Mutations in laminin α2-subunit (Lmα2, encoded by
Karen K. McKee, Stephanie C. Crosson, Sarina Meinen, Judith R. Reinhard, Markus A. Rüegg, Peter D. Yurchenco
Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies against complexes between human platelet factor 4 (hPF4) and heparin. A better understanding of the events that initiate the prothrombotic state may improve approaches to antithrombotic management. Here, we visualized thrombus formation in an in vivo murine model and an endothelialized microfluidic system that simulate the pathogenesis of HIT. hPF4 released from platelets predominantly bound to peri-injury endothelium and formed HIT antigenic complexes that were dissociated by heparin. In mice expressing both hPF4+ and human platelet IgG Fc receptor IIA (FcγRIIA), infusion of the HIT-like monoclonal antibody KKO increased fibrin and platelet deposition at sites of injury, followed immediately by antigen formation on proximate endothelial cells. After a few minutes, HIT antigen was detected within the thrombus itself at the interface between the platelet core and the surrounding shell. We observed similar results in the humanized, endothelialized microfluidic system. hPF4 and KKO selectively bound to photochemically injured endothelium at sites where surface glycocalyx was reduced. These studies support the concept that the perithrombus endothelium is the predominant site of HIT antigen assembly. This suggests that disrupting antigen formation along the endothelium or protecting the endothelium may provide a therapeutic opportunity to prevent thrombotic complications of HIT, while sparing systemic hemostatic pathways.
Vincent Hayes, Ian Johnston, Gowthami M. Arepally, Steven E. McKenzie, Douglas B. Cines, Lubica Rauova, Mortimer Poncz
Peptides derived from pre-proglucagon (GCG peptides) act in both the periphery and the CNS to change food intake, glucose homeostasis, and metabolic rate while playing a role in anxiety behaviors and physiological responses to stress. Although the actions of GCG peptides produced in the gut and pancreas are well described, the role of glutamatergic GGC peptide–secreting hindbrain neurons in regulating metabolic homeostasis has not been investigated. Here, we have shown that chemogenetic stimulation of GCG-producing neurons reduces metabolic rate and food intake in fed and fasted states and suppresses glucose production without an effect on glucose uptake. Stimulation of GCG neurons had no effect on corticosterone secretion, body weight, or conditioned taste aversion. In the diet-induced obese state, the effects of GCG neuronal stimulation on gluconeogenesis were lost, while the food intake–lowering effects remained, resulting in reductions in body weight and adiposity. Our work suggests that GCG peptide–expressing neurons can alter feeding, metabolic rate, and glucose production independent of their effects on hypothalamic pituitary-adrenal (HPA) axis activation, aversive conditioning, or insulin secretion. We conclude that GCG neurons likely stimulate separate populations of downstream cells to produce a change in food intake and glucose homeostasis and that these effects depend on the metabolic state of the animal.
Ronald P. Gaykema, Brandon A. Newmyer, Matteo Ottolini, Vidisha Raje, Daniel M. Warthen, Philip S. Lambeth, Maria Niccum, Ting Yao, Yiru Huang, Ira G. Schulman, Thurl E. Harris, Manoj K. Patel, Kevin W. Williams, Michael M. Scott
MicroRNAs (miRNAs) are negative modulators of gene expression that fine-tune numerous biological processes. miRNA loss-of-function rarely results in highly penetrant phenotypes, but rather, influences cellular responses to physiologic and pathophysiologic stresses. Here, we have reported that a single member of the evolutionarily conserved miR-7 family, miR-7a2, is essential for normal pituitary development and hypothalamic-pituitary-gonadal (HPG) function in adulthood. Genetic deletion of
Kashan Ahmed, Mary P. LaPierre, Emanuel Gasser, Rémy Denzler, Yinjie Yang, Thomas Rülicke, Jukka Kero, Mathieu Latreille, Markus Stoffel
Autoimmune responses to meiotic germ cell antigens (MGCA) that are expressed on sperm and testis occur in human infertility and after vasectomy. Many MGCA are also expressed as cancer/testis antigens (CTA) in human cancers, but the tolerance status of MGCA has not been investigated. MGCA are considered to be uniformly immunogenic and nontolerogenic, and the prevailing view posits that MGCA are sequestered behind the Sertoli cell barrier in seminiferous tubules. Here, we have shown that only some murine MGCA are sequestered. Nonsequestered MCGA (NS-MGCA) egressed from normal tubules, as evidenced by their ability to interact with systemically injected antibodies and form localized immune complexes outside the Sertoli cell barrier. NS-MGCA derived from cell fragments that were discarded by spermatids during spermiation. They egressed as cargo in residual bodies and maintained Treg-dependent physiological tolerance. In contrast, sequestered MGCA (S-MGCA) were undetectable in residual bodies and were nontolerogenic. Unlike postvasectomy autoantibodies, which have been shown to mainly target S-MGCA, autoantibodies produced by normal mice with transient Treg depletion that developed autoimmune orchitis exclusively targeted NS-MGCA. We conclude that spermiation, a physiological checkpoint in spermatogenesis, determines the egress and tolerogenicity of MGCA. Our findings will affect target antigen selection in testis and sperm autoimmunity and the immune responses to CTA in male cancer patients.
