Real-time Intravital Imaging Research Articles

Heme-Oxygenase 1 Mediated Activation of Cyp3A11 Protects Against Non-Steroidal Pain Analgesics Induced Acute Liver Damage in Sickle Cell Disease Mice

Pain constitutes a significant comorbidity associated with sickle cell disease (SCD). Analgesics serve as the primary method for pain management; however, the long-term effects of these drugs on the liver of SCD patients remain not completely understood. Using real-time intravital imaging, we analyzed the effect of non-steroidal analgesics (NSA) in the liver of control and SS (SCD) mice. Remarkably, we found completely opposing effects in the liver of control and SS mice post-NSA treatment. Whereas SS mice were able to better tolerate the NSA treatment acutely compared to their littermate controls, in the long term, these mice showed delayed resolution of liver injury and exacerbated fibrosis compared to control mice. Mechanistically, we found that SS mice were protected from cytotoxicity caused by NSA at baseline due to the significant activation of hepatic Kupffer cells, which produced heme-oxygenase 1 (HO-1). HO-1 promoted the activation of the cytoprotective enzyme Cyp3A11, which inhibited hepatic damage caused by NSA. However, in the long term, depletion of hepatic Kupffer cells led to reduced expression of HO-1, which blocked the activation of Cyp3A11, resulting in fibrosis and a delay in the resolution of liver injury and inflammation. These preclinical data provide a strong proof-of-concept for HO-1 as well as Cyp3A11 as cytoprotectors against NSA-induced liver damage in the Townes model of SCD and support further development of these compounds as potential novel therapies for end-organ damage in SCD.

Exceptional Uptake, Limited Protein Expression: Liver Macrophages Lost in Translation of Synthetic mRNA.

Most gene therapies exert their actions via manipulation of hepatocytes (parenchymal cells) and the reasons behind the suboptimal performance of synthetic mRNA in non-parenchymal cells (NPC) such as Kupffer cells (KC), and liver macrophages, remain unclear. Here, the spatio-temporal distribution of mRNA encoding enhanced green fluorescent protein (Egfp), siRNA, or both co-encapsulated into lipid nanoparticles (LNP) in the liver in vivo using real-time intravital imaging is investigated. Although both KC and hepatocytes demonstrate comparable high and rapid uptake of mRNA-LNP and siRNA-LNP in vivo, the translation of Egfp mRNA occurs exclusively in hepatocytes during intravital imaging. Despite attempts such as inhibiting intracellular ribonuclease, substituting uridine bases in mRNA with pseudouridine, and using a different ionizable lipid in the LNP mixture, no substantial increase in Egfp translation by NPC is possible. The investigation reveals that hepatocytes, which are distinct from other liver cells due to their polyploidy, exhibit significantly elevated levels of total RNA and protein, along with a higher proportion of ribosomal protein per individual cell. Consequently, fundamental cellular differences account for the low mRNA translation observed in NPC. The findings therefore suggest that cellular biology imposes a natural limitation on synthetic mRNA translation that is strongly influenced by cellular ploidy.

Abstract 4125528: Interleukin-6-Modulated Inflammation Augments Deep Vein Thrombosis Organization in Mice

Background: A disorganized thrombus jeopardizes its stability, leading to an augmented risk of fatal acute complications following deep vein thrombosis (DVT). One of its plausible causes is impaired immune cell responses. While the importance of the cytokine interleukin-6 (IL-6) in immune responses is well established, its action towards DVT organization is poorly elucidated. Accordingly, we aimed to investigate the role of IL-6 in the acute immunoinflammatory orchestration of DVT organization in vivo. Methods and Results: We performed total ligation of the infrarenal inferior vena cava (IVC) in 8–12-week-old male C57BL/6J mice a day after starting continuous, subcutaneous mini-osmotic pump infusion of recombinant IL-6 (rIL-6; 1.2 µg/day, n = 21) or vehicle (n = 21), after which DVT pathology was examined. Exogenous IL-6 administration significantly decreased DVT mass at day 2 (16.6±1.9 vs. 23.0±0.9 mg/cm; p<0.01) and day 4 (16.2±1.6 vs. 21.4±0.8 mg/cm; p<0.05) compared to vehicle-treated mice. Intriguingly, rIL-6-treated mice’s DVT exhibited distal tropism of a macroscopically white lesion with abundant neutrophils and platelet accumulation, indicating enhanced DVT organization (Figures A–B). Quantitative polymerase chain reaction (qPCR) analysis revealed the distal edge upregulation of leukocyte chemoattractants, leukocyte-platelet interaction markers, and extracellular matrix regulator genes, confirming that the IL-6-driven inflammatory response was linked to augmented DVT organization. Spatiotemporally, real-time intravital imaging on stasis- and irradiation-induced saphenous vein thrombosis among intraperitoneal rIL-6 (0.3 µg/mouse)-treated mice demonstrated marked acute leukocyte and platelet recruitment with subsequent aggregation to the distal edge of the thrombus (Figures C–D), which consequently yielded an enhanced thrombus resolution at the distal edge. Conclusions: Our findings suggest that IL-6-driven inflammation results in distal tropism of leukocyte-platelet infiltration, which is important for modulating thrombus organization and subsequent DVT amelioration. This study presents a novel insight into the immunothrombosis process, with potential clinical implications for DVT.

