Influenza A virus–specific (IAV-specific) T cell responses are important correlates of protection during primary and subsequent infections. The generation and maintenance of robust IAV-specific T cell responses relies on T cell interactions with dendritic cells (DCs). In this study, we explore the role of the nucleotide-binding domain leucine-rich repeat–containing receptor family member NLRC4 in modulating the DC phenotype during IAV infection. Nlrc4–/– mice had worsened survival and increased viral titers during infection, normal innate immune cell recruitment, and IAV-specific CD8+ T cell responses, but severely blunted IAV-specific CD4+ T cell responses compared with WT mice. The defect in the pulmonary IAV–specific CD4+ T cell response was not a result of defective priming or migration of these cells in Nlrc4–/– mice but was instead due to an increase in FasL+ DCs, resulting in IAV-specific CD4+ T cell death. Together, our data support a role for NLRC4 in regulating the phenotype of lung DCs during a respiratory viral infection and thereby influencing the magnitude of protective T cell responses.
Emma E. Hornick, Jargalsaikhan Dagvadorj, Zeb R. Zacharias, Ann M. Miller, Ryan A. Langlois, Peter Chen, Kevin L. Legge, Gail A. Bishop, Fayyaz S. Sutterwala, Suzanne L. Cassel
Bronchopulmonary dysplasia (BPD) remains a major respiratory illness in extremely premature infants. The biological mechanisms leading to BPD are not fully understood, although an arrest in lung development has been implicated. The current study aimed to investigate the occurrence of autophagy in the developing mouse lung and its regulatory role in airway branching and terminal sacculi formation. We found 2 windows of epithelial autophagy activation in the developing mouse lung, both resulting from AMPK activation. Inhibition of AMPK-mediated autophagy led to reduced lung branching in vitro. Conditional deletion of beclin 1 (Becn1) in mouse lung epithelial cells (Becn1Epi-KO), either at early (E10.5) or late (E16.5) gestation, resulted in lethal respiratory distress at birth or shortly after. E10.5 Becn1Epi-KO lungs displayed reduced airway branching and sacculi formation accompanied by impaired vascularization, excessive epithelial cell death, reduced mesenchymal thinning of the interstitial walls, and delayed epithelial maturation. E16.5 Becn1Epi-KO lungs had reduced terminal air sac formation and vascularization and delayed distal epithelial differentiation, a pathology similar to that seen in infants with BPD. Taken together, our findings demonstrate that intrinsic autophagy is an important regulator of lung development and morphogenesis and may contribute to the BPD phenotype when impaired.
Behzad Yeganeh, Joyce Lee, Leonardo Ermini, Irene Lok, Cameron Ackerley, Martin Post
Identifying nonaddictive opioid medications is a high priority in medical science, but μ-opioid receptors (MORs) mediate both the analgesic and addictive effects of opioids. We found a significant pharmacodynamic difference between morphine and methadone that is determined entirely by heteromerization of MORs with galanin Gal1 receptors (Gal1Rs), rendering a profound decrease in the potency of methadone. This finding was explained by the weaker proficiency of methadone in activating the dopaminergic system as compared with morphine and predicted a dissociation of the therapeutic and euphoric effects of methadone, which was corroborated by a significantly lower incidence of self-reports of feeling “high” in methadone-medicated patients. These results suggest that μ-opioid–Gal1R heteromers mediate the dopaminergic effects of opioids. The results further suggest a lower addictive liability of some opioids, such as methadone, due to their selective low potency for the μ-opioid–Gal1R heteromer.
Ning-Sheng Cai, César Quiroz, Jordi Bonaventura, Alessandro Bonifazi, Thomas O. Cole, Julia Purks, Amy S. Billing, Ebonie Massey, Michael Wagner, Eric D. Wish, Xavier Guitart, William Rea, Sherry Lam, Estefanía Moreno, Verònica Casadó-Anguera, Aaron D. Greenblatt, Arthur E. Jacobson, Kenner C. Rice, Vicent Casadó, Amy H. Newman, John W. Winkelman, Michael Michaelides, Eric Weintraub, Nora D. Volkow, Annabelle M. Belcher, Sergi Ferré
The polarization of macrophages is regulated by transcription factors, such as NF-κB and activator protein 1 (AP-1). In this manuscript, we delineated the role of the transcription factor Fos-related antigen 1 (Fra-1) during macrophage activation and development of arthritis. Network level interaction analysis of microarray data derived from Fra-1– or Fra-2–deficient macrophages revealed a central role of Fra-1, but not of Fra-2, in orchestrating the expression of genes related to wound response, Toll-like receptor activation, and interleukin signaling. ChIP sequencing and standard ChIP analyses of macrophages identified arginase 1 (Arg1) as a target of Fra-1. Luciferase reporter assays revealed that Fra-1 downregulated Arg1 expression by direct binding to the promoter region. Using macrophage-specific Fra-1– or Fra-2–deficient mice, we observed enhanced expression and activity of Arg1 and a reduction of arthritis in the absence of Fra-1, but not of Fra-2. This phenotype was reversed by treatment with the arginase inhibitor Nω-hydroxy-nor-ʟ-arginine, while ʟ-arginine supplementation increased arginase activity and alleviated arthritis, supporting the notion that reduced arthritis in macrophage-specific Fra-1–deficient mice resulted from enhanced Arg1 expression and activity. Moreover, patients with active rheumatoid arthritis (RA) showed increased Fra-1 expression in the peripheral blood and elevated Fra-1 protein in synovial macrophages compared with RA patients in remission. In addition, the Fra-1/ARG1 ratio in synovial macrophages was related to RA disease activity. In conclusion, these data suggest that Fra-1 orchestrates the inflammatory state of macrophages by inhibition of Arg1 expression and thereby impedes the resolution of inflammation.
Nicole Hannemann, Shan Cao, Daniel Eriksson, Anne Schnelzer, Jutta Jordan, Martin Eberhardt, Ulrike Schleicher, Jürgen Rech, Andreas Ramming, Steffen Uebe, Arif Ekici, Juan D. Cañete, Xiaoxiang Chen, Tobias Bäuerle, Julio Vera, Christian Bogdan, Georg Schett, Aline Bozec
Calcineurin acts as a calcium-activated phosphatase that dephosphorylates various substrates, including members of the nuclear factor of activated T cells (NFAT) family, to trigger their nuclear translocation and transcriptional activity. However, the detailed mechanism regulating the recruitment of NFATs to calcineurin remains poorly understood. Here, we report that calcineurin A (CNA), encoded by PPP3CB or PPP3CC, is constitutively ubiquitinated on lysine 327, and this polyubiquitin chain is rapidly removed by ubiquitin carboxyl-terminal hydrolase 16 (USP16) in response to intracellular calcium stimulation. The K29-linked ubiquitination of CNA impairs NFAT recruitment and transcription of NFAT-targeted genes. USP16 deficiency prevents calcium-triggered deubiquitination of CNA in a manner consistent with defective maintenance and proliferation of peripheral T cells. T cell–specific USP16 knockout mice exhibit reduced severity of experimental autoimmune encephalitis and inflammatory bowel disease. Our data reveal the physiological function of CNA ubiquitination and its deubiquitinase USP16 in peripheral T cells. Notably, our results highlight a critical mechanism for the regulation of calcineurin activity and a novel immunosuppressive drug target for the treatment of autoimmune diseases.
