Neuroinflammation is a recognized complication of immunotherapeutic approaches such as immune checkpoint inhibitor treatment, chimeric antigen receptor therapy, and graft versus host disease (GVHD) occurring after allogeneic hematopoietic stem cell transplantation. While T cells and inflammatory cytokines play a role in this process, the precise interplay between the adaptive and innate arms of the immune system that propagates inflammation in the central nervous system remains incompletely understood. Using a murine model of GVHD, we demonstrate that type 2 cannabinoid receptor (CB2R) signaling plays a critical role in the pathophysiology of neuroinflammation. In these studies, we identify that CB2R expression on microglial cells induces an activated inflammatory phenotype which potentiates the accumulation of donor-derived proinflammatory T cells, regulates chemokine gene regulatory networks, and promotes neuronal cell death. Pharmacological targeting of this receptor with a brain penetrant CB2R inverse agonist/antagonist selectively reduces neuroinflammation without deleteriously affecting systemic GVHD severity. Thus, these findings delineate a therapeutically targetable neuroinflammatory pathway and has implications for the attenuation of neurotoxicity after GVHD and potentially other T cell-based immunotherapeutic approaches.
Alison Moe, Aditya Rayasam, Garrett Sauber, Ravi K. Shah, Ashley Doherty, Cheng-Yin Yuan, Aniko Szabo, Bob M. Moore II, Marco Colonna, Weiguo Cui, Julian Romero, Anthony E. Zamora, Cecilia J. Hillard, William R. Drobyski
Widespread alterations in RNA alternative splicing (AS) have been identified in adult gliomas. However, their regulatory mechanism, biological significance, and therapeutic potential remain largely elusive. Here, using a computational approach with both bulk and single cell RNA-sequencing, we uncover a prognostic AS signature linked with neural developmental hierarchies. Using advanced iPSC glioma models driven by glioma driver mutations, we show that this AS signature could be enhanced by EGFRvIII and inhibited by in situ IDH1 mutation. Functional validation of two isoform switching events in CERS5 and MPZL1 shows regulations of sphingolipid metabolism and SHP2 signaling, respectively. Analysis of upstream RNA binding proteins reveals PTBP1 as a key regulator of the AS signature where targeting of PTBP1 suppresses tumor growth and promotes the expression of a neuron marker TUJ1 in glioma stem-like cells. Overall, our data highlights the role of AS in impacting glioma malignance and heterogeneity and its potential as a therapeutic vulnerability for treating adult gliomas.
Xiao Song, Deanna Tiek, Shunichiro Miki, Tianzhi Huang, Minghui Lu, Anshika Goenka, Rebeca P. Iglesia, Xiaozhou Yu, Runxin Wu, Maya N. Walker, Chang Zeng, Hardik Shah, Shao Huan Samuel Weng, Allen Huff, Wei Zhang, Tomoyuki Koga, Christopher G. Hubert, Craig M. Horbinski, Frank F. Furnari, Bo Hu, Shi-Yuan Cheng
Cancer cells exhibit heightened secretory states that drive tumor progression. Here, we identify a chromosome 3q amplicon that serves as a platform for secretory regulation in cancer. The 3q amplicon encodes multiple Golgi-resident proteins, including the scaffold Golgi integral membrane protein 4 (GOLIM4) and the ion channel ATPase Secretory Pathway Ca2+ Transporting 1 (ATP2C1). We show that GOLIM4 recruits ATP2C1 and Golgi phosphoprotein 3 (GOLPH3) to coordinate calcium-dependent cargo loading and Golgi membrane bending and vesicle scission. GOLIM4 depletion disrupts the protein complex, resulting in a secretory blockade that inhibits the progression of 3q-amplified malignancies. In addition to its role as a scaffold, GOLIM4 maintains intracellular manganese (Mn) homeostasis by binding excess Mn in the Golgi lumen, which initiates the routing of Mn-bound GOLIM4 to lysosomes for degradation. We show that Mn treatment inhibits the progression of multiple types of 3q-amplified malignancies by degrading GOLIM4, resulting in a secretory blockade that interrupts pro-survival autocrine loops and attenuates pro-metastatic processes in the tumor microenvironment. Potentially underlying the selective activity of Mn against 3q-amplified malignancies, ATP2C1 co-amplification increases Mn influx into the Golgi lumen, resulting in a more rapid degradation of GOLIM4. These findings show that functional cooperativity between co-amplified genes underlies heightened secretion and a targetable secretory addiction in 3q-amplified malignancies.