Lung microvascular occlusion by platelet-rich neutrophil-platelet aggregates promotes cigarette smoke-induced severe flu.

Cigarette smoking is associated with a higher risk of ICU admissions among patients with flu. However, the etiological mechanism by which cigarette smoke (CS) exacerbates flu remains poorly understood. Here, we show that a mild dose of influenza A virus promotes a severe lung injury in mice preexposed to CS but not room air for 4 weeks. Real-time intravital (in vivo) lung imaging revealed that the development of acute severe respiratory dysfunction in CS- and flu-exposed mice was associated with the accumulation of platelet-rich neutrophil-platelet aggregates (NPAs) in the lung microcirculation within 2 days following flu infection. These platelet-rich NPAs formed in situ and grew larger over time to occlude the lung microvasculature, leading to the development of pulmonary ischemia followed by the infiltration of NPAs and vascular leakage into the alveolar air space. These findings suggest, for the first time to our knowledge, that an acute onset of platelet-driven thrombo-inflammatory response in the lung contributes to the development of CS-induced severe flu.

Impaired Hemoglobin Clearance By Liver Sinusoidal Endothelium Promotes Liver Damage in Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic disorder that affects 100,000 African Americans and millions of people worldwide. Intra-erythrocytic polymerization of sickle hemoglobin (HbS) promotes erythrocyte sickling, impaired rheology, ischemia and hemolysis, leading to the development of progressive liver injury in SCD. Liver resident macrophages and monocytes are known to enable the clearance of HbS, however, the role of liver sinusoidal endothelial cells (LSECs) in HbS clearance and liver injury in SCD remains unknown. Using real-time intravital ( in vivo) imaging of the liver as well as primary culture of human and mice LSECs, we show for the first time that liver injury is associated with accumulation of HbS and iron in the LSECs, leading to the development of senescence in LSECs. Hepatic monocytes were observed to attenuate LSEC-senescence by accelerating HbS clearance in the liver of SS mice, however, this protection was impaired in P-selectin-deficient SCD mice secondary to reduced monocyte recruitment in the liver. These findings are the first to suggest that LSECs contribute to HbS clearance and LSEC-senescence promotes progressive liver injury in SS mice. Our results reveal a novel insight into the pathogenesis of hemolysis induced chronic liver injury in SCD caused by LSEC senescence. Identifying the regulators of LSEC mediated HbS clearance may lead to new therapies to prevent the progression of liver and other organ injuries in SCD.

Impaired hemoglobin clearance by sinusoidal endothelium promotes vaso-occlusion and liver injury in sickle cell disease.

