Invariant natural killer T (iNKT) cells are implicated in viral clearance, however their role in HCV infection remains controversial. Here, iNKT cells were studied during different stages of HCV infection. iNKT cells from patients with acute HCV infection and people who inject drugs (PWID) with chronic or spontaneously resolved HCV infection were characterized by flow cytometry. In a longitudinal analysis during acute HCV infection, frequencies of activated CD38+ iNKT cells reproducibly declined in spontaneously resolving patients whereas they were persistently elevated in patients progressing to chronic infection. During the first year of infection, the frequency of activated CD38+ or CD69+ iNKT cells strongly correlated with alanine transaminase levels with particularly pronounced correlations in spontaneously resolving patients. Increased frequencies of activated iNKT cells in chronic HCV infection were confirmed in cross-sectional analyses of PWID with chronic or spontaneously resolved HCV infection, however, no apparent functional differences were observed with various stimulation protocols. Our data suggest that iNKT cells are activated during acute hepatitis C and that activation is sustained in chronic infection. The correlation between the frequency of activated iNKT cells and ALT may point towards a role of iNKT cells in liver damage.
Tina Senff, Christopher Menne, Christine Cosmovici, Lia Laura Lewis-Ximenez, Jasneet Aneja, Ruth Broering, Arthur Y. Kim, Astrid M. Westendorf, Ulf Dittmer, Norbert Scherbaum, Georg M. Lauer, Jörg Timm
Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, yet the underlying genetic mechanisms remain poorly understood. Here, we performed whole genome sequencing analysis on 146 non-syndromic TOF parent-offspring trios of Chinese ethnicity. Comparison of de novo variants and recessive genotypes of this dataset to a European cohort identified both overlapping and novel gene loci, and revealed differential functional enrichment between cohorts. To assess the impact of these mutations on early cardiac development, we integrated single-cell and spatial transcriptomics of early human heart development with our genetic findings. We discovered that the candidate gene expression was enriched in the myogenic progenitors of the cardiac outflow tract. Moreover, subsets of the candidate genes were found in specific gene co-expression modules along cardiomyocyte differentiation trajectory. These integrative functional analyses help dissect the pathogenesis of TOF, revealing cellular hotspots in early heart development resulting in cardiac malformations.
Clara Sze Man Tang, Mimmi Mononen, Wai-Yee Lam, Sheng Chih Jin, Xuehan Zhuang, Maria-Mercè Garcia-Barcelo, Qiongfen Lin, Yujia Yang, Makoto Sahara, Elif Eroglu, Kenneth R. Chien, Haifa Hong, Paul K.H. Tam, Peter J. Gruber
Acute cardiac injury is prevalent in critical COVID-19 and associated with increased mortality. Its etiology remains debated, as initially presumed causes--- myocarditis and cardiac necrosis--- have proven uncommon. To elucidate the pathophysiology of COVID-19-associated cardiac injury, we conducted a prospective study of the first 69 consecutive COVID-19 decedents at Columbia University Irving Medical Center in New York City. Of six acute cardiac histopathologic features, microthrombi was the most commonly detected amongst our cohort (n=48, 70%). We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak erythrocyte sedimentation rate and c-reactive protein were independently associated with increased odds of microthrombi, supporting an immunothrombotic etiology. Using single nuclei RNA-sequencing analysis on 3 patients with and 4 patients without cardiac microthrombi, we discovered an enrichment of pro-thrombotic/anti-fibrinolytic, extracellular matrix remodeling, and immune-potentiating signaling amongst cardiac fibroblasts in microthrombi-positive, relative to microthrombi-negative, COVID-19 hearts. Non-COVID-19 non-failing hearts were used as reference controls. Our study identifies a specific transcriptomic signature in cardiac fibroblasts as a salient feature of microthrombi-positive COVID-19 hearts. Our findings warrant further mechanistic study as cardiac fibroblasts may represent a potential therapeutic target for COVID-19-associated cardiac microthrombi.
