TLR2 and its co-receptors determine responses of macrophages and dendritic cells to lipoproteins of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) signals through Toll-like receptor 2 (TLR2) to regulate antigen presenting cells (APCs). Mtb lipoproteins, including LpqH, LprA, LprG and PhoS1, are TLR2 agonists, but their co-receptor requirements are unknown. We studied Mtb lipoprotein-induced responses in TLR2(-/-), TLR1(-/-), TLR6(-/-), CD14(-/-) and CD36(-/-) macrophages. Responses to LprA, LprG, LpqH and PhoS1 were completely dependent on TLR2. LprG, LpqH, and PhoS1 were dependent on TLR1, but LprA did not require TLR1. None of the lipoproteins required TLR6, although a redundant contribution by TLR6 cannot be excluded. CD14 contributed to detection of LprA, LprG and LpqH, whereas CD36 contributed only to detection of LprA. Studies of lung APC subsets revealed lower TLR2 expression by CD11b(high)/CD11c(low) lung macrophages than CD11b(low)/CD11c(high) alveolar macrophages, which correlated with hyporesponsiveness of lung macrophages to LpqH. Thus, lung APC subsets differ in TLR expression, which may determine differences in responses to Mtb.

Figure 1. Mycobacterial lipoproteins are TLR2 agonists

HEK293.TLR2 cells (filled symbols) or control HEK293.pcDNA3 cells (empty symbols) were exposed to mycobacterial lipoproteins for 15 h, and the concentration of IL-8 in culture supernatant was assayed by ELISA. Data represent mean +/− SD for triplicate samples, except where error is too small to be visualized. For concentrations above 10 nM, LprG, LpqH, LprA and Pam₃CSK₄ produced responses in HEK293.TLR2 cells that were significantly different from responses in HEK293.pcDNA3 cells (p < 0.01 for all such comparisons).

Figure 2. TLR1 is more important than TLR6 for response to mycobacterial lipoproteins

Bone marrow-derived macrophages from wild-type, TLR1^−/−, TLR2^−/−, or TLR6^−/− mice were stimulated for 12 h with mycobacterial lipoproteins in standard medium. Culture supernatants were assayed for TNF-alpha by ELISA. Data are representative of three independent experiments (two for PhoS1) that used independent preparations of lipoproteins and different preparations of bone marrow derived macrophages. Data are shown as the mean +/− SD of triplicate assays (*, p < 0.05; **, p < 0.01; and ***, p < 0.001 for comparison of wild-type vs. TLR1^−/− or TLR2^−/− cells).

Figure 3. CD14 contributes to the detection of LpqH, LprA and LprG, but not PhoS1 or FSL-1

Bone marrow-derived macrophages derived from wild-type or CD14^−/− mice were stimulated with a range of concentrations of agonist in standard medium (LPS) or SFM (all other agonists). TNF-alpha in culture supernatant was measured by ELISA. Data are representative of three independent experiments (two for PhoS1). Data represent mean +/− SD for triplicate samples (*, p < 0.05; **, p < 0.01 for comparison of CD14^−/− vs. wild-type cells).

Figure 4. CD36 contributes to the detection LprA, but not LpqH, LprG, or PhoS1

Bone marrow-derived macrophages derived from wild-type or CD36 knock-out mice were stimulated with a range of concentrations of agonist for 12 h in standard medium (LPS) or SFM (all other ligands). Culture supernatant was assayed for TNF-alpha by ELISA. Data are representative of three independent experiments (two for PhoS1). Data represent mean +/− SD for triplicate samples (*, p < 0.05 for comparison of CD36^−/− vs. wild-type cells).

Figure 5. Isolation and characterization of lung APCs by flow cytometry

Crude lung APCs were gated by scatter properties (A) and GR1-negativity (shaded peak is isotype, dotted line is stain) (B). APC subsets were characterized by expression of CD11b and CD11c (C and D). Alveolar macrophages (AM) are defined as CD11b^low/CD11c^high. Lung macrophages (LM) are defined as CD11b^high/CD11c^low and consisted of 2 morphologies. Lung DCs are defined as CD11b^high/CD11c^high. GR1^high events were excluded from analysis. PMN, morphology of GR1-positive events.

Figure 6. Differing levels of TLR2 on lung APC subsets

Surface staining for TLR2 (A), CD14 (B) and CD36 (C) is shown with dotted lines, and isotype control staining is shown in gray. Specific MFI values (see text) were calculated as MFI with specific Ab minus MFI with isotype control Ab. In panel D, cells were stimulated with LpqH for 16 h, and TNF-alpha in culture supernatant was measured by ELISA. Results are representative of 3 independent experiments. Data points represent the mean +/− SD of results from at least triplicate cell cultures. Due to limited cell numbers, fewer LpqH concentrations were assessed with lung macrophages. At the LpqH concentration points that were common to both cell types (10 and 100 nM), the responses were significantly different (**, p < 0.01; ***, p < 0.001).