Lysine-63 (K63)–linked polyubiquitination of TRAF3 coordinates the engagement of pattern recognition receptors to recruited adaptor proteins and downstream activator TBK1 in pathways that induce type I interferon (IFN). Whether auto-ubiquitination or other E3 ligases mediate K63-linked TRAF3 polyubiquitination remains unclear. We demonstrated that mice deficient in E3 ligase gene Hectd3 remarkably increased host defense against infection by intracellular bacteria F. novicida, Mycobacterium, and Listeria by limiting bacterial dissemination. In the absence of HECTD3, type I IFN response was impaired during bacterial infection both in vivo and in vitro. HECTD3 regulated type I IFN production by mediating K63-linked polyubiquitination of TRAF3 at residue K138. The catalytic domain of HECTD3 regulated TRAF3 K63 polyubiquitination, which enabled TRAF3–TBK1 complex formation. Our study offers novel insights into mechanisms of TRAF3 modulation and provides potential therapeutic targets against infections by intracellular bacteria and inflammatory diseases.
Fubing Li, Yang Li, Huichun Liang, Tao Xu, Yanjie Kong, Maobo Huang, Ji Xiao, Xi Chen, Houjun Xia, Yingying Wu, Zhongmei Zhou, Xiaomin Guo, Chunmiao Hu, Chuanyu Yang, Xu Cheng, Ceshi Chen, Xiaopeng Qi
Myeloid-derived suppressor cells (MDSCs) densely accumulate into tumors and potently suppress anti-tumor immune responses promoting tumor development. Targeting MDSCs in tumor immunotherapy has been hampered by lack of understanding of the molecular pathways that govern MDSC differentiation and function. Herein, we identify autophagy as a crucial pathway for MDSC-mediated suppression of anti-tumor immunity. Specifically, MDSCs in melanoma patients and mouse melanoma exhibited increased levels of functional autophagy. Ablation of autophagy in myeloid cells, significantly delayed tumor growth and endowed anti-tumor immune responses. Notably, tumor-infiltrating autophagy-deficient monocytic MDSCs (M-MDSCs) demonstrated impaired suppressive activity in vitro and in vivo, while transcriptome analysis revealed significant differences in genes related to lysosomal function. Accordingly, autophagy-deficient M-MDSCs exhibited impaired lysosomal degradation thereby enhancing surface expression of MHC class II molecules, resulting in efficient activation of tumor-specific CD4+ T cells. Finally, targeting of the membrane-associated RING-CH1 (MARCH1) E3 ubiquitin ligase, that mediates the lysosomal degradation of MHC II, in M-MDSCs, attenuated their suppressive function, and resulted in significantly decreased tumor volume followed by development of a robust anti-tumor immunity. Collectively, these findings depict autophagy as a novel molecular target of MDSC-mediated suppression of anti-tumor immunity.
Themis Alissafi, Aikaterini Hatzioannou, Konstantinos Mintzas, Roza Maria Barouni, Aggelos Banos, Sundary Sormendi, Alexandros Polyzos, Maria Xilouri, Ben Wielockx, Helen Gogas, Panayotis Verginis
EZH2-mediated epigenetic regulation of T cell differentiation and regulatory T cell function has been described previously; however, the role of EZH2 in T cell–mediated anti-tumor immunity, especially in the context of immune checkpoint therapy, is not understood. Here, we showed that genetic depletion of EZH2 in regulatory T cells (FoxP3creEZH2fl/fl mice) leads to robust anti-tumor immunity. In addition, pharmacological inhibition of EZH2 in human T cells using CPI-1205 elicited phenotypic and functional alterations of the regulatory T cells and enhanced cytotoxic activity of effector T cells. We observed that ipilimumab (anti–CTLA-4) increased EZH2 expression in peripheral T cells from treated patients. We hypothesized that inhibition of EZH2 expression in T cells would increase the effectiveness of anti–CTLA-4 therapy, which we tested in murine models. Collectively, our data demonstrated that modulating EZH2 expression in T cells can improve anti-tumor responses elicited by anti–CTLA-4 therapy, which provides a strong rationale for a combination trial of CPI-1205 plus ipilimumab.
Sangeeta Goswami, Irina Apostolou, Jan Zhang, Jill Skepner, Swetha Anandhan, Xuejun Zhang, Liangwen Xiong, Patrick Trojer, Ana Aparicio, Sumit K. Subudhi, James P. Allison, Hao Zhao, Padmanee Sharma
Acute pancreatitis (AP), a human disease in which the pancreas digests itself, has substantial mortality with no specific therapy. The major causes of AP are alcohol abuse and gallstone complications, but it also occurs as an important side effect of the standard Asparaginase-based therapy for childhood acute lymphoblastic leukaemia. Previous investigations into the mechanisms underlying pancreatic acinar cell death induced by alcohol metabolites, bile acids or Asparaginase indicated that loss of intracellular ATP generation is a significant factor. In isolated mouse pancreatic acinar cells or cell clusters, we now report that removal of extracellular glucose had little effect on this ATP loss, suggesting that glucose metabolism was severely inhibited under these conditions. Surprisingly, we show that replacing glucose with galactose prevented or markedly reduced the loss of ATP and any subsequent necrosis. Addition of pyruvate had a similar protective effect. We also studied the effect of galactose in vivo in mouse models of AP induced either by a combination of fatty acids and ethanol or Asparaginase. In both cases, galactose markedly reduced acinar necrosis and inflammation. Based on these data we suggest that galactose feeding may be used to protect against AP.
