Research
Abstract
One of the most fundamental and challenging questions in the cancer field is how immunity is transformed from tumor immunosurveillance to tumor-promoting inflammation. Here, we identify the tumor suppressor PDZ-LIM domain-containing protein 2 (PDLIM2) as a checkpoint of alveolar macrophages (AMs) important for lung tumor suppression. During lung tumorigenesis, PDLIM2 expression in AMs is down-regulated by reactive oxygen species (ROS)-activated transcription repressor BTB and CNC homology 1 (BACH1). PDLIM2 down-regulation leads to constitutive activation of the transcription factor signal transducer and activator of transcription 3 (STAT3), driving AM pro-tumorigenic polarization/activation and differentiation from monocytes attracted from the circulation to suppress cytotoxic T lymphocytes (CTLs) and promote lung cancer. PDLIM2 down-regulation also decreases AM phagocytosis. These findings establish ROS/BACH1/PDLIM2/STAT3 as a signaling pathway driving AMs for lung tumor promotion.
Authors
Liwen Li, Fan Sun, Lei Han, Xujie Liu, Yadong Xiao, Alyssa D. Gregory, Steven D. Shapiro, Gutian Xiao, Zhaoxia Qu
Abstract
Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) damage is associated with Primary Open Angle Glaucoma (POAG). Myocilin mutations resulting in elevated IOP are the most common genetic cause of POAG. We have previously shown that mutant myocilin accumulates in the endoplasmic reticulum (ER) and induces chronic ER stress, leading to TM damage and IOP elevation. However, it is not understood how chronic ER stress leads to TM dysfunction and loss. Here, we report that mutant myocilin activates autophagy but it is functionally impairecd in cultured human trabecular meshwork (TM) cells and in a mouse model of myocilin-associated POAG (Tg-MYOCY437H). Genetic and pharmacological inhibition of autophagy worsens mutant myocilin accumulation and exacerbates IOP elevation in Tg-MYOCY437H mice. Remarkably, impaired autophagy is associated with chronic ER stress-induced transcriptional factor, CHOP. Deletion of CHOP corrects impaired autophagy, enhances recognition and degradation of mutant myocilin by autophagy,and reduces glaucoma in Tg-MYOCY437H mice. Stimulating autophagic flux via Tat-beclin 1 peptide or torin 2, promotes autophagic degradation of mutant myocilin and reduces elevated IOP in Tg-MYOCY437H mice. Together, our studies provide a novel treatment strategy for myocilin-associated POAG by correcting impaired autophagy in the TM.
Authors
Ramesh B. Kasetti, Prabhavathi Maddineni, Charles C. Kiehlbauch, Shruti Patil, Charles C. Searby, Beth Levine, Val C. Sheffield, Gulab S. Zode
Abstract
Extracellular vesicles (EVs) are implicated in the crosstalk between adipocytes and other metabolic organs, and an altered biological cargo has been observed in EVs from human obese adipose tissue (AT). Yet, the role of adipocyte-derived EVs in pancreatic β-cells remains to be determined. Here, we explored the effects of EVs, released from both rodent and human isolated adipocytes and human AT explants, on survival and function of pancreatic β-cells and human pancreatic islets. EVs from healthy 3T3-L1 adipocytes increased survival and proliferation and promoted insulin secretion in INS-1E β-cells and human pancreatic islets, both untreated or exposed to cytokines or glucolipotoxicity, while EVs from inflamed adipocytes caused β-cell death and dysfunction. Human lean adipocyte-derived EVs produced similar beneficial effects, while EVs from obese AT explants were harmful for human EndoC-βH3 β-cells. We observed differential expression of microRNAs in EVs from healthy and inflamed adipocytes, as well as alteration in signaling pathways and expression of β-cell genes, adipokines and CKs in recipient β-cells. These in vitro results suggest that, depending on the physiopathological state of AT, adipocyte-derived EVs may influence β-cell fate and function.
Authors
Iacopo Gesmundo, Barbara Pardini, Eleonora Gargantini, Giacomo Gamba, Giovanni Birolo, Alessandro Fanciulli, Dana Banfi, Noemi Congiusta, Enrica Favaro, Maria Chiara Deregibus, Gabriele Togliatto, Gaia Zocaro, Maria Felice Brizzi, Raul M. Luque, Justo P. Castaño, Maria Alessandra Bocchiotti, Simone Arolfo, Stefania Bruno, Rita Nano, Mario Morino, Lorenzo Piemonti, Huy Ong, Giuseppe Matullo, Juan M. Falcón-Pérez, Ezio Ghigo, Giovanni Camussi, Riccarda Granata
Abstract
Cantu Syndrome (CS) is caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) KATP channel subunits, the most common mutations being SUR2[R1154Q] and SUR2[R1154W], carried by ~30% of patients. We used CRISPR/Cas9 genome engineering to introduce the equivalent of human SUR2[R1154Q] mutation to the mouse ABCC9 gene. Along with minimal CS disease features, R1154Q cardiomyocytes and vascular smooth muscle showed much lower KATP current density and pinacidil activation than WT cells. Almost complete loss of SUR2-dependent protein and KATP in homozygous R1154Q ventricles revealed an underlying diazoxide-sensitive SUR1-dependent KATP channel activity. Surprisingly, sequencing of SUR2 cDNA revealed divergent transcripts, one encoding full length SUR2 protein, and the other with in-frame deletion of 93 bases (corresponding to 31 amino acids encoded by exon 28) that was present in ~40% and ~90% of transcripts from hetero- and homozygous R1154Q tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in non-functional channels. SUR2[R1154Q] CS patient tissue and iPSC-derived cardiomyocytes showed only full length SUR2 transcripts, although further studies will be required to fully test whether SUR2[R1154Q] or other CS mutations might result in aberrant splicing and variable expressivity of disease features in human CS.
