SHP-1 And Heat Shock Participates Toll-like Receptor Signaling In Monocytes And Macrophages

Innate immune cells recognize pathogen-associated molecule patterns (PAMPs) conserved in microbes by pattern recognition receptors (PRRs) to initiate host immune responses against infectious pathogens. As an important kind of PRRs, Toll-like receptors (TLRs) play critical roles in the activation of immune system. In this study, we investigated the role of SHP-1 and heat shock in Toll like receptor signal pathway in monocytes and macrophages.Part I : SHP-1 participates in TLR9 signal pathway in macrophagesCpG motif is abundant in bacteria DNA, and is methylated and suppressed in mammalian DNA. These differences between bacteria and mammalian host DNA make CpG motif function as PAMP to activate protective host immune responses. CpG oligodeoxynucleotides (CpG ODN) containing CpG motif can mimic the activity of bacterial DNA to activate immune cells, including B cells, NK cells, macrophages and dendritic cells, to produce a variety of cytokines and chemokines. CpG ODN also induces maturation of dendritic cells and facilitates activation of Th1 immune response. The adjuvant activity of CpG ODN has been proven useful in treatment of cancer, infectious and allergy diseases.CpG ODN recognition and signaling are mediated by a member of Toll-like receptor (TLR) family, named TLR9. TLRs act as PRRs in detecting PAMPs and play important roles in triggering host defense immune response. Upon recognizing respective ligand, TLR recruits adapter protein, myeloid differentiation protein 88 (MyD88), and activates mitogen-activated protein kinases (MAPKs) and nuclear factor-KB (NF-kB) through MyD88/IRAK /TRAF6/TAK1 kinases cascade. Activation of both MAPK and NF-kBpathways is necessary for full activation of immune cells by TLR signaling.SHP-1 (Src homology 2 domain containing phosphatase 1), initially designated as SHPTP-1, SHP, HCP and PTP1C, is a cytosolic protein tyrosine phosphatase, contains two N-terminal-located SH2 domains, a single phosphatase domain and a C-terminal tail encompassing two sites for tyrosine phosphorylation. SHP-1 is expressed highly in hematopoietic cells and at a moderate level in many other types of cells, playing positive as well as negative roles in signal transduction. In this study, we investigated the role of SHP-1 in CpG 0DN/TLR9 signaling in murine macrophage cell line RAW264.7.Using RAW264.7 cells stably transfected with SHP-1 , we found that SHP-1 overexpression enhanced CpG ODN-induced IL-6 production. This result was further verified in cells transiently transfected with interfering SHP-1 RNA (SHPi). However, the disruption of catalytic activity of SHP-1(C453S) (mu-SHP-1) did not impair the regulatory effects of SHP-1 on CpG ODN-induced IL-6 secretion, suggesting that the expression level of SHP-1 positively regulate CpG ODN-induced activation of macrophages.MAPK signal pathway plays important roles in CpG ODN-induced cytokine production. Effects of SHP-1, mu-SHP-1 overexpression, SHPi on CpG ODN-induced MAPK and NF-kB activation in macrophages were studied. CpG ODN-induced phosphorylation of p38 was enhanced in SHP-1 transfected RAW264.7 cells 5min after CpG ODN stimulation as compared with that in mock-transfected cells. This result was further verified in cells transiently transfected with interfering SHP-1 RNA (SHPi). Mu-SHP-1 also remained unchanged, just like SHP-1 to enhance the phosphorylation of p38. Using NF-kB activity luciferase reporter system, we also found that SHP-1 overexpression increased NF-kB reported gene expression in macrophages as compared with that in mock-transfected cells. The results demonstrated that SHP-1 overexpression enhances p38 and NF-kB activation in macrophages stimulated with CpG ODN.To study how SHP-1 participates in CpG 0DN/TLR9 signaling, interactions of SHP-1 with TRAF6 and IKK were investigated by immunoprecipitation. We showed that SHP-1 associated with TRAF6 in a CpG ODN-dependent manner, and associated with IKK in aCpG ODN-independent manner in macrophages. The results suggest that SHP-1 plays a role in downstream of CpG ODN/TLR9 pathway by recruitmen...