Evaluation of T lymphocyte frequency provides prognostic information for patients with oral squamous cell cancer (OSCC). However, the effect of simultaneously evaluating T cell frequency and assessing suppressive elements and defects in antigen-processing machinery (APM) has not been clarified. Simultaneous characterization of CD3+, CD8+, FoxP3+, CD163+, and PD-L1+ cells using multispectral imaging was performed on sections from 119 patients with HPV– OSCC. Expression of β2-microglobulin, MHC class I heavy chain, and large multifunctional peptidase 10 was quantified, and all data were correlated with patient outcome. We found that, consistent with previous reports, high numbers of CD8+ T cells at the invasive margin correlated significantly with prolonged overall survival (OS), while the number of FoxP3+ or PD-L1+ cells did not. Compiling the number of FoxP3+ or PD-L1+ cells within 30 μm of CD8+ T cells identified a significant association with a high number of suppressive elements close to CD8+ T cells and reduced OS. Integrating this information into a cumulative suppression index (CSI) increased correlation with OS. Incorporating tumor expression levels of APM components with CSI further improved prognostic power. This multiparametric immune profiling may be useful for stratifying patients with OSCC for clinical trials.
Zipei Feng, Daniel Bethmann, Matthias Kappler, Carmen Ballesteros-Merino, Alexander Eckert, R. Bryan Bell, Allen Cheng, Tuan Bui, Rom Leidner, Walter J. Urba, Kent Johnson, Clifford Hoyt, Carlo B. Bifulco, Juergen Bukur, Claudia Wickenhauser, Barbara Seliger, Bernard A. Fox
The Mediator complex regulates gene transcription by linking basal transcriptional machinery with DNA-bound transcription factors. The activity of the Mediator complex is mainly controlled by a kinase submodule that is composed of 4 proteins, including MED12. Although ubiquitously expressed, Mediator subunits can differentially regulate gene expression in a tissue-specific manner. Here, we report that MED12 is required for normal cardiac function, such that mice with conditional cardiac-specific deletion of MED12 display progressive dilated cardiomyopathy. Loss of MED12 perturbs expression of calcium-handling genes in the heart, consequently altering calcium cycling in cardiomyocytes and disrupting cardiac electrical activity. We identified transcription factors that regulate expression of calcium-handling genes that are downregulated in the heart in the absence of MED12, and we found that MED12 localizes to transcription factor consensus sequences within calcium-handling genes. We showed that MED12 interacts with one such transcription factor, MEF2, in cardiomyocytes and that MED12 and MEF2 co-occupy promoters of calcium-handling genes. Furthermore, we demonstrated that MED12 enhances MEF2 transcriptional activity and that overexpression of both increases expression of calcium-handling genes in cardiomyocytes. Our data support a role for MED12 as a coordinator of transcription through MEF2 and other transcription factors. We conclude that MED12 is a regulator of a network of calcium-handling genes, consequently mediating contractility in the mammalian heart.
Kedryn K. Baskin, Catherine A. Makarewich, Susan M. DeLeon, Wenduo Ye, Beibei Chen, Nadine Beetz, Heinrich Schrewe, Rhonda Bassel-Duby, Eric N. Olson
Over the last several years, one of the major advances in the field of alcoholic liver disease research was the discovery that binge alcohol consumption induced neutrophilia and hepatic neutrophil infiltration in chronically ethanol-fed mice and human subjects with excessive alcohol use (EAU); however, the underlying mechanisms remain obscure. Here, we demonstrated that chronic EAU patients with a history of recent excessive drinking (EAU + RD) had higher serum levels of mitochondrial DNA (mtDNA)-enriched microparticles (MPs) than EAU without recent drinking (EAU – RD) and healthy controls, which correlated positively with circulating neutrophils. Similarly, mice with chronic-plus-binge (E10d + 1B) ethanol feeding also had markedly elevated serum levels of mtDNA-enriched MPs, with activation of hepatic ER stress and inflammatory responses. Inhibition of ER stress by gene KO or inhibitors attenuated ethanol-induced elevation of mtDNA-enriched MPs, neutrophilia, and liver injury. The data from the study of hepatocyte-specific deletion of the protein kinase RNA-like ER kinase (Perk) gene in mice and of cultured hepatocytes demonstrated that hepatocytes were the main source of mtDNA-enriched MPs after ethanol feeding. Finally, administration of mtDNA-enriched MPs isolated from E10d+1B-fed mice caused neutrophilia in mice. In conclusion, E10d + 1B ethanol consumption activates hepatic ER stress–dependent mtDNA-enriched MP release, leading to neutrophilia and liver injury.
