Atrial fibrillation (AF) alters atrial-cardiomyocyte (ACM) Ca2+-handling, promoting ectopic-beat formation. Here, we examined the effects of AF-associated remodeling on Ca2+-related action-potential (AP) dynamics and consequences for AF-susceptibility. AF was maintained electrically (x1 week) in dogs by right-atrial (RA) tachypacing. ACMs isolated from AF-dogs showed increased Ca2+-release refractoriness, spontaneous Ca2+-spark frequency and cycle-length (CL) threshold for Ca2+ and APD alternans versus controls. Similarly, AF increased the in-situ CL-threshold for Ca2+/APD-alternans and spatial dispersion in Ca2+-release recovery kinetics, leading to spatially-discordant alternans associated with reentrant rotor formation and susceptibility to AF induction/maintenance. The clinically-available agent dantrolene reduced Ca2+-leak and CL-threshold for Ca2+/APD-alternans in both ACMs and AF-dog RA, while suppressing AF-susceptibility; caffeine increased Ca2+-leak, CL-threshold for Ca2+/APD-alternans in control-dog ACMs and RA-tissues. In vivo, the atrial repolarization alternans CL-threshold was increased in AF vs control, as was AF-vulnerability. Intravenous dantrolene restored repolarization alternans-threshold and reduced AF-vulnerability. Immunoblots showed significantly reduced expression of total and phosphorylated ryanodine-receptors and calsequestrin in AF, along with unchanged phospholamban/SERCA expression. Thus, in addition to promoting spontaneous ectopy, AF-induced Ca2+-handling abnormalities favor AF-occurrence by enhancing vulnerability to repolarization-alternans, thereby promoting the initiation and maintenance of reentrant activity; the clinically-available compound dantrolene provides a lead-molecule to target this mechanism.
Tao Liu, Feng Xiong, Xiao-yan Qi, Jiening Xiao, Louis Villeneuve, Issam Abu-Taha, Dobromir Dobrev, Congxin Huang, Stanley Nattel
BK channels are expressed in intercalated (ICs) and principal (PCs) cells in the cortical collecting duct (CCD) of the mammalian kidney and have been proposed to be responsible for flow-induced K+ secretion (FIKS) and K+ adaptation. To examine the IC-specific role of BK channels, we generated a mouse with targeted disruption of the pore-forming BK alpha subunit (BKα) in ICs (IC-BKα-KO). Whole cell charybdotoxin (ChTX)-sensitive K+ currents were readily detected in control ICs, but largely absent in ICs of IC-BKα-KO mice. When placed on a high K+ (HK) diet for 13 days, blood [K+] was significantly greater in IC-BKα-KO mice vs. controls in males only, although urinary K+ excretion rates following isotonic volume expansion were similar in males and females. FIKS was present in microperfused CCDs isolated from controls, but was absent in IC-BKα-KO CCDs of both sexes. Also, flow-stimulated ENaC-mediated Na+ absorption was greater in CCDs from female IC-BKα-KO mice than in CCDs from males. Our results confirm a critical role of IC BK channels in FIKS. Sex contributes to the capacity for adaptation to a HK diet in IC-BKα-KO mice.
Rolando Carrisoza-Gaytan, Evan C. Ray, Daniel Flores, Allison L. Marciszyn, Peng Wu, Leah Liu, Arohan R. Subramanya, WenHui Wang, Shaohu Sheng, Lubika J. Nkashama, Jingxin Chen, Edwin K. Jackson, Stephanie M. Mutchler, Szilvia Heja, Donald E. Kohan, Lisa M. Satlin, Thomas R. Kleyman
Immune suppressive donor regulatory T cells (Tregs) can prevent graft-versus-host disease (GVHD) or solid organ allograft rejection. We previously demonstrated inhibiting STAT3 phosphorylation (pSTAT3) augments FOXP3 expression, stabilizing induced Tregs (iTregs). Here we report human pSTAT3-inhibited iTregs prevent human skin graft rejection and xenogeneic GVHD yet spare donor anti-leukemia immunity. pSTAT3-inhibited iTregs express increased levels of skin-homing CLA antigen, immune suppressive GARP and PD-1, and IL-9 that supports tolerizing mast cells. Further, pSTAT3-inhibited iTregs significantly reduce alloreactive conventional T cells, Th1, and Th17 cells implicated in GVHD and tissue rejection, and impair infiltration by pathogenic Th2 cells. Mechanistically, pSTAT3 inhibition of iTregs provokes a shift in metabolism from oxidative phosphorylation (OxPhos) to glycolysis and reduced electron transport chain activity. Strikingly, co-treatment with coenzyme Q10 (coQ10) restores OxPhos in pSTAT3-inhibited iTregs and augments their suppressive potency. These findings support the rationale for clinically testing the safety and efficacy of metabolically tuned, human pSTAT3-inhibited iTregs to control alloreactive T cells.
