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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

DENV infection promotes NF-κB nuclear translocation and activation. (a) NF-κB reporter assay quantified activation of NF-κB in DENV 2-infected cells for the indicated MOIs 6 h after infection. (b) Immunostaining followed by immunofluorescence microscopy reveals the expression and intracellular localization of the NF-κB p65 (green) and DENV E protein (red) 6 h after infection with DENV 2; DAPI (blue) was used for nuclear staining. Images are representative of three independent experiments. (c) NF-κB reporter assay quantified activation of NF-κB in DENV 2-infected cells pretreated with CAPE. DMSO was used for the negative control. ∗P < 0.05 and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. ns: not significant. The quantitative data shown represent the mean ± SD values of three independent experiments.
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fig2: DENV infection promotes NF-κB nuclear translocation and activation. (a) NF-κB reporter assay quantified activation of NF-κB in DENV 2-infected cells for the indicated MOIs 6 h after infection. (b) Immunostaining followed by immunofluorescence microscopy reveals the expression and intracellular localization of the NF-κB p65 (green) and DENV E protein (red) 6 h after infection with DENV 2; DAPI (blue) was used for nuclear staining. Images are representative of three independent experiments. (c) NF-κB reporter assay quantified activation of NF-κB in DENV 2-infected cells pretreated with CAPE. DMSO was used for the negative control. ∗P < 0.05 and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. ns: not significant. The quantitative data shown represent the mean ± SD values of three independent experiments.

Mentions: We next investigated the effects of DENV infection on NF-κB activation. A luciferase reporter assay showed a significant increase of NF-κB activation at the high MOIs of DENV infection (Figure 2(a)). To explore the activation of NF-κB, we used immunocytochemical staining to detect the nuclear translocation of NF-κB in the context of DENV infection. In the infection group, the p65 subunit of NF-κB translocated into the nucleus in E protein-positive cells (Figure 2(b)) at 6 h after infection. Furthermore, a luciferase reporter assay showed that inhibiting NF-κB with CAPE, an inhibitor of NF-κB, blocked DENV-induced NF-κB activation (Figure 2(c)). These results demonstrate that DENV infection activates NF-κB in RAW264.7 cells.


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)

DENV infection promotes NF-κB nuclear translocation and activation. (a) NF-κB reporter assay quantified activation of NF-κB in DENV 2-infected cells for the indicated MOIs 6 h after infection. (b) Immunostaining followed by immunofluorescence microscopy reveals the expression and intracellular localization of the NF-κB p65 (green) and DENV E protein (red) 6 h after infection with DENV 2; DAPI (blue) was used for nuclear staining. Images are representative of three independent experiments. (c) NF-κB reporter assay quantified activation of NF-κB in DENV 2-infected cells pretreated with CAPE. DMSO was used for the negative control. ∗P < 0.05 and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. ns: not significant. 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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fig2: DENV infection promotes NF-κB nuclear translocation and activation. (a) NF-κB reporter assay quantified activation of NF-κB in DENV 2-infected cells for the indicated MOIs 6 h after infection. (b) Immunostaining followed by immunofluorescence microscopy reveals the expression and intracellular localization of the NF-κB p65 (green) and DENV E protein (red) 6 h after infection with DENV 2; DAPI (blue) was used for nuclear staining. Images are representative of three independent experiments. (c) NF-κB reporter assay quantified activation of NF-κB in DENV 2-infected cells pretreated with CAPE. DMSO was used for the negative control. ∗P < 0.05 and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with DENV. ns: not significant. The quantitative data shown represent the mean ± SD values of three independent experiments.
Mentions: We next investigated the effects of DENV infection on NF-κB activation. A luciferase reporter assay showed a significant increase of NF-κB activation at the high MOIs of DENV infection (Figure 2(a)). To explore the activation of NF-κB, we used immunocytochemical staining to detect the nuclear translocation of NF-κB in the context of DENV infection. In the infection group, the p65 subunit of NF-κB translocated into the nucleus in E protein-positive cells (Figure 2(b)) at 6 h after infection. Furthermore, a luciferase reporter assay showed that inhibiting NF-κB with CAPE, an inhibitor of NF-κB, blocked DENV-induced NF-κB activation (Figure 2(c)). These results demonstrate that DENV infection activates NF-κB in RAW264.7 cells.

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