucose Metabolism Reactome. Muscles from these patients express lower PFKFB3 protein and their muscle progenitor cells possess decreased glycolytic flux capacity in vitro. Here we show supplementary glycolytic flux as sufficient to protect against ischemic myopathy in instances where reduced blood flow related mitochondrial function is compromised pre-clinically. Additionally, our data reveal reduced glycolytic flux as a common characteristic of CLI patient limb skeletal muscle.
Terence E. Ryan, Cameron A. Schmidt, Michael D. Tarpey, Adam J. Amorese, Dean Yamaguchi, Emma Goldberg, Melissa R. Iñigo, Reema Karnekar, Allison R. O’Rourke, James M. Ervasti, Patricia Brophy, Thomas Green, P. Darrell Neufer, Kelsey H. Fisher-Wellman, Espen Spangenburg, Joseph McClung
Regulatory T cells (Tregs) are crucial for maintaining maternal immune-tolerance against the semi-allogeneic fetus. We investigated the elusive transcriptional profile and functional adaptation of human uterine Tregs (uTregs) during pregnancy. Uterine biopsies, from placental bed (=maternal-fetal interface) and incision site (=control), and blood were obtained from women with uneventful pregnancies undergoing Caesarean section. Tregs and CD4+ non-Tregs were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell level by flow cytometry. Placental bed uterine Tregs (uTregs) showed elevated expression of Treg signature markers, including FOXP3, CTLA-4 and TIGIT. Their transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation, with increased expression of immune checkpoints GITR, TNFR2, OX-40, 4-1BB, genes associated with suppressive capacity (HAVCR2, IL10, LAYN, PDCD1), and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored non-Treg Th1 polarization and tissue-residency. The particular transcriptional signature of placental bed uTregs overlapped strongly with that of tumor-infiltrating Tregs, and was remarkably pronounced at the placental bed compared to uterine control site. Concluding, human uTregs acquire a differentiated effector Treg profile similar to tumor-infiltrating Tregs, specifically at the maternal-fetal interface. This introduces the novel concept of site-specific transcriptional adaptation of Tregs within one organ.
Judith Wienke, Laura Brouwers, Leone M. van der Burg, Michal Mokry, Rianne C. Scholman, Peter G. J. Nikkels, Bas B. van Rijn, Femke van Wijk
Classical dynamins are large GTPases regulating membrane and cytoskeleton dynamics and are linked to different pathological conditions ranging from neuromuscular diseases to encephalopathy and cancer. Dominant DNM2 (dynamin 2) mutations lead to either mild adult onset or severe neonatal centronuclear myopathy (ADCNM). Our objectives were to better understand the pathomechanism of severe ADCNM and test a potential therapy. Here, we created the Dnm2SL/+ mouse line harboring the common S619L mutation found in patients with severe ADCNM and impairing the conformational switch regulating dynamin self-assembly and membrane remodeling. The Dnm2SL/+ mouse faithfully reproduces severe ADCNM hallmarks with early impaired muscle function and force together with myofibers hypotrophy. It revealed swollen mitochondria lacking cristae as the main ultrastructural defect and potential cause of the disease. Patient analysis confirmed this structural hallmark. In addition, DNM2 reduction with antisense oligonucleotides after disease onset efficiently reverted locomotor and force defects after only 3 weeks of treatment. Most histological defects including mitochondria alteration were partially or fully rescued. Overall, this study highlights an efficient approach to revert the severe form of dynamin-related centronuclear myopathy. These data also reveal that the dynamin conformational switch is key for muscle function and should be targeted for future therapeutic developments.
