Research
Metabolic reprogramming augments potency of human pSTAT3-inhibited iTregs to suppress alloreactivity
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
Takayoshi Yamauchi, Toshifumi Hoki, Takaaki Oba, Hidehito Saito, Kristopher Attwood, Michael S. Sabel, Alfred E. Chang, Kunle Odunsi, Fumito Ito
Abstract
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.
Authors
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
Abstract
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.
Authors
Inah Hwang, Heng Pan, Jun Yao, Olivier Elemento, Hongwu Zheng, Jihye Paik
Abstract
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.
Authors
Zhixin Jiang, Jin Zhou, Xin Qin, Huiling Zheng, Bo Gao, Xin-guang Liu, Guoxiang Jin, Zhongjun Zhou
Abstract
Familial Hypocalciuric Hypercalcemia (FHH) is a genetic condition associated with hypocalciuria, hypercalcemia and in some cases inappropriately high levels of circulating parathyroid hormone (PTH). FHH is associated with inactivating mutations in CaSR encoding the Ca2+ sensing receptor (CaSR), a G protein coupled receptor (GPCR) and GNA11 encoding G protein subunit alpha 11 (Gα11), implicating defective GPCR signaling as the root pathophysiology for FHH. However, the downstream mechanism by which CaSR activation inhibits PTH production/secretion is incompletely understood. Here, we show that mice lacking the transient receptor potential canonical channel 1 (TRPC1) develop chronic hypercalcemia, hypocalciuria, and elevated PTH levels mimicking human FHH. Ex vivo and in vitro studies reveal that TRPC1 serves a necessary and sufficient mediator to suppress PTH secretion from parathyroid glands (PTG) downstream of CaSR in response to high extracellular Ca2+ concentration. Gα11 physically interacts with both the N- and C-termini of TRPC1 and enhances CaSR-induced TRPC1 activity in transfected cells. These data identify TRPC1-mediated Ca2+ signaling as an essential component of the cellular apparatus controlling PTH secretion in the PTG downstream of CaSR.
Authors
Marta Onopiuk, Bonnie Eby, Vasyl Nesin, Peter Ngo, Megan Lerner, Caroline M. Gorvin, Victoria J. Stokes, Rajesh V. Thakker, Maria Luisa Brandi, Wenhan Chang, Mary Beth Humphrey, Leonidas Tsiokas, Kai Lau
Abstract
Septic cardiomyopathy is a life-threatening organ dysfunction caused by sepsis. Ribonuclease 1 (RNase 1) belongs to a group of host-defense peptides that specifically cleave extracellular RNA (eRNA). The activity of RNase1 is inhibited by ribonuclease-inhibitor 1 (RNH1). The role of RNase 1 in septic cardiomyopathy and associated cardiac apoptosis, however, is completely unknown. Here, we showed that sepsis resulted in a significant increase in RNH1 and eRNA serum levels compared to those of healthy subjects (p < 0.05). Treatment with RNase 1 resulted in a significant decrease of apoptosis, induced by the intrinsic pathway, and TNF expression in murine cardiomyocytes exposed to either necrotic cardiomyocytes or serum of septic patients for 16 h (p < 0.05). Furthermore, treatment of septic mice with RNase 1 resulted in a reduction in cardiac apoptosis, TNF expression and septic cardiomyopathy (p < 0.05). These data demonstrate that eRNA plays a crucial role in the pathophysiology of the organ (cardiac) dysfunction in sepsis and RNase and RNH1 may be new therapeutic targets/strategies to reduce the cardiac injury and dysfunction caused by sepsis.
Authors
Elisabeth Zechendorf, Caroline E O'Riordan, Lara Stiehler, Natalie Wischmeyer, Fausto Chiazza, Debora Collotta, Bernd Denecke, Sabrina Ernst, Gerhard Müller-Newen, Sina M. Coldewey, Bianka Wissuwa, Massimo Collino, Tim-Philipp Simon, Tobias Schuerholz, Christian Stoppe, Gernot Marx, Christoph Thiemermann, Lukas Martin
Abstract
Over 55,000 people in the US are diagnosed with pancreatic ductal adenocarcinoma (PDAC) yearly, and fewer than 20% of these patients survive a year beyond diagnosis. Chemotherapies are considered or used in nearly every PDAC case, but there is limited understanding of the complex signaling responses underlying resistance to these common treatments. Here, we take an unbiased approach to study protein kinase network changes following chemotherapies in patient-derived xenograft (PDX) models of PDAC to facilitate design of rational drug combinations. Proteomics profiling following chemotherapy regimens reveals that activation of JNK-JUN signaling occurs after 5-fluorouracil plus leucovorin (5-FU) and FOLFOX (5-FU plus oxaliplatin (OX)), but not after OX alone or gemcitabine. Cell and tumor growth assays with the irreversible inhibitor JNK-IN-8 and genetic manipulations demonstrate that JNK and JUN each contribute to chemoresistance and cancer cell survival after FOLFOX. Active JNK1 and JUN are specifically implicated in these effects, and synergy with JNK-IN-8 is linked to FOLFOX-mediated JUN activation, cell cycle dysregulation, and DNA damage response. This study highlights the potential for JNK-IN-8 as a biological tool and potential combination therapy with FOLFOX in PDAC and reinforces the need to tailor treatment to functional characteristics of individual tumors.
Authors
Matthew B. Lipner, Xianlu L. Peng, Chong Jin, Yi Xu, Yanzhe Gao, Michael P. East, Naim U. Rashid, Richard A. Moffitt, Silvia G. Herrera Loeza, Ashley B. Morrison, Brian T. Golitz, Cyrus Vaziri, Lee M. Graves, Gary L. Johnson, Jen Jen Yeh
Abstract
Infections due to carbapenem-resistant Klebsiella pneumoniae have emerged as a global threat due to its wide-spread antimicrobial resistance. Transplant recipients and patients with hematologic malignancies have high mortality rate suggesting host factors in susceptibility. We developed a model of pulmonary infection using ST258 C4, KPC-2 clone, which are predominant Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria, and demonstrated that Rag2-/-Il2rg-/- mice, but not wildtype C57BL/6 or Rag2-/- mice, were susceptible to this opportunistic infection. Using single-cell RNA-seq in infected Rag2-/- mice, we identified distinct clusters of Ifng+ NK cells and Il17a+, Il22+, and inducible T-cell costimulatory molecule (ICOS)+ group 3 innate lymphoid cells (ILCs) that were critical for host resistance. As solid organ transplantation is a risk factor, we generated a more clinically relevant model using FK506 in wildtype C57BL/6 mice. We further demonstrated that immunotherapy with recombinant IL-22 treatment ameliorated the ST258 pulmonary infection in both FK506 treated WT mice and Rag2-/-Il2rg-/- mice via hepatic IL-22ra1 signaling. These data support the development of host directed immunotherapy as an adjunct treatment to new antibiotics.
Authors
Naoki Iwanaga, Ivy Sandquist, Alanna Wanek, Janet E. McCombs, Kejing Song, Jay Kolls
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