scholarly journals Macrophage Selenoproteins Restrict Intracellular Replication of Francisella tularensis and Are Essential for Host Immunity

2021 ◽  
Vol 12 ◽  
Author(s):  
Rachel L. Markley ◽  
Katherine H. Restori ◽  
Bhuvana Katkere ◽  
Sarah E. Sumner ◽  
McKayla J. Nicol ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Post Infection ◽  
Pathogenic Bacteria ◽  
Ex Vivo ◽  
Alveolar Epithelial Cells ◽  
Bacterial Disease ◽  
Systemic Spread ◽  
Bacterial Replication ◽  
Deficient Mice

The essential micronutrient Selenium (Se) is co-translationally incorporated as selenocysteine into proteins. Selenoproteins contain one or more selenocysteines and are vital for optimum immunity. Interestingly, many pathogenic bacteria utilize Se for various biological processes suggesting that Se may play a role in bacterial pathogenesis. A previous study had speculated that Francisella tularensis, a facultative intracellular bacterium and the causative agent of tularemia, sequesters Se by upregulating Se-metabolism genes in type II alveolar epithelial cells. Therefore, we investigated the contribution of host vs. pathogen-associated selenoproteins in bacterial disease using F. tularensis as a model organism. We found that F. tularensis was devoid of any Se utilization traits, neither incorporated elemental Se, nor exhibited Se-dependent growth. However, 100% of Se-deficient mice (0.01 ppm Se), which express low levels of selenoproteins, succumbed to F. tularensis-live vaccine strain pulmonary challenge, whereas 50% of mice on Se-supplemented (0.4 ppm Se) and 25% of mice on Se-adequate (0.1 ppm Se) diet succumbed to infection. Median survival time for Se-deficient mice was 8 days post-infection while Se-supplemented and -adequate mice was 11.5 and >14 days post-infection, respectively. Se-deficient macrophages permitted significantly higher intracellular bacterial replication than Se-supplemented macrophages ex vivo, corroborating in vivo observations. Since Francisella replicates in alveolar macrophages during the acute phase of pneumonic infection, we hypothesized that macrophage-specific host selenoproteins may restrict replication and systemic spread of bacteria. F. tularensis infection led to an increased expression of several macrophage selenoproteins, suggesting their key role in limiting bacterial replication. Upon challenge with F. tularensis, mice lacking selenoproteins in macrophages (TrspM) displayed lower survival and increased bacterial burden in the lung and systemic tissues in comparison to WT littermate controls. Furthermore, macrophages from TrspM mice were unable to restrict bacterial replication ex vivo in comparison to macrophages from littermate controls. We herein describe a novel function of host macrophage-specific selenoproteins in restriction of intracellular bacterial replication. These data suggest that host selenoproteins may be considered as novel targets for modulating immune response to control a bacterial infection.

Apoptosis and DNA damage in type 2 alveolar epithelial cells cultured from hyperoxic rats

1998 ◽  
Vol 274 (5) ◽  
pp. L714-L720 ◽  
Author(s):  
Sue Buckley ◽  
Lora Barsky ◽  
Barbara Driscoll ◽  
Kenneth Weinberg ◽  
Kathryn D. Anderson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Epithelial Cells ◽  
Cellular Response ◽  
Ex Vivo ◽  
Keratinocyte Growth Factor ◽  
Alveolar Epithelial Cells ◽  
Alveolar Epithelial ◽  
Dna Laddering

Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.

scholarly journals The Characterization of chIFITMs in Avian Coronavirus Infection In Vivo, Ex Vivo and In Vitro

Genes ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 918
Author(s):  
Angela Steyn ◽  
Sarah Keep ◽  
Erica Bickerton ◽  
Mark Fife
Keyword(s):  
Post Infection ◽  
Viral Infections ◽  
Ex Vivo ◽  
Large Family ◽  
Respiratory Pathogen ◽  
Restriction Factors ◽  
Respiratory Illnesses ◽  
Bird Mortality