Yu Zhang, Rong-bei Liu, Qian Cao, Ke-qi Fan, Ling-jie Huang, Jian-shuai Yu, Zheng-jun Gao, Tao Huang, Jiang-yan Zhong, Xin-tao Mao, Fei Wang, Peng Xiao, Yuan Zhao, Xin-hua Feng, Yi-yuan Li, Jin Jin
The lung is a specialized barrier organ that must tightly regulate interstitial fluid clearance and prevent infection in order to maintain effective gas exchange. Lymphatic vessels are important for these functions in other organs, but their roles in the lung have not been fully defined. In the present study, we evaluated how the lymphatic vasculature participates in lung homeostasis. Studies using mice carrying a lymphatic reporter allele revealed that, in contrast to other organs, lung lymphatic collecting vessels lack smooth muscle cells entirely, suggesting that forward lymph flow is highly dependent on movement and changes in pressure associated with respiration. Functional studies using C-type lectin domain family 2–deficient (CLEC2-deficient) mice in which lymph flow is impaired because of loss of lympho-venous hemostasis, or using inducible lung-specific ablation of lymphatic endothelial cells in a lung transplant model revealed that loss of lymphatic function leads to an inflammatory state characterized by the formation of tertiary lymphoid organs (TLOs). In addition, impaired lymphatic flow in mice resulted in hypoxia and features of lung injury that resembled emphysema. These findings reveal both a lung-specific mechanism of lymphatic physiology and a lung-specific consequence of lymphatic dysfunction that may contribute to chronic lung diseases that arise in association with TLO formation.
Hasina Outtz Reed, Liqing Wang, Jarrod Sonett, Mei Chen, Jisheng Yang, Larry Li, Petra Aradi, Zoltan Jakus, Jeanine D’Armiento, Wayne W. Hancock, Mark L. Kahn
Cortical bones account for more than 80% of human bone mass. The periosteum, a thin tissue that covers almost the entire bone surface, is essential for bone formation and regeneration. However, its osteogenic and bone regenerative abilities are not well studied. In this study, we found that macrophage-lineage cells recruit periosteum-derived cells (PDCs) for cortical bone formation. Knockout of colony-stimulating factor-1 eliminated macrophage-lineage cells and resulted in loss of PDCs with impaired periosteal bone formation. Moreover, macrophage-lineage tartrate-resistant acid phosphatase–positive (TRAP+) cells induced transcriptional expression of periostin and recruitment of PDCs to the periosteal surface through secretion of PDGF-BB, where the recruited PDCs underwent osteoblast differentiation coupled with type H vessel formation. We also found that subsets of Nestin+ and LepR+CD45–Ter119–CD31– cells (LepR+ PDCs) possess multipotent and self-renewal abilities and contribute to cortical bone formation. Nestin+ PDCs are found primarily during bone development, whereas LepR+ PDCs are essential for bone homeostasis in adult mice. Importantly, conditional knockout of Pdgfr-β in LepR+ cells impaired periosteal bone formation and regeneration. These findings uncover the essential role of periosteal macrophage-lineage cells in regulating periosteum homeostasis and regeneration.
Bo Gao, Ruoxian Deng, Yu Chai, Hao Chen, Bo Hu, Xiao Wang, Shouan Zhu, Yong Cao, Shuangfei Ni, Mei Wan, Liu Yang, Zhuojing Luo, Xu Cao
Preclinical studies demonstrate that rapid-acting antidepressants, including ketamine, require stimulation of mTORC1 signaling. This pathway is regulated by neuronal activity and endocrine and metabolic signals, notably including the amino acid leucine, which activates mTORC1 signaling via binding to the upstream regulator sestrin. Here, we examined the antidepressant actions of NV-5138, a highly selective small molecule modulator of sestrin that penetrates the blood-brain barrier. The results demonstrate that a single dose of NV-5138 produced rapid and long-lasting antidepressant effects and rapidly reversed anhedonia caused by chronic stress exposure. The antidepressant actions of NV-5138 required brain-derived neurotrophic factor (BDNF) release, as the behavioral responses were blocked by infusion of a BDNF-neutralizing Ab into the medial prefrontal cortex (mPFC) or, in mice, with a knockin of a BDNF polymorphism that blocked activity-dependent BDNF release. NV-5138 administration also rapidly increased synapse number and function in the mPFC and reversed the synaptic deficits caused by chronic stress. Together, the results demonstrate that NV-5138 produces rapid synaptic and antidepressant behavioral responses via activation of the mTORC1 pathway and BDNF signaling, indicating that pharmacological modulation of sestrin may be an attractive approach for the development of rapid-acting antidepressants.