Yan Cai, Ming-Jiang Xu, Erik H. Koritzinsky, Zhou Zhou, Wei Wang, Haixia Cao, Peter S.T. Yuen, Ruth A. Ross, Robert A. Star, Suthat Liangpunsakul, Bin Gao
Pediatric dilated cardiomyopathy (DCM) is the most common indication for heart transplantation in children. Despite similar genetic etiologies, medications routinely used in adult heart failure patients do not improve outcomes in the pediatric population. The mechanistic basis for these observations is unknown. We hypothesized that pediatric and adult DCM comprise distinct pathological entities, in that children do not undergo adverse remodeling, the target of adult heart failure therapies. To test this hypothesis, we examined LV specimens obtained from pediatric and adult donor controls and DCM patients. Consistent with the established pathophysiology of adult heart failure, adults with DCM displayed marked cardiomyocyte hypertrophy and myocardial fibrosis compared with donor controls. In contrast, pediatric DCM specimens demonstrated minimal cardiomyocyte hypertrophy and myocardial fibrosis compared with both age-matched controls and adults with DCM. Strikingly, RNA sequencing uncovered divergent gene expression profiles in pediatric and adult patients, including enrichment of transcripts associated with adverse remodeling and innate immune activation in adult DCM specimens. Collectively, these findings reveal that pediatric and adult DCM represent distinct pathological entities, provide a mechanistic basis to explain why children fail to respond to adult heart failure therapies, and suggest the need to develop new approaches for pediatric DCM.
Meghna D. Patel, Jayaram Mohan, Caralin Schneider, Geetika Bajpai, Enkhsaikhan Purevjav, Charles E. Canter, Jeffrey Towbin, Andrea Bredemeyer, Kory J. Lavine
Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.
Luke M. Judge, Juan A. Perez-Bermejo, Annie Truong, Alexandre J.S. Ribeiro, Jennie C. Yoo, Christina L. Jensen, Mohammad A. Mandegar, Nathaniel Huebsch, Robyn M. Kaake, Po-Lin So, Deepak Srivastava, Beth L. Pruitt, Nevan J. Krogan, Bruce R. Conklin
APOL1 variants in African populations mediate resistance to trypanosomal infection but increase risk for kidney diseases through unknown mechanisms. APOL1 is expressed in glomerular podocytes and does not vary with underlying kidney disease diagnoses or APOL1 genotypes, suggesting that the kidney disease–associated variants dysregulate its function rather than its localization or abundance. Structural homology searches identified vesicle-associated membrane protein 8 (VAMP8) as an APOL1 protein interactor. VAMP8 colocalizes with APOL1 in the podocyte, and the APOL1:VAMP8 interaction was confirmed biochemically and with surface plasmon resonance. APOL1 variants attenuate this interaction. Computational modeling of APOL1’s 3-dimensional structure, followed by molecular dynamics simulations, revealed increased motion of the C-terminal domain of reference APOL1 compared with either variant, suggesting that the variants stabilize a closed or autoinhibited state that diminishes protein interactions with VAMP8. Changes in ellipticity with increasing urea concentrations, as assessed by circular dichroism spectroscopy, showed higher conformational stability of the C-terminal helix of the variants compared with the reference protein. These results suggest that reference APOL1 interacts with VAMP8-coated vesicles, a process attenuated by variant-induced reduction in local dynamics of the C-terminal. Disordered vesicular trafficking in the podocyte may cause injury and progressive chronic kidney diseases in susceptible African Americans subjects.
Sethu M. Madhavan, John F. O’Toole, Martha Konieczkowski, Laura Barisoni, David B. Thomas, Santhi Ganesan, Leslie A. Bruggeman, Matthias Buck, John R. Sedor
Graft-versus-host disease (GVHD) induces pathological damage in peripheral target organs leading to well-characterized, organ-specific clinical manifestations. Patients with GVHD, however, can also have behavioral alterations that affect overall cognitive function, but the extent to which GVHD alters inflammatory and biochemical pathways in the brain remain poorly understood. In the current study, we employed complementary murine GVHD models to demonstrate that alloreactive donor T cells accumulate in the brain and affect a proinflammatory cytokine milieu that is associated with specific behavioral abnormalities. Host IL-6 was identified as a pivotal cytokine mediator, as was host indoleamine 2,3-dioxygenase (IDO-1), which was upregulated in GVHD in an IL-6–dependent manner in microglial cells and was accompanied by dysregulated tryptophan metabolism in the dorsal raphe nucleus and prefrontal cortex. Blockade of the IL-6 signaling pathway significantly reduced donor T cell accumulation, inflammatory cytokine gene expression, and host microglial cell expansion, but did not reverse GVHD-induced tryptophan metabolite dysregulation. Thus, these results indicate that inhibition of IL-6 signaling attenuates neuroinflammation, but does not reverse all of the metabolic abnormalities in the brain during GVHD, which may also have implications for the treatment of neurotoxicity occurring after other T cell–based immune therapies with IL-6–directed approaches.
