(A) Total RNA was extracted from Jurkat cells infected with AA100jm, dotO mutant, Corby, or flaA mutant (MOI of 100) for the indicated find more time intervals and used for RT-PCR. (B) Jurkat cells were infected with the indicated concentrations of L. Tozasertib clinical trial pneumophila for 4 h. Total RNA was extracted and used for RT-PCR. (C) Total RNA was extracted from CD4+ T cells infected with Corby (MOI of 50) for 3 h and used for RT-PCR. (D) Jurkat cells were infected with live L. pneumophila Corby or flaA mutant (MOI of 100) for 4 h or incubated with L. pneumophila under the indicated treatment for 4 h. PFA, paraformaldehyde.

Total RNA was extracted and used for RT-PCR. Representative examples of three experiments with similar results. To determine the Bucladesine clinical trial correlation between IL-8 expression level and L. pneumophila bacterial proteins, heat-killed Corby was used to infect Jurkat

cells at a multiplicity of infection (MOI) of 100. At 4 h, IL-8 was not expressed in Jurkat cells infected with the heat-killed strain (Fig. 2D). Furthermore, IL-8 gene expression was not induced when paraformaldehyde-fixed L. pneumophila was used (Fig. 2D). However, bacteria heated at 56°C for 30 min induced IL-8 expression. These results suggest that the surface proteins of bacteria but not lipopolysaccharide are required for IL-8 induction. Considered together, it seems that Legionella flagellin is involved in IL-8 expression in T cells. Flagellin is recognized by toll-like receptor 5 (TLR5) [8]. Thus, we also examined the expression of TLR2, TLR3, TLR4, and TLR5 mRNAs in Jurkat and CD4+ T cells. All TLR mRNAs examined were expressed in Jurkat and CD4+ T cells (Fig. 3A and 3B). Furthermore, their expression levels did not change by L. pneumophila infection in CD4+ T cells

(Fig. 3B) and Jurkat cells (data not shown). PJ34 HCl Figure 3 TLR mRNA expression in T cells. (A) Expression of TLR mRNA in Jurkat cells. Total RNA was extracted from Jurkat cells and used for RT-PCR. (B) CD4+ T cells were infected without or with Corby (MOI of 50) for 3 h. Total RNA was extracted from CD4+ T cells and used for RT-PCR. Representative examples of three experiments with similar results. IL-8 production from Jurkat cells during infection with L. pneumophila We used enzyme-linked immunosorbent assay (ELISA) to determine IL-8 protein levels in culture supernatants of Jurkat cells at 8, 12, or 24 h after infection with either the parental strain Corby or flaA mutant strain at an MOI of 100. IL-8 was induced by Corby in a time-dependent manner. On the other hand, the amount of IL-8 produced by Jurkat cells infected with the flaA mutant strain was significantly less than that by cells infected with the wild-type strain (Fig. 4A). Corby-induced IL-8 production by Jurkat cells was MOI-dependent (Fig. 4B). Corby also induced a significant amount of IL-8 from CD4+ T cells (Fig. 4C). Figure 4 IL-8 production from Jurkat cells during infection with L. pneumophila strains.