Activation of Murine Macrophages via TLR2 and TLR4 Is Negatively Regulated by a Lyn/PI3K Module and Promoted by SHIP1

Abstract

Src family kinases are involved in a plethora of aspects of cellular signaling. We demonstrate in this study that the Src family kinase Lyn negatively regulates TLR signaling in murine bone marrow-derived macrophages (BMMΦs) and in vivo. LPS-stimulated Lyn−/− BMMΦs produced significantly more IL-6, TNF-α, and IFN-α/β compared with wild type (WT) BMMΦs, suggesting that Lyn is able to control both MyD88- and TRIF-dependent signaling pathways downstream of TLR4. CD14 was not involved in this type of regulation. Moreover, Lyn attenuated proinflammatory cytokine production in BMMΦs in response to the TLR2 ligand FSL-1, but not to ligands for TLR3 (dsRNA) or TLR9 (CpG 1668). In agreement with these in vitro experiments, Lyn-deficient mice produced higher amounts of proinflammatory cytokines than did WT mice after i. v. injection of LPS or FSL-1. Although Lyn clearly acted as a negative regulator downstream of TLR4 and TLR2, it did not, different from what was proposed previously, prevent the induction of LPS tolerance. Stimulation with a low dose of LPS resulted in reduced production of proinflammatory cytokines after subsequent stimulation with a high dose of LPS in both WT and Lyn−/− BMMΦs, as well as in vivo. Mechanistically, Lyn interacted with PI3K; in correlation, PI3K inhibition resulted in increased LPS-triggered cytokine production. In this line, SHIP1−/− BMMΦs, exerting enhanced PI3K-pathway activation, produced fewer cytokines than did WT BMMΦs. The data suggest that the Lyn-mediated negative regulation of TLR signaling proceeds, at least in part, via PI3K.