kansasii infected cells (Figure 5A). The impact of non-pathogenic mycoabcteria on IL-12 gene expression was also much higher when compared to facultative-pathogenic mycobacteria

(Figure 5B). Indeed, infection of the IL-12 p40 reporter cell line [12] at an MOI of 10:1 with M. smegmatis or M. fortuitum resulted in p40 promoter-driven GFP expression in about 30% of the cells, whereas only 5-10% of the cells became GFP positive after infection with the facultative-pathogenic mycobacteria (p < 0.001, Figure 5B). In conclusion, our results demonstrate a stronger induction of two pro-inflammatory cytokines (TNF and IL-12) after macrophage infection S3I-201 order with two species of non-pathogenic mycobacteria when compared to facultative-pathogenic mycobacteria. Figure 5 Differences in TNF secretion and IL-12 induction between facultative-pathogenic and non-pathogenic mycobacteria infected macrophages. A. BALB/c BMDMs were infected at MOIs of 1:1, 3:1, and

10:1 with M. smegmatis (Msme), M. fortuitum (Mfort), M. kansasii (Mkan), M. bovis BCG, or left untreated (UT). Cells were infected in triplicates for 2 h then washed and incubated in infection media with 100 μg/ml gentamycin for an additional 20 h. Culture supernatants were then collected and the amounts of secreted TNF was determined using ELISA. The values are the mean and standard deviation of triplicate readings and they are representative of three independent experiments. B. The induction of Il-12 gene expression was analyzed by infecting RAW/pIL-12-GFP macrophages with the indicated bacteria for 2 h at an MOI of 10:1. The GFP-expression was analyzed on 5,000 cells 16 h later and the mean and standard deviation of LY3009104 in vivo Digestive enzyme three independent experiments is shown. We showed that non-pathogenic mycobacteria induce a strong apoptotic response and TNF secretion in BALB/c macrophages (Figures 1B and 5A) when compared to facultative-pathogenic

mycobacteria. H 89 apoptosis of eukaryotic cells can follow either a caspase-dependent or caspase-independent pathway. All caspase-dependent pathways lead to activation of effector caspase-3/6/7 [33]. In order to determine which pathway was involved in the macrophage apoptotic response to non-pathogenic mycobacterial infection, we pretreated BALB/c BMDMs with caspase-3 inhibitor, TNF neutralizing antibody, pentoxifylline (a chemical inhibitor of TNF synthesis), the appropriate controls, or left the cells untreated then infected them with M. smegmatis at MOI of 10:1 for 2 hours. The cells were then incubated in media with gentamycin for an additional 20 hours. Host cell apoptosis was determined on 10,000 cells using the hypodiploid flow cytometry assay. In a representative experiment, cells treated with the caspase-3 inhibitor showed a significant decrease in apoptosis (1.2%) when compared to the untreated M. smegmatis infected control (20.0%) and to cells treated with an inactive chemical analogue of the caspase-3 inhibitor (16.