Xiaochao Tan, Shike Wang, Guan-Yu Xiao, Chao Wu, Xin Liu, Biyao Zhou, Jiang Yu, Dzifa Yawa Duose, Yuanxin Xi, Jing Wang, Kunika Gupta, Apar Pataer, Jack A. Roth, Michael P. Kim, Fengju Chen, Chad J. Creighton, William K. Russell, Jonathan M. Kurie
CD8+ T cell dysfunction impedes anti-tumor immunity in solid cancers but the underlying mechanisms are diverse and poorly understood. Extracellular matrix (ECM) composition has been linked to impaired T cell migration and enhanced tumor progression; however, impacts of individual ECM molecules on T cell function in the tumor microenvironment (TME) are only beginning to be elucidated. Upstream regulators of aberrant ECM deposition and organization in solid tumors are equally ill-defined. Therefore, we investigated how ECM composition modulates CD8+ T cell function in undifferentiated pleomorphic sarcoma (UPS), an immunologically active desmoplastic tumor. Using an autochthonous murine model of UPS and data from multiple human patient cohorts, we discovered a multifaceted mechanism wherein the transcriptional co-activator YAP1 promotes collagen VI (COLVI) deposition in the UPS TME. In turn, COLVI induces CD8+ T cell dysfunction and immune evasion by remodeling fibrillar collagen and inhibiting T cell autophagic flux. Unexpectedly, collagen I (COLI) opposed COLVI in this setting, promoting CD8+ T cell function and acting as a tumor suppressor. Thus, CD8+ T cell responses in sarcoma depend upon oncogene-mediated ECM composition and remodeling.
Ashley M. Fuller, Hawley C. Pruitt, Ying Liu, Valerie M. Irizarry-Negron, Hehai Pan, Hoogeun Song, Ann DeVine, Rohan S. Katti, Samir Devalaraja, Gabrielle E. Ciotti, Michael V. Gonzalez, Erik F. Williams, Ileana Murazzi, Dimitris Ntekoumes, Nicolas Skuli, Hakon Hakonarson, Daniel J. Zabransky, Jose G. Trevino, Ashani Weeraratna, Kristy Weber, Malay Haldar, Joseph A. Fraietta, Sharon Gerecht, T.S. Karin Eisinger-Mathason
While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids carrying retinal disease-causing variants in NR2E3, as well as isogenic and unrelated controls. Organoids were sampled using single-cell RNA sequencing across the developmental window encompassing photoreceptor specification, emergence, and maturation. Using scRNAseq data, we reconstruct the rod photoreceptor developmental lineage and identify a branchpoint unique to the disease state. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes. Using joint multimodal single-cell sequencing, we further identify putative regulatory sites where rod-specific factors act to steer photoreceptor cell development. Finally, we show that rod-committed photoreceptor cells form and persist throughout life in a patient with NR2E3-associated disease. Importantly, these findings are strikingly different than those observed in Nr2e3 rodent models. Together, these data provide a roadmap of human photoreceptor development and leverage patient iPSCs to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation in health and disease.
Nathaniel K. Mullin, Laura R. Bohrer, Andrew P. Voigt, Lola P. Lozano, Allison T. Wright, Vera L. Bonilha, Robert F. Mullins, Edwin M. Stone, Budd A. Tucker
The immune system can control cancer progression. However, even though some innate immune sensors of cellular stress are expressed intrinsically in epithelial cells, their potential role in cancer aggressiveness and subsequent overall survival in humans is mainly unknown. Here, we show that NLR family CARD Domain Containing 4 (NLRC4) is downregulated in epithelial tumor cells of colorectal cancer (CRC) patients by using spatial tissue imaging. Strikingly, only the loss of tumor NLRC4 but not stromal is associated with poor immune infiltration (mainly dendritic and CD4+/CD8+ T cells) and accurately predicts progression to metastatic Stage IV and decrease of overall survival. By combining multi-omics approaches, we show that restoring NLRC4 expression in human colorectal cancer cells triggers a broad inflammasome-independent immune reprogramming consisting of Type-I IFN signaling genes and the release of chemokines and myeloid growth factors involved in the tumor infiltration and activation of dendritic cells (DCs) and T cells. Consistently, such reprogramming in cancer cells is sufficient to directly mature human DCs towards a Th1 antitumor immune response through IL-12 production in vitro. In multiple human carcinomas (colorectal, lung, and skin), we confirmed that NLRC4 expression in patient tumors is strongly associated with Type-I IFN genes, immune infiltrates and high microsatellite instability. Thus, we shed light on the epithelial innate immune sensor NLRC4 as a novel therapeutic target to promote an efficient antitumor immune response against the aggressiveness of various carcinomas.