Kenneth S.K. Tung, Jessica Harakal, Hui Qiao, Claudia Rival, Jonathan C.H. Li, Alberta G.A. Paul, Karen Wheeler, Patcharin Pramoonjago, Constance M. Grafer, Wei Sun, Robert D. Sampson, Elissa W.P. Wong, Prabhakara P. Reddi, Umesh S. Deshmukh, Daniel M. Hardy, Huanghui Tang, C. Yan Cheng, Erwin Goldberg
Microvascular endothelial cells (ECs) are increasingly recognized as organ-specific gatekeepers of their microenvironment. Microvascular ECs instruct neighboring cells in their organ-specific vascular niches through angiocrine factors, which include secreted growth factors (angiokines), extracellular matrix molecules, and transmembrane proteins. However, the molecular regulators that drive organ-specific microvascular transcriptional programs and thereby regulate angiodiversity are largely elusive. In contrast to other ECs, which form a continuous cell layer, liver sinusoidal ECs (LSECs) constitute discontinuous, permeable microvessels. Here, we have shown that the transcription factor GATA4 controls murine LSEC specification and function. LSEC-restricted deletion of
Cyrill Géraud, Philipp-Sebastian Koch, Johanna Zierow, Kay Klapproth, Katrin Busch, Victor Olsavszky, Thomas Leibing, Alexandra Demory, Friederike Ulbrich, Miriam Diett, Sandhya Singh, Carsten Sticht, Katja Breitkopf-Heinlein, Karsten Richter, Sanna-Maria Karppinen, Taina Pihlajaniemi, Bernd Arnold, Hans-Reimer Rodewald, Hellmut G. Augustin, Kai Schledzewski, Sergij Goerdt
Munc13 proteins are essential regulators of neurotransmitter release at nerve cell synapses. They mediate the priming step that renders synaptic vesicles fusion-competent, and their genetic elimination causes a complete block of synaptic transmission. Here we have described a patient displaying a disorder characterized by a dyskinetic movement disorder, developmental delay, and autism. Using whole-exome sequencing, we have shown that this condition is associated with a rare, de novo Pro814Leu variant in the major human Munc13 paralog UNC13A (also known as Munc13-1). Electrophysiological studies in murine neuronal cultures and functional analyses in
Noa Lipstein, Nanda M. Verhoeven-Duif, Francesco E. Michelassi, Nathaniel Calloway, Peter M. van Hasselt, Katarzyna Pienkowska, Gijs van Haaften, Mieke M. van Haelst, Ron van Empelen, Inge Cuppen, Heleen C. van Teeseling, Annemieke M.V. Evelein, Jacob A. Vorstman, Sven Thoms, Olaf Jahn, Karen J. Duran, Glen R. Monroe, Timothy A. Ryan, Holger Taschenberger, Jeremy S. Dittman, Jeong-Seop Rhee, Gepke Visser, Judith J. Jans, Nils Brose
An intracellular complement system (ICS) has recently been described in immune and nonimmune human cells. This system can be activated in a convertase-independent manner from intracellular stores of the complement component C3. The source of these stores has not been rigorously investigated. In the present study, Western blotting identified a band corresponding to C3 in freshly isolated human peripheral blood cells that was absent in corresponding cell lines. One difference between native cells and cell lines was the time absent from a fluid-phase complement source; therefore, we hypothesized that loading C3 from plasma was a route of establishing intracellular C3 stores. We found that many types of human cells specifically internalized C3(H2O), the hydrolytic product of C3, and not native C3, from the extracellular milieu. Uptake was rapid, saturable, and sensitive to competition with unlabeled C3(H2O), indicating a specific mechanism of loading. Under steady-state conditions, approximately 80% of incorporated C3(H2O) was returned to the extracellular space. These studies identify an ICS recycling pathway for C3(H2O). The loaded C3(H2O) represents a source of C3a, and its uptake altered the cytokine profile of activated CD4+ T cells. Importantly, these results indicate that the impact of soluble plasma factors should be considered when performing in vitro studies assessing cellular immune function.
