Syk Regulates AP-1 Dependent Response to mmLDL Figure 3. c-Jun and c-Fos DNA binding in WT and Syk2/2 macrophages stimulated with mmLDL. BMDM from WT or Syk2/2 mice (A and B) and J774 expressing control of Syk-specific shRNA (C and D) were incubated for 1 hour with media or 50 mg/ml mmLDL. Nuclear extracts were isolated and used in a transcription factor DNA binding plate-based assay. Mean 6 SEM (n = 4). *, p,0.05; **, p,0.005 WT vs. Syk2/2. doi:10.1371/journal.pone.0032378.g003 Next, for testing the effect of Syk deficiency on secretion of CXCL2 (MIP-2) and IL6 protein, we used two types of primary macrophages: Syk2/2 BMDM and peritoneal macrophages from Sykflox/flox mice infected in vitro with a Cre adenovirus (or with a GFP adenovirus as a control). In agreement with the mRNA levels (Fig. 5), mmLDL-induced secretion of CXCL2 was absolutely Syk- dependent, while secretion of IL-6 was reduced by 40–80% in Syk-deficient macrophages (Fig. 6). Graphs in Fig. 6 show the cytokine levels 6 hours after mmLDL stimulation, and graphs in Fig. S4 show cytokine levels after 24 hours. Discussion Using mmLDL as an activator of TLR4-dependent responses in primary macrophages, in this study we demonstrated that Syk regulates ERK1/2-dependent phosphorylation of c-Fos and JNK- and IKKe-dependent phosphorylation of c-Jun, leading to AP-1- dependent expression of proinflammatory cytokines. Although involvement of Syk in macrophage responses to LPS have been reported [29,30], uncovering the mechanism of Syk in TLR4- dependent transcriptional regulation became possible due to the fact that mmLDL, unlike LPS, does not induce any apparent MyD88-mediated responses [4,5]. In this study, we also observed MyD88-independent phosphorylation of ERK1/2 induced by mmLDL in macrophages (Fig. S5). The mechanism explaining lack of MyD88 activation by mmLDL is yet to be found. However, the use of mmLDL presents a tool for dissecting MyD88- independent pathways in experiments in which absence of a strong MyD88/NF-kB component does not mask other pathways downstream from TLR4. As we suggested in a recent review article [8], pattern recognition receptors (PRRs), such as TLR4, likely evolved in invertebrates and early vertebrates to control homeostasis and development. Studies from our group and the studies of others have demonstrated that oxidation-specific epitopes formed in apoptotic cells and in oxidized lipoproteins (mmLDL is an example) are recognized by PRRs of innate immunity (summa- rized in [8]). Responses to these host-derived damage-associated molecular patterns (DAMPs) involve homeostatic surveillance of the tissues, characterized by extensive cytoskeletal changes and macropinocytosis/phagocytosis and moderate secretion of inflam- matory cytokines. Our results showing mmLDL activation of the TLR4/Syk signaling pathway, resulting in macropinocytosis [5] and moderate AP-1 dependent secretion of CXCL2 and IL-6 (this study) support this hypothesis. Because many of the same oxidation-specific PRRs also recognize pathogen-associated molecular patterns (PAMPs), we speculated that the ability to protect against infectious pathogens provided a strong secondary selecting pressure for PRRs. For the purpose of host defense against microbial pathogens, stronger cellular responses were required to such infectious pathogens than those resulting from host-derived activation. Indeed, LPS-induced PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e32378