Charlotte Domblides, Steven Crampton, Hong Liu, Juliet M. Bartleson, Annie Nguyen, Claudia Champagne, Emily E. Landy, Lindsey Spiker, Christopher Proffitt, Sunil Bhattarai, Anissa P. Grawe, Matias Fuentealba Valenzuela, Lydia Lartigue, Isabelle Mahouche, Jeremy Dupaul-Chicoine, Kazuho Nishimura, Félix Lefort, Marie Decraecker, Valérie Velasco, Sonia Netzer, Vincent Pitard, Christian Roy, Isabelle Soubeyran, Victor Racine, Patrick Blanco, Julie Déchanet-Merville, Maya Saleh, Scott W. Canna, David Furman, Benjamin Faustin
Molecular profiling of clear cell RCC (ccRCC) tumors of clinical trial patients has identified distinct transcriptomic signatures with predictive value, yet data in non-clear cell variants (nccRCC) are lacking. We examined the transcriptional profiles of RCC tumors representing key molecular pathways, from a multi-institutional, real-world patient cohort, including ccRCC (n = 508) and centrally-reviewed nccRCC (n = 149) samples. ccRCC had increased angiogenesis signature scores compared to the heterogeneous group of nccRCC tumors (mean z-score 0.37 vs –0.99, P < 0.001), while cell cycle, fatty acid oxidation (FAO)/AMPK signaling, fatty acid synthesis (FAS)/pentose phosphate signature scores were increased in one or more nccRCC subtypes. Among both ccRCC and nccRCC tumors, T-effector scores statistically correlated with increased immune cell infiltration and were more commonly associated with immunotherapy-related markers (PD-L1+/TMB-High/MSI-High). In conclusion, this study provides evidence of differential gene transcriptional profiles among ccRCC vs nccRCC tumors, providing new insights for optimizing personalized and histology-specific therapeutic strategies for patients with advanced RCC.
Pedro Barata, Shuchi Gulati, Andrew Elliott, Hans J. Hammers, Earle F. Burgess, Benjamin A. Gartrell, Sourat Darabi, Mehmet A. Bilen, Arnab Basu, Daniel M. Geynisman, Nancy A. Dawson, Matthew R. Zibelman, Tian Zhang, Shuanzeng Wei, Charles J. Ryan, Elisabeth I. Heath, Kelsey A. Poorman, Chadi Nabhan, Rana R. McKay
BACKGROUND. Early antiretroviral therapy initiation (ARTi) in HIV-1 restricts reservoir size and diversity while preserving immune function, potentially improving opportunities for immunotherapeutic cure strategies. For antibody-based cure approaches, the development of autologous neutralizing antibodies (anAb) after acute/early ARTi is relevant, but poorly understood. METHODS. We characterize antibody responses in a cohort of 23 participants following ARTi in acute HIV (<60 days after infection) and early HIV (60-128 days after infection). RESULTS. Plasma virus sequences at the time of ARTi revealed evidence of escape from anAbs after early, but not acute, ARTi. HIV-1 Envs representing the transmitted/founder virus(es) (acute ARTi) or escape variants (early ARTi) were tested for sensitivity to longitudinal plasma IgG. After acute ARTi, no anAb responses developed over months to years of suppressive ART. In two of the three acute ARTi participants who experienced viremia after ARTi, however, anAbs arose shortly thereafter. After early ARTi, anAbs targeting those early variants developed between 12 and 42 weeks of ART and continued to increase in breadth and potency thereafter. CONCLUSIONS. Results indicate a threshold of virus replication (~60 days) required to induce anAbs, after which they continue to expand on suppressive ART to better target the range of reservoir variants. TRIAL REGISTRATION. NCT02656511 FUNDING. National Institutes of Health grants U01AI169767; R01AI162646; UM1AI164570; UM1AI164560; U19AI096109; K23GM112526; T32AI118684, P30-AI-045008, P30 AI027763, R24 AI067039. Gilead Sciences grant INUS2361354; Viiv healthcare grant A126326.
