Figure 4. AP-1 activation in Syk-expressing CHO cells. (A) CHO cells were transfected with 1 mg Flag-Syk (or empty vector), 200 ng AP- 1-Luc reporter, and 200 ng b-galactosidase plasmid. After 36 hours, cells were incubated with media or mmLDL for 6 hours and luciferase activity was measured and normalized to b-galactosidase activity. Mean6SEM (n = 3). *, p,0.05 vector vs. Syk, and media vs. mmLDL. (B) CHO Cells were transfected with 300 ng empty vector or Flag-Syk. After 24 hours, cells were incubated with mmLDL for 6 hours and cell lysates were tested for expression of Syk (Flag) and for p-JNK, p-c-Jun and GAPDH. doi:10.1371/journal.pone.0032378.g004 TLR4 activation results in an extremely strong MyD88-dependent NF-kB transcriptional response. This implies that the intracellular signaling pathways linking a PRR response to an endogenous DAMP may be different from those involved in the same PRR response to microbial PAMPs. This paper provides such an example showing MyD88-independent signaling via Syk in mmLDL-activated macrophages, resulting in a relatively muted cytokine response. However, chronic exposure to increased levels of oxidation-specific DAMPs, such as mmLDL in hypercholester- olemic subjects, likely induces low-grade but sustained activation of PRRs, e.g. TLR4 and its Syk signaling component, and thus, may contribute to the inflammatory state in atherosclerotic lesions. Understanding cellular mechanisms of Syk regulation has the immediate importance because the oral Syk inhibitor fostamatinib is currently in a phase III clinical trial in patients with rheumatoid arthritis. Several companies develop a number of new Syk inhibitors for various clinical applications [1]. Although targeting Syk is currently designed to benefit conditions with excessively Syk Regulates AP-1 Dependent Response to mmLDL Figure 5. mRNA expression of Cxcl2 and Il-6 in WT and Syk2/2 macrophages stimulated with mmLDL. BMDM from WT or Syk2/2 mice (A and B) and J774 cells expressing control or Syk-specific shRNA (C and D) were incubated for 1 hour with media or 50 mg/ml mmLDL. Total RNA was isolated and expression of Cxcl2 and Il-6 mRNA was measured by qPCR and normalized to the GAPDH mRNA levels. Mean 6 SEM (n = 3). *, p,0.05; **, p,0.005 WT vs. Syk2/2. doi:10.1371/journal.pone.0032378.g005 Figure 6. Secretion of CXCL2 (MIP-2) and IL-6 by WT and Syk2/2 macrophages stimulated with mmLDL. BMDM from WT or Syk2/2 mice (A and B) and resident peritoneal macrophages from Sykflox/flox mice infected with adenovirus expressing GFP or Cre (for 48 hours at 500 MOI) (C and D) were incubated for 6 hours with media or 50 mg/ml mmLDL. Cell culture media were collected and CXCL2 and IL-6 protein levels were measured by ELISA. Mean 6 SEM (n = 3). *, p,0.05; ****, p,0.00005 WT vs. Syk2/2. doi:10.1371/journal.pone.0032378.g006 PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e32378