scholarly journals Activation of Mechanistic Target of Rapamycin (mTOR) in Human Endothelial Cells Infected with Pathogenic Spotted Fever Group Rickettsiae

2020 ◽  
Vol 21 (19) ◽  
pp. 7179
Author(s):  
Abha Sahni ◽  
Hema P. Narra ◽  
Sanjeev K. Sahni
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Spotted Fever ◽  
Target Of Rapamycin ◽  
Spotted Fever Group ◽  
Microvascular Endothelium ◽  
Rickettsia Species ◽  
Mechanistic Target Of Rapamycin ◽  
P70 S6k

Attributed to the tropism for host microvascular endothelium lining the blood vessels, vascular inflammation and dysfunction represent salient features of rickettsial pathogenesis, yet the details of fundamentally important pathogen interactions with host endothelial cells (ECs) as the primary targets of infection remain poorly appreciated. Mechanistic target of rapamycin (mTOR), a serine/threonine protein kinase of the phosphatidylinositol kinase-related kinase family, assembles into two functionally distinct complexes, namely mTORC1 (Raptor) and mTORC2 (Rictor), implicated in the determination of innate immune responses to intracellular pathogens via transcriptional regulation. In the present study, we investigated activation status of mTOR and its potential contributions to host EC responses during Rickettsia rickettsii and R. conorii infection. Protein lysates from infected ECs were analyzed for threonine 421/serine 424 phosphorylation of p70 S6 kinase (p70 S6K) and that of serine 2448 on mTOR itself as established markers of mTORC1 activation. For mTORC2, we assessed phosphorylation of protein kinase B (PKB or Akt) and protein kinase C (PKC), respectively, on serine 473 and serine 657. The results suggest increased phosphorylation of p70 S6K and mTOR during Rickettsia infection of ECs as early as 3 h and persisting for up to 24 h post-infection. The steady-state levels of phospho-Akt and phospho-PKC were also increased. Infection with pathogenic rickettsiae also resulted in the formation of microtubule-associated protein 1A/1B-light chain 3 (LC3-II) puncta and increased lipidation of LC3-II, a response significantly inhibited by introduction of siRNA targeting mTORC1 into ECs. These findings thus yield first evidence for the activation of both mTORC1 and mTORC2 during EC infection in vitro with Rickettsia species and suggest that early induction of autophagy in response to intracellular infection might be regulated by this important pathway known to function as a central integrator of cellular immunity and inflammation.

scholarly journals Significant Growth by Rickettsia Species within Human Macrophage-Like Cells Is a Phenotype Correlated with the Ability to Cause Disease in Mammals

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 228
Author(s):  
M. Nathan Kristof ◽  
Paige E. Allen ◽  
Lane D. Yutzy ◽  
Brandon Thibodaux ◽  
Christopher D. Paddock ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Spotted Fever ◽  
Growth Characteristic ◽  
Human Macrophage ◽  
Rickettsia Conorii ◽  
Spotted Fever Group ◽  
Rocky Mountain Spotted Fever ◽  
Phagocytic Cells ◽  
Pathogenic Potential ◽  
Rickettsia Species

Rickettsia are significant sources of tick-borne diseases in humans worldwide. In North America, two species in the spotted fever group of Rickettsia have been conclusively associated with disease of humans: Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, and Rickettsia parkeri, the cause of R. parkeri rickettsiosis. Previous work in our lab demonstrated non-endothelial parasitism by another pathogenic SFG Rickettsia species, Rickettsia conorii, within THP-1-derived macrophages, and we have hypothesized that this growth characteristic may be an underappreciated aspect of rickettsial pathogenesis in mammalian hosts. In this work, we demonstrated that multiple other recognized human pathogenic species of Rickettsia, including R. rickettsii, R. parkeri, Rickettsia africae, and Rickettsiaakari can grow within target endothelial cells as well as within PMA-differentiated THP-1 cells. In contrast, Rickettsia bellii, a Rickettsia species not associated with disease of humans, and R. rickettsii strain Iowa, an avirulent derivative of pathogenic R. rickettsii, could invade both cell types but proliferate only within endothelial cells. Further analysis revealed that similar to previous studies on R. conorii, other recognized pathogenic Rickettsia species could grow within the cytosol of THP-1-derived macrophages and avoided localization with two different markers of lysosomal compartments; LAMP-2 and cathepsin D. R. bellii, on the other hand, demonstrated significant co-localization with lysosomal compartments. Collectively, these findings suggest that the ability of pathogenic rickettsial species to establish a niche within macrophage-like cells could be an important factor in their ability to cause disease in mammals. These findings also suggest that analysis of growth within mammalian phagocytic cells may be useful to predict the pathogenic potential of newly isolated and identified Rickettsia species.

