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. 2006 May 15;176(10):5720-4.
doi: 10.4049/jimmunol.176.10.5720.

Cutting edge: apoptosis-regulating signal kinase 1 is required for reactive oxygen species-mediated activation of IFN regulatory factor 3 by lipopolysaccharide

Edward Chiang  1 , Oanh Dang, Keith Anderson, Atsushi Matsuzawa, Hidenori Ichijo, Michael David
Affiliations

Cutting edge: apoptosis-regulating signal kinase 1 is required for reactive oxygen species-mediated activation of IFN regulatory factor 3 by lipopolysaccharide

Edward Chiang et al. J Immunol. .

Abstract

IFN regulatory factor (IRF) 3 participates in the transcriptional induction of IFN-alpha, IFN-beta, and a subset of IFN-stimulated genes (ISGs) as a result of viral infection. In addition, bacterial cell wall components such as LPS activate IRF3 in a p38-dependent manner. In this study we show that IRF3-mediated ISG induction by LPS requires the production of reactive oxygen species (ROS) by the NADPH-dependent oxidase NOX4. Furthermore, we present evidence that LPS-mediated ROS production leads to activation of apoptosis-regulating-signal kinase (ASK) 1, a MAPK kinase kinase family member capable of activating the MAP kinase 6/p38 axis. ASK1 kinase activity proved essential for IRF3-mediated ISG induction by LPS. Thus, our results presented here suggest a novel role for ROS and ASK1 in the innate immune response as signaling intermediates in the IRF3 activation pathway.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1
FIGURE 1
Delayed, ROS-dependent activation of ISG induction by LPS A, U373 cells were treated with 1 µg/ml LPS or 1000 U/ml IFNβ for the indicated times in the presence of 50 µg/ml cycloheximide, and ISG54 mRNA was analyzed by RPA. B, U373 cells were pretreated for 1 h with 20mM l-NAC (LNAC) before stimulation with 1 µg/ml LPS or 1000 U/ml IFNβ for 2 h. CTL, control.
FIGURE 2
FIGURE 2
ROS-dependent activation of ISG induction by LPS. A, U373 cells were treated with the indicated inhibitors for 30 min before stimulation with 1 µg/ml LPS for 6 h. Human RANTES mRNA levels were analyzed by RT-PCR. B, Same as A, except that ISG54 mRNA levels were analyzed by RPA. C, WT, eNOS-, or iNOS-deficient peritoneal macrophages were stimulated with 1 µg/ml LPS for 6 h. Murine RANTES mRNA levels were analyzed by RPA.
FIGURE 3
FIGURE 3
DPI inhibits LPS-mediated ISG induction and IRF3 DNA binding: A, U373 cells were treated with DPI for 1 h in the presence of 50 µg/ml cycloheximide and stimulated with 1 µg/ml LPS for 6 h or 1000 U/ml IFNβ for 1 h, and ISG54 mRNA levels were analyzed by RPA. B, Same as A, except that extracts were analyzed for IRF3 binding to the ISRE. C, U373 cells were stimulated with 1µg/ml LPS for 6 h in the presence of 10 µM DPI before immunostaining for IRF3 (top panel). Nuclei were counterstained with DAPI (bottom panel). D, U373 cells transfected with TRIF and ISRE luciferase were treated with DPI at the indicated concentrations for 12 h before analysis of luciferase activity (a representative of three independent experiments is shown). E, RAW 264.7 cells were transfected with siRNA against either LacZ or murine NOX4. After 48 h, cells were stimulated with 100 ng/ml LPS for 6 h, and ISG54 mRNA levels were analyzed by real-time PCR. ISG54 expression was normalized using β-actin as an internal control. F,RAW264.7 cells were transfected with the siRNA against LacZ or murine NOX4. After 48 h, cells were stimulated with LPS for 6 h, and nuclear extracts were analyzed for the presence of IRF3. DAPI, 4′,6′-diamidino-2-phenylindole.
FIGURE 4
FIGURE 4
Kinase activity of ASK1 is required for ISRE-mediated transcription by LPS. A, U373 cells were transfected with ISRE luciferase and the indicated amount of TRX plasmid. After 16 h cells were stimulated with 1 µg/ml LPS for 24 h, and luciferase production was analyzed. B, Cells were treated as in A; however, WT and kinase-inactive ASK1 plasmid was used instead of TRX. CTL, control.
FIGURE 5
FIGURE 5
Activation and requirement of ASK1 in LPS signaling: A, U373 cells were stimulated with 1 µg/ml LPS or TNF-α for the indicated times, and blots were probed with anti-phospho-T845 ASK1 or ASK1 Abs. CTL, control. B, Peritoneal macrophages from WT and ASK1−/− mice were stimulated with 1 µg/ml LPS for the indicated times, and ISG54 and GAPDH mRNA levels were analyzed by RT-PCR. C, WT and ASK1−/− MEFs were transfected with murine TRIF (mTRIF) and/or IRF3 and ISRE luciferase. After 24 h, luciferase activity was analyzed. A representative of three independent experiments is shown. b-gal, β-galactosidase.
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