Association of host cell glutathione metabolism with Chlamydia pneumoniae infection phenotype

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Profiling Antichlamydial Compounds in Monocyte and Macrophage Cell Models

Chlamydia pneumoniae is an obligate intracellular human pathogen whose primary site of infection is the respiratory tract. In addition to respiratory tract infections, such as sinusitis and pharyngitis, C. pneumoniae has been related to several chronic inflammatory diseases of which atherosclerosis is the most widely studied. In order to contribute to the pathogenesis e.g. in atheromatous arteries, C. pneumoniae must disseminate from lungs to other tissues. This translocation occurs via peripheral blood mononuclear cells (PBMCs), mainly monocytes and macrophages. The presence of C. pneumoniae has been reported to induce inflammatory molecule production and alter redox balance in these cells, while also promoting their foam cell formation, migration and adherence. The ability of C. pneumoniae to persist inside the PBMCs, and thus become refractory to conventional antibiotics complicates the management of the associated underlying infection in chronic inflammatory diseases. Most C. pneumoniae susceptibility studies are currently based on the use of permissive cell lines, such as epithelial and endothelial cell lines in which the bacteria are actively replicating, which leaves the persistent infections unnoticed. Therefore, in addition to new active compounds against C. pneumoniae, new methods to study their effects against the persistent chlamydial infection are urgently needed. In this study, dibenzocyclooctadiene lignans, new antichlamydial compounds originating from berries of medicinal plant, Schisandra chinensis, were profiled regarding different aspects of C. pneumoniae-PBMC interactions. Human THP-1, a monocyte and macrophage cell line, and a murine macrophage cell line RAW264.7 were used in the studies to examine the effects of the schisandrin lignans on C. pneumoniae infection and their influence on host cell responses. Oxidative stress is the basic pathological mechanism underlying a spectrum of chronic inflammatory diseases, and it largely contributes to the consequences of C. pneumoniae infection as well. In this work, C. pneumoniae was found to induce proinflammatory cytokine, interleukin (IL)-12 secretion in the human monocytic cell line. It also increased the intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels in macrophages, and it had an impact on the concentration of glutathione (GSH), the major small-molecule antioxidant, in macrophages. Gene expression analysis of murine macrophages revealed that C. pneumoniae suppressed the peroxisome proliferator-activated receptor γ (PPARγ) transcription in the cells, which influences lipid metabolism and inflammatory responses in these cells. Schisandrin lignans had an impact on C. pneumoniae-induced alterations in these cell models. Schisandrin B and schisandrin C reduced the elevated interleukin (IL) -12 cytokine levels, as well as the LPS induced IL-6 and IL-12 levels. Schisandrin lignans also affected cellular oxidative balance by elevating the basal ROS levels while simultaneously reducing the ROS or NO levels induced by infection or LPS. The redox balance alteration was also shown within the GSH levels, which were reduced by the lignans in THP-1 monocytes and macrophages but elevated in RAW264.7 cells. Schisandrin B additionally upregulated the transcription of genes involved in GSH synthesis, GCL and GGT-1. Schisandrin B and schisandrin C also reduced the C. pneumoniae-induced macrophage foam cell formation and altered the related expression of PPARγ and ABCA1 genes. In this work, a new platform for studying the C. pneumoniae transfer between lung epithelial cells and phagocytes, is also introduced. The platform can be used in lead compound profiling studies against C. pneumoniae infection. Mitogen-activated protein kinase (MAPK) inhibitors were found to inhibit the transfer of the infection, serving as reference compounds in future profiling studies. This work provides new information about the C. pneumoniae infection in monocyte-macrophage cell models, while also offering valuable insight on the antichlamydial lead compounds, dibenzocyclooctadiene lignans. These lignans were shown to suppress the C. pneumoniae induced pathological changes in the host cells. With newly identified antichlamydial