scholarly journals Nitric Oxide Inhibits Coxiella burnetii Replication and Parasitophorous Vacuole Maturation

2002 ◽  
Vol 70 (9) ◽  
pp. 5140-5147 ◽  
Author(s):  
Dale Howe ◽  
Lorraine F. Barrows ◽  
Nicole M. Lindstrom ◽  
Robert A. Heinzen
Keyword(s):  
Nitric Oxide ◽  
Coxiella Burnetii ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Nitric Oxide Donor ◽  
Nitrite Production ◽  
Tnf Α ◽  
Infected Cells ◽  
Ifn Γ ◽  
Cytokine Stimulation

ABSTRACT Nitric oxide is a recognized cytotoxic effector against facultative and obligate intracellular bacteria. This study examined the effect of nitric oxide produced by inducible nitric oxide synthase (iNOS) up-regulated in response to cytokine stimulation, or by a synthetic nitric oxide donor, on replication of obligately intracellular Coxiella burnetii in murine L-929 cells. Immunoblotting and nitrite assays revealed that C. burnetii infection of L-929 cells augments expression of iNOS up-regulated in response to gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Infection in the absence of cytokine stimulation did not result in demonstrable up-regulation of iNOS expression or in increased nitrite production. Nitrite production by cytokine-treated cells was significantly inhibited by the iNOS inhibitor S-methylisothiourea (SMT). Treatment of infected cells with IFN-γ and TNF-α or the synthetic nitric oxide donor 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (DETA/NONOate) had a bacteriostatic effect on C. burnetii replication. Inhibition of replication was reversed upon addition of SMT to the culture medium of cytokine-treated cells. Microscopic analysis of infected cells revealed that nitric oxide (either cytokine induced or donor derived) inhibited formation of the mature (large) parasitophorous vacuole that is characteristic of C. burnetii infection of host cells. Instead, exposure of infected cells to nitric oxide resulted in the formation of multiple small, acidic vacuoles usually containing one C. burnetii cell. Removal of nitrosative stress resulted in the coalescence of small vacuoles to form a large vacuole harboring multiple C. burnetii cells. These experiments demonstrate that nitric oxide reversibly inhibits replication of C. burnetii and formation of the parasitophorous vacuole.

scholarly journals Nitric Oxide-Mediated Inhibition of the Ability ofRickettsia prowazekii To Infect Mouse Fibroblasts and Mouse Macrophagelike Cells

1998 ◽  
Vol 66 (2) ◽  
pp. 558-566 ◽  
Author(s):  
Jenifer Turco ◽  
Hua Liu ◽  
Sheldon F. Gottlieb ◽  
Herbert H. Winkler
Keyword(s):  
Nitric Oxide ◽  
Degradation Products ◽  
Host Cells ◽  
Raw264.7 Cells ◽  
Rickettsial Infection ◽  
L929 Cells ◽  
Nitrite Production ◽  
Tnf Α ◽  
Nos Inhibitors ◽  
Ifn Γ