Xènia Massana Muñoz, Christine Kretz, Roberto Silva-Rojas, Julien Ochala, Alexia Menuet, Norma B. Romero, Belinda S. Cowling, Jocelyn Laporte
Acute graft-versus-host disease (aGVHD) can occur after hematopoietic cell transplant in patients undergoing treatment for hematological malignancies or inborn errors. Although CD4 T helper (Th) cells play a major role in aGVHD, the mechanisms by which they contribute, particularly within the intestines, have remained elusive. We have identified a novel subset of Th cells that accumulated in the intestines and produced the serine protease granzyme A (GrA). GrA+ Th cells were distinct from other Th lineages and exhibited a non-cytolytic phenotype. In vitro, GrA+ Th cells differentiated in the presence of IL-4, IL-6, and IL-21 and were transcriptionally unique from cells cultured with either IL-4 or the IL-6/IL-21 combination alone. In vivo, both STAT3 and STAT6 were required for GrA+ Th cell differentiation and played roles in maintenance of the lineage identity. Importantly, GrA+ Th cells promoted aGVHD-associated morbidity and mortality and contributed to crypt destruction within intestines but were not required for the beneficial graft-versus-leukemia effect. Our data indicate that GrA+ Th cells represent a distinct Th subset and are critical mediators of aGVHD.
Sungtae Park, Brad Griesenauer, Hua Jiang, Djamilatou Adom, Pegah Mehrpouya-Bahrami, Srishti Chakravorty, Majid Kazemian, Tanbeena Imam, Rajneesh Srivastava, Tristan A. Hayes, Julian Pardo, Sarath Chandra Janga, Sophie Paczesny, Mark H. Kaplan, Matthew R. Olson
Background: Control of the tuberculosis (TB) pandemic remains hindered, in part, by a lack of simple and accurate measures of treatment efficacy. Current gold standard markers rely on sputum-based assays that are slow and challenging to implement. Previous work identified urinary N1, N12-diacetylspermine (DiAcSpm), neopterin, hydroxykynurenine, N-acetylhexosamine, ureidopropionic acid, sialic acid, and m/z 241.0903 as potential biomarkers of active pulmonary TB (ATB). Here, we evaluated their ability to serve as biomarkers of TB treatment response and mycobacterial load. Methods: We analyzed urine samples prospectively collected from two cohorts with ATB: 34 participants from African countries treated with first line TB therapy (HRZE) and followed for one year, and 35 participants from Haiti treated with either HRZE or an experimental drug followed for 14 days. Blinded samples were analyzed by untargeted high-performance liquid chromatography-coupled-time of flight-mass spectrometry. Results: Urinary levels of all seven molecules exhibited significant decreases by week 26 of successful treatment (p=0.01-p<0.0001), and positive correlations with sputum mycobacterial load (p<0.0001). Urinary levels of DiAcSpm exhibited significant decreases in participants treated with HRZE as early as 14 days (p<0.0001) but were unchanged in participants receiving ineffective therapy (p=0.14). Conclusion: Reductions in urinary DiAcSpm, neopterin, hydroxykynurenine, N-acetylhexosamine, ureidopropionic acid, sialic acid, and m/z 241.0903 correlated with successful anti-TB treatment and sputum mycobacterial load. Levels of DiAcSpm exhibited reductions capable of differentiating treatment success from failure as early as two weeks after the initiation of chemotherapy, commending its further development as a potentially simple, non-invasive biomarker of treatment response and bacterial load. Funding: This work was supported by the Clinical and Translational Science Center at Weill Cornell College of Medicine (NIH/NCATS 1 UL1 TR002384-02 and KL2TR000458), the Department of Defense (PR170782), the National Institute of Allergy and Infectious Disease grants (NIAID T32AI007613-16, K24 AI098627 and K23 AI131913), the NIH Fogarty International Center grants (R24 TW007988 and TW010062), the National Institute of Health grant (R01 GM135926 ), the Abby and Howard P. Milstein Program in Chemical Biology and Translational Medicine, and the Tuberculosis Research Units Networks (TBRU-N, AI111143).