Michael I. Brener, Michelle L. Hulke, Nobuaki Fukuma, Stephanie Golob, Robert S. Zilinyi, Zhipeng Zhou, Christos Tzimas, Ilaria Russo, Claire McGroder, Ryan D. Pfeiffer, Alexander Chong, Geping Zhang, Daniel Burkhoff, Martin B. Leon, Mathew S. Maurer, Jeffrey W. Moses, Anne-Catrin Uhlemann, Hanina Hibshoosh, Nir Uriel, Matthias J. Szabolcs, Björn Redfors, Charles C. Marboe, Matthew R. Baldwin, Nathan R. Tucker, Emily J. Tsai
We performed next generation sequencing in patients with familial steroid sensitive nephrotic syndrome (SSNS) and identified a homozygous segregating variant (p.H310Y) in the gene encoding clavesin-1 (CLVS1) in a consanguineous family with three affected individuals. Knockdown of the clavesin gene in zebrafish (clvs2) produced edema phenotypes due to disruption of podocyte structure and loss of glomerular filtration barrier integrity that can be rescued by WT CLVS1 but not the p.H310Y variant. Analysis of cultured human podocytes with CRISPR-Cas9 mediated CLVS1 knockout or homozygous H310Y knockin revealed deficits in clathrin-mediated endocytosis and increased susceptibility to apoptosis that could be rescued with corticosteroid treatment, mimicking the steroid-responsiveness observed in SSNS patients. The p.H310Y variant also disrupts binding of clavesin-1 to alpha-tocopherol transfer protein, resulting in increased reactive oxygen species (ROS) accumulation in CLVS1-deficient podocytes. Treatment of CLVS1 knockout or homozygous H310Y knockin podocytes with pharmacological ROS inhibitors restored viability to control levels. Taken together, this data identifies CLVS1 as a candidate gene for SSNS, provides insight into therapeutic effects of corticosteroids on podocyte cellular dynamics and adds to the growing evidence on the importance of endocytosis and oxidative stress regulation to podocyte function.
Brandon M. Lane, Megan Chryst-Stangl, Guanghong Wu, Mohamed Shalaby, Sherif El Desoky, Claire C. Middleton, Kinsie Huggins, Amika Sood, Alejandro Ochoa, Andrew F. Malone, Ricardo Vancini, Sara E. Miller, Gentzon Hall, So Young Kim, David N. Howell, Jameela A. Kari, Rasheed Gbadegesin
Approximately 80% of pancreatic cancer patients suffer from cachexia and one-third die due to cachexia-related complications such as respiratory failure and cardiac arrest. Although there has been considerable research into cachexia mechanisms and interventions, there are, to date, no FDA-approved therapies. A major contributing factor could be the failure of animal models to accurately recapitulate the human condition. In this study, we generated an aged model of pancreatic cancer cachexia to compare cachexia progression in young versus aged tumor-bearing mice. Comparative skeletal muscle transcriptome analyses identified 3-methyladenine (3-MA) as a candidate anti-wasting compound. In vitro analyses confirmed anti-wasting capacity while in vivo analysis revealed potent anti-tumor effects. Transcriptome analyses of 3-MA-treated tumor cells implicated Perp as a 3-MA target gene. We subsequently 1) observed significantly higher expression of Perp in cancer cell lines compared to control cells, 2) noted a survival disadvantage associated with elevated Perp, and 3) found that 3-MA-associated Perp reduction inhibited tumor cell growth. Finally, we provide in vivo evidence that survival benefits conferred by 3-MA administration are independent of its effect on tumor progression. Taken together, we report a novel mechanism linking 3-MA to Perp inhibition, and further implicate PERP as a novel tumor promoting factor in pancreatic cancer.