Sickle cell disease (SCD) is a monogenic disorder that affects 100,000 African-Americans and millions of people worldwide. Intra-erythrocytic polymerization of sickle hemoglobin (HbS) promotes erythrocyte sickling, impaired rheology, ischemia and hemolysis, leading to the development of progressive liver injury in SCD. Liver-resident macrophages and monocytes are known to enable the clearance of HbS; however, the role of liver sinusoidal endothelial cells (LSEC) in HbS clearance and liver injury in SCD remains unknown. Using real-time intravital (in vivo) imaging in mice liver as well as flow cytometric analysis and confocal imaging of primary human LSEC, we show for the first time that liver injury in SCD is associated with accumulation of HbS and iron in the LSEC, leading to senescence of these cells. Hemoglobin uptake by LSEC was mediated by micropinocytosis. Hepatic monocytes were observed to attenuate LSEC senescence by accelerating HbS clearance in the liver of SCD mice; however, this protection was impaired in P-selectin-deficient SCD mice secondary to reduced monocyte recruitment in the liver. These findings are the first to suggest that LSEC contribute to HbS clearance and HbS-induced LSEC senescence promotes progressive liver injury in SCD mice. Our results provide a novel insight into the pathogenesis of hemolysis-induced chronic liver injury in SCD caused by LSEC senescence. Identifying the regulators of LSEC-mediated HbS clearance may lead to new therapies to prevent the progression of liver injury in SCD.

Intravital Imaging of Leukocyte-Endothelial Interaction in Hindlimb Ischemia/Reperfusion Injury by Intravital Multiphoton Microscopy.

Ischemia/reperfusion injury in skeletal muscle leads to sterile inflammation and affects structure and function permanently. However, the main understanding of the molecular and cellular mechanisms mainly relies on in vitro and ex vivo investigations. Recent advances in intravital microscopy allow for insights into dynamic processes at the cellular and subcellular level under both physiological and pathophysiological conditions. Real-time intravital imaging by two-photon microscopy (2P-IVM) has emerged as a powerful tool in the evaluation of the cell-cell interaction and molecular biology of leukocytes in live animals. Acute ischemic injury in limbs may occur due to crush syndrome, compartment syndrome, and vascular diseases and injury as in acute peripheral arterial occlusion, caused by a diverse array of pathological conditions. Iatrogenic revascularization and restoration of perfusion results paradoxically in aggravated tissue injury. Furthermore, the effects of IR-injured skeletal muscle in clinical conditions such as compartment syndrome or crush syndrome may induce rhabdomyolysis and are associated with so-called remote injuries as acute kidney dysfunction. Here, we discuss the considerations for and describe a 2P-IVM method designed for visualization of leukocyte-endothelial interaction. This chapter will provide a detailed experimental setup and a step-by-step protocol for the dynamic imaging of leukocyte-endothelial-interaction in an ischemia/reperfusion injury model.

Real-Time Intravital Imaging of Acoustic Cluster Therapy-Induced Vascular Effects in the Murine Brain.

The blood-brain barrier (BBB) is an obstacle for cerebral drug delivery. Controlled permeabilization of the barrier by external stimuli can facilitate the delivery of drugs to the brain. Acoustic Cluster Therapy (ACT®) is a promising strategy for transiently and locally increasing the permeability of the BBB to macromolecules and nanoparticles. However, the mechanism underlying the induced permeability change and subsequent enhanced accumulation of co-injected molecules requires further elucidation. In this study, the behavior of ACT® bubbles in microcapillaries in the murine brain was observed using real-time intravital multiphoton microscopy. For this purpose, cranial windows aligned with a ring transducer centered around an objective were mounted to the skull of mice. Dextrans labeled with 2 MDa fluorescein isothiocyanate (FITC) were injected to delineate the blood vessels and to visualize extravasation. Activated ACT® bubbles were observed to alter the blood flow, inducing transient and local increases in the fluorescence intensity of 2 MDa FITC-dextran and subsequent extravasation in the form of vascular outpouchings. The observations indicate that ACT® induced a transient vascular leakage without causing substantial damage to the vessels in the brain. The study gave novel insights into the mechanism underlying ACT®-induced enhanced BBB permeability which will be important considering treatment optimization for a safe and efficient clinical translation of ACT®.

Hypoxia-Induced miR-210 Promotes Endothelial Cell Permeability and Angiogenesis via Exosomes in Pancreatic Ductal Adenocarcinoma.