Qianjing Xia, Myung Hee Lee, Kathleen F. Walsh, Kathrine McAulay, James M. Bean, Daniel W. Fitzgerald, Kathryn M. Dupnik, Warren D. Johnson, Jean W. Pape, Kyu Y. Rhee, Flonza Isa
Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies to the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological enzyme-linked immunosorbent assay (ELISA) using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 RT-PCR confirmed SARS-CoV-2 hospitalized patients, as well as 464 healthy and non-COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies to the two different viral proteins showed a moderate correlation. Antibodies to N-protein were detected at a rate of 3.6% in healthy and non-COVID-19 sera collected during the pandemic in 2020, whereas 1.6% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD binding antibodies exhibited neutralizing capacity, but only 74% of N-protein positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein binding antibodies does not always correlate with presence of S-RBD neutralizing antibodies, and cautions against the extensive use of N-protein based serology testing for determination of potential COVID-19 immunity.
Kathleen M. McAndrews, Dara P. Dowlatshahi, Jianli Dai, Lisa M. Becker, Janine Hensel, Laura M. Snowden, Jennifer M. Leveille, Michael R. Brunner, Kylie Holden, Nikolas S. Hopkins, Alexandria Harris, Jerusha J. Kumpati, Michael A. Whitt, J. Jack Lee, Luis Ostrosky-Zeichner, Ramesha Papanna, Valerie LeBleu, James Allison, Raghu Kalluri
Angiogenesis is essential for cardiac functional recovery after myocardial infarction (MI). HSPA12B is predominately expressed in endothelial cells and required for angiogenesis. Yes-associated protein (YAP) plays an important role in tumor angiogenesis. This study investigated the cooperative role of HSPA12B and YAP in angiogenesis post-MI. Silencing of either HSPA12B or YAP impairs hypoxia-promoted endothelial cell proliferation and angiogenesis. Deficiency of HSPA12B suppresses YAP expression and nuclear translocation following hypoxia. Knockdown of YAP attenuates hypoxia-stimulated HSPA12B nuclear translocation and abrogates HSPA12B-promoted endothelial cell angiogenesis. Mechanistically, hypoxia induced an interaction between endothelial HSPA12B and YAP. ChIP assay shows that HSPA12B is a target gene of YAP/transcriptional enhanced associated domain4 (TEAD4) and a co-activator in YAP-associated angiogenesis. In vivo studies using the MI model show that endothelial specific deficiency of HSPA12B (eHspa12b-/-) or YAP (eYap-/-) impairs angiogenesis and exacerbates cardiac dysfunction, when compared with wild type (WT) mice. MI increased YAP expression and nuclear translocation in WT hearts, but not in eHspa12b-/- hearts. HSPA12B expression and nuclear translocation were up-regulated in WT MI hearts, but not in eYap-/- MI myocardium. Our data demonstrated that endothelial HSPA12B is a novel target and co-activator for YAP/TEAD4 and cooperates with YAP to regulate endothelial angiogenesis post-MI.
Min Fan, Kun Yang, Xiaohui Wang, Yana Wang, Fei Tu, Tuanzhu Ha, Li Liu, David L. Williams, Chuanfu Li
Age-associated systemic, chronic inflammation is partially attributed to increased self (auto)-reactivity, resulting from disruption of central tolerance in the aged, involuted thymus. This involution causally results from gradually decreased expression of the transcription factor FOXN1 in thymic epithelial cells (TECs), while exogenous FOXN1 in TECs can partially rescue age-related thymic involution. Given the findings that TECs induced from FOXN1-overexpressing embryonic fibroblasts can generate an ectopic de novo thymus under the kidney capsule and intra-thymically injected naturally young TECs can lead to middle-aged thymus regrowth, we attempted to extend these two findings by combining them as a novel thymic rejuvenation strategy with two types of promoter-driven (Rosa26CreERT and FoxN1Cre) Cre-mediated FOXN1-reprogrammed embryonic fibroblasts (FREFs). We engrafted these two-types of FREFs directly into the aged murine thymus. We found significant regrowth of the native aged thymus with rejuvenated architecture and function in both males and females, exhibiting increased thymopoiesis and reinforced thymocyte negative selection, along with reduced senescent T cells and auto-reactive T cell-mediated inflammation in old mice. Therefore, this strategy has preclinical significance and presents a strategy to potentially rescue decreased thymopoiesis and perturbed negative selection to significantly, albeit partially, restore defective central tolerance and reduce subclinical autoimmune symptoms in the elderly.
