Role Of Toll Like Receptor 9 In Innate Immune Response Of Mice Skin

As the first line of the innate immune system, skin plays a crucial role in defence against microbial infection. It not only provides a physical barrier against microbial pathogens but also secretes cytokines, chemokines and antimicrobial peptides to mediate innate and acquired immune responses.However, the exact cellular mechanisms of this defence are not well understood. Toll like receptors (TLRs), a newly recognized 11-member family of vertebrate pattern recognition receptors (PRRs), have been identified as crucial mediators of innate immune recognition. Among them, TLR2 and TLR4 recognize lipopolysaccharide (LPS) and lipoproteins, respectively. TLR9, which is restricted to antigen presenting cells (APCs), is specific for unmethylated CpG oligodeoxynucleotides (CpG motifs) that are present in bacterial DNA. Bacterial CpG motifs act as PAMPs to activate TLR9-positive cells, through the Toll/IL-1-receptor signaling pathway, which in turn results in activation of transcription factors like NF-κB,JNK,P38 and ERK. This subsequently leads to the upregulation of co-stimulatory cell surface molecules and major histocompatibility complex (MHC) class I and II molecules, and the secretion of large amounts of proinflammatory cytokines such as IL-1, IL-6, TNF-( and IL-12. Thus, interactions between CpG DNA and TLR9 effectively bridge innate and acquired immunities. Although both TLR2 and TLR4 have been detected in normal human skin, little is known about the expression and function of TLR9, a CpG motif receptor, in skin.In this study, reverse transcription-polymerase chain reaction and in situ hybridization analysis were used to identify TLR9 mRNA expression in mouse skin. Results showed that TLR9 mRNA was not detected in normal mouse skin, but its presence in skin could be induced by intradermal injection of either normal saline, or the bacteria-based CpG motif in a time- and volume-dependent manner. TLR9 mRNA was detected at 6 and 9 hours, but not at 12 hours after intradermal injection of 20 (l NS or detected after treatment for 6 hours with 15 (l or 20 (l NS, but not after treatment with 0, 5 or 10 (l NS. In situ hybridization studies further demonstrated that clusters of TLR9 mRNA positive cells occurred in mouse skin after intradermal injection of 20 (l NS for 6 hours. In addition, H&E staining also detected that a lot of leucocytes infiltrated in skin in the same condition. It was speculated that TLR9 expression was the result of inflitration by leucocytes that express TLR9 by in situ hybridization and H&E staining detection. Furthermore, intradermal injection of CpG motif induced increased expression of mRNAs for proinflammatory cytokines such as interleukin (IL) 1, IL-6, IL-12 and tumor necrosis factor ( (TNF-(). This suggests that TLR9, while not present basally in skin, can be induced by physical trauma and then mediate responses to CpG motif.The above results suggest that the TLR9 expression was the result of infiltration by blood leucocytes that express TLR9, rather than the de novo synthesis by resident skin cells. To investigate the migration and function of these TLR9 positive cells induced by physical trauma and the role of CpG motif in this process is helpful for elucidating the function of TLR9 in innate immune response in skin. Chemokine receptors mRNA expression in skin and cytokines mRNA expression in draining lymph nodes (LNs) were detected by Real-time PCR after intradermal injection of normal saline, GpC motif or the bacteria-based CpG motif in skin. Chemokine receptor (CCR) 5 and CCR6 have been identified as the crucial receptors function in trafficking of immature peripheral blood mononuclear cells to tissues. When these cells are induced to mature, expression of CCR5 and CCR6 is gradually lost while expression of lymphoid chemokine receptors, CCR7 and CXCR4, is rapidly induced. It has been shown that IL-12 and IFN-( direct the differentiation of undifferentiated T helper (Th0) cells to T helper type 1 (Th1) cells and induces a cell-mediated immune response, and IL-4 and IL-10 are Th...