Gregory D. Whitehill, Jaimy Joy, Francesco E. Marino, Ryan J. Krause, Suvadip Mallick, Hunter M. Courtney, Kyewon Park, John W. Carey, Rebecca Hoh, Heather Hartig, Vivian Pae, Sannidhi Sarvadhavabhatla, Maria Sophia B. Donaire, Steven G. Deeks, Rebecca M. Lynch, Sulggi A. Lee, Katharine J. Bar
BACKGROUND. Features of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies contribute to a procoagulant state in CM. METHODS. Plasma from children with uncomplicated malaria (UM, n = 124) and CM (n = 136) was analyzed by ELISA for a panel of 8 autoantibodies including anti-Platelet Factor 4/polyanion (anti-PF4/P), anti-Phospholipid, anti-Phosphatidylserine, anti-Myeloperoxidase, anti-Proteinase 3, anti-dsDNA, anti-Beta-2-Glycoprotein I (β2GPI), and anti-Cardiolipin. Non-malaria coma (NMC, n = 49) and healthy controls (HC, n = 56) were assayed for comparison. Associations with clinical and immune biomarkers were determined using univariate and logistic regression analyses. RESULTS. Median anti-PF4/P and anti-PS IgG levels were elevated with malaria infection relative to HC (P < 0.001) and NMC (PF4/P: P < 0.001). Anti-PF4/P IgG levels were elevated in CM (median = 0.27, IQR: 0.19–0.41) compared to UM (median = 0.19, IQR: 0.14–0.22, P ≤ 0.0001). Anti-PS IgG levels did not differ between UM and CM (P = 0.39). When CM cases were stratified by malaria retinopathy (Ret) status, levels of anti-PF4/P IgG correlated negatively with peripheral platelet count in Ret+ CM cases (Rs = 0.201, P = 0.04) and associated positively with mortality (OR = 15.2, 95% CI: 1.02–275, P = 0.048). Plasma from CM patients induced a greater platelet activation capacity in an ex-vivo assay relative to plasma from UM patients (P = 0.02). Platelet activation was associated with anti-PF4/P IgG levels (Rs = 0.293, P = 0.035). CONCLUSIONS. Thrombosis mediated by elevated anti-PF4/P autoantibodies may be one mechanism contributing to the clinical complications of CM.
Iset M. Vera, Anne Kessler, Visopo Harawa, Ajisa Ahmadu, Thomas E. Keller, Stephen T.J. Ray, Terrie E. Taylor, Stephen J. Rogerson, Wilson L. Mandala, Morayma Reyes Gil, Karl B. Seydel, Kami Kim
Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation (OXPHOS). Metabolic inflexibility, defined as an impaired ability to switch between these fuels, is implicated in a number of metabolic diseases. Here we explore the mechanism by which physical inactivity promotes metabolic inflexibility in skeletal muscle. We developed a mouse model of sedentariness, small mouse cage (SMC) that, unlike other classic models of disuse in mice, faithfully recapitulated metabolic responses that occur in humans. Bioenergetic phenotyping of skeletal muscle mitochondria displayed metabolic inflexibility induced by physical inactivity, demonstrated by a reduction in pyruvate-stimulated respiration (JO2) in absence of a change in palmitate-stimulated JO2. Pyruvate resistance in these mitochondria was likely driven by a decrease in phosphatidylethanolamine (PE) abundance in the mitochondrial membrane. Reduction in mitochondrial PE by heterozygous deletion of phosphatidylserine decarboxylase (PSD) was sufficient to induce metabolic inflexibility measured at the whole-body level, as well as at the level of skeletal muscle mitochondria. Low mitochondrial PE in C2C12 myotubes was sufficient to increase glucose flux towards lactate. We further implicate that resistance to pyruvate metabolism is due to attenuated mitochondrial entry via mitochondrial pyruvate carrier (MPC). These findings suggest a mechanism by which mitochondrial PE directly regulates MPC activity to modulate metabolic flexibility in mice.