activities and novel study methods, we can explore more impactful approaches to overcoming C. pneumoniae-induced chronic inflammatory diseases.Chlamydia pneumoniae on solunsisäinen hengitystiepatogeeni. Infektioiden, kuten poskiontelontulehduksen, nielutulehduksen ja keuhkokuumeen lisäksi C. pneumoniae on liitetty moniin kroonisiin tulehduksellisiin sairauksiin. Eniten näistä on tutkittu bakteerin yhteyttä ateroskleroosiin. Jotta patogeeni pystyisi osallistumaan esimerkiksi aterooman muodostumiseen valtimoissa on sen kyettävä siirtymään ensisijaiselta infektiopaikaltaan, hengityselimistöstä, muihin kudoksiin. Tämä siirtymä tapahtuu mononukleaaristen valkosolujen, enimmäkseen monosyyttien ja makrofagien välityksellä. C. pneumoniae -infektion on näissä soluissa todettu lisäävän tulehdusvälittäjäaineiden tuotantoa, vaikuttavan solunsisäiseen hapetus-pelkistystasapainoon sekä edistävän migraatiota ja adheesiota ja vaahtosolujen muodostusta. Monosyyteissä ja makrofageissa C. pneumoniae -infektio voi muuntua persistenttiin muotoon, jolloin bakteerien replikaatio pysähtyy ja niistä tulee vastustuskykyisiä perinteisille antibiooteille. Tämä vaikeuttaa infektion hävittämistä ja edesauttaa kroonisten tulehdussairauksien syntyä. Suurin osa C. pneumoniae -bakteerin herkkyysmäärityksistä perustuu permissiivisten solujen, kuten epiteeli- tai endoteelisolujen käyttöön, joissa C. pneumoniae replikoituu aktiivisesti. Tällöin persistentti infektio jää helposti huomaamatta. Tämän vuoksi uusien aktiivisten yhdisteiden lisäksi tarvitaan uusia menetelmiä persistentin C. pneumoniae -infektion tutkimiseen. Tässä työssä tutkittiin uusia antiklamydiaalisia yhdisteitä, dibentsosyklo-oktadieenilignaaneja, jotka on alun perin eristetty Schisandra chinensis -lääkekasvin marjoista. Näiden lignaanien kykyä vaikuttaa C. pneumoniae -infektion aiheuttamiin muutoksiin tutkittiin monosyyteissä ja makrofageissa. Työssä käytettiin ihmisen monosyytti-makrofagisolulinjaa THP-1 sekä hiiren makrofagisolulinjaa RAW264.7. Useat C. pneumoniae -infektion vaikutukset soluissa liittyvät oksidatiiviseen stressiin, joka on myös keskeinen patologinen tekijä kroonisissa tulehdussairauksissa. Saamiemme tulosten perusteella infektio lisäsi solunsisäisten reaktiivisten happi- ja typpiradikaalien määrää sekä vaikutti glutationin (GSH), merkittävän pienmolekyylirakenteisen antioksidantin, pitoisuuteen soluissa. Lisäksi osoitettiin, että C. pneumoniae -infektio lisäsi tulehdusta edistävän sytokiinin, interleukiini-12:n (IL-12) tuotantoa ihmisen monosyyttisolulinjassa. Hiiren makrofageissa tehdyssä geeniekspressioanalyysissä C. pneumoniae -infektio vähensi PPARγ:n transkriptiota, mikä vaikuttaa lipidimetaboliaan ja tulehdusvasteisiin solussa. S. chinensis -lignaanit vaikuttivat C. pneumoniae –infektion aiheuttamiin vasteisiin tutkituissa solumalleissa. Schisandriini B ja schisandriini C laskivat solujen IL-12-tasoja sekä lipopolysakkaridin (LPS) vaikutuksesta kohonneita IL-6- ja IL-12-tasoja. Lignaanit vaikuttivat myös solujen hapetus-pelkistystasapainoon nostamalla solunsisäisiä ROS-tasoja, mutta toisaalta laskemalla niitä LPS-stimulaation tai C. pneumoniae -infektion jälkeen. Vaikutus hapetus-pelkistystasapainoon ilmeni myös muuttuneina GSH-tasoina, jotka laskivat lignaanien vaikutuksesta THP-1-monosyyteissä ja makrofageissa mutta nousivat RAW264.7-makrofageissa. Schisandriini B kohotti myös GSH-synteesiin liittyvien geenien, GCL:n ja GGT-1:n, ekspressiotasoja. Sekä schisandriini B että schisandriini C vähensivät C. pneumoniae -infektiosta johtuvaa vaahtosolujen muodostusta sekä muuttivat siihen liittyvien geenien, PPARγ:n ja ABCA1:n, ekspressiotasoja. Tässä työssä esitellään myös uusi menetelmä, jolla voidaan tutkia C. pneumoniae -bakteerin siirtymistä keuhkoepiteelin ja fagosyyttien välillä. Menetelmää voidaan hyödyntää uusien C. pneumoniae -infektioon vaikuttavien johtoyhdisteiden karakterisoinnissa. Osana tutkimusta osoitimme lisäksi MAPK-inhibiittoreiden soveltuvuuden referenssiyhdisteiksi menetelmää käytettäessä. Tämä työ tarjoaa uutta tietoa C. pneumoniae -infektioista monosyytti-makrofagisolumalleissa. Tutkimus valotti myös C. pneumoniae -infektioita vastaan tehokkaaksi havaittujen schisandriinilignaanien solutason vaikutuksia. Väitöstyössä esitellyt löydökset ja menetelmät edesauttavat C. pneumoniae -infektioihin liitettyjen kroonisten tulehdussairauksien tutkimusta ja tuovat meidät näin lähemmäksi uusia hoitokeinoja

