The macrophages were then infected by BCG for 24 hr. We used an LDH assay to analyse the viability of macrophages in the presence of SP600125. The data revealed that there was no significant difference in LDH release among the groups, suggesting that the viabilities of Alvelestat in vivo macrophages among the groups were similar (Fig. 2b). Consistent with previous studies, with the addition of SP600125,

NO production in BCG-infected macrophages was significantly reduced by about 74% when compared with solvent control. The inhibitor also significantly reduced IL-17A-enhanced NO production by about 66% (Fig. 2c). The specificity of SP600125 towards JNK, ERK1/2 and p38 MAPK was also analysed. It was observed that only the BCG-induced phosphorylation of JNK, but not ERK1/2 or p38 MAPK, was inhibited by SP600125 (Fig. 2d, lane 2 versus lane 6; lane 3 versus lane

7). The data suggested ICG-001 datasheet that SP600125 was able to specifically block the activation of JNK. Taken together, we confirmed the involvement of JNK in IL-17A-enhanced NO production in BCG-infected macrophages. The expression of iNOS has been shown to be regulated at the post-transcriptional level via the JNK signalling pathway, which contributes to stabilization of iNOS mRNA.[27] Our data showed that IL-17 was able to enhance BCG-induced phosphorylation of JNK (Fig. 2a). Therefore, we are interested to assess whether IL-17A is able to affect the stability of BCG-induced iNOS mRNA. Using qPCR analysis, our data showed that the half-life of iNOS mRNA in BCG-infected macrophages was about 101 min. In the presence of IL-17A, the half-life of BCG-induce iNOS mRNA was prolonged to about 227 min (Fig. 3). Our results indicated that IL-17A was able to enhance the stability of BCG-induced iNOS mRNA, thereby allowing for increased NO production. Nuclear factor-κB is a key transcription factor that drives the expression of iNOS.[28, 29] The BCG-induced activation of the NF-κB pathway in macrophages

requires degradation of IκBα in the cytoplasm, which allows the release of NF-κB and subsequent translocation many of NF-κB into the nucleus for initiation of gene expression.[19, 30, 31] To investigate whether IL-17A pre-treatment affects BCG-activated NF-κB pathways, we analysed the degradation of IκBα in the cytoplasm and translocation of NF-κB p65 into the nucleus. We pre-treated the macrophages with IL-17A for 24 hr, followed by BCG infection for 15 min. Cytoplasmic proteins and nuclear proteins were extracted for Western blot analysis of IκBα and NF-κB p65, respectively. Our results showed that infection of macrophages by BCG caused degradation of IκBα and also translocation of NF-κB p65 into the nucleus (Fig. 4, lane 2). However, neither process was affected by IL-17A pre-treatment (Fig. 4, lane 3). Our results suggested that IL-17A had no effects on the activation of the NF-κB pathway during BCG infection.