Aneesha Dasgupta, Paige C. Arneson-Wissink, Rebecca E. Schmitt, Dong Seong Cho, Alexandra M. Ducharme, Tara L. Hogenson, Eugene W. Krueger, William R. Bamlet, Lizhi Zhang, Gina L. Razidlo, Martin E. Fernandez-Zapico, Jason D. Doles
BACKGROUND. >1,500 variants in the ABCA4 locus underlie a heterogeneous spectrum of retinal disorders ranging from aggressive childhood-onset chorioretinopathy to milder, late-onset macular disease. Genotype-phenotype correlation studies have been limited in clinical applicability as patient cohorts are typically small and seldom capture the full natural history of individual genotypes. To overcome these limitations, we constructed a genotype-phenotype correlation matrix that provides quantifiable probabilities of long-term disease outcomes associated with specific ABCA4 genotypes from a large, age-restricted patient cohort. METHODS. The study included 112 unrelated patients ≥50 years of age in whom 2 pathogenic variants were identified after sequencing of the ABCA4 locus. Clinical characterization was performed using the results of best-corrected visual acuity, retinal imaging and full-field electroretinogram testing. RESULTS. Four distinct prognostic groups were defined according to the spatial severity of disease features across the fundus. Recurring genotypes were observed in milder prognoses including those associated with a newly defined class of rare hypomorphic alleles. PVS1 (predicted null) variants were enriched in the most severe prognoses; however, missense variants comprised a larger than expected fraction of these patients. Analysis of allele combinations and their respective prognostic severity, showed that certain variants such as p.(Gly1961Glu), and both rare and frequent hypomorphic alleles, are “clinically dominant” with respect to patient phenotypes irrespective of the allele in trans. CONCLUSION. These results provide much needed structure to the complex genetic and clinical landscape of ABCA4 disease and adds a tool to the clinical repertoire to quantitatively assess individual genotype-specific prognoses in patients.
Winston Lee, Jana Zernant, Pei-Yin Su, Takayuki Nagasaki, Stephen H. Tsang, Rando Allikmets
Nonphlogistic migration of macrophages contributes to the clearance of pathogens and apoptotic cells: critical steps for the resolution of inflammation and return to homeostasis. Angiotensin-(1-7) [Ang-(1-7)] is an heptapeptide of the Renin-Angiotensin system that acts through Mas receptor (MasR). Ang-(1-7) has recently emerged as a novel pro-resolving mediator, yet Ang-(1-7) resolution mechanisms are not fully determined. Herein, Ang-(1-7) stimulated migration of human and murine monocytes/macrophages in a MasR, CCR2 and MEK/ERK1/2-dependent manner. Pleural injection of Ang-(1-7) promoted nonphlogistic mononuclear cell influx alongside increased levels of CCL2, IL-10 and macrophage polarization towards a regulatory phenotype. Ang-(1-7) induction of CCL2 and mononuclear cell migration was also dependent on MasR and MEK/ERK. Noteworthy, MasR was upregulated during resolution phase of inflammation and their pharmacological inhibition or genetic deficiency impaired mononuclear cell recruitment during self-resolving models of LPS pleurisy and E. coli peritonitis. Inhibition/absence of MasR was associated with reduced CCL2 levels, impaired phagocytosis of bacteria, efferocytosis and delayed resolution of inflammation. In summary, we have uncovered a novel pro-resolving feature of Ang-(1-7), namely the recruitment of mononuclear cells favoring efferocytosis, phagocytosis and resolution of inflammation. Mechanistically, cell migration was dependent on MasR, CCR2 and the MEK/ERK pathway.
Isabella Zaidan, Luciana P. Tavares, Michelle A. Sugimoto, Kátia M. Lima, Graziele L. Negreiros-Lima, Lívia C.R. Teixeira, Thais C. Miranda, Bruno V.S. Valiate, Allysson Cramer, Juliana Priscila Vago, Gabriel H. Campolina-Silva, Jéssica A.M. Souza, Laís C. Grossi, Vanessa Pinho, Maria Jose Campagnole-Santos, Robson A .S. Santos, Mauro M. Teixeira, Izabela Galvão, Lirlândia P. Sousa
Immune checkpoint therapy targeting the PD-1/PD-L1 axis is a novel development in anticancer therapy and has been applied to clinical medicine. However, there are still some problems, including a relatively low response rate, innate mechanisms of resistance against immune checkpoint blockades, and the absence of reliable biomarkers to predict responsiveness. In this study of in vitro and in vivo models, we demonstrate that PD-L1-vInt4, a splicing variant of PD-L1, plays a role as a decoy in anti-PD-L1 antibody treatment. First, we showed that PD-L1-vInt4 was detectable in clinical samples and that it was possible to visualize the secreting variants with IHC. By overexpressing the PD-L1-secreted splicing variant on MC38 cells, we observed that an immune-suppressing effect was not induced by their secretion alone. We then demonstrated that PD-L1-vInt4 secretion resisted anti-PD-L1 antibody treatment, compared with wild type PD-L1, which was explicable by the PD-L1-vInt4’s decoying of the anti-PD-L1 antibody. The decoying function of PD-L1 splicing variants may be one of the reasons for cancers being resistant to anti-PD-L1 therapy. Measuring serum PD-L1 levels might be helpful in deciding the therapeutic strategy.