Piyarat Siripoksup, Guoshen Cao, Ahmad A. Cluntun, J. Alan Maschek, Quentinn Pearce, Marisa J. Lang, Mi-Young Jeong, Hiroaki Eshima, Patrick J. Ferrara, Precious C. Opurum, Ziad S. Mahmassani, Alek D. Peterlin, Shinya Watanabe, Maureen A. Walsh, Eric B. Taylor, James E. Cox, Micah J. Drummond, Jared Rutter, Katsuhiko Funai
Renal interstitial fibrosis is an important mechanism in the progression of chronic kidney disease (CKD) to end-stage kidney disease. However, we lack specific treatments to slow or halt renal fibrosis. Ribosome profiling identified upregulation of a secreted micropeptide, C4orf48 (Cf48), in mouse diabetic nephropathy. Cf48 RNA and protein levels were upregulated in tubular epithelial cells in human and experimental CKD. Serum Cf48 levels were increased in human CKD and correlated with loss of kidney function, increasing CKD stage, and the degree of active interstitial fibrosis. Cf48 overexpression in mice accelerated renal fibrosis, while Cf48 gene deletion or knockdown by antisense oligonucleotides significantly reduced renal fibrosis in CKD models. In vitro, recombinant Cf48 (rCf48) enhanced TGF-β1-induced fibrotic responses in renal fibroblasts and epithelial cells independent of Smad3 phosphorylation. Cellular uptake of Cf48 and its pro-fibrotic response in fibroblasts operated via the transferrin receptor. RNA immunoprecipitation-sequencing identified Cf48 binding to mRNA of genes involved in the fibrotic response, including Serpine1, Acta2, Ccn2, and Col4a1. rCf48 binds to the 3′-untranslated region of Serpine1 and increases mRNA half-life. We identify the secreted Cf48 micropeptide as a potential enhancer of renal fibrosis which operates as an RNA-binding peptide to promote the production of extracellular matrix.
Jiayi Yang, Hongjie Zhuang, Jinhua Li, Ana B. Nunez-Nescolarde, Ning Luo, Huiting Chen, Andy Li, Xinli Qu, Qing Wang, Jinjin Fan, Xiaoyan Bai, Zhiming Ye, Bing Gu, Yue Meng, Xingyuan Zhang, Di Wu, Youyang Sia, Xiaoyun Jiang, Wei Chen, Alexander N. Combes, David J. Nikolic-Paterson, Xueqing Yu
Just as the androgen receptor (AR), the estrogen receptor α (ERα) is expressed in the prostate and is thought to influence prostate cancer (PCa) biology. Yet, the incomplete understanding of ERα functions in PCa hinders our ability to fully comprehend its clinical relevance and restricts the repurposing of estrogen-targeted therapies for the treatment of this disease. Using two human PCa tissue microarray cohorts, we first demonstrated that nuclear ERα expression was heterogeneous among patients, being only detected in half of tumors. Positive nuclear ERα levels were correlated with disease recurrence, progression to metastatic PCa, and patient survival. Using in vitro and in vivo models of the normal prostate and PCa, bulk and single-cell RNA-Seq analyses revealed that estrogens partially mimic the androgen transcriptional response and induce specific biological pathways linked to proliferation and metabolism. Bioenergetic flux assays and metabolomics confirmed the regulation of cancer metabolism by estrogens, supporting proliferation. Using cancer cell lines and patient-derived organoids, selective estrogen receptor modulators, a pure anti-estrogen, and genetic approaches impaired cancer cell proliferation and growth in an ERα-dependent manner. Overall, our study revealed that, when expressed, ERα functionally reprograms PCa metabolism, is associated with disease progression, and could be targeted for therapeutic purposes.
