Silencing TLR4 Down-regulates NOD1-mediated Inflammatory Responses by <I>Aspergillus fumigatus</I> Conidia Stimulation in RAW264.7 Macrophages

Abstract

This paper aims to study the role of NOD1 and TLR4 signal transduction in RAW264.7 monocyte-macrophages in the state of normal or immunosuppression when stimulated by <i>Aspergillus fumigatus</i> conidia and to investigate the expression of NOD1 signal transduction after silencing the gene of TLR4. First, we established an immune suppression model, and then assayed expression levels of NOD1, TLR4, RIP2, MyD88 mRNA and TNF-α protein using methods of RT-PCR and Western blot, when macrophages were stimulated for 12 h by <i>Aspergillus fumigatus</i> conidia after tranfected for 24 h with TLR4-siRNA by using technology of RNAi. RAW264.7 cells were cultured for 12 h with RPMI1640 culture medium containing immunosuppressive agents (methylprednisolone sodium succinate) at a concentration of 0.04mg/mL. The immune suppression model of macrophages was established with 90% survival rate. The signaling pathway of NOD1 and TLR4 in macrophages was activated by given stimulus of <i>Aspergillus fumigatus</i> conidia and exerted the effect of anti-<i>Aspergillus fumigatus</i> spores stimulation through releasing pro-inflammatory cytokines TNF-α. The macrophages cannot start their effective immune response when they were in the state of immunosuppression. Silencing efficiency of macrophates was up to 83% after transfection with TLR4-siRNA. Silencing the gene of TLR4 down-regulates the effect of NOD1 signal transduction in RAW264.7 cells on anti-<i>Aspergillus fumigatus</i> conidia stimulation.