Kelly Walton, Mario R. Fernandez, Elizabeth M. Sagatys, Jordan Reff, Jongphil Kim, Marie Catherine Lee, John Kiluk, Jane Yuet Ching Hui, David McKenna, Meghan Hupp, Colleen Forster, Michael A. Linden, Nicholas J. Lawrence, Harshani R. Lawrence, Joseph Pidala, Steven Z. Pavletic, Bruce R. Blazar, Said M. Sebti, John L. Cleveland, Claudio Anasetti, Brian C. Betts
Chronic beryllium disease (CBD) is a metal hypersensitivity/autoimmune disease in which damage-associated molecular patterns (DAMPs) promote a break in T cell tolerance and expansion of Be2+/self-peptide reactive CD4+ T cells. In this study, we investigated the mechanism of cell death induced by beryllium particles (Be) in alveolar macrophages (AMΦs) and its impact on DAMP release. We found that phagocytosis of Be led to AM cell death independently of caspase, RIP1K, RIP3K or ROS activity. Prior to cell death, Be-exposed AMΦs secreted TNFalpha that boosted intracellular stores of IL-1alpha followed by caspase 8-dependent fragmentation of DNA. IL-1alpha and nucleosomal DNA were subsequently released from AMΦs upon loss of plasma membrane integrity. In contrast, necrotic AMs released only unfragmented DNA and necroptotic AMΦs released only IL-1alpha. In mice exposed to Be, TNFalpha promoted release of both DAMPs and was required for the mobilization of immunogenic DCs, expansion of Be-reactive CD4+ T cells and pulmonary inflammation in a mouse model of CBD. Thus, early autocrine effects of particle-induced TNFalpha on AMs led to a break in peripheral tolerance. This novel mechanism may underlie the known relationship between fine particle inhalation, TNFalpha and loss of peripheral tolerance in T cell-mediated autoimmune disease and hypersensitivities.
Morgan K. Collins, Abigail M. Shotland, Morgan F. Wade, Shaikh M. Atif, Denay J.K. Richards, Manolo Torres-Llompart, Douglas G. Mack, Allison K. Martin, Andrew P. Fontenot, Amy S. McKee
While blockade of PD-1/PD-L1 immune checkpoint revolutionized cancer treatment, how it works on tumor-infiltrating CD8+ T cells recognizing the same antigen at various differentiation stages remains elusive. Here, we found that the chemokine receptor CX3CR1 identified three distinct differentiation states of intratumoral CD8+ T-cell subsets. Adoptively transferred antigen-specific CX3CR1neg CD8+ T cells generated phenotypically and functionally distinct CX3CR1int and CX3CR1hi subsets in the periphery. Notably, expression of co-inhibitory receptors and Tcf1 inversely correlated with the degree of T-cell differentiation defined by CX3CR1. Despite significantly lower expression of co-inhibitory receptors and potent cytolytic activity, in vivo depletion of the CX3CR1hi subset did not alter the antitumor efficacy of adoptively transferred CD8+ T cells. Furthermore, differentiated CX3CR1int and CX3CR1hi subsets were impaired in their ability to undergo proliferation upon re-stimulation, and had no impact on established tumors upon second adoptive transfer compared with the CX3CR1neg subset that remained effective. Accordingly, anti-PD-L1 therapy preferentially rescued proliferation and cytokine production of the CX3CR1neg subset, and significantly enhanced antitumor efficacy of adoptively transferred CD8+ T cells. These findings provide a better understanding of the phenotypic and functional heterogeneity of tumor-infiltrating CD8+ T cells, and can be exploited to develop more effective immunotherapy.
