RESUMO
BACKGROUND: The ubiquitin regulatory X (UBX) domain-containing proteins (UBXNs) are putative adaptors for ubiquitin ligases and valosin-containing protein; however, their in vivo physiological functions remain poorly characterised. We recently showed that UBXN3B is essential for activating innate immunity to DNA viruses and controlling DNA/RNA virus infection. Herein, we investigate its role in adaptive immunity. METHODS: We evaluated the antibody responses to multiple viruses and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza in tamoxifen-inducible global and constitutive B cell-specific Ubxn3b knockout mice; quantified various immune populations, B lineage progenitors/precursors, B cell receptor (BCR) signalling and apoptosis by flow cytometry, immunoblotting and immunofluorescence microscopy. We also performed bone marrow transfer, single-cell and bulk RNA sequencing. FINDINGS: Both global and B cell-specific Ubxn3b knockout mice present a marked reduction in small precursor B-II (>60%), immature (>70%) and mature B (>95%) cell numbers. Transfer of wildtype bone marrow to irradiated global Ubxn3b knockouts restores normal B lymphopoiesis, while reverse transplantation does not. The mature B population shrinks rapidly with apoptosis and higher pro and activated caspase-3 protein levels were observed following induction of Ubxn3b knockout. Mechanistically, Ubxn3b deficiency leads to impaired pre-BCR signalling and cell cycle arrest. Ubxn3b knockout mice are highly vulnerable to respiratory viruses, with increased viral loads and prolonged immunopathology in the lung, and reduced production of virus-specific IgM/IgG. INTERPRETATION: UBXN3B is essential for B lymphopoiesis by maintaining constitutive pre-BCR signalling and cell survival in a cell-intrinsic manner. FUNDING: United States National Institutes of Health grants, R01AI132526 and R21AI155820.
Assuntos
Linfócitos B , Linfopoese , Camundongos Knockout , Animais , Linfopoese/genética , Camundongos , Linfócitos B/imunologia , Linfócitos B/metabolismo , COVID-19/imunologia , SARS-CoV-2/fisiologia , Transdução de Sinais , Apoptose , Receptores de Antígenos de Linfócitos B/metabolismo , HumanosRESUMO
The cytoplasmic RIG-I-like receptors (RLRs) recognize viral RNA and initiate innate antiviral immunity. RLR signaling also triggers glycolytic reprogramming through glucose transporters (GLUTs), whose role in antiviral immunity is elusive. Here, we unveil that insulin-responsive GLUT4 inhibits RLR signaling independently of glucose uptake in adipose and muscle tissues. At steady state, GLUT4 is docked at the Golgi matrix by ubiquitin regulatory X domain 9 (UBXN9, TUG). Following RNA virus infection, GLUT4 is released and translocated to the cell surface where it spatially segregates a significant pool of cytosolic RLRs, preventing them from activating IFN-ß responses. UBXN9 deletion prompts constitutive GLUT4 trafficking, sequestration of RLRs, and attenuation of antiviral immunity, whereas GLUT4 deletion heightens RLR signaling. Notably, reduced GLUT4 expression is uniquely associated with human inflammatory myopathies characterized by hyperactive interferon responses. Overall, our results demonstrate a noncanonical UBXN9-GLUT4 axis that controls antiviral immunity via plasma membrane tethering of cytosolic RLRs.
RESUMO
This protocol aims to establish a method for identifying small molecular antagonists of ß2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. ß2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling ß2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of ß2 integrins, is utilized to quantify ß2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil ß2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on ß2 integrin inhibition are assessed within 3 h. Molecules that directly target ß2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.
Assuntos
Antígenos CD18 , Moléculas de Adesão Celular , Humanos , Antígenos CD18/química , Antígenos CD18/metabolismo , Adesão Celular , Citometria de Fluxo , Moléculas de Adesão Celular/metabolismo , Neutrófilos/metabolismoRESUMO
Macrophages are central players in systemic inflammation associated with obesity and aging, termed meta-inflammation and inflammaging. Activities of macrophages elicited by the two chronic conditions display shared and distinct patterns mechanistically, resulting in multifaceted actions for their pathogenic roles. Drastically expanded tissue macrophage populations under obesity and aging stress attribute to both enhanced recruitment and local expansion. Importantly, molecular networks governing the multifaceted actions of macrophages are directly altered by environmental cues and subsequently contribute to metabolic reprogramming, resulting in meta-inflammation in obesity or inflammaging in aging. In this review, we will summarize how meta-inflammation and inflammaging affect macrophages and the molecular mechanisms involved in these processes.
