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Blood, Vol. 113, Issue 14, 3235-3244, April 2, 2009
TLR2-dependent eosinophil interactions with mycobacteria: role of  -defensins
Blood Driss et al.
113: 3235
Supplemental materials for: Driss et al
Cytokine array
Multiple cytokines and chemokines in supernatants were simultaneously detected with Human Cytokine Array Panel following the recommendation of the manufacturer A (R&D Systems).
Files in this Data Supplement:
- Table S1. Characteristics of eosinophil donors and eosinophil membrane phenotype presented in Fig. 2B (PDF, 23.9 KB) -
ND, normal donors; SK, skin diseases; HES, hypereosinophilic syndromes.
- Figure S1. LAM and LM attract human eosinophils (JPG, 22.8 KB)
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Eosinophil migration in response to LAM and LM purified from BCG. Error bars represent Standard Error of the Mean (± SEM) from three independent experiments. *, p < 0.05, **, p < 0.01.
- Figure S2. Eosinophil response to BCG (JPG, 43.8 KB)
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(A) BCG-induced ROS production by eosinophils, purified from normal donor and eosinophilic patient (nº13 and 7 respectively in Table 1), is indicated for different BCG : eosinophil ratios 0 : 1 (−), 5 : 1 (+), 10 : 1(◇), 20 : 1(●). Results correspond to one representative of five experiments for each group and are expressed in counts per second (CPS). (B) Eosinophils were incubated with medium alone or BCG (10:1) for 18h. Cell-free supernatants were collected and analyzed by a cytokine protein microarray. Four dots in the top row (left and right) and two dots in the bottom row (left) indicate positive controls provided by the manufacturer. Results are representative from two independent experiments. 1. IL-13; 2. IL-16; 3. MIF; 4. MIP1-α; 5. MIP1-β; 6. IL-8.
- Figure S3. Eosinophil activation by TLR ligands and mycobacterial components (JPG, 34.7 KB)
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(A) Functionality of TLR2 and TLR4 on purified peripheral blood eosinophils. Eosinophils were incubated with or without blocking anti-TLR2 (20µg/ml) or anti-TLR4 (20µg/ml) antibodies following by activation with Pam3CSK4 (0.5 µg/ml) or LPS (0.1 µg/ml) respectively. Representative time course ROS production following the activation of eosinophil from eosinophilic patient is represented. Results are expressed as ΔROS cps values (values from medium stimulation are subtracted from values obtained with each TLR ligand). (B). EPO release by eosinophils, incubated with or without blocking anti-TLR2 (20µg/ml) or anti-TLR4 (20µg/ml) antibodies, upon Pam3CSK4 and LPS activation respectively. Results are expressed as ΔEPO cps values. Results are expressed as mean ± SEM (n = 4, 1 normal donors and 3 eosinophilic patients). (C) EPO release following eosinophil activation by LAM and LM. Results are espressed as ΔEPO cps values and are expressed as mean ± SEM (n = 3). * p < 0.05; **, p < 0.01.
- Figure S4. MyD88 and p38 MAP kinase pathway involvement during the LM-induced eosinophil activation (JPG, 29.2 KB)
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(A) Eosinophils were preincubated with serially diluted Myd88 inhibitor 1 µM, 10 µM or 100 µM or with control peptide 100 µM at 37°C for 30 min, further activated with LM (1 µg/ml) or with culture medium and EPO release was determined. Results are expressed as EPO cps (counts per second) values and as mean ± SEM (n = 3). (C) Purified eosinophils were pre-treated with inhibitor to p38 MAP kinase inhibitor (SB 203580) for 30 minutes at 37°C, before the addition of LM (1 µg/ml). EPO release by eosinophils was analysed by chemiluminescence. Bar graph represents the EPO cps (counts per second) values. Results are expressed as mean ± SEM (n = 3). * p < 0.05.
- Figure S5. Synergistic effect of α-defensins and ECP in eosinophil cytotoxicity (JPG, 38.3 KB)
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(A) BCG growth inhibition by purified α-defensins and ECP. CFU after incubation of BCG with ECP alone (50 ng/ml), α-defensins alone (50 µg/ml) and ECP and α-defensins together at 37°C for 6 hours. Data are shown as the mean of three independent experiments ± SEM, expressed in percentage of CFU control ((CFU with purified proteins/CFU with medium) × 100). Significant differences between conditions studied and controls are indicated as followed: *, p < 0.05, **, p < 0.01. (B) Intracellular BCG growth inhibition. Eosinophils were not treated or treated with a blocking anti-α-defensin (△) or anti-ECP (○) or the two antibodies (▲) or an isotype control (+) and were infected with BCG (5 : 1 ■). Values are mean ± SEM of three independent experiments. Significant differences between conditions studied and controls are indicated as followed: *, p < 0.05, **, p < 0.01.
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