A platform for studying the transfer of Chlamydia pneumoniae infection between respiratory epithelium and phagocytes

The obligate intracellular bacterium, Chlamydia pneumoniae, has been identified as a risk factor for several chronic inflammatory diseases in addition to respiratory tract infections. The dissemination of C. pneumoniae from respiratory tract to secondary sites of infection occurs via infected monocyte / macrophage line cells, in which C. pneumoniae can persist as an antibiotic-refractory phenotype. To allow more detailed studies on the epithelium-monocyte/macrophage transition of the infection, new in vitro bioassays are needed. To this end, a coculture system with human continuous cell lines was established. Respiratory epithelial HL cells were infected with C. pneumoniae and THP-1 monocytes were added into the cultures at 67 h post infection. After a 5 h coculture, THP-1 cells were collected with a biotinylated HLA antibody and streptavidin-coated magnetic beads and C. pneumoniae genome copy numbers in THP-1 determined by quantitative PCR. The assay was optimized for cell densities, incubation time, THP-1 separation technique and buffer composition, and its robustness was demonstrated by a Z' value of 0.6. The mitogen-activated protein kinase (MAPK) inhibitors: SP600125 (JNK inhibitor), SB203580 (p38 inhibitor) and FR180204 (ERK inhibitor) suppressed the transfer of C. pneumoniae from HL to THP-1 cells, making them suitable positive controls for the assay. Based on analysis of separate steps of the process, the MAPK inhibitors suppress the bacterial entry to THP-1 cells. The transfer of C. pneumoniae from epithelium to phagocytes represents a crucial step in the establishment of persistent infections by this pathogen, and the presented methods enables future studies to block this process by therapeutic means.Peer reviewe

Impact of dibenzocyclooctadiene lignans from Schisandra chinensis on the redox status and activation of human innate immune system cells

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Assaying Chlamydia pneumoniae Persistence in Monocyte-Derived Macrophages Identifies Dibenzocyclooctadiene Lignans as Phenotypic Switchers