Assuntos
Inflamação , Macrófagos , Humanos , Inflamação/metabolismo , Macrófagos/metabolismo , Envelhecimento/genética , Obesidade/genética , Obesidade/metabolismo , Contagem de LeucócitosRESUMO
Neutrophils are the most abundant leukocyte in human blood. They are critical for fighting infections and are involved in inflammatory diseases. Mitochondria are indispensable for eukaryotic cells, as they control the biochemical processes of respiration and energy production. Mitochondria in neutrophils have been underestimated since glycolysis is a major metabolic pathway for fuel production in neutrophils. However, several studies have shown that mitochondria are greatly involved in multiple neutrophil functions as well as neutrophil-related diseases. In this review, we focus on how mitochondrial components, metabolism, and related genes regulate neutrophil functions and relevant diseases.
Assuntos
Mitocôndrias , Neutrófilos , Humanos , Mitocôndrias/metabolismo , Neutrófilos/metabolismoRESUMO
Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting ß2 integrin activation. Using a microfluidic chamber, we found that Nexinhib20 inhibited IL-8-induced ß2 integrin-dependent human neutrophil adhesion under flow. Using a dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and ß2 integrin activation after IL-8 stimulation. Western blots of Ras-related C3 botulinum toxin substrate 1 (Rac-1)-GTP pull-down assays confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. An in vitro competition assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.
Assuntos
Antígenos CD18 , Neutrófilos , Animais , Antígenos CD18/metabolismo , Cálcio/metabolismo , Adesão Celular , Guanosina , Guanosina Trifosfato/metabolismo , Humanos , Interleucina-8/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Polifosfatos , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Neutrophil migration and activation are essential for defense against pathogens. However, this process may also lead to collateral tissue injury. We used microRNA overexpression as a platform and discovered protein-coding genes that regulate neutrophil migration. Here we show that miR-99 decreased the chemotaxis of zebrafish neutrophils and human neutrophil-like cells. In zebrafish neutrophils, miR-99 directly targets the transcriptional factor RAR-related orphan receptor alpha (roraa). Inhibiting RORα, but not the closely related RORγ, reduced chemotaxis of zebrafish and primary human neutrophils without causing cell death, and increased susceptibility of zebrafish to bacterial infection. Expressing a dominant-negative form of Rorα or disrupting the roraa locus specifically in zebrafish neutrophils reduced cell migration. At the transcriptional level, RORα regulates transmembrane signaling receptor activity and protein phosphorylation pathways. Our results, therefore, reveal previously unknown functions of miR-99 and RORα in regulating neutrophil migration and anti-microbial defense.
Assuntos
MicroRNAs , Peixe-Zebra , Animais , Movimento Celular/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Neutrófilos/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Investigation on the long-term thermal response of precast high-strength concrete (PHC) energy pile is relatively rare. This paper combines field experiments and numerical simulations to investigate the long-term thermal properties of a PHC energy pile in a layered foundation. The major findings obtained from the experimental and numerical studies are as follows: First, the thermophysical ground properties gradually produce an influence on the long-term temperature variation. For the soil layers with relatively higher thermal conductivity, the ground temperature near to the energy pile presents a slowly increasing trend, and the ground temperature response at a longer distance from the center of the PHC pile appears to be delayed. Second, the short- and long-term thermal performance of the PHC energy pile can be enhanced by increasing the thermal conductivity of backfill soil. When the thermal conductivities of backfill soil in the PHC pile increase from 1 to 4 W/(m K), the heat exchange amounts of energy pile can be enhanced by approximately 30%, 79%, 105%, and 122% at 1 day and 20%, 47%, 59%, and 66% at 90 days compared with the backfill water used in the site. However, the influence of specific heat capacity of the backfill soil in the PHC pile on the short-term or long-term thermal response can be ignored. Furthermore, the variation of the initial ground temperature is also an important factor to affect the short-and-long-term heat transfer capacity and ground temperature variation. Finally, the thermal conductivity of the ground has a significant effect on the long-term thermal response compared with the short-term condition, and the heat exchange rates rise by about 5% and 9% at 1 day and 21% and 37% at 90 days as the thermal conductivities of the ground increase by 0.5 and 1 W/(m K), respectively.