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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 induces TLR3-regulated NO and iNOS expression, but not TNF-α expression, following NF-κB activation. (a) In the absence and presence of CAPE, an NF-κB reporter assay was performed to measure NF-κB activation in Poly(I:C)-treated RAW264.7 cells. NF-κB reporter assay (b), ELISA (c), Griess reagent (d), and western blot analysis (e) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected RAW264.7 cells pretreated with TLR3 inhibitor. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with Poly(I:C) or DENV. ns: not significant. 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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fig6: DENV infection induces TLR3-regulated NO and iNOS expression, but not TNF-α expression, following NF-κB activation. (a) In the absence and presence of CAPE, an NF-κB reporter assay was performed to measure NF-κB activation in Poly(I:C)-treated RAW264.7 cells. NF-κB reporter assay (b), ELISA (c), Griess reagent (d), and western blot analysis (e) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected RAW264.7 cells pretreated with TLR3 inhibitor. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with Poly(I:C) or DENV. ns: not significant. 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: According to Tsai et al., TLR3 plays a major role in cellular activation compared with other TLRs during DENV infection [15]. TLR3 is a well-defined receptor for viral RNA, which can induce NF-κB activation during viral infection [13]. We further confirmed the involvement of TLR3 in NF-κB activation and inflammatory response. First, we used the TLR3 agonist polyinosinic-polycytidylic acid poly (Poly(I:C)) to determine whether TLR3 signaling can activate NF-κB. The results revealed that Poly(I:C) activated luciferase activity of NF-κB and that this activity was inhibited by CAPE (Figure 6(a)). Moreover, pharmacological inhibition of TLR3 suppressed NF-κB activation (Figure 6(b)). Surprisingly, there was no difference in TNF-α between the presence and absence of TLR3 inhibitor during DENV infection (Figure 6(c)). In contrast, DENV-induced NO (Figure 6(d)) and iNOS (Figure 6(e)) biosynthesis was significantly attenuated by TLR3 inhibition. These results demonstrate that TLR3-activated NF-κB is required for iNOS/NO biosynthesis, but not TNF-α expression, during DENV infection.


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 induces TLR3-regulated NO and iNOS expression, but not TNF-α expression, following NF-κB activation. (a) In the absence and presence of CAPE, an NF-κB reporter assay was performed to measure NF-κB activation in Poly(I:C)-treated RAW264.7 cells. NF-κB reporter assay (b), ELISA (c), Griess reagent (d), and western blot analysis (e) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected RAW264.7 cells pretreated with TLR3 inhibitor. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with Poly(I:C) or DENV. ns: not significant. 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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fig6: DENV infection induces TLR3-regulated NO and iNOS expression, but not TNF-α expression, following NF-κB activation. (a) In the absence and presence of CAPE, an NF-κB reporter assay was performed to measure NF-κB activation in Poly(I:C)-treated RAW264.7 cells. NF-κB reporter assay (b), ELISA (c), Griess reagent (d), and western blot analysis (e) quantified the activation of NF-κB and the expression of TNF-α and iNOS/NO in DENV 2-infected RAW264.7 cells pretreated with TLR3 inhibitor. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, compared with untreated cells. #P < 0.05, compared with Poly(I:C) or DENV. ns: not significant. 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: According to Tsai et al., TLR3 plays a major role in cellular activation compared with other TLRs during DENV infection [15]. TLR3 is a well-defined receptor for viral RNA, which can induce NF-κB activation during viral infection [13]. We further confirmed the involvement of TLR3 in NF-κB activation and inflammatory response. First, we used the TLR3 agonist polyinosinic-polycytidylic acid poly (Poly(I:C)) to determine whether TLR3 signaling can activate NF-κB. The results revealed that Poly(I:C) activated luciferase activity of NF-κB and that this activity was inhibited by CAPE (Figure 6(a)). Moreover, pharmacological inhibition of TLR3 suppressed NF-κB activation (Figure 6(b)). Surprisingly, there was no difference in TNF-α between the presence and absence of TLR3 inhibitor during DENV infection (Figure 6(c)). In contrast, DENV-induced NO (Figure 6(d)) and iNOS (Figure 6(e)) biosynthesis was significantly attenuated by TLR3 inhibition. These results demonstrate that TLR3-activated NF-κB is required for iNOS/NO biosynthesis, but not TNF-α expression, during DENV infection.

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