Induction of Direct Antimicrobial Activity Through Mammalian Toll-Like Receptors Sybille Thoma-Uszynski, Steffen Stenger, Osamu Takeuchi, Maria Teresa Ochoa, Matthias Engele, Peter A. Sieling, Peter F. Barnes, Martin Röllinghoff, Pal L. Bölcskei, Manfred Wagner, Shizuo Akira, Michael V. Norgard, John T. Belisle, Paul J. Godowski, Barry R. Bloom, and Robert L. Modlin

Medium version | Full size version

Supplemental Figure 2. Lipoprotein-induced NO production is mediated by TLR2. Peritoneal macrophages from wild-type (wt), TLR2^-/-, or TLR4^-/- mice were elicited by intraperitoneal injection with 2 ml of 4% thioglycollate medium (Difco). After 3 days, peritoneal exudate cells were isolated from the peritoneal cavity by washing with ice-cold Hanks' balanced salt solution (HBSS) (Gibco). Cells were cultured for 2 hours, nonadherent cells were removed by washing with HBSS, and cells were cultured in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum. At the time of stimulation with ligand, IFN-g?(30 U/ml) was added. NO was measured by the Griess reaction.

Medium version | Full size version

Supplemental Figure 3. Activation of human monocytes with bacterial lipoproteins reduced the viability of intracellular M. tuberculosis to an extent comparable in magnitude to the effects of activated mouse macrophages. Although lipoproteins stimulated interleukin 12 (IL-12) release from human monocyte-derived dendritic cells (DCs), no antimicrobial activity was detected by these cells. To generate DCs, monocytes were incubated in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) (1000 U/ml) and IL-4 (1000 U/ml) for 7 days. By flow cytometry, the resulting population of immature DCs expressed CD1a, MHC class II, and CD11c.

Medium version | Full size version

Supplemental Figure 4. TLR2 expression in tuberculous lymphadenitis by immunoperoxidase. Photomicrographs (a), (b), and (c) are from the same specimen and demonstrate the presence of TLR2 (red label) on large ovoid cells within granulomas, typical of monocytes. In photomicrographs from another specimen [shown in (d) and text Fig. 3b], the TLR2-positive cells are larger, typical of well-differentiated macrophages. In the third specimen, photomicrograph (e), TLR2-positive cells are in the infiltrate surrounding a focus of caseation necrosis. Photomicrographs (e) taken with 10× objective; (a and d), 20× objective; (b and c), 40× objective.

Medium version | Full size version

Supplemental Figure 5. Identification of TLR2-positive cells in lesions of tuberculous lymphadenitis by confocal laser microscopy. TLR2-positive cells are positive for CD14 and CD68, but negative for CD3.Double immunofluorescence was performed by incubating sections with mouse monoclonal antibody against human CD14 (clone RPA-MI, IgG2b, Zymed), antibody against human CD68 (clone Y1/82A, IgG2b, PharMingen) or antibody against CD3 (clone B355.1, IgG3 Biomeda, Foster City, CA). Sections were then incubated with isotype-specific fluorescein isothiocyanate (FITC)-conjugated goat antibody against mouse IgG2b or IgG3 (Caltag, Burlingame, CA) as appropriate, then antibody against TLR2 (clone 2392, IgG1) followed by tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat antibody against mouse IgG1 (Southern Biotechnology, Birmingham, AL). Controls included staining with isotype-matched irrelevant antibodies. Slides were examined with a Leica-TCS-SP inverted confocal laser scanning microscope equipped with krypton and argon lasers. Sections were excited with 488-nm blue light for FITC and 568-nm yellow light for TRITC. Fluorescent images were recorded sequentially through separate optical detectors with a 500- to 550-nm band pass setting for FITC and 590 long-pass setting for TRITC, respectively. Pairs of images were superimposed for colocalization analysis.

Medium version | Full size version

Supplemental Figure 6. Kinetics of antimycobacterial activity of the 19-kD lipoprotein. Fresh monocytes (2 × 10⁵) were pulse-infected with M. tuberculosis (MOI 1, 4 hours). Extracellular bacteria were removed by washing. The 19-kD lipoprotein (2 mg/ml) was added as indicated. After different time points cells were lysed, and the number of CFUs was determined by plating fourfold dilutions in duplicates.

Medium version | Full size version