Michelle Elvington, M. Kathryn Liszewski, Paula Bertram, Hrishikesh S. Kulkarni, John P. Atkinson
A highly orchestrated gene expression program establishes the properties that define mature adipocytes, but the contribution of posttranscriptional factors to the adipocyte phenotype is poorly understood. Here we have shown that the RNA-binding protein PSPC1, a component of the paraspeckle complex, promotes adipogenesis in vitro and is important for mature adipocyte function in vivo. Cross-linking and immunoprecipitation followed by RNA sequencing revealed that PSPC1 binds to intronic and 3′-untranslated regions of a number of adipocyte RNAs, including the RNA encoding the transcriptional regulator EBF1. Purification of the paraspeckle complex from adipocytes further showed that PSPC1 associates with the RNA export factor DDX3X in a differentiation-dependent manner. Remarkably, PSPC1 relocates from the nucleus to the cytoplasm during differentiation, coinciding with enhanced export of adipogenic RNAs. Mice lacking PSPC1 in fat displayed reduced lipid storage and adipose tissue mass and were resistant to diet-induced obesity and insulin resistance due to a compensatory increase in energy expenditure. These findings highlight a role for PSPC1-dependent RNA maturation in the posttranscriptional control of adipose development and function.
Jiexin Wang, Prashant Rajbhandari, Andrey Damianov, Areum Han, Tamer Sallam, Hironori Waki, Claudio J. Villanueva, Stephen D. Lee, Ronni Nielsen, Susanne Mandrup, Karen Reue, Stephen G. Young, Julian Whitelegge, Enrique Saez, Douglas L. Black, Peter Tontonoz
Defective apoptotic death of activated macrophages has been implicated in the pathogenesis of rheumatoid arthritis (RA). However, the molecular signatures defining apoptotic resistance of RA macrophages are not fully understood. Here, global transcriptome profiling of RA macrophages revealed that the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) critically regulates diverse pathologic processes in synovial macrophages including the cell cycle, apoptosis, and proliferation. Transcriptomic analysis of NFAT5-deficient macrophages revealed the molecular networks defining cell survival and proliferation. Proinflammatory M1-polarizing stimuli and hypoxic conditions were responsible for enhanced NFAT5 expression in RA macrophages. An in vitro functional study demonstrated that NFAT5-deficient macrophages were more susceptible to apoptotic death. Specifically, CCL2 secretion in an NFAT5-dependent fashion bestowed apoptotic resistance to RA macrophages in vitro. Injection of recombinant CCL2 into one of the affected joints of
Susanna Choi, Sungyong You, Donghyun Kim, Soo Youn Choi, H. Moo Kwon, Hyun-Sook Kim, Daehee Hwang, Yune-Jung Park, Chul-Soo Cho, Wan-Uk Kim
Tissue inflammation is a key component of obesity-induced insulin resistance, with a variety of immune cell types accumulating in adipose tissue. Here, we have demonstrated increased numbers of B2 lymphocytes in obese adipose tissue and have shown that high-fat diet–induced (HFD-induced) insulin resistance is mitigated in B cell-deficient (Bnull) mice. Adoptive transfer of adipose tissue B2 cells (ATB2) from wild-type HFD donor mice into HFD Bnull recipients completely restored the effect of HFD to induce insulin resistance. Recruitment and activation of ATB2 cells was mediated by signaling through the chemokine leukotriene B4 (LTB4) and its receptor LTB4R1. Furthermore, the adverse effects of ATB2 cells on glucose homeostasis were partially dependent upon T cells and macrophages. These results demonstrate the importance of ATB2 cells in obesity-induced insulin resistance and suggest that inhibition of the LTB4/LTB4R1 axis might be a useful approach for developing insulin-sensitizing therapeutics.