scholarly journals Suppressor of cytokine signalling protein SOCS1 and UBP43 regulate the expression of type I interferon-stimulated genes in human microvascular endothelial cells infected with Rickettsia conorii

2013 ◽  
Vol 62 (7) ◽  
pp. 968-979 ◽  
Author(s):  
Punsiri M. Colonne ◽  
Abha Sahni ◽  
Sanjeev K. Sahni
Keyword(s):  
Endothelial Cells ◽  
Transcriptional Activation ◽  
Spotted Fever ◽  
Microvascular Endothelial Cells ◽  
Rickettsia Conorii ◽  
Type I ◽  
Spotted Fever Group ◽  
Dependent Manner ◽  
Microvascular Endothelium ◽  
Microvascular Endothelial

Rickettsia conorii, the causative agent of Mediterranean spotted fever, preferentially infects human microvascular endothelium and activates pro-inflammatory innate immune responses as evidenced by enhanced expression and secretion of cytokines and chemokines. Our recent studies reveal that human microvascular endothelial cells (HMECs) infected with R. conorii also launch ‘antiviral’ host defence mechanisms typically governed by type I interferons. To summarize, infected HMECs secrete IFN-β to activate STAT1 in an autocrine/paracrine manner and display increased expression of IFN-stimulated genes, for example ISG15, which in turn activate innate responses to interfere with intracellular replication of rickettsiae. We now present evidence that UBP43 and SOCS1, known negative regulators of JAK/STAT signalling, are also induced in R. conorii-infected HMECs, of which UBP43 but not SOCS1 functions to negatively regulate STAT1 activation. Interestingly, UBP43 induction is almost completely abolished in the presence of IFN-β-neutralizing antibody, implicating an important role for UBP43 as a feedback inhibitor for IFN-β-mediated STAT1 activation. In contrast, SOCS1 expression is only partially affected by IFN-β neutralization, implicating potential involvement of as-yet-unidentified IFN-independent mechanism(s) in SOCS1 induction during R. conorii infection. A number of IFN-stimulated genes, including ISG15, OAS1, MX1, IRF1, IRF9 and TAP1 are also induced in an IFN-β-dependent manner, whereas GBP1 remains unaffected by IFN-β neutralization. Increased STAT1 phosphorylation in HMECs subjected to UBP43 knockdown led to transcriptional activation of OAS1, MX1 and GBP1, confirming the negative regulatory role of UBP43. Although IRF1, IRF9 and TAP1 were induced by IFN-β, siRNA-mediated silencing of UBP43 or SOCS1 did not significantly affect their transcriptional activation. Expression of ISG15 was, however, increased in HMECs transfected with siRNA for UBP43 and SOCS1. Thus, unique regulatory patterns of induced expression of UBP43, SOCS1 and IFN-stimulated genes represent pathogen-specific responses underlying IFN-β-mediated host endothelial signalling during the pathogenesis of spotted fever group rickettsiosis.

scholarly journals Quantitative Analyses of Variations in the Injury of Endothelial Cells Elicited by 11 Isolates of Rickettsia rickettsii

2001 ◽  
Vol 8 (4) ◽  
pp. 788-796 ◽  
Author(s):  
Marina E. Eremeeva ◽  
Gregory A. Dasch ◽  
David J. Silverman
Keyword(s):  
North Carolina ◽  
Endothelial Cells ◽  
Reduced Glutathione ◽  
Spotted Fever ◽  
Ixodid Ticks ◽  
Spotted Fever Group ◽  
Cellular Injury ◽  
Rickettsia Rickettsii ◽  
Cytopathic Effects ◽  
Group I

ABSTRACT Eleven isolates of spotted fever group rickettsiae from the blood of patients or ixodid ticks from North and South America were characterized. All isolates were identified as Rickettsia rickettsii using restriction fragment length polymorphism analysis of a 532-bp rOmpA gene fragment obtained by PCR. The ability of the R. rickettsii isolates to elicit cytopathic effects and parameters of oxidative injury were examined in cultured human EA.hy 926 endothelial cells. Cytopathic effects were determined by direct observation of infected cultures, by measuring the release of cytoplasmic lactate dehydrogenase (LDH), and by determination of intracellular pools of peroxide and reduced glutathione. Four biotypes of R. rickettsii were defined. Group I included two highly cytopathic isolates from Montana, Bitterroot and Sheila Smith, and three isolates from Maryland, North Carolina, and Brazil. These isolates rapidly damaged cells, released large amounts of cytoplasmic LDH, caused accumulation of intracellular peroxide, and depleted intracellular pools of reduced glutathione. Group II contained three isolates, two from Montana, Hlp#2 and Lost Horse Canyon, and an isolate from Colombia, which were similar to group I but caused either lower responses in LDH release or smaller changes in intracellular peroxide levels. The group III isolates, Sawtooth from Montana and 84JG from North Carolina, caused lower cellular injury by all measures. Group IV isolate Price T from Montana was the least cytopathic and caused minimal alterations of all parameters measured. Understanding the molecular basis for the varied cellular injury caused by different isolates of R. rickettsii may contribute to improved treatment of Rocky Mountain spotted fever and to the rapid identification of those isolates which are more likely to cause fulminant disease.

