Abstract
Macrophages (MØs) are sentinels of the immune system that use pattern recognition receptors such as Toll-like receptors (TLR) to detect invading pathogens and immune receptors such as FcγR to sense the host’s immune state. Crosstalk between these two signaling pathways allows the MØ to tailor the cell’s overall response to prevailing conditions. However, the molecular mechanisms underlying TLR-FcγR crosstalk are only partially understood. Therefore, we employed an immunologically-relevant MØ stimulus, an inactivated gram-negative bacterium that bears TLR2 agonists but no TLR4 agonist (iBTLR2) opsonized with a monoclonal antibody (mAb-iBTLR2), as a tool to study FcγR regulation of TLR2-driven production of IL-6, a key inflammatory cytokine. We chose this particular agonist as an investigational tool because MØ production of any detectable IL-6 in response to mAb-iBTLR2 requires both TLR2 and FcγR signaling, making it an excellent system for the study of receptor synergy. Using genetic, pharmacological and immunological approaches, we demonstrate that the murine MØ IL-6 response to mAb-iBTLR2 requires activation of both the TLR/NF-κB and FcγR/ITAM signaling pathways. mAb-iBTLR2 engagement of TLR2 drives NF-κB activation and up-regulation of IL-6 mRNA but fails to result in IL-6 cytokine production/release. Here, Src family kinase-driven FcγR ITAM signaling is necessary to enable IL-6 mRNA incorporation into polysomes and translation. These results reveal a novel mechanism by which FcγR ITAM signaling synergizes with TLR signaling, by “licensing” cytokine mRNA ribosome binding/translation to drive a strong murine MØ cytokine response.
Highlights
Macrophage (MØ) pattern recognition receptors such as the Toll-like receptors (TLRs) are capable of driving production of key inflammation-associated cytokines such as IL-6
To generate the ligand used throughout these studies, inactivated F. tularensis LVS bacteria were opsonized with a murine IgG2a anti-LPS mAb
The level of mAb opsonization was determined by staining of mAb-iBTLR2 with a fluorescently labeled anti-mouse IgG antibody and analysis of the stained bacteria by flow cytometry (Fig 1A)
Summary
Macrophage (MØ) pattern recognition receptors such as the Toll-like receptors (TLRs) are capable of driving production of key inflammation-associated cytokines such as IL-6. The cellular and molecular mechanisms of FcγR enhancement/modulation of TLR-driven cytokine production are only understood at a cursory level. For the analysis of receptor crosstalk or synergy, it is most appropriate to use an experimental agonist that possesses a pair of receptor ligands that each on their own drive a sub-maximal response. Under such conditions, receptor crosstalk becomes the main driving factor behind induction of a readily detectable immune response. An inactivated form of the gram-negative bacteria Francisella tularensis LVS, which has been opsonized with an IgG antiLPS mAb is such an agonist and is an excellent choice as a tool to study TLR-FcγR crosstalk
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