Shuang Peng, Julia V. Gerasimenko, Tetyana M. Tsugorka, Oleksiy Gryshchenko, Sujith Samarasinghe, Ole H. Petersen, Oleg V. Gerasimenko
Painful signals are transmitted by mutisynaptic glutamatergic pathways. Their first synapse between primary nociceptors and excitatory spinal interneurons gates sensory load. Glutamate release herein is orchestrated by Ca2+ sensor proteins with neuronal calcium-binding protein 2 (NECAB2) being particularly abundant. However, neither the importance of NECAB2+ neuronal contingents in dorsal root ganglia (DRG) and spinal cord nor function-determination by NECAB2 has been defined. A combination of histochemistry and single-cell RNA-seq showed NECAB2 in small/medium-sized C- and Aδ D-hair low threshold mechanoreceptors in DRG, as well as in protein kinase γ-positive excitatory spinal interneurons. NECAB2 was downregulated by peripheral nerve injury, offering the hypothesis that NECAB2 loss-of-funtion could limit pain sensation. Indeed, Necab2–/– mice reached a pain-free state significantly faster after peripheral inflammation than wild-type littermates. Genetic access to transiently-activated neurons revealed that a mediodorsal cohort of NECAB2+ neurons mediates inflammatory pain in mouse spinal dorsal horn. Here, besides dampening excitatory transmission in spinal interneurons, NECAB2 limited pronociceptive brain-derived neurotrophic factor release from sensory afferents. Hox8b-dependent reinstatement of NECAB2 expression in Necab2–/– mice then demonstrated that spinal/DRG NECAB2 alone could control inflammation-induced sensory hyperensitivity. Overall, we identify NECAB2 as a critical component of pro-nociceptive pain signaling whose inactivation offers substantial pain relief.
Ming-Dong Zhang, Jie Su, Csaba Adori, Valentina Cinquina, Katarzyna Malenczyk, Fatima Girach, Changgeng Peng, Patrik Ernfors, Peter Löw, Lotta Borgius, Ole Kiehn, Masahiko Watanabe, Mathias Uhlén, Nicholas Mitsios, Jan Mulder, Tibor Harkany, Tomas Hökfelt
T cells must migrate in order to encounter antigen-presenting cells (APCs) and to execute their varied functions in immune defense and inflammation. ATP release and autocrine signaling through purinergic receptors contribute to T cell activation at the immune synapse that T cells form with APCs. Here, we show that T cells also require ATP release and purinergic signaling for their migration to APCs. We found that the chemokine SDF-1α triggered mitochondrial ATP production, rapid bursts of ATP release, and increased migration of primary human CD4+ T cells. This process depended on pannexin-1 ATP release channels and autocrine stimulation of P2X4 receptors. SDF-1α stimulation caused localized accumulation of mitochondria with P2X4 receptors near the front of cells, resulting in a feed-forward signaling mechanism that promotes cellular Ca2+ influx and sustains mitochondrial ATP synthesis at levels needed for pseudopod protrusion, T cell polarization, and cell migration. Inhibition of P2X4 receptors blocked the activation and migration of T cells in vitro. In a mouse lung transplant model, P2X4 receptor antagonist treatment prevented the recruitment of T cells into allograft tissue and the rejection of lung transplants. Our findings suggest that P2X4 receptors are therapeutic targets for immunomodulation in transplantation and inflammatory diseases.
Carola Ledderose, Kaifeng Liu, Yutaka Kondo, Christian J. Slubowski, Thomas Dertnig, Sara Denicoló, Mona Arbab, Johannes Hubner, Kirstin Konrad, Mahtab Fakhari, James A. Lederer, Simon C. Robson, Gary A. Visner, Wolfgang G. Junger
PRDM16 is a transcriptional co-regulator involved in translocations in acute myeloblastic leukemia (AML), myelodysplastic syndromes and T acute lymphoblastic leukemia that is highly expressed in and required for the maintenance of hematopoietic stem cells (HSCs), and can be aberrantly expressed in AML. Prdm16 is expressed as full-length (fPrdm16) and short (sPrdm16) isoforms, the latter lacking the N-terminal PR-domain. The role of both isoforms in normal and malignant hematopoiesis is unclear. We show here that fPrdm16 was critical for HSC maintenance, induced multiple genes involved in GTPase signaling and repressed inflammation, while sPrdm16 supported B-cell development biased towards marginal zone B-cells and induced an inflammatory signature. In a mouse model of human MLL-AF9 leukemia fPrdm16 extended latency, while sPrdm16 shortened latency and induced a strong inflammatory signature, including several cytokines and chemokines that are associated with myelodysplasia and with a worse prognosis in human AML. Finally, in human NPM1-mutant and in MLL-translocated AML high expression of PRDM16, which negatively impacts outcome, was associated with inflammatory gene expression, thus corroborating the mouse data. Our observations demonstrate distinct roles for Prdm16 isoforms in normal HSCs and AML, and identify sPrdm16 as one of the drivers of prognostically adverse inflammation in leukemia.
