05 for Msme PI-LAM and p < 0.001 for Mfort PI-LAM; Figure 4A). All of the LAMs had minimal interaction with TLR-4 (less than 2 fold induction), when compared to LPS-treated cells which increased CD25 expression about 7 fold (Figure 4B). Figure 4 PI-LAMs activate

cells in a TLR-2-dependent manner. A. CHO/CD14/TLR-2 and B. CHO/CD14/TLR-4 reporter cell lines were incubated with the indicated lipoglycans at 20 P5091 chemical structure μg/ml or LPS at 1 μg/ml for 16 h. Cellular activation was measured by determining the expression of CD25 at the cell surface by using anti-CD25 monoclonal antibodies and flow cytometry. The mean fluorescence intensities were determined and the fold induction over untreated cells was calculated and the mean and standard deviation of three independent experiments is shown. Overall, the results of the current study are very consistent with reported results demonstrating that the PI-LAM of an unidentified, fast-growing mycobacterial SCH727965 supplier species induces host cell cytokine secretion and apoptosis [24]. We extended these results to include PI-LAM of M. smegmatis and another PI-LAM of M. fortuitum [27], both of which induced host cell apoptosis and cytokine secretion. These results thus confirmed the general principle that PI-modified LAMs are pro-inflammatory. Furthermore, both of these PI-LAMs interact

with macrophage TLR-2 but not TLR-4 receptors suggesting that the PI-component is the ligand of the TLR-2. Interestingly, despite the existence of a mycolic acid rich PI3K inhibitor outermembrane in myocbacteria, it seems that LAM are still able to reach the outermost layers of the envelop to be exposed at the cell surface of the bacterium and thus exert their function as immunomodulins [29–31]. Non-pathogenic mycobacteria induce apoptosis via TNF and caspase-3 signaling pathways TNF is a central pro-inflammatory cytokine that mediates and regulates innate immunity. TNF binding to TNF-R1 may lead to activation of

NF- B, followed by gene transcription, production of inflammatory mediators and survival proteins. On the other hand, TNF binding may also initiate JNK protein kinase activation followed by activation of caspase-8 and downstream effector caspases such as caspase-3 resulting in apoptosis of the cell Hydroxychloroquine molecular weight [32]. In order to analyze the importance of TNF in apoptosis induction by the non-pathogenic mycoabcteria BALB/c BMDMs were infected with M. smegmatis, M. fortuitum, BCG, and M. kansasii at three MOIs (1:1, 3:1, and 10:1) for two hours and then incubated in medium with gentamycin for an additional 20 hours. The amounts of secreted TNF in the culture supernatants were measured using ELISA. BALB/c macrophages infected with M. smegmatis secreted 10 to 18 fold more TNF than macrophages infected with BCG or M. kansasii, which did not secrete significant amounts of TNF. M.