Exosomes have been proven to play important diagnostic, regulatory, or communication roles in tumorigenesis, tumor progression, or metastasis; in recent studies, lots of molecules, including miRNAs, were found to be aberrantly expressed in tumor exosomes and were correlated with tumor development. However, studies about the expression, relationship, or control mechanisms of miRNAs in exosomes in pancreatic ductal adenocarcinoma (PDAC) are scarce and urgently needed. The aim of this article was to identify and investigate abnormally expressed miRNAs in PDAC exosomes in vivo and in vitro. Microarray studies were used to detect aberrantly expressed miRNAs in PDAC exosomes, and miR-210 expression in cells or exosomes was further analyzed by qRT-PCR. Bioinformatics analyses, dual-luciferase assays, WB, and other assays were utilized to explore the miRNA molecular mechanisms. The living cell coculture model and immunofluorescence analysis were employed to image the communication between PDAC cells and endothelial cells. Other biological experiments in the study include a real-time intravital imaging system, EdU, transwell, xenograft models, and so on. miR-210 is significantly expressed in PDAC exosomes and malignant cells. High miR-210 significantly facilitated tumor angiogenesis, cell invasion, and proliferation in PDAC cells. Further mechanistic detection revealed that miR-210 negatively regulated EFNA3 expression and participated in the PI3K/AKT/VEGFA or Wnt/Β-catenin/RHOA pathways, thus promoting tumor angiogenesis and cellular permeability. PDAC cells promote endothelial angiogenesis or permeability via miR-210 transmission by tumor exosomes. Exosomal miR-210 promotes PDAC progression in vivo. Further detection of PDAC plasma exosomal miR-210 suggests that exosomal miR-210 expression was high and significantly associated with vascular invasion and TNM stage and was an independent risk factor for PDAC overall survival. PDAC cell-secreted exosomes could promote angiogenesis and cellular permeability of neighboring endothelial angiogenesis or permeability via miR-210 transmission. Exosomal miR-210 may play important roles in tumor biology and may be a useful prognostic marker in PDAC.

Revealing the nanometric structural changes in myocardial infarction models by time-lapse intravital imaging.

In the development of bioinspired nanomaterials for therapeutic applications, it is very important to validate the design of nanomaterials in the disease models. Therefore, it is desirable to visualize the change of the cells in the diseased site at the nanoscale. Heart diseases often start with structural, morphological, and functional alterations of cardiomyocyte components at the subcellular level. Here, we developed straightforward technique for long-term real-time intravital imaging of contracting hearts without the need of cardiac pacing and complex post processing images to understand the subcellular structural and dynamic changes in the myocardial infarction model. A two-photon microscope synchronized with electrocardiogram signals was used for long-term in vivo imaging of a contracting heart with subcellular resolution. We found that the structural and dynamic behaviors of organelles in cardiomyocytes closely correlated with heart function. In the myocardial infarction model, sarcomere shortening decreased from ∼15% (healthy) to ∼8% (diseased) as a result of impaired cardiac function, whereas the distances between sarcomeres increased by 100 nm (from 2.11 to 2.21 μm) in the diastolic state. In addition, T-tubule system regularity analysis revealed that T-tubule structures that were initially highly organized underwent significant remodeling. Morphological remodeling and changes in dynamic activity at the subcellular level are essential to maintain heart function after infarction in a heart disease model.

VlsE, the nexus for antigenic variation of the Lyme disease spirochete, also mediates early bacterial attachment to the host microvasculature under shear force.

Hematogenous dissemination is a critical step in the evolution of local infection to systemic disease. The Lyme disease (LD) spirochete, which efficiently disseminates to multiple tissues, has provided a model for this process, in particular for the key early event of pathogen adhesion to the host vasculature. This occurs under shear force mediated by interactions between bacterial adhesins and mammalian cell-surface proteins or extracellular matrix (ECM). Using real-time intravital imaging of the Lyme spirochete in living mice, we previously identified BBK32 as the first LD spirochetal adhesin demonstrated to mediate early vascular adhesion in a living mouse; however, deletion of bbk32 resulted in loss of only about half of the early interactions, suggesting the existence of at least one other adhesin (adhesin-X) that promotes early vascular interactions. VlsE, a surface lipoprotein, was identified long ago by its capacity to undergo rapid antigenic variation, is upregulated in the mammalian host and required for persistent infection in immunocompetent mice. In immunodeficient mice, VlsE shares functional overlap with OspC, a multi-functional protein that displays dermatan sulfate-binding activity and is required for joint invasion and colonization. In this research, using biochemical and genetic approaches as well as intravital imaging, we have identified VlsE as adhesin-X; it is a dermatan sulfate (DS) adhesin that efficiently promotes transient adhesion to the microvasculature under shear force via its DS binding pocket. Intravenous inoculation of mice with a low-passage infectious B. burgdorferi strain lacking both bbk32 and vlsE almost completely eliminated transient microvascular interactions. Comparative analysis of binding parameters of VlsE, BBK32 and OspC provides a possible explanation why these three DS adhesins display different functionality in terms of their ability to promote early microvascular interactions.