The coronaviruses are a large family of enveloped RNA viruses that commonly cause gastrointestinal or respiratory illnesses in the infected host. Avian coronavirus infectious bronchitis virus (IBV) is a highly contagious respiratory pathogen of chickens that can affect the kidneys and reproductive systems resulting in bird mortality and decreased reproductivity. The interferon-inducible transmembrane (IFITM) proteins are activated in response to viral infections and represent a class of cellular restriction factors that restrict the replication of many viral pathogens. Here, we characterize the relative mRNA expression of the chicken IFITM genes in response to IBV infection, in vivo, ex vivo and in vitro using the pathogenic M41-CK strain, the nephropathogenic QX strain and the nonpathogenic Beaudette strain. In vivo we demonstrate a significant upregulation of chIFITM1, 2, 3 and 5 in M41-CK- and QX-infected trachea two days post-infection. In vitro infection with Beaudette, M41-CK and QX results in a significant upregulation of chIFITM1, 2 and 3 at 24 h post-infection. We confirmed a differential innate response following infection with distinct IBV strains and believe that our data provide new insights into the possible role of chIFITMs in early IBV infection.

scholarly journals Asymptomatic Carriage of Group A Streptococcus Is Associated with Elimination of Capsule Production

2014 ◽  
Vol 82 (9) ◽  
pp. 3958-3967 ◽  
Author(s):  
Anthony R. Flores ◽  
Brittany E. Jewell ◽  
Randall J. Olsen ◽  
Samuel A. Shelburne ◽  
Nahuel Fittipaldi ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Group A Streptococcus ◽  
Ex Vivo ◽  
Human Subjects ◽  
Full Genome Sequencing ◽  
Isogenic Strain ◽  
Capsule Production ◽  
Key Factor ◽  
Group A

ABSTRACTHumans commonly carry pathogenic bacteria asymptomatically, but despite decades of study, the underlying molecular contributors remain poorly understood. Here, we show that a group A streptococcus carriage strain contains a frameshift mutation in thehasAgene resulting in loss of hyaluronic acid capsule biosynthesis. This mutation was repaired by allelic replacement, resulting in restoration of capsule production in the isogenic derivative strain. The “repaired” isogenic strain was significantly more virulent than the carriage strain in a mouse model of necrotizing fasciitis and had enhanced growthex vivoin human blood. Importantly, the repaired isogenic strain colonized the mouse oropharynx with significantly greater bacterial burden and had significantly reduced ability to internalize into cultured epithelial cells than the acapsular carriage strain. We conducted full-genome sequencing of 81 strains cultured serially from 19 epidemiologically unrelated human subjects and discovered the common theme that mutations negatively affecting capsule biosynthesis arisein vivoin thehasoperon. The significantly decreased capsule production is a key factor contributing to the molecular détente between pathogen and host. Our discoveries suggest a general model for bacterial pathogens in which mutations that downregulate or ablate virulence factor production contribute to carriage.

Anticoagulant and antithrombotic properties of platelet protease nexin-1

Blood ◽  
2010 ◽  
Vol 115 (1) ◽  
pp. 97-106 ◽  
Author(s):  
Yacine Boulaftali ◽  
Frédéric Adam ◽  
Laurence Venisse ◽  
Véronique Ollivier ◽  
Benjamin Richard ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Type Plasminogen Activator ◽  
Healthy Donors ◽  
Protease Nexin ◽  
Deficient Mice

AbstractProtease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

Platelet ADP receptors contribute to the initiation of intravascular coagulation

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 594-600 ◽  
Author(s):  
Catherine Leon ◽  
Meike Alex ◽  
Antje Klocke ◽  
Eberhard Morgenstern ◽  
Christine Moosbauer ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Intravascular Coagulation ◽  
Adp Receptors ◽  
Adp Receptor ◽  
Deficient Mice ◽  
Storage Pools

Abstract While the adenosine 5′-diphosphate (ADP) pathway is known to enhance thrombus formation by recruiting platelets and leukocytes to the primary layer of collagen-adhering platelets, its role for the initiation of coagulation has not been revealed. Ex vivo inhibition of the P2Y12 ADP receptor by clopidogrel administration diminished the rapid exposure of tissue factor (TF), the major initiator of coagulation, in conjugates of platelets with leukocytes established by the contact of whole blood with fibrillar collagen. Under in vitro conditions, the P2Y12 and P2Y1 ADP receptors were both found to be implicated in the exposure of TF in collagen-activated whole blood. Immunoelectron-microscopy revealed that collagen elicited the release of TF from its storage pools within the platelets. Functional activation of the intravascular TF was reduced by inhibition of the ADP receptors, partially due to the disruption of the platelet-neutrophil adhesions. Injection of collagen into the venous system of mice increased the number of thrombin-antithrombin complexes, indicative for the formation of thrombin in vivo. In P2Y1-deficient mice, the ability of collagen to enhance the generation of thrombin was impaired. In conclusion, the platelet ADP pathway supports the initiation of intravascular coagulation, which is likely to contribute to the concomitant formation of fibrin at the site of the growing thrombus.