ABSTRACT The role of the nitric oxide synthase (NOS) pathway in inhibiting the ability of Rickettsia prowazekii to initially infect (invade) mouse cytokine-treated, fibroblastic L929 cells and macrophagelike RAW264.7 cells and the ability of nitric oxide (NO) to damage isolated rickettsiae were investigated. Substantial amounts of nitrite (a degradation product of NO) were produced and the initial rickettsial infection was suppressed in cultures of L929 cells treated with crude lymphokine preparations (LK) or with gamma interferon (IFN-γ) plus tumor necrosis factor alpha (TNF-α) but not in L929 cell cultures treated with IFN-γ alone or TNF-α alone. The NOS inhibitors N G-methyl-l-arginine and aminoguanidine both inhibited nitrite production and prevented the suppression of the initial rickettsial infection. Antibody-mediated neutralization of the IFN-γ in the LK also inhibited both nitrite production and suppression of the initial rickettsial infection. Cultures of RAW264.7 cells treated with IFN-γ plus lipopolysaccharide exhibited suppression of the initial rickettsial infection, and the suppression was relieved by aminoguanidine. Addition of oxyhemoglobin (a scavenger of extracellular NO) during the rickettsial infection alleviated the suppression of the initial rickettsial infection observed in appropriately treated L929 cells and RAW264.7 cells. In addition, the oxyhemoglobin restored the rickettsia-mediated, rapid killing of the treated RAW264.7 cells. Incubation of isolated rickettsiae with NO inhibited their ability to infect L929 and IFN-γ-treated RAW264.7 cells and to rapidly kill IFN-γ-treated RAW264.7 cells. In contrast, incubation of L929 cells with a solution that contained NO and/or degradation products of NO did not affect their ability to be infected by rickettsiae. The data are consistent with the hypothesis that NO released from appropriately stimulated potential host cells kills extracellular rickettsiae and thus prevents the rickettsiae from infecting the cells.

scholarly journals The secreted protein kinase CstK from Coxiella burnetii influences vacuole development and interacts with the GTPase-activating host protein TBC1D5

2020 ◽  
Vol 295 (21) ◽  
pp. 7391-7403 ◽  
Author(s):  
Eric Martinez ◽  
Sylvaine Huc-Brandt ◽  
Solène Brelle ◽  
Julie Allombert ◽  
Franck Cantet ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Coxiella Burnetii ◽  
Membrane Trafficking ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Host Protein ◽  
Host Cells ◽  
Intracellular Replication ◽  
Gtpase Activating Protein ◽  
Infected Cells

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system–mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host–pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro. This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein–protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.

scholarly journals Inducible Nitric Oxide Synthase Can Be Induced in the Absence of Active Nuclear Factor-κB in Rat Mesangial Cells

1999 ◽  
Vol 10 (4) ◽  
pp. 721-729 ◽  
Author(s):  
OSAMU NAKASHIMA ◽  
YOSHIO TERADA ◽  
SEIJI INOSHITA ◽  
MICHIO KUWAHARA ◽  
SEI SASAKI ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mesangial Cells ◽  
Nuclear Factor Κb ◽  
Nitrite Production ◽  
Tnf Α ◽  
Inos Promoter ◽  
Ifn Γ ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide ◽  
Inos Induction

Abstract. The contribution of nuclear factor-κB (NF-κB) and interferon-γ (IFN-γ) signaling to nitric oxide generation is not completely understood. The effect of NF-κB release and its inhibition on nitrite production and the involvement of Janus kinase 2 (JAK2) in inducible nitric oxide synthase (iNOS) induction were investigated. The following assays were performed. (1) Nitrite produced by rat mesangial cells in primary culture was measured in incubations with tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS), with or without IFN-γ. Cells were stimulated with TNF-α or LPS plus IFN-γ in the presence of NF-κB inhibitors, herbimycin A (HerA), or the more specific JAK2 inhibitor AG490. (2) Immunoblotting was performed against the p65 and p50 subunits of NF-κB and iNOS. (3) Electrophoretic mobility shift assays were performed against NF-κB in the presence of NF-κB inhibitors or AG490. (4) iNOS promoter activity was measured in the presence of AG490 or JAK2 antisense oligonucleotides. TNF-α or LPS alone did not induce nitrite production, but with IFN-γ these compounds did induce nitrite production. Pyrrolidine dithiocarbamate (PDTC),N-acetyl-L-cysteine, dexamethasone (Dex), HerA, and AG490 partially inhibited LPS/IFN-γ- or TNF-α/IFN-γ-induced nitrite production. p65 was inhibited by the three NF-κB inhibitors described above, whereas p50 was not. PDTC and Dex completely inhibited the p65/p50 heterodimer, but HerA and AG490 had little effect on p65/p50. AG490 and JAK2 antisense oligonucleotides suppressed iNOS promoter activity. It can be concluded that (1) iNOS can be induced without active NF-κB; (2) Dex, acetylsalicylic acid, and PDTC inhibit only p65; and (3) JAK2 is involved in iNOS induction, and the contribution of JAK2 to nitrite production is greater than that of NF-κB.

scholarly journals Differential Responses of Bovine Macrophages to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

2002 ◽  
Vol 70 (10) ◽  
pp. 5556-5561 ◽  
Author(s):  
Douglas J. Weiss ◽  
Oral A. Evanson ◽  
Andreas Moritz ◽  
Ming Qi Deng ◽  
Mitchell S. Abrahamsen
Keyword(s):  
Nitric Oxide ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subsp ◽  
Necrosis Factor Alpha ◽  
Gm Csf ◽  
Intracellular Degradation ◽  
Tnf Α ◽  
Infected Cells ◽  
Ifn Γ ◽  
Mycobacterium Avium Subsp Paratuberculosis

ABSTRACT Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically similar organisms; however, they differ in their virulence for cattle. M. avium subsp. paratuberculosis causes a chronic intestinal infection leading to a chronic wasting disease termed paratuberculosis or Johne's disease, whereas M. avium subsp. avium causes only a transient infection. We compared the response of bovine monocyte-derived macrophages to ingestion of M. avium subsp. paratuberculosis and M. avium subsp. avium organisms by determining organism survival, superoxide and nitric oxide production, and expression of the cytokines tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), interleukin-8 (IL-8), IL-10, IL-12, and granulocyte-monocyte colony-stimulating factor (GM-CSF). Unlike M. avium subsp. paratuberculosis, macrophages were able to kill approximately half of the M. avium subsp. avium organisms after 96 h of incubation. This difference in killing efficiency was not related to differences in nitric oxide or superoxide production. Compared to macrophages activated with IFN-γ and lipopolysaccharide, macrophages incubated with M. avium subsp. paratuberculosis showed greater expression of IL-10 and GM-CSF (all time points) and IL-8 (72 h) and less expression of IL-12 (72 h), IFN-γ (6 h), and TNF-α (6 h). When cytokine expression by macrophages incubated with M. avium subsp. paratuberculosis was compared to those of macrophages incubated with M. avium subsp. avium, M. avium subsp. paratuberculosis-infected cells showed greater expression of IL-10 (6 and 24 h) and less expression of TNF-α (6 h). Therefore, the combination of inherent resistance to intracellular degradation and suppression of macrophage activation through oversecretion of IL-10 may contribute to the virulence of M. avium subsp. paratuberculosis in cattle.

scholarly journals Comparing the protective effects of resveratrol, curcumin and sulforaphane against LPS/IFN-γ-mediated inflammation in doxorubicin-treated macrophages

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Haidy A. Saleh ◽  
Eman Ramdan ◽  
Mohey M. Elmazar ◽  
Hassan M. E. Azzazy ◽  
Anwar Abdelnaser
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Response ◽  
Raw 264.7 Cells ◽  
Inos Mrna ◽  
No Production ◽  
Raw 264.7 ◽  
Mrna Levels ◽  
Protective Effects ◽  
Tnf Α ◽  
Ifn Γ

