Enhanced Toll-like receptor responses in the absence of signaling adaptor DAP12

Abstract

DAP12 is a signaling adaptor containing an immunoreceptor tyrosine-based activation motif (ITAM) that pairs with receptors on myeloid cells and natural killer cells. We examine here the responses of mice lacking DAP12 to stimulation through Toll-like receptors (TLRs). Unexpectedly, DAP12-deficient macrophages produced higher concentrations of inflammatory cytokines in response to a variety of pathogenic stimuli. Additionally, macrophages deficient in spleen tyrosine kinase (Syk), which signals downstream of DAP12, showed a phenotype identical to that of DAP12-deficient macrophages. DAP12-deficient mice were more susceptible to endotoxic shock and had enhanced resistance to infection by the intracellular bacterium Listeria monocytogenes. These data suggest that one or more DAP12-pairing receptors negatively regulate signaling through TLRs.TLRs are pattern recognition receptors used by cells of the innate immune system to detect the presence of a wide variety of pathogens. The 11 TLRs known in mammals allow the innate immune system to respond to a range of pathogen products, including proteins, lipoproteins, polysaccharides and nucleic acids 1 . In macrophages, TLR ligation leads to the production of proinflammatory cytokines, including tumor necrosis factor (TNF), interleukin 6 (IL-6) and IL-12. This cytokine production is crucial to a productive innate and adaptive immune response, leading to pathogen clearance. Additionally, when cytokine production is not properly regulated, inflammatory disorders such as rheumatoid arthritis or septic shock may result.The TLR signaling pathway differs somewhat for each TLR 1 . In common are a series of signaling proteins beginning with the adaptor protein MyD88, which associates with TLRs through homotypic interactions of Toll-IL-1 receptor (TIR) domains. When associated with a TLR, MyD88 recruits members of the IL-1 receptor-associated kinase (IRAK) family of serine-threonine kinases, which then phosphorylate and activate the ubiquitin ligase TNF receptor-associated factor 6 (TRAF6). Activation of TRAF6 leads to activation of the three mitogen-activated protein kinase (MAPK) pathways, p38 MAPK, ERK and JNK, and results in translocation of the transcription factor NF-κB into the nucleus. This occurs through the phosphorylation and degradation of IκBα, a protein that binds and retains NF-κB in the cytoplasm in resting cells. NF-κB is a key transcription factor in the induction of proinflammatory cytokines in macrophages, including TNF, IL-6 and IL-12 p40. Some TLRs also use other TIR-containing adaptor proteins, including TIRAP (also called Mal), TRIF (also Correspondence should be addressed to L.L.L. ( lanier@itsa.ucsf.edu).. Note: Supplementary information is available on the Nature Immunology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access RESULTS Increased cytokine production by DAP12-deficient macrophagesTo investigate the role of macrophage DAP12-associated receptors in the responses to...

CLEC‐2 signaling via Syk in myeloid cells can regulate inflammatory responses

“…5C, left panel) while IL-12/IL-23p40 levels were increased in Syk À/À DC supernatants (Fig. 5C, right panel), consistent with published data [33].…”

“…5C, left panel) while IL-12/IL-23p40 levels were increased in Syk À/À DC supernatants (Fig. 5C, right panel), consistent with published data [33].We next evaluated the contribution of the NFAT pathway to CLEC-2 modulation of LPS responses. The calcineurin inhibitor FK506 reduced IL-10 production in LPS and anti-CLEC-2 Fab-treated DCs to levels similar to DCs treated only with LPS (Fig.…”

Cutting Edge: Inhibition of TLR and FcR Responses in Macrophages by Triggering Receptor Expressed on Myeloid Cells (TREM)-2 and DAP12

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B-cell adaptor for PI3K (BCAP) negatively regulates Toll-like receptor signaling through activation of PI3K

“…BM-derived macrophages were made as described (28 . For measurement of intracellular TNF and IL-12 p40 by flow cytometry, 2 × 10 5 macrophages were plated in 24-well non-TC-treated plates overnight and then stimulated for 6 h with CpG DNA in the presence of Brefeldin A (10 μg/mL) for the final 2 h. Macrophages were blocked with 2.4G2 for 10 min and fixed in 4% paraformaldehyde (wt/vol), followed by permeabilization using Perm/Wash buffer (BD Bioscience).…”