Impaired “outside-in” integrin αIIbβ3 signaling and thrombus stability in TSSC6-deficient mice

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 1911-1918 ◽  
Author(s):  
Matt W. Goschnick ◽  
Lai-Man Lau ◽  
Janet L. Wee ◽  
Yong S. Liu ◽  
P. Mark Hogarth ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Fluorescein Isothiocyanate ◽  
Clot Retraction ◽  
In Vivo Evaluation ◽  
Integrin Αiibβ3 ◽  
Normal Platelet ◽  
Granule Secretion ◽  
Platelet Spreading ◽  
Deficient Mice

AbstractWe investigated the role of the hematopoietic-specific tetraspanin superfamily member, TSSC6, in platelet function using wild-type mice and TSSC6-deficient mice. TSSC6 is expressed on the surface of murine platelets and is up-regulated by thrombin stimulation, indicating an intracellular pool of TSSC6. Immunoprecipitation/Western blot studies reveal a constitutive physical association of TSSC6 with the integrin αIIbβ3 complex under strong detergent conditions. In vivo evaluation of hemostasis by tail bleeding revealed increased bleeding time, volume of blood lost, and evidence of tail rebleeds in TSSC6 null mice, indicating unstable hemostasis. Using ex vivo techniques, we showed that TSSC6-deficient platelets exhibited impaired kinetics of clot retraction, platelet aggregation at lower doses of PAR-4, and collagen and platelet spreading on fibrinogen in the presence of normal integrin αIIbβ3 expression. TSSC6-deficient platelets showed normal alpha granule secretion, normal “insideout” integrin αIIbβ3 signaling (fluorescein isothiocyanate [FITC]–fibrinogen and JON/A binding), and normal platelet adhesion on fibrinogen. Furthermore, we show that absence of platelet TSSC6 affects the secondary stability of arterial thrombi in vivo upon vascular injury. These data demonstrate that TSSC6 appears to regulate integrin αIIbβ3 “outside-in” signaling events in platelets and is necessary for stability of arterial thrombi in vivo.

scholarly journals Targeted Deletion of the Lipopolysaccharide (LPS)-binding Protein Gene Leads to Profound Suppression of LPS Responses Ex Vivo, whereas In Vivo Responses Remain Intact

1997 ◽  
Vol 186 (12) ◽  
pp. 2051-2056 ◽  
Author(s):  
Mark M. Wurfel ◽  
Brian G. Monks ◽  
Robin R. Ingalls ◽  
Russell L. Dedrick ◽  
Russell Delude ◽  
...  
Keyword(s):  
Lipid Transfer Protein ◽  
Ex Vivo ◽  
Binding Protein ◽  
Embryonic Stem ◽  
Cellular Responses ◽  
Tnf Α ◽  
Deficient Mice ◽  
Lps Binding

Gram-negative bacterial lipopolysaccharide (LPS) stimulates phagocytic leukocytes by interacting with the cell surface protein CD14. Cellular responses to LPS are markedly potentiated by the LPS-binding protein (LBP), a lipid-transfer protein that binds LPS aggregates and transfers LPS monomers to CD14. LBP also transfers LPS to lipoproteins, thereby promoting the neutralization of LPS. LBP present in normal plasma has been shown to enhance the LPS responsiveness of cells in vitro. The role of LBP in promoting LPS responsiveness in vivo was tested in LBP-deficient mice produced by gene targeting in embryonic stem cells. Whole blood from LBP-deficient animals was 1,000-fold less responsive to LPS as assessed by the release of tumor necrosis factor (TNF)-α. Blood from gene-targeted mice was devoid of immunoreactive LBP, essentially incapable of transferring LPS to CD14 in vitro, and failed to support cellular responses to LPS. These activities were restored by the addition of exogenous recombinant murine LBP to the plasma. Despite these striking in vitro findings, no significant differences in TNF-α levels were observed in plasma from wild-type and LBP-deficient mice injected with LPS. These data suggest the presence of an LBP-independent mechanism for responding to LPS. These LBP knockout mice may provide a tool for discovering the nature of the presumed second mechanism for transferring LPS to responsive cells.