Camille Lafront, Lucas Germain, Gabriel H. Campolina-Silva, Cindy Weidmann, Line Berthiaume, Hélène Hovington, Hervé Brisson, Cynthia Jobin, Lilianne Frégeau-Proulx, Raul Cotau, Kevin Gonthier, Aurélie Lacouture, Patrick Caron, Claire Ménard, Chantal Atallah, Julie Riopel, Éva Latulippe, Alain Bergeron, Paul Toren, Chantal Guillemette, Martin Pelletier, Yves Fradet, Clémence Belleannée, Frédéric Pouliot, Louis Lacombe, Éric Lévesque, Étienne Audet-Walsh
Tissue regeneration is limited in several organs including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest existing remodeling capacity. This study uncovered endogenous tissue remodeling mechanisms in the kidney that were activated by the loss of body fluid and salt and regulated by a unique niche of a minority renal cell type called the macula densa (MD). Here we identified neuronal differentiation features of MD cells that sense the local and systemic environment, secrete angiogenic, growth and extracellular matrix remodeling factors, cytokines and chemokines, and control resident progenitor cells. Serial intravital imaging, MD nerve growth factor receptor and Wnt mouse models and transcriptome analysis revealed cellular and molecular mechanisms of these MD functions. Human and therapeutic translation studies illustrated the clinical potential of MD factors including CCN1 as a urinary biomarker and therapeutic target in chronic kidney disease. The concept that a neuronally differentiated key sensory and regulatory cell type responding to organ-specific physiological inputs controls local progenitors to remodel or repair tissues may be applicable to other organs and diverse tissue regenerative therapeutic strategies.
Georgina Gyarmati, Urvi Nikhil Shroff, Anne Riquier-Brison, Dorinne Desposito, Wenjun Ju, Sean D. Stocker, Audrey Izuhara, Sachin Deepak, Alejandra Becerra Calderon, James L. Burford, Hiroyuki Kadoya, Ju-Young Moon, Yibu Chen, Markus M. Rinschen, Nariman Ahmadi, Lester Lau, Daniel Biemesderfer, Aaron W. James, Liliana Minichiello, Berislav Zlokovic, Inderbir S. Gill, Matthias Kretzler, János Peti-Peterdi
Individuals with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of aging related health conditions and all-cause mortality, but whether CHIP impacts risk of infection is much less clear. Using UK Biobank data, we revealed a positive association between CHIP and incident pneumonia in 438,421 individuals. We show that inflammation enhanced pneumonia risk, as CHIP carriers with a hypomorphic IL6 receptor polymorphism were protected. To better characterize the pathways of susceptibility, we challenged hematopoietic Tet Methylcytosine Dioxygenase 2 knockout (Tet2–/–) and floxed control mice (Tet2f/f) with Streptococcus pneumoniae. As with human CHIP carriers, Tet2–/– mice had hematopoietic abnormalities resulting in the expansion of inflammatory monocytes and neutrophils in peripheral blood. Yet, these cells were insufficient in defending against S. pneumoniae and resulted in increased pathology, impaired bacterial clearance, and higher mortality in Tet2–/– mice. We delineated the transcriptional landscape of Tet2–/– neutrophils and found that while inflammation-related pathways were upregulated in Tet2–/– neutrophils, migration and motility pathways were compromised. Using live-imaging techniques, we demonstrated impairments in motility, pathogen uptake and neutrophil extracellular trap (NET) formation by Tet2–/– neutrophils. Collectively, we show that CHIP is a risk factor for bacterial pneumonia related to innate immune impairments.
Candice Quin, Erica N. DeJong, Elina K. Cook, Yi Zhen Luo, Caitlyn Vlasschaert, Sanathan Sadh, Amy J.M. McNaughton, Marco M. Buttigieg, Jessica A Breznik, Allison E. Kennedy, Kevin Zhao, Jeffrey Mewburn, Kimberly J. Dunham-Snary, Charles C.T. Hindmarch, Alexander G. Bick, Stephen L. Archer, Michael J. Rauh, Dawn M.E. Bowdish
BACKGROUND. The molecular signature of pediatric acute respiratory distress syndrome (ARDS) is poorly described, and the degree to which hyperinflammation or specific tissue injury contributes to outcomes is unknown. Therefore, we profiled inflammation and tissue injury dynamics over the first 7 days of ARDS, and associated specific biomarkers with mortality, persistent ARDS, and persistent multiple organ dysfunction syndrome (MODS). METHODS. In a single-center prospective cohort of intubated pediatric ARDS, we collected plasma on days 0, 3, and 7. Nineteen biomarkers reflecting inflammation, tissue injury, and damage associated molecular patterns were measured. We assessed the relationship between biomarkers and trajectories with mortality, persistent ARDS, or persistent MODS using multivariable mixed effect models. RESULTS. In 279 subjects (64 [23%] non-survivors), hyperinflammatory cytokines, tissue injury markers, and DAMPs were higher in non-survivors. Survivors and non-survivors showed different biomarker trajectories. IL-1α, sTNFR1, ANG2, and SPD increased in non-survivors, while DAMPs remained persistently elevated. ANG2 and P3NP were associated with persistent ARDS, whereas multiple cytokines, tissue injury markers, and DAMPs were associated with persistent MODS. Corticosteroid use did not impact the association of biomarker levels or trajectory with mortality. CONCLUSIONS. Pediatric ARDS survivors and non-survivors had distinct biomarker trajectories, with cytokines, endothelial and alveolar epithelial injury, and DAMPs elevated in non-survivors. Mortality markers overlapped with markers associated with persistent MODS, rather than persistent ARDS.
