We also demonstrate that although TNF-α gene induction was not significantly different in Mal−/− cells when compared with WT cells following poly(I:C) stimulation, a significant decrease in LPS-mediated TNF-α gene induction was evident (Fig. 1B). Next, we sought to investigate the role of Mal in the translational regulation of IFN-β and TNF-α by ELISA. As shown in Fig. 1C, we show that although stimulation of WT BMDM with poly(I:C) resulted in IFN-β induction, a significantly https://www.selleckchem.com/products/PD-0332991.html greater induction of IFN-β was evident in Mal−/− BMDM. Correlating with

real-time PCR data and the previous reports 16–18, LPS and poly(I:C)-induced IFN-β production was significantly decreased in TRIF-deficient BMDM when compared with WT BMDM (Fig. 1C). In accordance with the previous studies showing that Mal P125H and the TIRAP inhibitory peptide block LPS induced IFN-β gene induction 15, 19, we show that LPS-induced IFN-β production was significantly decreased in Mal-deficient BMDM when compared with WT BMDM (Fig. 1C). We also show that TNF-α and IL-6 induction were not significantly different in Mal−/− cells when compared with WT cells following poly(I:C) stimulation (Fig. 1E and F). As expected, click here we demonstrate an impairment of TNF-α and IL-6 induction in Mal- and TRIF-deficient BMDM cells stimulated with LPS

(Fig. 1E and F). To rule out the possibility that enhanced IFN-β in Mal−/− cells may be attributed to the BMDM immortalisation procedure per se, ex vivo BMDM from WT and Mal−/− mice were stimulated with either poly(I:C) or LPS and cytokines were measured by ELISA. Similar to data generated using the immortalised BMDM, poly(I:C)-induced IFN-β production was significantly enhanced in Mal-deficient BMDM when compared with WT BMDM (Fig. 1D). We also show that treatment of BMDM with a Mal inhibitory peptide significantly augmented poly(I:C)-mediated IFN-β gene induction when compared with cells treated with the control-inhibitory

peptide (Fig. 1G). Furthermore, C57BL/6, Mal-deficient and TRIF-deficient BMDM did not exhibit differences in TLR3 mRNA receptor expression, indicating that reported differences in gene induction are not attributable to perturbations in TLR3 Pyruvate dehydrogenase lipoamide kinase isozyme 1 expression levels (Table 1). Contrary to the previous reports 20, the data presented herein demonstrate that poly(I:C)-mediated induction of IFN-β in murine macrophages is TLR3 dependent, as TRIF, the critical adaptor involved in TLR3 signal transduction, is essential for poly(I:C)-mediated IFN-β induction. Also, correlating with the previous reports 21 poly(I:C)-mediated induction of IFN-β, CCL5/Rantes and TNF-α was similar in WT and MAVS−/− BMDM (Supporting Information Fig. 2), suggesting that the TLR and retinoic acid-inducible gene-I-like receptor (RLR) pathways work in parallel to sense viruses.