Intravital Imaging with Two-Photon Microscopy: A Look into the Kidney

Fluorescence microscopy has represented a crucial technique to explore the cellular and molecular mechanisms in the field of biomedicine. However, the conventional one-photon microscopy exhibits many limitations when living samples are imaged. The new technologies, including two-photon microscopy (2PM), have considerably improved the in vivo study of pathophysiological processes, allowing the investigators to overcome the limits displayed by previous techniques. 2PM enables the real-time intravital imaging of the biological functions in different organs at cellular and subcellular resolution thanks to its improved laser penetration and less phototoxicity. The development of more sensitive detectors and long-wavelength fluorescent dyes as well as the implementation of semi-automatic software for data analysis allowed to gain insights in essential physiological functions, expanding the frontiers of cellular and molecular imaging. The future applications of 2PM are promising to push the intravital microscopy beyond the existing limits. In this review, we provide an overview of the current state-of-the-art methods of intravital microscopy, focusing on the most recent applications of 2PM in kidney physiology.

Intravital Imaging of Hepatic Blood Biliary Barrier in Live Mice.

Understanding the kinetics and spatiotemporal interactions of living cells within the tissue environment requires real-time imaging. The introduction of two-photon microscopy has substantially boosted the power of live intravital imaging, making it possible to obtain information of individual cells in near-physiologic conditions within intact tissues nondestructively. Intravital imaging of the liver has proved useful in understanding its 3D structure, function, and dynamic cellular interactions. Recently we have shown that integrity of the blood-bile barrier in different physiologic and pathophysiologic conditions can be imaged in real time using intravital microscopy. Here we discuss the real-time intravital imaging method to visualize blood-bile barrier integrity in the murine liver. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Live imaging in the mouse liver Support Protocol: Monitoring vital signs of the mouse during live liver imaging Basic Protocol 2: Visualizing blood and bile transport using intravital microscopy.

State-of-the-art microscopy to understand islets of Langerhans: what to expect next?

The discovery of Langerhans and microscopic description of islets in the pancreas were crucial steps in the discovery of insulin. Over the past 150 years, many discoveries in islet biology and type 1 diabetes have been made using powerful microscopic techniques. In the past decade, combination of new probes, animal and tissue models, application of new biosensors and automation of light and electron microscopic methods and other (sub)cellular imaging modalities have proven their potential in understanding the beta cell under (patho)physiological conditions. The imaging evolution, from fluorescent jellyfish to real‐time intravital functional imaging, the revolution in automation and data handling and the increased resolving power of analytical imaging techniques are now converging. Here, we review innovative approaches that address islet biology from new angles by studying cells and molecules at high spatiotemporal resolution and in live models. Broad implementation of these cellular imaging techniques will shed new light on cause/consequence of (mal)function in islets of Langerhans in the years to come.

Released Myeloperoxidase Attenuates Neutrophil Migration and Accumulation in Inflamed Tissue.

Neutrophil (PMN) recruitment to sites of insult is critical for host defense, however excessive PMN activity and tissue accumulation can lead to exacerbated inflammation and injury. Myeloperoxidase (MPO) is a PMN azurophilic granule enzyme, which together with H2O2, forms a powerful antimicrobial system designed to kill ingested bacteria. Intriguingly, in addition to intracellular killing of invading microorganisms and extracellular tissue damage due generation of ROS, soluble MPO has been directly implicated in modulating cellular responses and tissue homeostasis. In the current work, we used several models of inflammation, murine and human PMNs and state-of-the-art intravital microscopy to examine the effect of MPO on PMN migration and tissue accumulation. We found that in the absence of functional MPO (MPO knockout, KO mice) inflammatory PMN tissue accumulation was significantly enhanced. We determined that the elevated numbers of PMNs in MPO knockout mice was not due to enhanced viability, but due to increased migratory ability. Acute PMN migration in models of zymosan-induced peritonitis or ligated intestinal loops induced by intraluminal administration of PMN-chemokine CXCL1 was increased over 2-fold in MPO KO compared to wild type (WT) mice. Using real-time intravital imaging of inflamed mouse cremaster muscle and ex vivo PMN co-culture with inflamed endothelial cells (ECs) we demonstrate that elevated migration of MPO KO mice was due to enhanced adhesive interactions. In contrast, addition of soluble recombinant MPO both in vivo and ex vivo diminished PMN adhesion and migration. Although MPO has been previously suggested to bind CD11b, we found no significant difference in CD11b expression in either resting or activated PMNs and further showed that the MPO binding to the PMN surface is not specific to CD11b. As such, our data identify MPO as a novel regulator of PMN trafficking in inflammation.