Ludovic Belle, Vivian Zhou, Kara L. Stuhr, Margaret Beatka, Emily M. Siebers, Jennifer M. Knight, Michael W. Lawlor, Casey Weaver, Misato Hashizume, Cecilia J. Hillard, William R. Drobyski
Atherosclerosis is considered both a metabolic and inflammatory disease; however, the specific tissue and signaling molecules that instigate and propagate this disease remain unclear. The liver is a central site of inflammation and lipid metabolism that is critical for atherosclerosis, and JAK2 is a key mediator of inflammation and, more recently, of hepatic lipid metabolism. However, precise effects of hepatic Jak2 on atherosclerosis remain unknown. We show here that hepatic Jak2 deficiency in atherosclerosis-prone mouse models exhibited accelerated atherosclerosis with increased plaque macrophages and decreased plaque smooth muscle cell content. JAK2’s essential role in growth hormone signalling in liver that resulted in reduced IGF-1 with hepatic Jak2 deficiency played a causal role in exacerbating atherosclerosis. As such, restoring IGF-1 either pharmacologically or genetically attenuated atherosclerotic burden. Together, our data show hepatic Jak2 to play a protective role in atherogenesis through actions mediated by circulating IGF-1 and, to our knowledge, provide a novel liver-centric mechanism in atheroprotection.
Tharini Sivasubramaniyam, Stephanie A. Schroer, Angela Li, Cynthia T. Luk, Sally Yu Shi, Rickvinder Besla, David W. Dodington, Adam H. Metherel, Alex P. Kitson, Jara J. Brunt, Joshua Lopes, Kay-Uwe Wagner, Richard P. Bazinet, Michelle P. Bendeck, Clinton S. Robbins, Minna Woo
The role of negative regulators or suppressors of the damage-associated molecular pattern–mediated (DAMP-mediated) stimulation of innate immune responses is being increasingly appreciated. However, the presence and function of suppressors of DAMP-mediated effects on T cells, and whether they can be targeted to mitigate T cell–dependent immunopathology remain unknown. Sialic acid–binding immunoglobulin-like lectin G (Siglec-G) is a negative regulator of DAMP-mediated responses in innate immune cells, but its T cell–autonomous role is unknown. Utilizing loss-of-function–based (genetic knockout) and gain-of-function–based (agonist) approaches, we demonstrate that in the presence of certain DAMPs, Siglec-G suppressed in vitro and in vivo T cell responses. We also demonstrate that its T cell–autonomous role is critical for modulating the severity of the T cell–mediated immunopathology, graft-versus-host disease (GVHD). Enhancing the Siglec-G signaling in donor T cells with its agonist, a CD24Fc fusion protein, ameliorated GVHD while preserving sufficient graft-versus-tumor (GVT) effects in vivo. Collectively, these data demonstrate that Siglec-G is a potentially novel negative regulator of T cell responses, which can be targeted to mitigate GVHD.
Tomomi Toubai, Corinne Rossi, Katherine Oravecz-Wilson, Cynthia Zajac, Chen Liu, Thomas Braun, Hideaki Fujiwara, Julia Wu, Yaping Sun, Stuart Brabbs, Hiroya Tamaki, John Magenau, Pang Zheng, Yang Liu, Pavan Reddy
Our previous work showed myocellular differences in pediatric and adult dilated cardiomyopathy (DCM). However, a thorough characterization of the molecular pathways involved in pediatric DCM does not exist, limiting the development of age-specific therapies. To characterize this patient population, we investigated the transcriptome profile of pediatric patients. RNA-Seq from 7 DCM and 7 nonfailing (NF) explanted age-matched pediatric left ventricles (LV) was performed. Changes in gene expression were confirmed by real-time PCR (RT-PCR) in 36 DCM and 21 NF pediatric hearts and in 20 DCM and 10 NF adult hearts. The degree of myocyte hypertrophy was investigated in 4 DCM and 7 NF pediatric hearts and in 4 DCM and 9 NF adult hearts. Changes in gene expression in response to pluripotency-inducing factors were investigated in neonatal rat ventricular myocytes (NRVMs). Transcriptome analysis identified a gene expression profile in children compared with adults with DCM. Additionally, myocyte hypertrophy was not observed in pediatric hearts but was present in adult hearts. Furthermore, treatment of NRVMs with pluripotency-inducing factors recapitulated changes in gene expression observed in the pediatric DCM heart. Pediatric DCM is characterized by unique changes in gene expression that suggest maintenance of an undifferentiated state.
Philip D. Tatman, Kathleen C. Woulfe, Anis Karimpour-Fard, Danielle A. Jeffrey, James Jaggers, Joseph C. Cleveland, Karin Nunley, Matthew R.G. Taylor, Shelley D. Miyamoto, Brian L. Stauffer, Carmen C. Sucharov
No posts were found with this tag.