Jiyoung Oh, Weikan Wang, Rachel Thomas, Dong-Ming Su
Abstract: S-Nitroso-L-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase (sGC)-independent effects are stereoselective - that is, not recapitulated by S-nitroso-D-cysteine (D-CSNO) – and are inhibited by chemical cogeners. However, candidate L-CSNO receptors have never been identified. Here, we have used two complementary affinity chromatography assays - followed by unbiased proteomic analysis - to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO-Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy, hydrogen deuterium exchange and, in Kv1.1/Kv1.2/Kvβ2 overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv 1.1 prevented this effect. In intact animals, L-CSNO injection at level of the carotid body (CB) dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO cogener S-methyl-L-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.
Benjamin Gaston, Laura A. Smith, Jürgen Bosch, James M. Seckler, Diana L. Kunze, Janna Kiselar, Nadzeya Marozkina, Craig Hodges, Patrick Wintrode, Kellen McGee, Tatiana Morozkina, Spencer T. Burton, Tristan Lewis, Timothy Strassmaier, Paulina Getsy, James N. Bates, Stephen J. Lewis
New strategies are needed to enhance the efficacy of anti-programmed cell death protein (PD-1) antibody (Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of CQ derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in melanoma. The combination resulted tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen primed T cells to macrophage conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma specific killing. Genetic or chemical PPT1 inhibition resulted an M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells (MDSCs) in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), tank-binding kinase 1 (TBK1) pathway activation and the secretion of interferon β (IFN-β) in macrophages which was a key component for augmented T cell-mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for a melanoma clinical trial testing this new immunotherapy combination and may also be effective in other cancers.
Gaurav Sharma, Rani Ojha, Estela Noguera-Ortega, Vito W. Rebecca, John Attanasio, Shujing Liu, Shengfu Piao, Jennifer J. Lee, Michael C. Nicastri, Sandra L. Harper, Amruta Ronghe, Vaibhav Jain, Jeffrey D. Winkler, David W. Speicher, Jerome Mastio, Phyllis A Gimotty, Xiaowei Xu, E. John Wherry, Dmitry I. Gabrilovich, Ravi K. Amaravadi
Tumor-associated macrophages (TAMs) affect cancer progression and therapy. Ovarian carcinoma often metastasizes to the peritoneal cavity. Here, we found two peritoneal macrophage subsets in mice bearing ID8 ovarian cancer based on the Tim-4 (T-cell immunoglobulin and mucin domain containing 4) expression. Tim-4+ TAMs were embryonically originated and locally sustained while Tim-4- TAMs were replenished from circulating monocytes. Tim-4+ TAMs, but not Tim-4- TAMs, promoted tumor growth in vivo. Relative to Tim-4- TAMs, Tim-4+ TAMs manifested high oxidative phosphorylation and adapted mitophagy to alleviate oxidative stress. High levels of arginase-1 in Tim-4+ TAMs contributed to potent mitophagy activities via weakened mTORC1 activation due to low arginine resultant from arginase-1-mediated metabolism. Furthermore, genetic deficiency of autophagy element FIP200 resulted in Tim-4+ TAM loss via ROS-mediated apoptosis, and elevated T cell-immunity and ID8 tumor inhibition in vivo. Moreover, human ovarian cancer-associated CRIg (complement receptor of the Immunoglobulin superfamily) positive macrophages were transcriptionally, metabolically, and functionally similar to murine Tim-4+ TAMs. Thus, targeting CRIg+ (Tim-4+) TAMs may potentially treat ovarian cancer patients with peritoneal metastasis.