Engineering of Reversible Luminescent Probes for Real-Time Intravital Imaging of Liver Injury and Repair

As the major organ for drug metabolism and detoxification, the liver is prone to damage and severely impaired functionality. The treatment of liver diseases is based on a clear understanding of the...

Myeloid cell-derived HOCl is a paracrine effector that trans-inhibits IKK/NF-κB in melanoma cells and limits early tumor progression.

The myeloperoxidase (MPO) system of myeloid-derived cells (MDCs) is central to cellular innate immunity. Upon MDC activation, MPO is secreted into phagosomes where it catalyzes the production of hypochlorous acid (HOCl), a potent chlorinating oxidant. Here, we demonstrated that the myeloid lineage-restricted MPO-HOCl system had antitumor effects in early melanoma growth in aged mice. Orthotopic melanomas grew more slowly in immunocompetent MPO+/+ host mice compared to age-matched syngeneic MPO-/- mice. Real-time intravital tumor imaging in vivo and in cell cocultures revealed a cell-cell proximity-dependent association between MDC-derived MPO enzyme activity and blockade of ligand-induced IκBα degradation in tumor cells. HOCl directly trans-inhibited IκB kinase (IKK) activity in tumor cells, thereby decreasing nuclear factor κB (NF-κB) transcriptional activation and inducing changes in the expression of genes involved in metabolic pathways, cell cycle progression, and DNA replication. By contrast, HOCl induced transcriptional changes in CD8+ T cells related to ion transport and the MAPK and PI3K-AKT signaling pathways that are associated with T cell activation. MPO increased the circulating concentrations of the myeloid cell-attracting cytokines CXCL1 and CXCL5, enhanced local infiltration by CD8+ cytotoxic T cells, and decreased tumor growth. Overall, these data reveal a role for MDC-derived HOCl as a small-molecule paracrine signaling factor that trans-inhibits IKK in melanoma tumor cells, mediating antitumor responses during early tumor progression.

Engineering of Reversible Luminescent Probes for Real-time Intravital Imaging of Liver Injury and Repair

Open AccessCCS Chemistry27 Feb 2021Engineering of Reversible Luminescent Probes for Real-time Intravital Imaging of Liver Injury and Repair Xiao Liu, Linhui He, Xiangyang Gong, Yue Yang, Dan Cheng, Juanjuan Peng, Lu Wang, Xiao-Bing Zhang and Lin Yuan Xiao Liu Google Scholar More articles by this author , Linhui He Google Scholar More articles by this author , Xiangyang Gong Google Scholar More articles by this author , Yue Yang Google Scholar More articles by this author , Dan Cheng Google Scholar More articles by this author , Juanjuan Peng Google Scholar More articles by this author , Lu Wang Google Scholar More articles by this author , Xiao-Bing Zhang Google Scholar More articles by this author and Lin Yuan Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202100679 SectionsSupplemental MaterialAboutPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail As the primary organ for drug metabolism and detoxification, the liver is prone to be damaged and its functionality is severely impaired. The treatment of liver diseases is based on a clear understanding of the process underlying liver injury and repair. However, intravital real-time imaging of liver injury and repair is still limited due to the lack of in vivo reversible visualization methods. To this end, we proposed a rational design strategy for the development of reversible upconversion luminescence nanoprobe that allows real-time and in vivo imaging of liver injury and repair processes. As a proof of demonstration, we first developed a small molecule probe NB3 which can reversibly respond to related analytes of early liver injury (peroxynitrite, ONOO-) and liver repair (glutathione, GSH). The small molecule probe was then integrated with a core-shell upconversion nanoparticle to form a sophisticated nanoprobe. Compared to traditional small molecule probes, this nanoprobe exhibited higher selectivity to ONOO−, longer retention time in liver, and a wider response dynamic range to GSH after oxidized by ONOO−. The novel nanoprobe facilitated the successful monitoring and discrimination among the different degrees of liver injury and repair in a mouse model. Download figure Download PowerPoint Previous articleNext article FiguresReferencesRelatedDetails Issue AssignmentNot Yet AssignedSupporting Information Copyright & Permissions© 2021 Chinese Chemical Society Downloaded 137 times Loading ...