Takayoshi Yamauchi, Toshifumi Hoki, Takaaki Oba, Hidehito Saito, Kristopher Attwood, Michael S. Sabel, Alfred E. Chang, Kunle Odunsi, Fumito Ito
Background: Prehospital plasma improves survival in severely injured trauma patients at risk for hemorrhagic shock and transported by air ambulance. We hypothesized that prehospital plasma would be associated with a reduction in immune imbalance and endothelial damage. Methods: We collected blood samples from 405 trauma patients enrolled in the Prehospital Air MedicalPlasma (PAMPer) trial upon hospital admission (0 hours) and 24 hours post admission across 6 U.S. sites(9 level-one trauma centers) with air medical transport services. We assayed samples for 21 inflammatory mediators and 7 markers of endothelial damage. We performed hierarchical clustering analysis (HCA) on principal components of these biomarkers of the immune response and endothelial injury. Regression analysis was used to control for known differences across study arms near the time of randomization and to assess any association with prehospital plasma administration. Results: HCA based on inflammatory mediator and endothelial damage marker concentrations distinguished two patient clusters, each with different injury patterns and outcomes. Patients in cluster A had greater injury severity and incidence of blunt trauma, traumatic brain injury, and mortality. Cluster A patients that received prehospital plasma as compared to standard care fluid resuscitation showed improved 30-day survival. Prehospital plasma did not improve survival in cluster B patients. In an adjusted analysis of themost seriously injured patients (ISS>30), plasma was associated with a an increase in circulating levels of adiponectin, IL-1β, IL-17A, IL-23, and IL-17E upon admission. One day following admission, prehospital plasmas was associated with a reduction in syndecan-1, TM, VEGF, IL-6, IP-10, MCP-1, and TNF-α, and an increase in IL-33, IL-21, IL-23, and IL-17E. Conclusion: This is the first human study to suggest that prehospital plasma may ameliorate the endotheliopathy of trauma and modulate an imbalance between pro-inflammatory (e.g. IL-6, TNF-α, and MCP-1) and protective (e.g. IL-33 and IL-17E) mediators. These effects of early plasma administration may contribute to improved survival in severely injured patients. Trial Registration: ClinicalTrials.gov NCT01818427 Funding: National Institutes of Health T32; U.S. Army Medical Research and Materiel Command W81XWH-12-2-0023; National Institutes of Health R35; National Institutes of Health 1R35GM119526-01; the Office of the Assistant Secretary of Defense for Health Affairs, through the Defense Medical Research and Development Program W81XWH-18-2-0051 and W81XWH-15-PRORP-OCRCA. Opinions, interpretations, conclusions and recommendations are those of the authors and not necessarily endorsed by the Department of Defense.
Danielle S. Gruen, Joshua B. Brown, Francis X. Guyette, Yoram Vodovotz, Par I. Johansson, Jakob Stensballe, Derek A. Barclay, Jinling Yin, Brian J. Daley, Richard S. Miller, Brian G. Harbrecht, Jeffrey A. Claridge, Herb A. Phelan, Matthew D. Neal, Brian Zuckerbraun, Timothy R. Billiar, Jason L. Sperry
Glucokinase (GK) is highly expressed in the hypothalamic paraventricular nucleus (PVN); however its role is currently unknown. We found that glucokinase in the PVN acts as part of a glucose sensing mechanism within the PVN that regulates glucose homeostasis by controlling glucagon like peptide 1 (GLP-1) release. GLP-1 is released from enteroendocrine L-cells in response to oral glucose. Here we identify a brain mechanism critical to the release of GLP-1 in response to oral glucose. We show that increasing expression of GK or injection of glucose into the PVN increases GLP-1 release in response to oral glucose. On the contrary decreasing expression of GK or injection of non-metabolisable glucose into the PVN prevents GLP-1 release. Our results demonstrate that glucosensitive GK neurones in the PVN, are critical to the response to oral glucose and subsequent release of GLP-1.