Wei Ying, Joshua Wollam, Jachelle M. Ofrecio, Gautam Bandyopadhyay, Dalila El Ouarrat, Yun Sok Lee, Da Young Oh, Pingping Li, Olivia Osborn, Jerrold M. Olefsky
The most frequent focal alterations in human retinoblastoma are mutations in the tumor-suppressor gene retinoblastoma (
Nan Wu, Deshui Jia, Breanna Bates, Ryan Basom, Charles G. Eberhart, David MacPherson
The mechanisms underlying the neurodevelopmental deficits associated with CHARGE syndrome, which include cerebellar hypoplasia, developmental delay, coordination problems, and autistic features, have not been identified. CHARGE syndrome has been associated with mutations in the gene encoding the ATP-dependent chromatin remodeler CHD7. CHD7 is expressed in neural stem and progenitor cells, but its role in neurogenesis during brain development remains unknown. Here we have shown that deletion of
Danielle E. Whittaker, Kimberley L.H. Riegman, Sahrunizam Kasah, Conor Mohan, Tian Yu, Blanca Pijuan Sala, Husam Hebaishi, Angela Caruso, Ana Claudia Marques, Caterina Michetti, María Eugenia Sanz Smachetti, Apar Shah, Mara Sabbioni, Omer Kulhanci, Wee-Wei Tee, Danny Reinberg, Maria Luisa Scattoni, Holger Volk, Imelda McGonnell, Fiona C. Wardle, Cathy Fernandes, M. Albert Basson
Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in
Svjetlana Lovric, Sara Goncalves, Heon Yung Gee, Babak Oskouian, Honnappa Srinivas, Won-Il Choi, Shirlee Shril, Shazia Ashraf, Weizhen Tan, Jia Rao, Merlin Airik, David Schapiro, Daniela A. Braun, Carolin E. Sadowski, Eugen Widmeier, Tilman Jobst-Schwan, Johanna Magdalena Schmidt, Vladimir Girik, Guido Capitani, Jung H. Suh, Noëlle Lachaussée, Christelle Arrondel, Julie Patat, Olivier Gribouval, Monica Furlano, Olivia Boyer, Alain Schmitt, Vincent Vuiblet, Seema Hashmi, Rainer Wilcken, Francois P. Bernier, A. Micheil Innes, Jillian S. Parboosingh, Ryan E. Lamont, Julian P. Midgley, Nicola Wright, Jacek Majewski, Martin Zenker, Franz Schaefer, Navina Kuss, Johann Greil, Thomas Giese, Klaus Schwarz, Vilain Catheline, Denny Schanze, Ingolf Franke, Yves Sznajer, Anne S. Truant, Brigitte Adams, Julie Désir, Ronald Biemann, York Pei, Elisabet Ars, Nuria Lloberas, Alvaro Madrid, Vikas R. Dharnidharka, Anne M. Connolly, Marcia C. Willing, Megan A. Cooper, Richard P. Lifton, Matias Simons, Howard Riezman, Corinne Antignac, Julie D. Saba, Friedhelm Hildebrandt
Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types.
Paul A. Beavis, Melissa A. Henderson, Lauren Giuffrida, Jane K. Mills, Kevin Sek, Ryan S. Cross, Alexander J. Davenport, Liza B. John, Sherly Mardiana, Clare Y. Slaney, Ricky W. Johnstone, Joseph A. Trapani, John Stagg, Sherene Loi, Lev Kats, David Gyorki, Michael H. Kershaw, Phillip K. Darcy
Ischemic heart disease resulting from myocardial infarction (MI) is the most prevalent form of heart disease in the United States. Post-MI cardiac remodeling is a multifaceted process that includes activation of fibroblasts and a complex immune response. T-regulatory cells (Tregs), a subset of CD4+ T cells, have been shown to suppress the innate and adaptive immune response and limit deleterious remodeling following myocardial injury. However, the mechanisms by which injured myocardium recruits suppressive immune cells remain largely unknown. Here, we have shown a role for Hippo signaling in the epicardium in suppressing the post-infarct inflammatory response through recruitment of Tregs. Mice deficient in epicardial YAP and TAZ, two core Hippo pathway effectors, developed profound post-MI pericardial inflammation and myocardial fibrosis, resulting in cardiomyopathy and death. Mutant mice exhibited fewer suppressive Tregs in the injured myocardium and decreased expression of the gene encoding IFN-γ, a known Treg inducer. Furthermore, controlled local delivery of IFN-γ following MI rescued Treg infiltration into the injured myocardium of YAP/TAZ mutants and decreased fibrosis. Collectively, these results suggest that epicardial Hippo signaling plays a key role in adaptive immune regulation during the post-MI recovery phase.