Ray Sagawa, Seiji Sakata, Bo Gong, Yosuke Seto, Ai Takemoto, Satoshi Takagi, Hironori Ninomiya, Noriko Yanagitani, Masayuki Nakao, Mingyon Mun, Ken Uchibori, Makoto Nishio, Yasunari Miyazaki, Yuichi Shiraishi, Seishi Ogawa, Keisuke Kataoka, Naoya Fujita, Kengo Takeuchi, Ryohei Katayama
Accumulating evidence has shown that cancer stroma and bone marrow-derived cells (BMDCs) in the tumor microenvironment (TME) play vital roles in tumor progression. However, the mechanism by which oral cancer stroma recruits any particular subset of BMDCs remains largely unknown. Here we sought to identify the subset of BMDCs that is recruited by cancer stroma. We established a sequential transplantation model in BALB/c nude mice, including (i) bone marrow transplantation of GFP-expressing cells and (ii) co-xenografting of patient-derived stroma (two cases, designated PDS1 and PDS2) with oral cancer cells (HSC-2). As controls, xenografting was performed with HSC-2 alone or in combination with normal human dermal fibroblasts (HDF). PDS1, PDS2, and HDF all promoted BMDCs migration in vitro and recruitment in vivo. Multicolor immunofluorescence revealed that the PDS co-xenografts recruited Arginase-1/CD11b/GR1/GFP quadruple-positive cells, which are myeloid-derived suppressor cells (MDSCs), to the TME, whereas the HDF co-xenograft did not. Screening using microarrays revealed that PDS1 and PDS2 expressed CCL2 mRNA (encoding C-C motif chemokine ligand 2) at higher levels than did HDF. Indeed, PDS xenografts contained significantly higher proportions of CCL2-positive stromal cells and CCR2/Arginase-1/CD11b/GR1 quadruple-positive MDSCs (as receiver cells) than the HDF co-xenograft. Consistently, a CCL2 synthesis inhibitor and a CCR2 antagonist significantly inhibited the PDS-driven migration of BM cells in vitro. Furthermore, intraperitoneal injection of the CCR2 antagonist to the PDS xenograft models significantly reduced the CCR2/Arginase-1/CD11b/GR1 quadruple-positive MDSCs infiltration to the TME. In conclusion, oral cancer stroma-secreted CCL2 is a key signal for recruiting CCR2-positive MDSCs from bone marrow to the TME.
May Wathone Oo, Hotaka Kawai, Kiyofumi Takabatake, Shuta Tomida, Takanori Eguchi, Kisho Ono, Qiusheng Shan, Toshiaki Ohara, Saori Yoshida, Haruka Omori, Shintaro Sukegawa, Keisuke Nakano, Kuniaki Okamoto, Akira Sasaki, Hitoshi Nagatsuka
A fibrotic stroma accumulates in advanced cancers, and invasive cancer cells migrate along collagen fibers that facilitate dissemination from the primary tumor. However, the ways in which tumor cells govern these processes remain unclear. Here, we report that the epithelial-to-mesenchymal transition–activating transcription factor ZEB1 increased type I collagen (Col1) secretion and enhanced tumor cell adherence to type 1 collagen (Col1). Mechanistically, ZEB1 increased the levels of α1β1 integrin (encoded by Itga1 and Itgb1) by inhibiting PP2A activity, which reduced nuclear accumulation of HDAC4 and thereby derepressed Itga1 gene transcription. In parallel, ZEB1 relieved Itga1 from microRNA-148a-mediated silencing. High levels of Itga1 enhanced tumor cell adherence to Col1 and were essential for Col1-induced tumor growth and metastasis. Furthermore, ZEB1 enhanced Col1 secretion by increasing the expression of a kinesin protein that facilitated transport and secretion of Col1-containing vesicles. Our findings elucidate a transcriptional mechanism by which lung adenocarcinoma cells coordinate a collagen deposition and adhesion process that facilitates tumor progression.
Xiaochao Tan, Priyam Banerjee, Xin Liu, Jiang Yu, Sieun Lee, Young-Ho Ahn, Chad J. Creighton, Jonathan M. Kurie
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