David J. Corrigan, Larry L. Luchsinger, Mariana Justino de Almeida, Linda J. Williams, Alexandros Strikoudis, Hans-Willem Snoeck
While the genomic binding of MYC protein correlates with active epigenetic marks on chromatin, it remains largely unclear how major epigenetic mechanisms functionally impact the tumorigenic potential of MYC. Here we showed that compared to the catalytic subunits, the core subunits, including DPY30, of the major H3K4 methyltransferase complexes were frequently amplified in human cancers, and selectively upregulated in Burkitt lymphoma. We showed that DPY30 promoted expression of endogenous MYC, and was also functionally important for efficient binding of MYC to its genomic targets by regulating chromatin accessibility. Dpy30 heterozygosity did not affect normal animal physiology including life span, but significantly suppressed Myc-driven lymphomagenesis, as cells failed to combat oncogene-triggered apoptosis due to insufficient epigenetic modulation and expression of a subset of anti-apoptotic genes. Dpy30 reduction also greatly impeded MYC-dependent cellular transformation without affecting normal cell growth. These results suggest that MYC hijacks a major epigenetic pathway — H3K4 methylation — to facilitate its molecular activity in target binding and to coordinate its oncogenic program for efficient tumorigenesis, meanwhile creating “epigenetic vulnerability.” DPY30 and the H3K4 methylation pathway are thus potential epigenetic targets for treating certain MYC-driven cancers.
Zhenhua Yang, Kushani Shah, Theodore Busby, Keith Giles, Alireza Khodadadi-Jamayran, Wei Li, Hao Jiang
Neuronatin (Nnat) is an imprinted gene implicated in human obesity and widely expressed in neuroendocrine and metabolic tissues in a hormone and nutrient-sensitive manner. However, its molecular and cellular functions and precise role in organismal physiology remain only partly defined. Here we demonstrate that mice lacking Nnat globally or specifically in β cells display impaired glucose-stimulated insulin secretion leading to defective glucose handling under conditions of nutrient-excess. In contrast, we report no evidence for any feeding or body weight phenotypes in global Nnat null mice. At the molecular level neuronatin augments insulin signal peptide cleavage by binding to the signal peptidase complex and facilitates translocation of the nascent preprohormone. Loss of neuronatin expression in β cells therefore reduces insulin content and blunts glucose-stimulated insulin secretion. Nnat expression, in turn, is glucose-regulated. This mechanism therefore represents a novel site of nutrient-sensitive control of β cell function and whole animal glucose homeostasis. These data also suggest a potential wider role for Nnat in the regulation of metabolism through the modulation of peptide processing events.
Steven J. Millership, Gabriela da Silva Xavier, Agharul I. Choudhury, Sergio Bertazzo, Pauline Chabosseau, Silvia M.A. Pedroni, Elaine E. Irvine, Alex Montoya, Peter Faull, William R. Taylor, Julie Kerr-Conte, Francois Pattou, Jorge Ferrer, Mark Christian, Rosalind M. John, Mathieu Latreille, Ming Liu, Guy A. Rutter, James Scott, Dominic J. Withers
In type 1 diabetes, cytotoxic CD8 T cells with specificity for β-cell autoantigens are found in the pancreatic islets where they are implicated in the destruction of insulin-secreting β cells. In contrast, the disease relevance of β-cell-reactive CD8 T cells that are detectable in the circulation, and their relationship to β-cell function, are not known. Here, we tracked multiple, circulating β-cell-reactive CD8 T cell subsets and measured β-cell function longitudinally for two years, starting immediately after diagnosis of type 1 diabetes. We found that change in β-cell-specific effector memory CD8 T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8 T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer specific protein 37 and CD16, and reduced expression of CD28) compared with their CD57-negative counterparts, and network association modelling indicated that the dynamics of β-cell-reactive CD57+ effector memory CD8 T cell subsets were strongly linked. Thus, coordinated changes in circulating β-cell-specific CD8 T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.
Lorraine Yeo, Alyssa Woodwyk, Sanjana Sood, Anna Lorenc, Martin Eichmann, Irma Pujol-Autonell, Rossella Melchiotti, Ania Skowera, Efthymios Fidanis, Garry M. Dolton, Katie Tungatt, Andrew K. Sewell, Susanne Heck, Alka Saxena, Craig A. Beam, Mark Peakman
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