Vimal Ramjee, Deqiang Li, Lauren J. Manderfield, Feiyan Liu, Kurt A. Engleka, Haig Aghajanian, Christopher B. Rodell, Wen Lu, Vivienne Ho, Tao Wang, Li Li, Anamika Singh, Dasan M. Cibi, Jason A. Burdick, Manvendra K. Singh, Rajan Jain, Jonathan A. Epstein
Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G>A (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G>A (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism.
Rathi Prasad, Irene Hadjidemetriou, Avinaash Maharaj, Eirini Meimaridou, Federica Buonocore, Moin Saleem, Jenny Hurcombe, Agnieszka Bierzynska, Eliana Barbagelata, Ignacio Bergadá, Hamilton Cassinelli, Urmi Das, GOSgene, Ruth Krone, Bulent Hacihamdioglu, Erkan Sari, Ediz Yesilkaya, Helen L. Storr, Maria Clemente, Monica Fernandez-Cancio, Nuria Camats, Nanik Ram, John C. Achermann, Paul P. Van Veldhoven, Leonardo Guasti, Debora Braslavsky, Tulay Guran, Louise A. Metherell
Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a
Irina Pleines, Joanne Woods, Stephane Chappaz, Verity Kew, Nicola Foad, José Ballester-Beltrán, Katja Aurbach, Chiara Lincetto, Rachael M. Lane, Galina Schevzov, Warren S. Alexander, Douglas J. Hilton, William J. Astle, Kate Downes, Paquita Nurden, Sarah K. Westbury, Andrew D. Mumford, Samya G. Obaji, Peter W. Collins, NIHR BioResource, Fabien Delerue, Lars M. Ittner, Nicole S. Bryce, Mira Holliday, Christine A. Lucas, Edna C. Hardeman, Willem H. Ouwehand, Peter W. Gunning, Ernest Turro, Marloes R. Tijssen, Benjamin T. Kile
Elevated expression of the chemokine receptor CCR4 in tumors is associated with poor prognosis in several cancers. Here, we have determined that CCR4 was highly expressed in human renal cell carcinoma (RCC) biopsies and observed abnormal levels of CCR4 ligands in RCC patient plasma. An antagonistic anti-CCR4 antibody had antitumor activity in the RENCA mouse model of RCC. CCR4 inhibition did not reduce the proportion of infiltrating leukocytes in the tumor microenvironment but altered the phenotype of myeloid cells, increased NK cell and Th1 cytokine levels, and reduced immature myeloid cell infiltrate and blood chemokine levels. In spite of prominent changes in the myeloid compartment, the anti-CCR4 antibody did not affect RENCA tumors in T cell–deficient mice, and treatment with an anti–class II MHC antibody abrogated its antitumor activity. We concluded that the effects of the anti-CCR4 antibody required the adaptive immune system and CD4+ T cells. Moreover, CCL17-induced IFN-γ production was reduced when Th1-polarized normal CD4+ T cells were exposed to the CCR4 ligand, evidencing the involvement of CCR4 in Th1/Th2 regulation. The anti-CCR4 antibody, alone or in combination with other immune modulators, is a potential treatment approach to human solid cancers with high levels of CCR4-expressing tumor-infiltrating leukocytes and abnormal plasma CCR4 ligand levels.
Chiara Berlato, Moddasar N. Khan, Tiziana Schioppa, Richard Thompson, Eleni Maniati, Anne Montfort, Maryam Jangani, Monica Canosa, Hagen Kulbe, Urs B. Hagemann, Alexander R. Duncan, Laura Fletcher, Robert W. Wilkinson, Thomas Powles, Sergio A. Quezada, Frances R. Balkwill
Disruption of the retinoblastoma (RB) tumor suppressor pathway, either through genetic mutation of upstream regulatory components or mutation of
Lindsey N. Kent, Sooin Bae, Shih-Yin Tsai, Xing Tang, Arunima Srivastava, Christopher Koivisto, Chelsea K. Martin, Elisa Ridolfi, Grace C. Miller, Sarah M. Zorko, Emilia Plevris, Yannis Hadjiyannis, Miguel Perez, Eric Nolan, Raleigh Kladney, Bart Westendorp, Alain de Bruin, Soledad Fernandez, Thomas J. Rosol, Kamal S. Pohar, James M. Pipas, Gustavo Leone