Yue Ma, Risheka Ratnasabapathy, Ivan De Backer, Chioma Izzi-Engbeaya, Marie-Sophie Nguyen-Tu, Joyceline Cuenco, Ben Jones, Christopher D. John, Brian Y. H. Lam, Guy A. Rutter, Giles Yeo, Waljit Dhillo, James Gardiner
Capicua (CIC), a member of the high mobility group (HMG)-box superfamily of transcriptional repressors, is frequently mutated in human oligodendrogliomas. But its function in brain development and tumorigenesis remains poorly understood. Here, we report that brain-specific deletion of Cic compromises developmental transition of neuroblast to immature neurons in mouse hippocampus and compromises normal neuronal differentiation. Combined gene expression and ChIP-seq analyses identified VGF as an important CIC-repressed transcriptional surrogate involved in neuronal lineage regulation. Aberrant VGF expression promotes neural progenitor cell proliferation by suppressing their differentiation. Mechanistically, we demonstrated that CIC represses VGF expression by tethering SIN3-HDAC to form a transcriptional corepressor complex. Mass spectrometry analysis of CIC-interacting proteins further identified BRG1 containing mSWI/SNF complex whose function is necessary for transcriptional repression by CIC. Together, this study uncovers a novel regulatory pathway of CIC-dependent neuronal differentiation and may implicate these molecular mechanisms in CIC-dependent brain tumorigenesis.
Inah Hwang, Heng Pan, Jun Yao, Olivier Elemento, Hongwu Zheng, Jihye Paik
Hydrocephalus is characterized by abnormal accumulation of cerebrospinal fluid (CSF) in the ventricular cavity. The circulation of CSF in brain ventricles is controlled by the coordinated beating of motile cilia at the surface of ependymal cells (ECs). Here we show that MT1-MMP is highly expressed in olfactory bulb, rostral migratory stream, and ventricular system. Mice deficient for Membrane type-1-MMP (MT1-MMP) develop typical phenotypes observed in hydrocephalus such as dome-shaped skull, dilated ventricles, corpus callosum agenesis and astrocyte hypertrophy during the first two weeks of postnatal development. MT1-MMP deficient mice exhibits reduced and disorganized motile cilia with the impaired maturation of ECs, leading to abnormal CSF flow. Consistent with the defects in motile cilia morphogenesis, the expressions of pro-multiciliogenic genes are significantly decreased with a concomitant hyper-activation of Notch signaling in the wall of lateral ventricles in Mmp14-/- brains. Inhibition of Notch signaling by γ-secretase inhibitor restores ciliogenesis in Mmp14-/- ECs. Taken together, these data suggest that MT1-MMP is required for ciliogenesis and ependymal cell maturation by suppressing Notch signaling during early brain development. Our findings implicate that MT1-MMP is critical for early brain development and loss of MT1-MMP activity gives rise to hydrocephalus.
Zhixin Jiang, Jin Zhou, Xin Qin, Huiling Zheng, Bo Gao, Xin-guang Liu, Guoxiang Jin, Zhongjun Zhou
Background: Specific features of the tumor microenvironment (TME) may provide useful prognostic information. We conducted a systematic investigation of the cellular composition and prognostic landscape of TME in gastric cancer. Methods: We evaluated the prognostic significance of major stromal and immune cells within TME. We proposed a composite TME-based risk score and tested it in six independent cohorts of 1,678 patients with gene expression or immunohistochemistry measurements. Further, we devised a new patient classification system based on TME characteristics. Results: We identified natural killer cells, fibroblasts, and endothelial cells as the most robust prognostic markers. The TME risk score combining these cell types was an independent prognostic factor when adjusted for clinicopathologic variables (gene expression: HR [95% CI]: 1.42 [1.22–1.66]; immunohistochemistry: 1.34 [1.24–1.45], P<0.0001). Higher TME risk scores consistently associated with worse survival within every pathologic stage (HR range: 2.18-3.11, P<0.02) and among patients who received surgery only. The TME risk score provided additional prognostic value beyond stage, and combination of the two improved prognostication accuracy (likelihood-ratio test χ2 = 235.4 vs. 187.6, P<0.0001; net reclassification index: 23%). The TME risk score can predict the survival benefit of adjuvant chemotherapy in non-metastatic patients (stage I-III) (interaction test P<0.02). Patients were divided into four TME subtypes that demonstrated distinct genetic and molecular patterns and complemented established genomic and molecular subtypes. Conclusion: We developed and validated a TME-based risk score as an independent prognostic and predictive factor, which has the potential to guide personalized management of gastric cancer.
Bailiang Li, Yuming Jiang, Guoxin Li, George A. Fisher, Ruijiang Li
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