Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells

Abstract

Background: Although pneumococcal pneumonia is one of the most common causes of death due to infectious diseases, little is known about pneumococci-lung cell interaction. Herein we tested the hypothesis that pneumococci activated pulmonary epithelial cell cytokine release by c-Jun-NH2-terminal kinase (JNK) METHODS: Human bronchial epithelial cells (BEAS-2B) or epithelial HEK293 cells were infected with S. pneumoniae R6x and cytokine induction was measured by RT-PCR, ELISA and Bioplex assay. JNK-phosphorylation was detected by Western blot and nuclear signaling was assessed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). JNK was modulated by the small molecule inhibitor SP600125 and AP1 by transfection of a dominant negative mutant.

Results: S. pneumoniae induced the release of distinct CC and CXC, as well as Th1 and Th2 cytokines and growth factors by human lung epithelial cell line BEAS-2B. Furthermore, pneumococci infection resulted in JNK phosphorylation in BEAS-2B cells. Inhibition of JNK by small molecule inhibitor SP600125 reduced pneumococci-induced IL-8 mRNA expression and release of IL-8 and IL-6. One regulator of the il8 promoter is JNK-phosphorylated activator protein 1 (AP-1). We showed that S. pneumoniae time-dependently induced DNA binding of AP-1 and its phosphorylated subunit c-Jun with a maximum at 3 to 5 h after infection. Recruitment of Ser63/73-phosphorylated c-Jun and RNA polymerase II to the endogenous il8 promoter was found 2 h after S. pneumoniae infection by chromatin immunoprecipitation. AP-1 repressor A-Fos reduced IL-8 release by TLR2-overexpressing HEK293 cells induced by pneumococci but not by TNFalpha. Antisense-constructs targeting the AP-1 subunits Fra1 and Fra2 had no inhibitory effect on pneumococci-induced IL-8 release.

Conclusion: S. pneumoniae-induced IL-8 expression by human epithelial BEAS-2B cells depended on activation of JNK and recruitment of phosphorylated c-Jun to the il8 promoter.

Figure 1

S. pneumoniae induce the release of distinct CC and CXC, as well as Th1 and Th2 cytokines and growth factors by human lung epithelial cells. BEAS-2B cells were infected with S. pneumoniae strain R6x (10⁶ cfu/ml) for 5, 10, or 20 h. Cytokine release in the supernatant was measured by Bioplex assay. *, p < 0.01 vs. uninfected control, #, p < 0.01 one time point vs. another, at least in three independent experiments.

Figure 2

S. pneumoniae induced JNK-dependent IL-8 and IL-6 release by human lung epithelial cells. (A) BEAS-2B cells were infected with 10⁶ cfu/ml S. pneumoniae R6x for the times and JNK2 phosphorylation was detected by Western blot. A representative of three independent experiments is shown and quantification of all three experiments is given. (B/D) BEAS-2B cells were preincubated with the indicated concentrations of JNK inhibitor SP600125 and then infected with 10⁶ cfu/ml S. pneumoniae R6x for 15 h. IL-8 (B) and IL-6 (D) concentrations were measured in the supernatant. *, p < 0.01 vs. control; #, p < 0.01 vs. infected cells without pre-incubation with inhibitors in three independent experiments. (C) BEAS-2B cells were preincubated with the indicated concentrations of JNK inhibitor SP600125 and then infected with 10⁶ cfu/ml S. pneumoniae R6x for 3 h. IL-8 and GAPDH mRNA was detected by RT-PCR. Representative gels of three independent experiments are shown.

Figure 3

S. pneumoniae induced AP-1 activation in human lung epithelial cells. BEAS-2B cells were infected with S. pneumoniae R6x (10⁶ or 10⁷ cfu/ml as indicated) (A/B/C) for the indicated times or TNFα (50 ng/ml, 0.5 h). DNA binding of AP-1 (A) was detected by EMSA and of phosphorylated c-Jun by transcription factor activation assay (B). *, p < 0.01 vs. control. Recruitment of Ser^63/73-phosphorylated c-Jun and RNA polymerase II to the endogenous il8 promoter was detected by chromatin immunoprecipitation (C). Representatives of at least three independent experiments are shown.

Figure 4

AP-1 repressor blocked S. pneumoniae-induced IL-8 release. HEK293 cells were transfected with plasmids encoding TLR2, as well as empty vector (CMV500), AP-1 repressor (CMV500-A-Fos), Fra1 antisense (AS Fra1), or Fra2 antisense (AS Fra2), respectively. Then, cells were infected with S. pneumoniae strain R6x (10⁵ cfu/ml) or stimulated with TNFα (50 ng/ml) for 15 h, and IL-8 concentration was measured in the supernatant. *, p < 0.01 vs. control; #, p < 0.01 vs. infected cells with empty vector at least in three independent experiments.