Isolation and characterization of human and rat cardiac microvascular endothelial cells

1993 ◽  
Vol 264 (2) ◽  
pp. H639-H652 ◽  
Author(s):  
M. Nishida ◽  
W. W. Carley ◽  
M. E. Gerritsen ◽  
O. Ellingsen ◽  
R. A. Kelly ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelium ◽  
Adult Rat ◽  
Isolation And Characterization ◽  
Ventricular Tissue ◽  
Microvascular Endothelial ◽  
Cardiac Microvascular Endothelial Cells

Although reciprocal intercellular signaling may occur between endocardial or microvascular endothelium and cardiac myocytes, suitable in vitro models have not been well characterized. In this report, we describe the isolation and primary culture of cardiac microvascular endothelial cells (CMEC) from both adult rat and human ventricular tissue. Differential uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) indicated that primary isolates of rat CMEC were quite homogeneous, unlike primary isolates of human ventricular tissue, which required cell sorting based on Ac-LDL uptake to create endothelial cell-enriched primary cultures. The endothelial phenotype of both primary isolates and postsort subcultured CMEC and their microvascular origin were determined by characteristic histochemical staining for a number of endothelial cell-specific markers, by the absence of cells with fibroblast or pericyte-specific cell surface antigens, and by rapid tube formation on purified basement membrane preparations. Importantly, [3H]-thymidine uptake was increased 2.3-fold in subconfluent rat microvascular endothelial cells 3 days after coculture with adult rat ventricular myocytes because of release of an endothelial cell mitogen(s) into the extracellular matrix, resulting in a 68% increase in cell number compared with CMEC in monoculture. Thus biologically relevant cell-to-cell interactions can be modeled with this in vitro system.

scholarly journals Rickettsia spp. in rodent-attached ticks and first evidence of Spotted fever Group Rickettsia species Candidatus Rickettsia uralica in Europe.

2020 ◽  
Author(s):  
Maria Vikentjeva ◽  
Julia Geller ◽  
Jaanus Remm ◽  
Irina Golovljova
Keyword(s):  
Spotted Fever ◽  
Ixodid Ticks ◽  
Distribution Area ◽  
Myodes Glareolus ◽  
Ixodes Persulcatus ◽  
Spotted Fever Group ◽  
Human Pathogens ◽  
Rickettsia Species ◽  
Questing Ticks ◽  
Spotted Fever Group Rickettsia

Abstract BACKGROUND Rickettsia spp. are human pathogens that cause a number of diseases and are transmitted by arthropods, including ixodid ticks. Estonia contributes a region, where the distribution area of two exophilic tick species of known medical importance, Ixodes persulcatus and I. ricinus, overlap. The presence of the nidicolous rodent-associated I. trianguliceps has recently been shown for Estonia. Although there is no Estonian data available on human disease caused by tick-borne Rickettsia spp., the presence of three Rickettsia species in non-nidicolous ticks, albiet at very dissimilar rates, was also previously reported. The aim of this studywas to screen, identify and characterize Rickettsia species in nidicolous and non-nidicolous ticks attached to rodents. RESULTS Nymphs and larvae of I. ricinus ( n = 1004), I . persulcatus ( n = 75) and I. trianguliceps ( n = 117) attached to rodents and shrews caught in different parts of Estonia were studied for the presence of Rickettsia spp. by nested PCR. Ticks were removed from 314 small animals of 5 species (bank voles Myodes glareolus , yellow necked mice Apodemus flavicollis , striped field mice A. agrarius, pine voles M. subterranius and common shrews S. araneus) . Rickettsial DNA was detected in 8,7% (103/1186) studied ticks. In addition to R. helvetica, previously found in questing ticks, this study reports the first identification of the recently described I. trianguliceps- associated Candidatus R. uralica in west of the Ural.

scholarly journals Lipid-dependent Akt-ivity: where, when, and how

2019 ◽  
Vol 47 (3) ◽  
pp. 897-908 ◽  
Author(s):  
Katharina M. Siess ◽  
Thomas A. Leonard
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Target Of Rapamycin ◽  
Endomembrane System ◽  
Essential Protein ◽  
Mechanistic Target Of Rapamycin ◽  
Subcellular Compartments ◽  
Membrane Bound ◽  
Phosphoinositide 3 Kinase