scholarly journals Role of Acinetobactin-Mediated Iron Acquisition Functions in the Interaction of Acinetobacter baumannii Strain ATCC 19606Twith Human Lung Epithelial Cells, Galleria mellonella Caterpillars, and Mice

2012 ◽  
Vol 80 (3) ◽  
pp. 1015-1024 ◽  
Author(s):  
Jennifer A. Gaddy ◽  
Brock A. Arivett ◽  
Michael J. McConnell ◽  
Rafael López-Rojas ◽  
Jerónimo Pachón ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Acinetobacter Baumannii ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Iron Acquisition ◽  
Alveolar Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Content Type ◽  
Alveolar Epithelial

Acinetobacter baumannii, which causes serious infections in immunocompromised patients, expresses high-affinity iron acquisition functions needed for growth under iron-limiting laboratory conditions. In this study, we determined that the initial interaction of the ATCC 19606Ttype strain with A549 human alveolar epithelial cells is independent of the production of BasD and BauA, proteins needed for acinetobactin biosynthesis and transport, respectively. In contrast, these proteins are required for this strain to persist within epithelial cells and cause their apoptotic death. Infection assays usingGalleria mellonellalarvae showed that impairment of acinetobactin biosynthesis and transport functions significantly reduces the ability of ATCC 19606Tcells to persist and kill this host, a defect that was corrected by adding inorganic iron to the inocula. The results obtained with theseex vivoandin vivoapproaches were validated using a mouse sepsis model, which showed that expression of the acinetobactin-mediated iron acquisition system is critical for ATCC 19606Tto establish an infection and kill this vertebrate host. These observations demonstrate that the virulence of the ATCC 19606Tstrain depends on the expression of a fully active acinetobactin-mediated system. Interestingly, the three models also showed that impairment of BasD production results in an intermediate virulence phenotype compared to those of the parental strain and the BauA mutant. This observation suggests that acinetobactin intermediates or precursors play a virulence role, although their contribution to iron acquisition is less relevant than that of mature acinetobactin.

scholarly journals Ex Vivo Enteroids Recapitulate In Vivo Citrulline Production in Mice

2018 ◽  
Vol 148 (9) ◽  
pp. 1415-1420 ◽  
Author(s):  
Xiaoying Wang ◽  
Yang Yuan ◽  
Inka C Didelija ◽  
Mahmoud A Mohammad ◽  
Juan C Marini
Keyword(s):  
Ex Vivo ◽  
Transcript Abundance ◽  
Mutant Mice ◽  
Ex Vivo Model ◽  
Endogenous Production ◽  
Gene Coding ◽  
Cycle Disorders ◽  
Deficient Mice

Abstract Background The endogenous production of arginine relies on the synthesis of citrulline by enteral ornithine transcarbamylase (OTC). Mutations in the gene coding for this enzyme are the most frequent cause of urea cycle disorders. There is a lack of correlation between in vivo metabolic function and DNA sequence, transcript abundance, or in vitro enzyme activity. Objective The goal of the present work was to test the hypothesis that enteroids, a novel ex vivo model, are able to recapitulate the in vivo citrulline production of wild-type (WT) and mutant mice. Methods Six-week-old male WT and OTC-deficient mice [sparse fur and abnormal skin (spf-ash) mutation] were studied. Urea and citrulline fluxes were determined in vivo, and OTC abundance was measured in liver and gut tissue. Intestinal crypts were isolated and cultured to develop enteroids. Ex vivo citrulline production and OTC abundance were determined in these enteroids. Results Liver OTC abundance was lower (mean ± SE: 0.16 ± 0.01 compared with 1.85 ± 0.18 arbitrary units; P < 0.001) in spf-ash mice than in WT mice, but there was no difference in urea production. In gut tissue, OTC was barely detectable in mutant mice; despite this, a lower but substantial citrulline production (67 ± 3 compared with 167 ± 8 µmol · kg−1 · h−1; P < 0.001) was shown in the mutant mice. Enteroids recapitulated the in vivo findings of a very low OTC content accompanied by a reduced citrulline production (1.07 ± 0.20 compared with 4.64 ± 0.44 nmol · µg DNA−1 · d−1; P < 0.001). Conclusions Enteroids recapitulate in vivo citrulline production and offer the opportunity to study the regulation of citrulline production in a highly manipulable system.

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