Simultaneous Blocking of Human Toll-Like Receptors 2 and 4 Suppresses Myeloid Dendritic Cell Activation Induced byMycobacterium bovisBacillus Calmette-Guérin Peptidoglycan

Abstract

The Mycobacterium bovis bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-␣) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-␣ production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-B activation as determined by a reporter assay. Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-B activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-B in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.Phagocytosis of Mycobacterium tuberculosis by antigen-presenting cells is usually accompanied by activation of the transcription factor NF-B, secretion of inflammatory and initial cytokines, release of the reactive nitrites, including NO, and secretion of several chemokines (9, 16). These responses involve the outputs of the signaling of pattern recognition receptors for microbes (16, 34). More than 10 members of the mammalian Toll-like receptor (TLR) family in the innate immune system have been identified as representatives of such receptors that primarily respond to microbial constituents to elicit the immune response in macrophages and dendritic cells (DCs) (25,34). M. tuberculosis-mediated adjuvant activity may be expressed through TLRs on DCs.Two of the human TLRs, TLR2 and TLR4, are involved in M. tuberculosis-mediated intracellular signaling in vitro (22,41). Means et al. (22) demonstrated that viable M. tuberculosis bacilli contain distinct ligands that activate cells via TLR2 and TLR4, while heat-killed M. tuberculosis fails to activate cells via TLR4. Several purified mycobacterial ligands have now been identified as TLR2 agonists. Underhill et al. (43) ...

Combinational recognition of bacterial lipoproteins and peptidoglycan by chicken Toll-like receptor 2 subfamily

“…Peptidoglycan (Staphylococcus aureus) was purchased from Fluka Chemie, Tokyo. Mycobacterial PGN was purified from BCG cell-wall skeleton (CWS) as described previously (15).…”

Extracts of Larix Leptolepis effectively augments the generation of tumor antigen-specific cytotoxic T lymphocytes via activation of dendritic cells in TLR-2 and TLR-4-dependent manner

“…Arabinogalactan is included in bacterial cell wall, for example BCG. However, Arabinogalacton in BCG did not activate BMDCs [47]. It is noteworthy that structure of polysaccaride is different among species even if similar ingredients.…”

“…TLR4, but not TLR2 use TRIF/TICAM pathway, which induces Type1 interferon as well as TLR3. Thus, TLR2 and TLR4 have been considered to act cooperatively when they recognize various ligands [38,47,48]. Therefore, it is possible to speculate that ELL partially stimulates innate immunity by two signal pathways mediated by TLR2 and TLR4.…”

In vitro cytokine stimulation assay for glycolipid biosurfactant from Rhodococcus ruber: role of monocyte adhesion

“…There are only few reports on human monocyte/ macrophage/neutrophil stimulation in vitro by TDM or other mycobacterial glycolipids using oil-in-water emulsions (Tsuji et al 2000;Uehori et al 2003;Franzetti et al 2010) and glycolipid-coated polystyrene beads (Puissegur et al 2007;Simons et al 2007); however, there are no similar publications describing TDM-coated microplate application. As was shown by Perez et al (2000), addition of TDMcoated polystyrene beads led to some interfering nonspecific stimulation of mice bone marrow-derived macrophages resulting in increased levels of TNF-a, IL-1b, IL-6 possibly induced by cell adhesion to the beads.…”

“…For immunological analysis, water insoluble glycolipids are dissolved in organic solvents, e.g. chloroform, methanol, or oil-in-water emulsions are prepared with the mineral oil and chemical surfactants, e.g., Tween 80 (Tsuji et al 2000;Uehori et al 2003;Novik et al 2005). As was shown by Yarkoni and Rapp (1978), the toxicity and biological activity of trehalose-6,6'-dimycolate (TDM) from M. tuberculosis depend on the size distribution of the emulsion oil droplets.…”