Patrolling Alveolar Macrophages Conceal Bacteria from the Immune System to Maintain Homeostasis

During respiration, humans breathe in more than 10,000 liters of non-sterile air daily, allowing some pathogens access to alveoli. Interestingly, alveoli outnumber alveolar macrophages (AMs), which favors alveoli devoid of AMs. If AMs, like most tissue macrophages, are sessile, then this numerical advantage would be exploited by pathogens unless neutrophils from the blood stream intervened. However, this would translate to omnipresent persistent inflammation. Developing invivo real-time intravital imaging of alveoli revealed AMs crawling in and between alveoli using the pores of Kohn. Importantly, these macrophages sensed, chemotaxed, and, with high efficiency, phagocytosed inhaled bacterial pathogens such as P.aeruginosa and S.aureus, cloaking the bacteria from neutrophils. Impairing AM chemotaxis toward bacteria induced superfluous neutrophil recruitment, leading to inappropriate inflammation and injury. In a disease context, influenza A virus infection impaired AM crawling via the type II interferon signaling pathway, and this greatly increased secondary bacterial co-infection.

Abstract 379: Myeloperoxidase-produced HOCl is a paracrine effector linking myeloid cells to NF-κB signaling in melanoma, mediating anti-tumor responses during early melanoma progression

Abstract The myeloperoxidase (MPO) system of myeloid-derived cells (MDCs) is central to cellular innate immunity and the microbicidal activities of phagocytes, while also impacting the pathogenesis of many disorders, including cancer. Upon MDC activation, MPO is secreted into phagosomes, catalyzing the production of hypochlorous acid (HOCl). HOCl is a potent chlorinating oxidant with significant biological effects, but the mechanisms by which HOCl impacts cancer growth remain unresolved. Herein, we demonstrate that the myeloid lineage-restricted MPO-HOCl system has anti-tumor effects during early melanoma progression. Orthotopic melanomas grew slower in immuno-competent host mice wild type for MPO+/+ compared to syngeneic MPO-null (MPO-/-) animals. Using a specific and potent inhibitor of MPO, 4-aminobenzoic acid hydrazide (4-ABAH), melanoma tumor growth in MPO+/+ mice continuously administered 4-ABAH, pharmacologically mimicked the MPO-/- phenotype. Real-time intravital tumor imaging in vivo using skinfold window chamber animal models as well as live cell co-culture studies revealed a cell-cell proximity-dependent association between MDC-derived MPO activity and blockade of ligand-induced IκBα degradation in tumor cells. Real-time bioluminescent imaging and quantitative monitoring of the canonical NF-κB pathway was assessed in melanoma cells stably expressing bioluminescent reporters: a transcriptionally-activated NF-κB-promoter-driven Firefly luciferase (FLuc) reporter or a fusion reporter under the control of an NF-κB-responsive promoter fused to IκBα-FLuc reporter. HOCl was shown to directly inhibit IκB kinase (IKK) activity, thereby decreasing NF-κB transcriptional activation within melanomas, increasing circulating levels of the myeloid-attracting cytokines CXCL1 and CXCL5, enhancing local infiltration of CD8+ cytotoxic T cells, and dampening tumor growth. Overall, these data reveal a novel role for MDC-derived HOCl as a small molecule paracrine signaling factor that trans-inhibits IKK in melanoma cells, mediating anti-tumor responses during early tumor progression. Citation Format: Tracy W. Liu, Seth T. Gammon, Ping Yang, David T. Fuentes, David Piwnica-Worms. Myeloperoxidase-produced HOCl is a paracrine effector linking myeloid cells to NF-κB signaling in melanoma, mediating anti-tumor responses during early melanoma progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 379.