Mycobacterium Tuberculosis-Induced Cell Death of Primary Human Monocytes and Macrophages Is Not Significantly Modulated by Tumor Necrosis Factor-Targeted.

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Mycobacterium Tuberculosis-Induced Cell Death of Primary Human Monocytes and Macrophages Is Not Significantly Modulated by Tumor Necrosis Factor-Targeted Biologicals Norbert Reiling, Dagmar Schneider, Stefan Ehlers Journal of Investigative Dermatology Symposium Proceedings Volume 12, Issue 1, Pages 26-33 (May 2007) DOI: 10.1038/sj.jidsymp.5650033 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Flow cytometric analysis of M. tuberculosis-induced cell death. Human monocytes (2 × 105) were incubated with M. tuberculosis (MOI 1:1) for (a) 24 hours, (b) 48 hours, and (c) 72 hours. At the indicated time points the cells were stained with TO-PRO-3 and analyzed in a flow cytometer. In all three graphs, forward scatter is depicted versus TO-PRO-3 fluorescence intensity. (a) At 24 hours, the majority of the cells are located in the lower right quadrant, representing TO-PRO-3 negative (viable) cells (R2). (b) At 48 hours after infection, many cells have decreased in size and move to the lower, left quadrant, still representing TO-PRO-3 negative cells. (c) At 72 hours almost all cells were located in the upper left quadrant, representing TO-PRO-3 positive (dead) cells (R1). Journal of Investigative Dermatology Symposium Proceedings 2007 12, 26-33DOI: (10.1038/sj.jidsymp.5650033) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The induction of cell death by M. tuberculosis in human monocytes is time and dose dependent. Monocytes (2 × 105) were incubated in the absence or presence of M. tuberculosis and cell death was analyzed as described in Materials and Methods. (a) Incubation at different multiplicities of infection (1:1, 3:1, and 10:1) for 48 hours. Staurosporine (Stau) (300nM) was used as a positive control. (b) Analysis at different time points (24 hours, 48 hours and 72 hours) at a MOI of 1:1. Means±SD of three independent experiments for each set of experiments (a and b) is shown. Med, medium. Journal of Investigative Dermatology Symposium Proceedings 2007 12, 26-33DOI: (10.1038/sj.jidsymp.5650033) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 TNF-targeted biologicals block TNF-induced cell death in fibroblast cultures. L929 fibroblasts at a density of 2.5 × 105 per well were incubated with 5 ng/ml TNF in the absence or presence of increasing concentrations of the TNF antagonists etanercept, PEGsTNFR1, adalimumab and infliximab for 20 hours. After crystal violet staining of the remaining cells, absorption was measured in a microplate reader at 550nm. (a–d) Means±SD of two sets of independent experiments are shown. Med, medium. Journal of Investigative Dermatology Symposium Proceedings 2007 12, 26-33DOI: (10.1038/sj.jidsymp.5650033) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 M. tuberculosis-induced cell death of human monocytes is not modulated by the addition of TNF antagonists in vitro. Human monocytes (2 × 105) were incubated for 48 hours with M. tuberculosis (MOI 1:1) in the absence or presence of increasing concentrations of the TNF antagonists (a) etanercept, (b) adalimumab, (c) PEGsTNFR1, and (d) infliximab. Cells were subsequently stained with TO-PRO-3 and analyzed in a flow cytometer. Means±SD of five independent experiments performed with cells from independent individuals are shown. Statistical analysis was performed using the Wilcoxon test. No significant differences between untreated and TNF antagonist treated cells were observed. Med, medium. Journal of Investigative Dermatology Symposium Proceedings 2007 12, 26-33DOI: (10.1038/sj.jidsymp.5650033) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 M. tuberculosis-induced cell death of human macrophages is not modulated by the addition of TNF antagonists in vitro. Human macrophages (2 × 105) were incubated for 48 hours with M. tuberculosis (MOI 1:1) in the absence or presence of increasing concentrations of the TNF antagonists (a) etanercept, (b) adalimumab, (c) PEGsTNFR1, and (d) infliximab. Cells were subsequently stained with TO-PRO-3 and analyzed in a flow cytometer. Means±SD of five independent experiments performed with cells from independent individuals are shown. Statistical analysis was performed using the Wilcoxon test. No significant differences between untreated and TNF antagonist-treated cells were observed. Med, medium. Journal of Investigative Dermatology Symposium Proceedings 2007 12, 26-33DOI: (10.1038/sj.jidsymp.5650033) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Primary human monocytes are highly resistant to TNF-induced cell death in vitro. Human monocytes (2 × 105) were incubated for 48 hours in the absence or presence of increasing concentrations of TNF as indicated. Cells were subsequently stained with TO-PRO-3 and analyzed in a flow cytometer. Means±SD of three independent experiments performed with cells from three independent individuals are shown. Med, medium. Journal of Investigative Dermatology Symposium Proceedings 2007 12, 26-33DOI: (10.1038/sj.jidsymp.5650033) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions