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Dengue Virus Infection Causes the Activation of Distinct NF-κB Pathways for Inducible Nitric Oxide Synthase and TNF-α Expression in RAW264.7 Cells.

Cheng YL, Lin YS, Chen CL, Wan SW, Ou YD, Yu CY, Tsai TT, Tseng PC, Lin CF - Mediators Inflamm. (2015)

Bottom Line: In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation.Heat-inactivated DENV failed to cause the identified inflammatory responses.Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan.

ABSTRACT
Infection with dengue virus (DENV) causes an increase in proinflammatory responses, such as nitric oxide (NO) generation and TNF-α expression; however, the molecular mechanism underlying this inflammatory activation remains undefined, although the activation of the transcription factor NF-κB is generally involved. In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation. NF-κB is known to be activated by DENV infection. Pharmacologically inhibiting NF-κB activation abolishes iNOS/NO biosynthesis and TNF-α production. With inhibition, the potential role of NF-κB in oxidative signaling regulation was prevented during DENV infection. Heat-inactivated DENV failed to cause the identified inflammatory responses. Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production. In contrast to TLR3, viral protein NS2B3 also independently contributed to NF-κB activation to regulate TNF-α production. These results show the distinct pathways for NF-κB activation caused by DENV infection individually for the regulation of iNOS/NO and TNF-α expression.

No MeSH data available.


Related in: MedlinePlus

Heat-inactivated DENV does not efficiently cause NF-κB activation, TNF-α production, or iNOS/NO biosynthesis. NF-κB reporter assay (a), ELISA (b), Griess' reagent (c), and western blot analysis (d) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected and heat-inactivated DENV 2-treated RAW264.7 cells. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. For western blot results, one set of representative data obtained from three independent experiments is shown. The relative ratio to β-actin based on densitometer quantification and analysis using ImageJ software is shown. For all experiments, the quantitative data shown represent the mean ± SD values of three independent experiments.
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fig5: Heat-inactivated DENV does not efficiently cause NF-κB activation, TNF-α production, or iNOS/NO biosynthesis. NF-κB reporter assay (a), ELISA (b), Griess' reagent (c), and western blot analysis (d) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected and heat-inactivated DENV 2-treated RAW264.7 cells. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. For western blot results, one set of representative data obtained from three independent experiments is shown. The relative ratio to β-actin based on densitometer quantification and analysis using ImageJ software is shown. For all experiments, the quantitative data shown represent the mean ± SD values of three independent experiments.

Mentions: To investigate whether DENV-induced NF-κB activation followed by TNF-α production and iNOS/NO biosynthesis occurs in a protein-mediated manner, RAW264.7 cells were inoculated with MOI of 50 of heat-inactivated DENV (HI-DENV). The results revealed that HI-DENV was less efficient at inducing NF-κB activity than live-DENV (Figure 5(a)). Similarly, the levels of TNF-α (Figure 5(b)), NO (Figure 5(c)), and iNOS expression (Figure 5(d)) were lower in the HI-DENV group compared with the live DENV group. These experiments show that DENV activates NF-κB followed by TNF-α and iNOS/NO biosynthesis through viral proteins.


Dengue Virus Infection Causes the Activation of Distinct NF-κB Pathways for Inducible Nitric Oxide Synthase and TNF-α Expression in RAW264.7 Cells.

Cheng YL, Lin YS, Chen CL, Wan SW, Ou YD, Yu CY, Tsai TT, Tseng PC, Lin CF - Mediators Inflamm. (2015)

Heat-inactivated DENV does not efficiently cause NF-κB activation, TNF-α production, or iNOS/NO biosynthesis. NF-κB reporter assay (a), ELISA (b), Griess' reagent (c), and western blot analysis (d) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected and heat-inactivated DENV 2-treated RAW264.7 cells. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. For western blot results, one set of representative data obtained from three independent experiments is shown. The relative ratio to β-actin based on densitometer quantification and analysis using ImageJ software is shown. For all experiments, the quantitative data shown represent the mean ± SD values of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Heat-inactivated DENV does not efficiently cause NF-κB activation, TNF-α production, or iNOS/NO biosynthesis. NF-κB reporter assay (a), ELISA (b), Griess' reagent (c), and western blot analysis (d) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected and heat-inactivated DENV 2-treated RAW264.7 cells. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. For western blot results, one set of representative data obtained from three independent experiments is shown. The relative ratio to β-actin based on densitometer quantification and analysis using ImageJ software is shown. For all experiments, the quantitative data shown represent the mean ± SD values of three independent experiments.
Mentions: To investigate whether DENV-induced NF-κB activation followed by TNF-α production and iNOS/NO biosynthesis occurs in a protein-mediated manner, RAW264.7 cells were inoculated with MOI of 50 of heat-inactivated DENV (HI-DENV). The results revealed that HI-DENV was less efficient at inducing NF-κB activity than live-DENV (Figure 5(a)). Similarly, the levels of TNF-α (Figure 5(b)), NO (Figure 5(c)), and iNOS expression (Figure 5(d)) were lower in the HI-DENV group compared with the live DENV group. These experiments show that DENV activates NF-κB followed by TNF-α and iNOS/NO biosynthesis through viral proteins.

Bottom Line: In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation.Heat-inactivated DENV failed to cause the identified inflammatory responses.Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan.

ABSTRACT
Infection with dengue virus (DENV) causes an increase in proinflammatory responses, such as nitric oxide (NO) generation and TNF-α expression; however, the molecular mechanism underlying this inflammatory activation remains undefined, although the activation of the transcription factor NF-κB is generally involved. In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation. NF-κB is known to be activated by DENV infection. Pharmacologically inhibiting NF-κB activation abolishes iNOS/NO biosynthesis and TNF-α production. With inhibition, the potential role of NF-κB in oxidative signaling regulation was prevented during DENV infection. Heat-inactivated DENV failed to cause the identified inflammatory responses. Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production. In contrast to TLR3, viral protein NS2B3 also independently contributed to NF-κB activation to regulate TNF-α production. These results show the distinct pathways for NF-κB activation caused by DENV infection individually for the regulation of iNOS/NO and TNF-α expression.

No MeSH data available.


Related in: MedlinePlus