Abstract Akt is an essential protein kinase activated downstream of phosphoinositide 3-kinase and frequently hyperactivated in cancer. Canonically, Akt is activated by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2, which phosphorylate it on two regulatory residues in its kinase domain upon targeting of Akt to the plasma membrane by PI(3,4,5)P3. Recent evidence, however, has shown that, in addition to phosphorylation, Akt activity is allosterically coupled to the engagement of PI(3,4,5)P3 or PI(3,4)P2 in cellular membranes. Furthermore, the active membrane-bound conformation of Akt is protected from dephosphorylation, and Akt inactivation by phosphatases is rate-limited by its dissociation. Thus, Akt activity is restricted to membranes containing either PI(3,4,5)P3 or PI(3,4)P2. While PI(3,4,5)P3 has long been associated with signaling at the plasma membrane, PI(3,4)P2 is gaining increasing traction as a signaling lipid and has been implicated in controlling Akt activity throughout the endomembrane system. This has clear implications for the phosphorylation of both freely diffusible substrates and those localized to discrete subcellular compartments.

Physiological concentration of atrial natriuretic peptide induces endothelial regeneration in vitro

2003 ◽  
Vol 284 (4) ◽  
pp. H1388-H1397 ◽  
Author(s):  
Hyun Kook ◽  
Hiroshi Itoh ◽  
Bong Seok Choi ◽  
Naoki Sawada ◽  
Kentaro Doi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Physiological Concentration ◽  
Cell Numbers ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Regeneration In Vitro

Both nitric oxide (NO) and natriuretic peptides produce apoptosis of vascular smooth muscle cells. However, there is evidence that NO induces endothelial cell proliferation, which suggests that there is a difference in the response of endothelial cells to natriuretic peptides. The purpose of this study was to investigate the effect of atrial natriuretic peptide (ANP) on human endothelial cell survival. ANP within the physiological concentration (10−11mol/l) induced a 52% increase in the number of human coronary arterial endothelial cells and a 63% increase in human umbilical vein endothelial cells at a low concentration of serum. The increase in cell numbers was blocked by pretreatment with RP8-CPT-cGMP (RP8), a cGMP-dependent protein kinase inhibitor, with wortmannin, an Akt/PKB inhibitor, and with PD-98059, an ERK1/2 inhibitor. In a Transwell migration test, ANP also increased the cell migration, and RP8, wortmannin, and PD-98059 blocked this increase. A wound healing assay was performed to examine the effects of ANP on regeneration in vitro. ANP increased both cell numbers and migration, but the effects were blocked by the above three kinase inhibitors. ANP increased the expression of phospho-Akt and of phospho-ERK1/2 within 1.5 h. These results suggest that ANP can potentiate endothelial regeneration by cGMP-dependent protein kinase stimulation and subsequent Akt and ERK1/2 activations.

scholarly journals CD36 mediates albumin transcytosis by dermal but not lung microvascular endothelial cells: role in fatty acid delivery

2019 ◽  
Vol 316 (5) ◽  
pp. L740-L750 ◽  
Author(s):  
Hira Raheel ◽  
Siavash Ghaffari ◽  
Negar Khosraviani ◽  
Victoria Mintsopoulos ◽  
Derek Auyeung ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fatty Acid ◽  
Scavenger Receptor ◽  
Subcutaneous Fat ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelium ◽  
Knockout Mouse Model ◽  
Microvascular Endothelial ◽  
Total Body

In healthy blood vessels, albumin crosses the endothelium to leave the circulation by transcytosis. However, little is known about the regulation of albumin transcytosis or how it differs in different tissues; its physiological purpose is also unclear. Using total internal reflection fluorescence microscopy, we quantified transcytosis of albumin across primary human microvascular endothelial cells from both lung and skin. We then validated our in vitro findings using a tissue-specific knockout mouse model. We observed that albumin transcytosis was saturable in the skin but not the lung microvascular endothelial cells, implicating a receptor-mediated process. We identified the scavenger receptor CD36 as being both necessary and sufficient for albumin transcytosis across dermal microvascular endothelium, in contrast to the lung where macropinocytosis dominated. Mutations in the apical helical bundle of CD36 prevented albumin internalization by cells. Mice deficient in CD36 specifically in endothelial cells exhibited lower basal permeability to albumin and less basal tissue edema in the skin but not in the lung. Finally, these mice also exhibited a smaller subcutaneous fat layer despite having identical total body weights and circulating fatty acid levels as wild-type animals. In conclusion, CD36 mediates albumin transcytosis in the skin but not the lung. Albumin transcytosis may serve to regulate fatty acid delivery from the circulation to tissues.

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