Houjun Xia, Shasha Li, Xiong Li, Weichao Wang, Yingjie Bian, Shuang Wei, Sara Grove, Weimin Wang, Linda Vatan, J. Rebecca Liu, Karen McLean, Ramandeep Rattan, Adnan R. Munkarah, Jun-Lin Guan, Ilona Kryczek, Weiping Zou
Electroconvulsive therapy is highly effective in neuropsychiatric disorders by unknown mechanisms. Microglial toxicity plays key role in neuroinflammatory and degenerative diseases, where there is critical shortage in therapies. This study examined the effects of electroconvulsive seizures (ECS) on chronic neuroinflammation and microglial neurotoxicity.Electric brain stimulation inducing full tonic-clonic seizures during chronic relapsing-progressive experimental autoimmune encephalomyelitis (EAE) reduced spinal immune cell infiltration, reduced myelin and axonal loss, and prevented clinical deterioration. Using the transfer EAE model we examined the effect of ECS on systemic immune response in donor mice versus ECS effect on CNS innate immune activity in recipient mice. ECS did not affect encephalitogenicity of systemic T cells, but targeted the CNS directly to inhibit T-cell induced neuroinflammation. In vivo and ex-vivo assays indicated that ECS suppressed microglial neurotoxicity, by reducing iNOS expression, nitric oxide and reactive oxygen species (ROS) production, and by reducing CNS oxidative stress. Microglia from ECS treated EAE mice expressed less T cell stimulatory and chemoattractant factors. Our finding indicate that Electroconvulsive therapy targets the CNS innate immune system to reduce neuroinflammation by attenuating microglial neurotoxicity. These findings signify a novel therapeutic approach for chronic neuroinflammatory, neuropsychiatric and neurodegenerative diseases.
Smadar Goldfarb, Nina Fainstein, Tamir Ben-Hur
Background Tuberculosis (TB) kills more people than any other infection and new diagnostic tests to identify active cases are urgently required. We aimed to discover and verify novel markers for TB in non-depleted plasma. Methods We applied an optimised quantitative proteomics discovery methodology based on multidimensional and orthogonal liquid chromatographic separation hyphenated with high-resolution mass spectrometry (q3D LC-MS) to study non-depleted plasma of 11 patients with active TB compared to 10 healthy control donors. Prioritised candidates were verified in an independent UK-based (n=118) and a South African cohorts (n=203). Results We generated the most comprehensive TB plasma proteome to date, profiling 5022 proteins spanning 11 orders-of-magnitude concentration range with diverse biochemical and molecular properties. We further analysed the predominantly low molecular weight sub-proteome; identifying 46 proteins with significantly increased and 90 with decreased abundance (peptide FDR ≤1%, q-value ≤0.05). Biological network analysis showed regulation of new pathways involving lipid and organophosphate ester transport. Verification was performed for novel candidate biomarkers (CFHR5, ILF2) in two independent cohorts. These proteins were elevated in both TB and other respiratory diseases (ORD). Receiver-operating-characteristics analyses using a 5-protein panel (CFHR5, LRG1, CRP, LBP and SAA1) exhibited discriminatory power in distinguishing between TB and ORD (AUC =0.81). Conclusions We report the most comprehensive TB plasma proteome to date, identifying numerous novel markers with verification in two independent cohorts, which led to a 5-protein biosignature with potential to improve TB diagnosis. With further development, these biomarkers have potential as a diagnostic triage test. Funding Colombia: Colciencias. UK: Medical Research Council, Innovate UK, National Institute for Health Research, Academy of Medical Sciences. Peru: Program for Advanced Research Capacities for AIDS. South Africa: Wellcome Centre for Infectious Diseases Research.
Diana J. Garay-Baquero, Cory H. White, Naomi F. Walker, Marc Tebruegge, Hannah F. Schiff, Cesar Ugarte-Gil, Stephen Morris-Jones, Ben G. Marshall, Antigoni Manousopoulou, John H. Adamson, Andres F. Vallejo, Magdalena K. Bielecka, Robert J. Wilkinson, Liku B. Tezera, Christopher H. Woelk, Spiros D. Garbis, Paul Elkington