AbstractDoxorubicin (DOX) chemotherapy is associated with the release of inflammatory cytokines from macrophages. This has been suggested to be, in part, due to DOX-mediated leakage of endotoxins from gut microflora, which activate Toll-like receptor 4 (TLR4) signaling in macrophages, causing severe inflammation. However, the direct function of DOX on macrophages is still unknown. In the present study, we tested the hypothesis that DOX alone is incapable of stimulating inflammatory response in macrophages. Then, we compared the anti-inflammatory effects of curcumin (CUR), resveratrol (RES) and sulforaphane (SFN) against lipopolysaccharide/interferon-gamma (LPS/IFN-γ)-mediated inflammation in the absence or presence of DOX. For this purpose, RAW 264.7 cells were stimulated with LPS/IFN-γ (10 ng/mL/10 U/mL) in the absence or presence of DOX (0.1 µM). Our results showed that DOX alone is incapable of stimulating an inflammatory response in RAW 264.7 macrophages. Furthermore, after 24 h of incubation with LPS/IFN-γ, a significant increase in tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) mRNA levels was observed. Similarly, nitric oxide (NO) production and TNF-α and IL-6 protein levels were significantly upregulated. Moreover, in LPS/IFN-γ-treated macrophages, the microRNAs (miRNAs) miR-146a, miR-155, and miR-21 were significantly overexpressed. Interestingly, upon testing CUR, RES, and SFN against LPS/IFN-γ-mediated inflammation, only SFN was able to significantly reverse the LPS/IFN-γ-mediated induction of iNOS, TNF-α and IL-6 and attenuate miR-146a and miR-155 levels. In conclusion, SFN, at the transcriptional and posttranscriptional levels, exhibits potent immunomodulatory action against LPS/IFN-γ-stimulated macrophages, which may indicate SFN as a potential treatment for DOX-associated inflammation.

Cytokine and inflammatory mediators are associated with cytotoxic, anti-inflammatory and apoptotic activity of honeybee venom

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Mohamed A. Salama ◽  
Mohamed A. Younis ◽  
Roba M. Talaat
Keyword(s):  
Nitric Oxide ◽  
Cell Lines ◽  
Cancer Cell ◽  
Hela Cells ◽  
No Production ◽  
Hep G2 ◽  
Normal Cells ◽  
Tnf Α ◽  
Ifn Γ ◽  
Mcf 7

AbstractObjectiveThe present study aimed to evaluate cytotoxic, apoptotic, and anti-inflammatory properties of bee venom (BV) as well as changes in cytokine secretion levels and nitric oxide (NO) production using three different cancer cell lines [liver (Hep-G2), breast (MCF-7), and cervical (HPV-18 infected HeLa cells)] and two normal cells (splenocytes and macrophages (MQ).MethodsCytotoxic activity of BV against tumor cell lines and normal splenocytes/MQ was tested by MTT assay. By ELISA (ELISA); Tumor necrosis factor (TNF-α), Interleukine (IL-10) and interferon (IFN-γ) were measured. Caspase three expressions was evaluated using reverse transcription-polymerase chain reaction (RT-PCR). Nitric oxide (NO) was estimated using a colorimetric assay.ResultsBV has a significant cytotoxic effect on all cell lines in a dose- and time-dependent manner; none of them was toxic for normal cells. Treating Hep-G2 cells with BV showed a reduction in IL-10, elevation in TNF-α with no change in IFN-γ level. MCF-7 cells have low IL-10 and TNF-α and high IFN-γ production level. Elevation of IL-10 and IFN-γ coincides with a reduction in TNF-α level was demonstrated in HeLa cells. The expression of Caspase three was dramatically increased with elevation in BV concentration in all tested cancer cell lines. A gradual decrease in NO production by MQ with increasing BV dose was observed.ConclusionTaken together, our results stressed on the importance of BV as a potent anti-tumor agent against various types of cancers (Liver, Breast, and Cervix). Further steps towards the use of BV for pharmacological purposes must be done.

scholarly journals Nitric oxide interacts with cholinoceptors to modulate insulin secretion by pancreatic β cells

2020 ◽  
Vol 472 (10) ◽  
pp. 1469-1480
Author(s):  
Bashair M. Mussa ◽  
Ankita Srivastava ◽  
Abdul Khader Mohammed ◽  
Anthony J. M. Verberne
Keyword(s):  
Nitric Oxide ◽  
Insulin Secretion ◽  
Cell Viability ◽  
Combined Treatment ◽  
No Production ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Tnf Α ◽  
Ifn Γ

