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
The gut microbiota is crucial for our health, and well-balanced interactions between the host’s immune system and the microbiota are essential to prevent chronic intestinal inflammation, as observed in inflammatory bowel diseases (IBD). A variant in protein tyrosine phosphatase non-receptor type 22 (PTPN22) is associated with reduced risk of developing IBD, but promotes the onset of autoimmune disorders. While the role of PTPN22 in modulating molecular pathways involved in IBD pathogenesis is well studied, its impact on shaping the intestinal microbiota has not been addressed in depth. Here, we demonstrate that mice carrying the PTPN22 variant (619W mice) were protected from acute dextran sulfate sodium (DSS) colitis, but suffered from pronounced inflammation upon chronic DSS treatment. The basal microbiota composition was distinct between genotypes, and DSS-induced dysbiosis was milder in 619W mice than in WT littermates. Transfer of microbiota from 619W mice after the first DSS cycle into treatment-naive 619W mice promoted colitis, indicating that changes in microbial composition enhanced chronic colitis in those animals. This indicates that presence of the PTPN22 variant affects intestinal inflammation by modulating the host’s response to the intestinal microbiota.
Marianne R. Spalinger, Thomas S.B. Schmidt, Marlene Schwarzfischer, Larissa Hering, Kirstin Atrott, Silvia Lang, Claudia Gottier, Annelies Geirnaert, Christophe Lacroix, Xuezhi Dai, David J. Rawlings, Andrew C. Chan, Christian von Mering, Gerhard Rogler, Michael Scharl
Glial cells have emerged as key players in the central control of energy balance and etiology of obesity. Astrocytes play a central role in neural communication via the release of gliotransmitters. Acyl-CoA–binding protein–derived (ACBP-derived) endozepines are secreted peptides that modulate the GABAA receptor. In the hypothalamus, ACBP is enriched in arcuate nucleus (ARC) astrocytes, ependymocytes, and tanycytes. Central administration of the endozepine octadecaneuropeptide (ODN) reduces feeding and improves glucose tolerance, yet the contribution of endogenous ACBP in energy homeostasis is unknown. We demonstrated that ACBP deletion in GFAP+ astrocytes, but not in Nkx2.1-lineage neural cells, promoted diet-induced hyperphagia and obesity in both male and female mice, an effect prevented by viral rescue of ACBP in ARC astrocytes. ACBP+ astrocytes were observed in apposition with proopiomelanocortin (POMC) neurons, and ODN selectively activated POMC neurons through the ODN GPCR but not GABAA, and suppressed feeding while increasing carbohydrate utilization via the melanocortin system. Similarly, ACBP overexpression in ARC astrocytes reduced feeding and weight gain. Finally, the ODN GPCR agonist decreased feeding and promoted weight loss in ob/ob mice. These findings uncover ACBP as an ARC gliopeptide playing a key role in energy balance control and exerting strong anorectic effects via the central melanocortin system.
Khalil Bouyakdan, Hugo Martin, Fabienne Liénard, Lionel Budry, Bouchra Taib, Demetra Rodaros, Chloé Chrétien, Éric Biron, Zoé Husson, Daniela Cota, Luc Pénicaud, Stephanie Fulton, Xavier Fioramonti, Thierry Alquier
We studied human T cell repertoire formation using high-throughput T cell receptor β (TCRβ) complementarity-determining region 3 (CDR3) sequencing in immunodeficient mice receiving human hematopoietic stem cells (HSCs) and human thymus grafts. Replicate humanized mice generated diverse and highly divergent repertoires. We observed repertoire narrowing and increased CDR3β sharing during thymocyte selection. Whereas hydrophobicity analysis implicated self-peptides in positive selection of the overall repertoire, positive selection favored shorter shared sequences that had reduced hydrophobicity at positions 6 and 7 of CDR3βs, suggesting weaker interactions with self-peptides than were observed with unshared sequences, possibly allowing escape from negative selection. Sharing was similar between autologous and allogeneic thymi and occurred between different cell subsets. Shared sequences were enriched for allo–cross-reactive CDR3βs and for type 1 diabetes–associated autoreactive CDR3βs. Single-cell TCR sequencing showed increased sharing of CDR3αs compared with CDR3βs between mice. Our data collectively implicate preferential positive selection for shared human CDR3βs that are highly cross-reactive. Although previous studies suggested a role for recombination bias in producing “public” sequences in mice, our study is the first to our knowledge to demonstrate a role for thymic selection. Our results implicate positive selection for promiscuous TCRβ sequences that probably evade negative selection, given their low affinity for self-ligands, in the abundance of “public” human TCRβ sequences.
Mohsen Khosravi-Maharlooei, Aleksandar Obradovic, Aditya Misra, Keshav Motwani, Markus Holzl, Howard R. Seay, Susan DeWolf, Grace Nauman, Nichole Danzl, Haowei Li, Siu-hong Ho, Robert Winchester, Yufeng Shen, Todd M. Brusko, Megan Sykes
Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their prevalence in various diseases including cancer. Drug development targeting IDPs is challenging because IDPs have dynamic structure features and conventional drug design is not applicable. NUPR1 is an IDP that plays an important role in pancreatic cancer. We previously reported that trifluoperazine (TFP), an antipsychotic agent, was capable of binding to NUPR1 and inhibiting tumor growth. Unfortunately, TFP showed strong central nervous system side effects. In the present work, we undertook a multidisciplinary approach to optimize TFP based on the synergy of computer modeling, chemical synthesis, and a variety of biophysical, biochemical, and biological evaluations. A family of TFP-derived compounds was produced and the most active one, ZZW-115, showed a dose-dependent tumor regression with no neurological effects and an ability to induce cell death mainly by necroptosis. This study opens a new perspective for drug development against IDPs, demonstrating the possibility of successful ligand-based drug design for such challenging targets.
Patricia Santofimia-Castaño, Yi Xia, Wenjun Lan, Zhengwei Zhou, Can Huang, Ling Peng, Philippe Soubeyran, Adrián Velázquez-Campoy, Olga Abián, Bruno Rizzuti, José L. Neira, Juan Iovanna
Tumor-infiltrating lymphocytes (TILs) are widely associated with positive outcomes, yet carry key indicators of a systemic failed immune response against unresolved cancer. Cancer immunotherapies can reverse their tolerance phenotypes while preserving tumor reactivity and neoantigen specificity shared with circulating immune cells. We performed comprehensive transcriptomic analyses to identify gene signatures common to circulating and TILs in the context of clear cell renal cell carcinoma. Modulated genes also associated with disease outcome were validated in other cancer types. Through comprehensive bioinformatics analyses, we identified practical diagnostic markers and actionable targets of the failed immune response. On circulating lymphocytes, 3 genes (LEF1, FASLG, and MMP9) could efficiently stratify patients from healthy control donors. From their associations with resistance to cancer immunotherapies and microbial infections, we uncovered not only pan-cancer, but pan-pathology, failed immune response profiles. A prominent lymphocytic matrix metallopeptidase cell migration pathway is central to a panoply of diseases and tumor immunogenicity, correlates with multi-cancer recurrence, and identifies a feasible noninvasive approach to pan-pathology diagnoses. The differentially expressed genes we have identified warrant future investigation into the development of their potential in noninvasive precision diagnostics and precision pan-disease immunotherapeutics.
Anne Monette, Antigoni Morou, Nadia A. Al-Banna, Louise Rousseau, Jean-Baptiste Lattouf, Sara Rahmati, Tomas Tokar, Jean-Pierre Routy, Jean-François Cailhier, Daniel E. Kaufmann, Igor Jurisica, Réjean Lapointe
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