The black lines define the assay cut-off of 3-fold induction or 70% reduction of transcript levels. Genes of interest are highlighted in black. (C) Inhibition of c-KIT recovers EGR1, chemokine, and cell adhesion transcript

PU-H71 ic50 levels in pathogenic Yersinia-infected THP1 cells. THP1 cells were pre-treated with 1μM OSI-930 for 18 h or were left ARN-509 untreated prior to infection with Y. pestis Ind195 at MOI 10 for 1 h. EGR1, VCAM1, CCL20, and IL-8 mRNA levels were determined by Taqman qPCR using total RNA isolated 24 h post-infection. Depicted RNA levels are relative to untreated THP1 control samples and were calculated using the 2-ΔΔCt formula. A ‘*” denotes that relative RNA levels were significantly different (p<0.05) compared to infected cells untreated with OSI930. Data is shown from three independent infection experiments performed Rigosertib supplier in duplicate. To further explore whether c-KIT function can regulate EGR1 and downstream inflammatory gene expression, we examined the effect of OSI-930 treatment on EGR1, VCAM1, CCL20, and IL-8 gene expression in Y. pestis-infected THP-1 cells using qPCR (Figure 4C). Inhibition of c-KIT kinase activity by OSI-930 (Figure 4C, dark gray bar) restored EGR1 transcription >2-fold in Y. pestis-infected THP-1 cells compared to infected

cells with functional c-KIT (Figure 4C, light gray bar). Similarly, OSI-930 treatment induced VCAM1, CCL20, and IL-8 transcription upon bacterial infection (Figure 4C, dark vs. light gray bars), suggesting that c-KIT function is required for the inhibition of key cytokines and adhesion molecules by pathogenic

Yersinia. Notably, treatment of THP-1 cells with OSI-930 alone did not significantly change EGR1 transcript levels (Figure 4C, white bar), indicating that however pharmacological inhibition of c-KIT did not initiate a non-specific immune response mediated by EGR1 in the absence of bacterial infection. Collectively, these findings suggest that there is a link between c-KIT function and suppression of the host immune response by pathogenic Yersinia and that transcriptional inhibition of EGR1 by Yersinia is dependent on c-KIT function. We next studied the role of Yersinia T3SS in suppression of the host immune response via c-KIT signaling. The expression profiles of EGR1, IL-8, and CCL20 were compared in THP-1 cells infected with pathogenic Y. enterocolitica WA and its non-pathogenic counterpart, Y. enterocolitica WA-01 (pYV-), cured of the pYV virulence plasmid (Figure 5A). Inhibition of c-KIT with OSI930 fully restored EGR1 levels in cells infected with virulent Y. enterocolitica and significantly recovered transcription of IL-8 and CCL20 at 5 h and 20 h post-infection (Figure 5A, dark grey bars). In contrast, we did not observe any significant effect by the c-KIT inhibitor OSI930 on EGR1, IL-8, and CCL20 transcription in THP-1 cells exposed to pYV- Y. enterocolitica.