Taro Kato, Santosh Pothula, Rong-Jian Liu, Catharine H. Duman, Rosemarie Terwilliger, George P. Vlasuk, Eddine Saiah, Seung Hahm, Ronald S. Duman
Increased urinary oxalate excretion (hyperoxaluria) promotes the formation of calcium oxalate crystals. Monogenic diseases due to hepatic enzyme deficiency result in chronic hyperoxaluria, promoting end-stage renal disease in children and young adults. Ethylene glycol poisoning also results in hyperoxaluria, promoting acute renal failure and frequently death. Stiripentol is an antiepileptic drug used to treat children affected by Dravet syndrome. It has been shown to inhibit neuronal lactate dehydrogenase 5 enzyme. As this isoenzyme is also the last step of hepatic oxalate production, we hypothesized that stiripentol would potentially reduce hepatic oxalate production and urine oxalate excretion. In vitro, stiripentol decreased the synthesis of oxalate by hepatocytes in a dose-dependent manner. In vivo, oral administration of stiripentol significantly reduced urine oxalate excretion in rats. Stiripentol protected the kidneys against calcium oxalate crystal deposits in acute ethylene glycol intoxication and chronic calcium oxalate nephropathy models. In both models, stiripentol significantly improved renal function. Patients affected by Dravet syndrome and treated with stiripentol had a lower urine oxalate excretion than control patients. A young girl affected by severe type I hyperoxaluria received stiripentol for several weeks, and urine oxalate excretion decreased by two-thirds. Stiripentol is a promising potential therapy against genetic hyperoxaluria and ethylene glycol poisoning.
Marine Le Dudal, Léa Huguet, Joëlle Perez, Sophie Vandermeersch, Elise Bouderlique, Ellie Tang, Carole Martori, Nicole Chemaly, Rima Nabbout, Jean-Philippe Haymann, Vincent Frochot, Laurent Baud, Georges Deschênes, Michel Daudon, Emmanuel Letavernier
The nuclear protein DEK is an endogenous DNA-binding chromatin factor regulating hematopoiesis. DEK is one of only 2 known secreted nuclear chromatin factors, but whether and how extracellular DEK regulates hematopoiesis is not known. We demonstrated that extracellular DEK greatly enhanced ex vivo expansion of cytokine-stimulated human and mouse hematopoietic stem cells (HSCs) and regulated HSC and hematopoietic progenitor cell (HPC) numbers in vivo and in vitro as determined both phenotypically (by flow cytometry) and functionally (through transplantation and colony formation assays). Recombinant DEK increased long-term HSC numbers and decreased HPC numbers through a mechanism mediated by the CXC chemokine receptor CXCR2 and heparan sulfate proteoglycans (HSPGs) (as determined utilizing Cxcr2–/– mice, blocking CXCR2 antibodies, and 3 different HSPG inhibitors) that was associated with enhanced phosphorylation of ERK1/2, AKT, and p38 MAPK. To determine whether extracellular DEK required nuclear function to regulate hematopoiesis, we utilized 2 mutant forms of DEK: one that lacked its nuclear translocation signal and one that lacked DNA-binding ability. Both altered HSC and HPC numbers in vivo or in vitro, suggesting the nuclear function of DEK is not required. Thus, DEK acts as a hematopoietic cytokine, with the potential for clinical applicability.
Maegan L. Capitano, Nirit Mor-Vaknin, Anjan K. Saha, Scott Cooper, Maureen Legendre, Haihong Guo, Rafael Contreras-Galindo, Ferdinand Kappes, Maureen A. Sartor, Christopher T. Lee, Xinxin Huang, David M. Markovitz, Hal E. Broxmeyer
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