Antibiotic-tolerant persister bacteria involve frequent treatment failures, relapsing infections and the need for extended antibiotic treatment. The virulence of an intracellular human pathogen C. pneumoniae is tightly linked to its propensity for persistence and means for its chemosensitization are urgently needed. In the current work, persistence of C. pneumoniae clinical isolate CV6 was studied in THP-1 macrophages using quantitative PCR and quantitative culture. A dibenzocyclooctadiene lignan schisandrin reverted C. pneumoniae persistence and promoted productive infection. The concomitant administration of schisandrin and azithromycin resulted in significantly improved bacterial eradication compared to sole azithromycin treatment. In addition, the closely related lignan schisandrin C was superior to azithromycin in eradicating the C. pneumoniae infection from the macrophages. The observed chemosensitization of C. pneumoniae was associated with the suppression of cellular glutathione pools by the lignans, implying to a previously unknown aspect of chlamydia–host interactions. These data indicate that schisandrin lignans induce a phenotypic switch in C. pneumoniae, promoting the productive and antibiotic-susceptible phenotype instead of persistence. By this means, these medicinal plant -derived compounds show potential as adjuvant therapies for intracellular bacteria resuscitation

Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus.

The present study investigated Staphylococcus aureus ATCC25923 surfaceomes (cell surface proteins) during prolonged growth by subjecting planktonic and biofilm cultures (initiated from exponential or stationary cells) to label-free quantitative surfaceomics and phenotypic confirmations. The abundance of adhesion, autolytic, hemolytic, and lipolytic proteins decreased over time in both growth modes, while an opposite trend was detected for many tricarboxylic acid (TCA) cycle, reactive oxygen species (ROS) scavenging, Fe-S repair, and peptidolytic moonlighters. In planktonic cells, these changes were accompanied by decreasing and increasing adherence to hydrophobic surface and fibronectin, respectively. Specific RNA/DNA binding (cold-shock protein CspD and ribosomal proteins) and the immune evasion (SpA, ClfA, and IsaB) proteins were notably more abundant on fully mature biofilms initiated with stationary-phase cells (SDBF) compared to biofilms derived from exponential cells (EDBF) or equivalent planktonic cells. The fully matured SDBF cells demonstrated higher viability in THP-1 monocyte/macrophage cells compared to the EDBF cells. Peptidoglycan strengthening, specific urea-cycle, and detoxification enzymes were more abundant on planktonic than biofilm cells, indicating the activation of growth-mode specific pathways during prolonged cultivation. Thus, we show that S. aureus shapes its surfaceome in a growth mode-dependent manner to reach high levofloxacin tolerance (>200-times the minimum biofilm inhibitory concentration). This study also demonstrates that the phenotypic state of the cells prior to biofilm formation affects the immune-evasion and persistence-related traits of S. aureus.Peer reviewe

Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus

The present study investigated Staphylococcus aureus ATCC25923 surfaceomes (cell surface proteins) during prolonged growth by subjecting planktonic and biofilm cultures (initiated from exponential or stationary cells) to label-free quantitative surfaceomics and phenotypic confirmations. The abundance of adhesion, autolytic, hemolytic, and lipolytic proteins decreased over time in both growth modes, while an opposite trend was detected for many tricarboxylic acid (TCA) cycle, reactive oxygen species (ROS) scavenging, Fe-S repair, and peptidolytic moonlighters. In planktonic cells, these changes were accompanied by decreasing and increasing adherence to hydrophobic surface and fibronectin, respectively. Specific RNA/DNA binding (cold-shock protein CspD and ribosomal proteins) and the immune evasion (SpA, ClfA, and IsaB) proteins were notably more abundant on fully mature biofilms initiated with stationary-phase cells (SDBF) compared to biofilms derived from exponential cells (EDBF) or equivalent planktonic cells. The fully matured SDBF cells demonstrated higher viability in THP-1 monocyte/macrophage cells compared to the EDBF cells. Peptidoglycan strengthening, specific urea-cycle, and detoxification enzymes were more abundant on planktonic than biofilm cells, indicating the activation of growth-mode specific pathways during prolonged cultivation. Thus, we show that S. aureus shapes its surfaceome in a growth mode-dependent manner to reach high levofloxacin tolerance (>200-times the minimum biofilm inhibitory concentration). This study also demonstrates that the phenotypic state of the cells prior to biofilm formation affects the immune-evasion and persistence-related traits of S. aureus

Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus

The present study investigated Staphylococcus aureus ATCC25923 surfaceomes (cell surface proteins) during prolonged growth by subjecting planktonic and biofilm cultures (initiated from exponential or stationary cells) to label-free quantitative surfaceomics and phenotypic confirmations. The abundance of adhesion, autolytic, hemolytic, and lipolytic proteins decreased over time in both growth modes, while an opposite trend was detected for many tricarboxylic acid (TCA) cycle, reactive oxygen species (ROS) scavenging, Fe-S repair, and peptidolytic moonlighters. In planktonic cells, these changes were accompanied by decreasing and increasing adherence to hydrophobic surface and fibronectin, respectively. Specific RNA/DNA binding (cold-shock protein CspD and ribosomal proteins) and the immune evasion (SpA, ClfA, and IsaB) proteins were notably more abundant on fully mature biofilms initiated with stationary-phase cells (SDBF) compared to biofilms derived from exponential cells (EDBF) or equivalent planktonic cells. The fully matured SDBF cells demonstrated higher viability in THP-1 monocyte/macrophage cells compared to the EDBF cells. Peptidoglycan strengthening, specific urea-cycle, and detoxification enzymes were more abundant on planktonic than biofilm cells, indicating the activation of growth-mode specific pathways during prolonged cultivation. Thus, we show that S. aureus shapes its surfaceome in a growth mode-dependent manner to reach high levofloxacin tolerance (>200-times the minimum biofilm inhibitory concentration). This study also demonstrates that the phenotypic state of the cells prior to biofilm formation affects the immune-evasion and persistence-related traits of S. aureus

Chlamydia pneumoniae Interferes with Macrophage Differentiation and Cell Cycle Regulation to Promote Its Replication

Chlamydia pneumoniae is a ubiquitous intracellular bacterium which infects humans via the respiratory route. The tendency of C. pneumoniae to persist in monocytes and macrophages is well known, but the underlying host-chlamydial interactions remain elusive. In this work, we have described changes in macrophage intracellular signaling pathways induced by C. pneumoniae infection. Label-free quantitative proteome analysis and pathway analysis tools were used to identify changes in human THP-1-derived macrophages upon C. pneumoniae CV6 infection. At 48-h postinfection, pathways associated to nuclear factor kappa B (NF-kappa B) regulation were stressed, while negative regulation on cell cycle control was prominent at both 48 h and 72 h. Upregulation of S100A8 and S100A9 calcium binding proteins, osteopontin, and purine nucleoside hydrolase, laccase domain containing protein 1 (LACC1) underlined the proinflammatory consequences of the infection, while elevated NF-kappa B2 levels in infected macrophages indicates interaction with the noncanonical NF-kappa B pathway. Infection-induced alteration of cell cycle control was obvious by the downregulation of mini chromosome maintenance (MCM) proteins MCM2-7, and the significance of host cell cycle regulation for C. pneumoniae replication was demonstrated by the ability of a cyclin-dependent kinase (CDK) 4/6 inhibitor Palbociclib to promote C. pneumoniae replication and infectious progeny production. The infection was found to suppress retinoblastoma expression in the macrophages in both protein and mRNA levels, and this change was reverted by treatment with a histone deacetylase inhibitor. The epigenetic suppression of retinoblastoma, along with upregulation of S100A8 and S100A9, indicate host cell changes associated with myeloid-derived suppressor cell (MDSC) phenotype.Peer reviewe