Fig. 6D shows phosphorylation and degradation of IκBα in Jurkat cells infected with the wild-type Corby but not the flaA mutant for 1, 2 and 4 h. The IκBα phosphorylation became evident at 1 h and decreased thereafter. Consistent with this, Corby-induced degradation of IκBα was observed at 1 h. NF-κB signaling AZD1390 order occurs either through the classical or alternative pathway [10]. In the classical pathway, NF-κB dimers, such as p50/p65, are maintained in the cytoplasm by interaction with IκBα. Whereas the classical NF-κB activation is IκB kinase β(IKKβ)- and

IKKγ-dependent and occurs through IκBα phosphorylation and subsequent proteasomal degradation, the alternative pathway depends on IKKα homodimers and NF-κB-inducing kinase (NIK) and results in regulated processing of the p100 precursor protein to p52 via phosphorylation and degradation of its IκB-terminus [10]. Indeed, the wild-type Corby but not the flaA mutant induced phosphorylation of p65 and upstream kinase IKKβ (Fig. 6D). Next, we examined the alternative pathway, which involves the cleavage of NF-κB2/p100 to p52. The level of p52 protein increased in

Jurkat cells infected with the wild-type Corby but not the flaA mutant (Fig. 6D), indicating that flagellin activates NF-κB via the alternative pathway. NF-κB signal is essential for induction of IL-8 expression by L. pneumophila To further confirm the involvement of IκBα degradation, we this website transfected the cells with transdominant mutant of IκBα in which two critical serine residues required for inducer-mediated phosphorylation were deleted [11]. As seen in Fig. 6E, overexpression of mutant Trichostatin A order IκBα greatly inhibited the Corby-induced IL-8 promoter activation.

This observation implicates the involvement of IκBα phosphorylation and degradation in flagellin-induced IL-8 expression. To address the mechanism of flagellin-mediated IL-8 expression, we investigated the role of NIK and IKK in L. pneumophila-induced IL-8 expression. Cotransfection with the dominant-negative mutant forms of NIK, IKKα, IKKβ, and IKKγ inhibited L. pneumophila-induced IL-8 expression (Fig. 6E). MyD88 is a universal adaptor for induction of cytokines by TLR2, TLR4, TLR5, TLR7, and TLR9. It is also required for activation of NF-κB by these TLRs [12]. Likewise, Inositol oxygenase overexpression of a dominant negative mutant form of MyD88 also inhibited L. pneumophila-induced IL-8 expression. Taken together, these findings clearly demonstrate that L. pneumophila induces IL-8 expression via activation of flagellin-dependent NF-κB signaling pathway. Because activation of the IL-8 promoter by L. pneumophila infection required the activation of NF-κB, we blocked NF-κB activation with Bay 11-7082, an inhibitor of IκBα phosphorylation [13]. Bay 11-7082 markedly inhibited L. pneumophila-induced phosphorylation and degradation of IκBα, as well as NF-κB DNA binding (Fig. 7A and 7B). Furthermore, Bay 11-7082 resulted in a dose-dependent reduction in L.