Fig 2: TLR2 is important in inducing IDO-1 activity in human monocyte-derived dendritic cells (mDCs). mDCs were plated (1 × 106 cells per well) and 1 h prior to challenge, anti-human TLR2 (1 µg/ml) neutralizing antibody or MyD88 peptide inhibitor (MyD88i; IMGENEX) (100 µM) was added, followed by stimuli: M. leprae (ML), membrane fraction (MLMA), or soluble fraction (MLSA) was added at 10 µg/ml, TLR2 agonist Pam3Cys was used as positive control (10 µg/ml) for 24 h at 37°C 5%CO2. Controls are nonstimulated cultures. Supernatants from these cultures were collected and IDO-1 activity was assessed by the kynurenine/tryptophan ratio (kyn/trp) by HPLC. The plot represents the mean ± sd of five independently performed experiments. *P < 0.05 (Mann-Whitney)

Fig 3: Importance of TNF in the induction of IDO-1 activity in M. leprae-stimulated human monocyte-derived dendritic cells (mDCs). mDCs were plated (1 × 106 cells per well) and 1 h prior to challenge, anti-human IL-10 (100 µg/ml) or anti-human TNF (500 µg/ml) neutralizing antibodies were added at cultures followed by stimuli: M. leprae (ML), membrane fraction of M. leprae (MLMA), or soluble fraction of M. leprae (MLSA) were added 10 µg/ml or not (NS—nonstimulated cell). After 24 h, supernatants were collected and IDO-1 activity was assessed by the kynurenine/tryptophan ratio (kyn/trp) by HPLC. The plot represents the mean ± sd of four independently performed experiments. *P < 0.05 (Kruskal-Wallis)

Fig 4: MLMA is able to increase the frequency of IDO-1+TNF+ human monocyte-derived dendritic cells (mDCs). DCs differentiated from monocytes were plated (1 × 106 cells per well) and stimulated with M. leprae (ML), membrane fraction of M. leprae (MLMA), and soluble fraction of M. leprae (MLSA) at 10 µg/ml, for 24 h, or not (NS—unstimulated cell). Cells were labeled with anti-IDO-1 PE and Alexa Fluor 700 TNF intracellular antibodies. Acquisition was performed in a BD FACS Aria IIu flow cytometer and analysis performed using FlowJo software. Analysis is representative of cells from a donor from four experiments. For each sample, a minimum of 10,000 events were analyzed. *P < 0.05 (Mann-Whitney)