Abstract Dysfunction of the pancreatic β cells leads to several chronic disorders including diabetes mellitus. Several mediators and mechanisms are known to be involved in the regulation of β cell secretory function. In this study, we propose that cytokine-induced nitric oxide (NO) production interacts with cholinergic mechanisms to modulate insulin secretion from pancreatic β cells. Using a rat insulinoma cell line INS-1, we demonstrated that β cell viability decreases significantly in the presence of SNAP (NO donor) in a concentration- and time-dependent manner. Cell viability was also found to be decreased in the presence of a combined treatment of SNAP with SMN (muscarinic receptor antagonist). We then investigated the impact of these findings on insulin secretion and found a significant reduction in glucose uptake by INS-1 cells in the presence of SNAP and SMN as compared with control. Nitric oxide synthase 3 gene expression was found to be significantly reduced in response to combined treatment with SNAP and SMN suggesting an interaction between the cholinergic and nitrergic systems. The analysis of gene and protein expression further pin-pointed the involvement of M3 muscarinic receptors in the cholinergic pathway. Upon treatment with cytokines, reduced cell viability was observed in the presence of TNF-α and IFN-γ. A significant reduction in insulin secretion was also noted after treatment with TNF-α and IFN-γ and IL1-β. The findings of the present study have shown for the first time that the inhibition of the excitatory effects of cholinergic pathways on glucose-induced insulin secretion may cause β cell injury and dysfunction of insulin secretion in response to cytokine-induced NO production.

scholarly journals Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Alicja M. Cygan ◽  
Terence C. Theisen ◽  
Alma G. Mendoza ◽  
Nicole D. Marino ◽  
Michael W. Panas ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Translocation ◽  
Affinity Purification ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Host Cells ◽  
Content Type ◽  
Cyclin E1 ◽  
Infected Cells

ABSTRACT Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins. IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

scholarly journals Both Inducible Nitric Oxide Synthase and NADPH Oxidase Contribute to the Control of Virulent Phase I Coxiella burnetii Infections

2004 ◽  
Vol 72 (11) ◽  
pp. 6666-6675 ◽  
Author(s):  
Robert E. Brennan ◽  
Kasi Russell ◽  
Guoquan Zhang ◽  
James E. Samuel
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Cell Lines ◽  
Inducible Nitric Oxide Synthase ◽  
Coxiella Burnetii ◽  
Wild Type ◽  
Primary Mouse ◽  
Ifn Γ ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

ABSTRACT Host control of Coxiella burnetii infections is believed to be mediated primarily by activated monocytes/macrophages. The activation of macrophages by cytokines leads to the production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that have potent antimicrobial activities. The contributions of ROI and RNI to the inhibition of C. burnetii replication were examined in vitro by the use of murine macrophage-like cell lines and primary mouse macrophages. A gamma interferon (IFN-γ) treatment of infected cell lines and primary macrophages resulted in an increased production of nitric oxide (NO) and hydrogen peroxide (H2O2) and a significant inhibition of C. burnetii replication. The inhibition of replication was reversed in the murine cell line J774.16 upon the addition of either the inducible nitric oxide synthase (iNOS) inhibitor NG-monomethyl-l-arginine (NGMMLA) or the H2O2 scavenger catalase. IFN-γ-treated primary macrophages from iNOS−/− and p47phox−/− mice significantly inhibited replication but were less efficient at controlling infection than IFN-γ-treated wild-type macrophages. To investigate the contributions of ROI and RNI to resistance to infection, we performed in vivo studies, using C57BL/6 wild-type mice and knockout mice lacking iNOS or p47phox. Both iNOS−/− and p47phox−/− mice were attenuated in the ability to control C. burnetii infection compared to wild-type mice. Together, these results strongly support a role for both RNI and ROI in the host control of C. burnetii infection.

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