Authors
Haixia Zhang, Alex M. Hanson, Tobias U. Scherf de Almeida, Christopher H. Emfinger, Conor McClenaghan, Theresa Harter, Zihan Yan, Paige E. Cooper, G. Schuyler Brown, Eric C. Arakel, Robert P. Mecham, Attila Kovacs, Carmen M. Halabi, Blanche Schwappach, Maria S. Remedi, Colin G. Nichols
Abstract
The development of prophylactic and therapeutic agents for coronavirus disease 2019 (COVID-19) is a current global health priority. Here, we investigated the presence of cross-neutralizing antibodies against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in dromedary camels that were Middle East respiratory syndrome (MERS)-CoV-seropositive but MERS-CoV-free. The tested 229 dromedaries had anti-MERS-CoV camel antibodies with variable cross-reactivity patterns against SARS-CoV-2 proteins, including the S trimer, M, N, and E proteins. Using SARS-CoV-2 competitive immunofluorescence immunoassays and pseudovirus neutralization assays, we found medium-to-high titers of cross-neutralizing antibodies against SARS-CoV-2 in these animals. Through linear B cell epitope mapping using phage immunoprecipitation sequencing and a SARS-CoV-2 peptide/proteome microarray, we identified a large repertoire of betacoronavirus cross-reactive antibody specificities in these dromedaries and demonstrated that the SARS-CoV-2-specific VHH antibody repertoire is qualitatively diverse. This analysis revealed not only several SARS-CoV-2 epitopes that are highly immunogenic in humans, including a neutralizing epitope, but also epitopes exclusively targeted by camel antibodies. The identified SARS-CoV-2 cross-neutralizing camel antibodies are not proposed as a potential treatment for COVID-19. Rather, their presence in non-immunized camels supports the development of SARS-CoV-2 hyperimmune camels, which could be a prominent source of therapeutic agents for the prevention and treatment of COVID-19.
Authors
Lotfi Chouchane, Jean-Charles Grivel, Elmoubasher Farag, Igor Pavlovski, Selma Maacha, Abbirami Sathappan, Hamad Al-Romaihi, Sirin Abuaqel, Manar Ata, Aouatef Ismail Chouchane, Sami Remadi, Najeeb M. Halabi, Arash Rafii, Mohammed Al-Thani, Nico Marr, Murugan Subramanian, Jingxuan Shan
Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size
Abstract
Limitations in cell proliferation are important for normal function of differentiated tissues, and essential for the safty of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing S-phase entry, or S-phase completion and determined growth potential, differentiation and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S-phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.
Authors
Lina Sui, Yurong Xin, Qian Du, Daniela Georgieva, Giacomo Diedenhofen, Leena Haataja, Qi Su, Michael V. Zuccaro, Jinrang Kim, Jiayu Fu, Yuan Xing, Yi He, Danielle Baum, Robin S. Goland, Yong Wang, Jose Oberholzer, Fabrizio Barbetti, Peter Arvan, Sandra Kleiner, Dieter Egli
Abstract
Interleukin-33 (IL-33) is a key mediator of chronic airway disease driven by type-2 immune pathways, yet the non-classical secretory mechanism for this cytokine remains undefined. We performed a comprehensive analysis in human airway epithelial cells, which revealed that tonic IL-33 secretion is dependent on the ceramide biosynthetic enzyme neutral sphingomyelinase 2 (nSMase2). IL-33 is co-secreted with exosomes by the nSMase2-regulated multivesicular endosome (MVE) pathway as surface-bound cargo. In support of these findings, human chronic obstructive pulmonary disease (COPD) specimens exhibited increased epithelial expression of the abundantly secreted IL33Δ34 isoform and augmented nSMase2 expression compared to non-COPD specimens. Using an Alternaria-induced airway disease model, we found the nSMase2 inhibitor GW4869 abrogated both IL-33 and exosome secretion as well as downstream inflammatory pathways. This work elucidates a novel aspect of IL-33 biology that may be targeted for therapeutic benefit in chronic airway diseases driven by type-2 inflammation.