Nadir Yehya, Thomas J. Booth, Gnana D. Ardhanari, Jill M. Thompson, L.K. Metthew Lam, Jacob E. Till, Mark V. Mai, Garrett Keim, Daniel J. McKeone, E. Scott Halstead, Patrick Lahni, Brian M. Varisco, Wanding Zhou, Erica L. Carpenter, Jason D. Christie, Nilam S. Mangalmurti
Cancer-derived small extracellular vesicles (sEVs) are capable of modifying tumor microenvironment and promoting tumor progression. Ovarian cancer (OvCa) is a lethal malignancy that preferentially spreads through the abdominal cavity. Thus, the secretion of such vesicles into the peritoneal fluid could be a determinant factor in the dissemination and behavior of this disease. We designed a prospective observational study to assess the impact of peritoneal fluid-derived sEVs (PFD-sEVs) in OvCa clinical outcome. For this purpose, two patient cohorts were enrolled, including OvCa cases who underwent a diagnostic or cytoreductive surgery, and non-oncological patients as controls, who underwent abdominal surgery for benign gynecological conditions. PFD-sEVs systematic extraction from surgical samples enabled us to observe significant quantitative and qualitative differences associated with cancer diagnosis, disease stage and platinum chemosensitivity. Proteomic profiling of PFD-sEVs led to the identification of molecular pathways and proteins of interest and to the biological validation of S100A4 and STX5. In addition, unsupervised analysis of PFD-sEVs proteomic profiles in high-grade serous ovarian carcinomas (HGSOC) revealed two clusters with different outcomes in terms of overall survival. In conclusion, comprehensive characterization of the PFD-sEVs content provided a prognostic value with potential implications in HGSOC clinical management.
Miguel Quiralte, Arantzazu Barquín, Mónica Yagüe Fernández, Paloma Navarro, Tatiana P. Grazioso, Elena Sevillano, Juan F. Rodriguez Moreno, Alejandra Balarezo-Saldivar, Héctor Peinado, Elena Izquierdo, Carlos Millán, Irene López Carrasco, Mario Prieto, Rodrigo Madurga de Lacalle, Ismael Fernández-Miranda, Sergio Ruiz-Llorente, Jesús García-Donas
Nuclear factor kappa-B (NFκB) is activated in arrhythmogenic cardiomyopathy (ACM) patient-derived iPSC-cardiac myocytes under basal conditions and inhibition of NFκB signaling prevents disease in Dsg2mut/mut mice, a robust mouse model of ACM. Here, we used genetic approaches and single cell RNA sequencing to define the contributions of immune signaling in cardiac myocytes and macrophages in the natural progression of ACM using Dsg2mut/mut mice. We found that NFκB signaling in cardiac myocytes drives myocardial injury, contractile dysfunction, and arrhythmias in Dsg2mut/mut mice. NFκB signaling in cardiac myocytes mobilizes macrophages expressing C-C motif chemokine receptor-2 (CCR2+ cells) to affected areas within the heart, where they mediate myocardial injury and arrhythmias. Contractile dysfunction in Dsg2mut/mut mice is caused both by loss of heart muscle and negative inotropic effects of inflammation in viable muscle. Single nucleus RNA sequencing and cellular indexing of transcriptomes and epitomes (CITE-seq) studies revealed marked pro-inflammatory changes in gene expression and the cellular landscape in hearts of Dsg2mut/mut mice involving cardiac myocytes, fibroblasts and CCR2+ macrophages. Changes in gene expression in cardiac myocytes and fibroblasts in Dsg2mut/mut mice were dependent on CCR2+ macrophage recruitment to the heart. These results highlight complex mechanisms of immune injury and regulatory crosstalk between cardiac myocytes, inflammatory cells and fibroblasts in the pathogenesis of ACM.
