The MyD88-mediated signaling cascade is important for MTb induction of NFAT5 expression in human monocytes.

<p>(A) Engagement of TLR2 significantly enhances NFAT5 mRNA synthesis in monocytes. THP-1 cells were incubated with the TLR2-specific ligand Pam3Cys (5 µg/ml), infected with MTb strain CDC1551 (1∶1 cells∶bacilli), or left untreated. After 16 hours, NFAT5 mRNA levels were measured. NFAT5 mRNA expression was significantly increased when the cells were incubated with Pam3Cys (*, p<0.05) or infected with MTb (**, p<0.01). (B) MyD88 is important for MTb induction of NFAT5 gene expression in human monocytes. THP-1 cells that constitutively express lentivirally-delivered shRNA targeting MyD88 or control shRNA targeting GFP were constructed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002620#s2" target="_blank">Materials and Methods</a>. Cells were infected with MTb strain CDC1551 (1∶1 cells∶bacilli) or left uninfected for 16 hours at 37°C, and RNA was analyzed. NFAT5 mRNA levels were significantly inhibited in the cells transduced with MyD88-specific shRNA (black bars) compared with the cells transduced with control shRNA (open bars) in both, the absence or presence of MTb infection. TNF and CD86 mRNAs, the expression of which are MyD88-dependent and -independent, respectively, during MTb infection, were measured as controls. (C, D) IRAK1 and TRAF6 are required for MTb induction of NFAT5 gene expression in human monocytes. THP-1 cells that constitutively express lentivirally-delivered IRAK1- or TRAF6-specific shRNA were constructed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002620#s2" target="_blank">Materials and Methods</a>. THP-1 cells expressing control shRNA were used as described in 7B. Cells were infected with MTb strain CDC1551 or left uninfected for 16 hours at 37°C, and RNA was analyzed. NFAT5 expression was significantly inhibited in the MTb-infected cells expressing either IRAK1 (C) or TRAF6 (D) shRNA (black bars) compared to cells expressing control shRNA (open bars). TNF and CD86 mRNAs, both of which require IRAK1 and TRAF6 to be induced by PRR activation, were measured as controls. (*, p<0.05; **, p<0.01).