Authors
Ella Katz-Kiriakos, Deborah F. Steinberg, Colin E. Kluender, Omar A. Osorio, Catie Newsom-Stewart, Arjun Baronia, Derek E. Byers, Michael J. Holtzman, Dawn Katafiasz, Kristina L. Bailey, Steven L. Brody, Mark J. Miller, Jennifer Alexander-Brett
Abstract
Tenascin-C, an extracellular matrix protein that has proinflammatory properties, is a recently described antibody target in rheumatoid arthritis. In this study, we utilized a systematic discovery process and identified five novel citrullinated tenascin-C (cit-TNC) T cell epitopes. CD4+ T cells specific for these epitopes were elevated in the peripheral blood of subjects with rheumatoid arthritis and showed signs of activation. Cit-TNC-specific T cells were also present among synovial fluid T cells and secreted interferon-γ. Two of these cit-TNC peptides were recognized by antibodies within the serum and synovial fluid of individuals with RA. Detectable serum levels of cit-TNC reactive antibodies were prevalent among subjects with RA and positively associated with cyclic citrullinated peptide (CCP) reactivity and the HLA shared epitope. Furthermore, cit-TNC reactive antibodies were correlated with rheumatoid factor and elevated in subjects with a history of smoking. Taken together this work confirms cit-TNC as an autoantigen that is targeted by autoreactive CD4+ T cells and autoantibodies in patients with RA. Furthermore, our findings suggest that a unique set of epitopes recognized by both CD4+ T cells and B cells have the potential to amplify autoimmunity and promote the development and progression of rheumatoid arthritis.
Authors
Jing Song, Anja Schwenzer, Alicia Wong, Sara Turcinov, Cliff Rims, Lorena Rodríguez-Martínez, David Arribas-Layton, Christina Gerstner, Virginia S. Muir, Kim S. Midwood, Vivianne Malmström, Eddie A. James, Jane H. Buckner
Abstract
An intact lung epithelial barrier is essential for lung homeostasis. The Na+, K+-ATPase (NKA), primarily serving as an ion transporter, also regulates epithelial barrier function via modulation of tight junctions. However, the underlying mechanism is not well-understood. Here, we showed that overexpression of the NKA β1 subunit upregulates the expression of tight junction proteins, leading to increased alveolar epithelial barrier function by an ion transport-independent mechanism. Using immunoprecipitation and mass spectrometry, we identified a number of unknown protein interactions of the β1 subunit, including a top candidate, myotonic dystrophy kinase-related cdc42-binding kinase α (MRCKα), a protein kinase known to regulate peripheral actin formation. Using a doxycycline-inducible gene expression system, we demonstrated that MRCKα and its downstream activation of myosin light chain is required for the regulation of alveolar barrier function by the NKA β1 subunit. Importantly, MRCKα is expressed in both human airways and alveoli and has reduced expression in patients with Acute Respiratory Distress Syndrome (ARDS), a lung illness that can be caused by multiple direct and indirect insults, including the infection of influenza virus and SARS-CoV-2. Our results have elucidated a novel mechanism by which NKA regulates epithelial tight junctions and identified potential drug targets for treating ARDS and other pulmonary diseases that are caused by barrier dysfunction.
Authors
Haiqing Bai, Rui Zhou, Michael Barravecchia, Rosemary Norman, Alan E. Friedman, Deborah Yu, Xin Lin, Jennifer L. Young, David A. Dean
Abstract
Preterm birth increases the risk for pulmonary hypertension and heart failure in adulthood. Oxygen therapy can damage the immature cardiopulmonary system and may be partially responsible for the cardiovascular disease in adults born preterm. We previously showed that exposing newborn mice to hyperoxia causes pulmonary hypertension by 1 year of age that is preceded by a poorly understood loss of pulmonary vein cardiomyocyte proliferation. We now show that hyperoxia also reduces cardiomyocyte proliferation and survival in the left atrium and causes diastolic heart failure by disrupting its filling of the left ventricle. Transcriptomic profiling showed that neonatal hyperoxia permanently suppressed fatty acid synthase (Fasn), stearoyl-CoA desaturase 1 (Scd1) and other fatty acid synthesis genes in the atria of mice, the HL-1 line of mouse atrial cardiomyocytes and left atrial tissue explanted from human infants. Suppressing Fasn or Scd1 reduced HL-1 cell proliferation and increased cell death while overexpressing these genes maintained their expansion in hyperoxia, suggesting oxygen directly inhibits atrial cardiomyocyte proliferation and survival by repressing Fasn and Scd1. Pharmacologic interventions that restore Fasn, Scd1 and other fatty acid synthesis genes in atrial cardiomyocytes may thus provide a way of ameliorating the adverse effects of supplemental oxygen on preterm infants.
Authors
Ethan David Cohen, Min Yee, George A. Porter, Jr., Erin E. Ritzer, Andrew N. McDavid, Paul S. Brookes, Gloria S. Pryhuber, Michael A. O'Reilly
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