Stephen P. Chelko, Vinay R. Penna, Morgan Engel, Emily A. Shiel, Ann M. Centner, Waleed Farra, Elisa N. Cannon, Maicon Landim-Vieira, Niccole Schaible, Kory Lavine, Jeffrey E. Saffitz
Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppresses the growth of CRPC xenografts but also induces tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplifies and modulates the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affects stromal gene expression, indicating that Mediator kinase activity in CRPC molds the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulates the MYC pathway, and Mediator kinase inhibition suppresses a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors show efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlates with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediate androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.
Jing Li, Thomas A. Hilimire, Liu Yueying, Lili Wang, Jiaxin Liang, Balázs Győrffy, Vitali Sikirzhytski, Hao Ji, Li Zhang, Chen Cheng, Xiaokai Ding, Kendall R. Kerr, Charles E. Dowling, Alexander A. Chumanevich, Zachary T. Mack, Gary P. Schools, Chang-uk Lim, Leigh Ellis, Xiaolin Zi, Donald C. Porter, Eugenia V. Broude, Campbell McInnes, George Wilding, Michael B. Lilly, Igor B. Roninson, Mengqian Chen
Aberrant expression of ETS transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS::FLI1 and EWS::ERG fusions in Ewing sarcoma (EwS) and TMPRSS2::ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS::FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs) and are highly elevated in plasma of EwS patients with metastatic disease. High HSAT2,3 levels in EWS::FLI1 or ERG expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs which transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.
Peter Ruzanov, Valentina Evdokimova, Manideep C. Pachva, Alon Minkovich, Zhenbo Zhang, Sofya Langman, Hendrik Gassmann, Uwe Thiel, Marija Orlic-Milacic, Syed H. Zaidi, Vanya Peltekova, Lawrence E. Heisler, Manju Sharma, Michael E. Cox, Trevor D. McKee, Mark Zaidi, Eve Lapouble, John D. McPherson, Olivier Delattre, Laszlo Radvanyi, Stefan E.G. Burdach, Lincoln D. Stein, Poul H. Sorensen
G protein-coupled receptor 37-like 1 (GPR37L1) is an orphan GPCR with largely unknown functions. Here we report that Gpr37l1/GRP37L1 ranks among the most highly expressed GPCR transcripts in mouse and human dorsal root ganglia (DRGs), selectively expressed in satellite glial cells (SGCs). Peripheral neuropathy induced by streptozotoxin (STZ) and paclitaxel (PTX) led to reduced GPR37L1 expression on the plasma membrane expression in mouse and human DRGs. Transgenic mice with Gpr37l1 deficiency exhibited impaired resolution of neuropathic pain symptoms following PTX and STZ-induced pain, whereas overexpression of Gpr37l1 in mouse DRGs reversed pain. GPR37L1 is co-expressed with potassium channels, including KCNJ10 (Kir4.1) in mouse SGCs and both KCNJ3 (Kir3.1) and KCNJ10 in human SGCs. GPR37L1 regulates the surface expression and function of the potassium channels. Notably, the pro-resolving lipid mediator maresin 1 (MaR1) serves as a ligand of GPR37L1 and enhances KCNJ10 or KCNJ3-mediated potassium influx in SGCs through GPR37L1. Chemotherapy suppressed KCNJ10 expression and function in SGCs, which MaR1 rescued through GPR37L1. Finally, genetic analysis revealed that the GPR37L1-E296K variant increased chronic pain risk by destabilizing the protein and impairing the protein’s function. Thus, GPR37L1 in SGCs offers a new therapeutic target for the protection of neuropathy and chronic pain.
Sangsu Bang, Changyu Jiang, Jing Xu, Sharat Chandra, Aidan McGinnis, Xin Luo, Qianru He, Yize Li, Zilong Wang, Xiang Ao, Marc Parisien, Lorenna Oliveira Fernandes de Araujo, Sahel Jahangiri Esfahani, Qin Zhang, Raquel Tonello, Temugin Berta, Luda Diatchenko, Ru-Rong Ji