Regulation Of Innate Immunity By Notch Signaling

Notch proteins are a family of large transmembrane receptors that regulate cell fate decisions during many developmental processes. Signals transduced through Notch receptors during cell–cell interactions directly regulate the expression of genes that are important to cell fate choices and differentiation. To date, four Notch receptors (Notch1-4) and five ligands (Delta1, -3, -4 and Jagged1, -2) have been identified in mammalians. Notch proteins play a pivotal role in regulating interpretation of environmental signals in a wide variety of cell types and species. After ligand binding, the intracellular region of Notch is proteolytically cleaved from the transmembrane portion and translocated to the nucleus, where it interacts with a transcriptional repressor, known as CSL. After binding Notch, CSL becomes a transcriptional activator, and in conjunction with the activity of other cofactors, including SKIP, PCAF, and/or GCN5, induces transcription of downstream targets.The ability of the innate immune system to recognize and respond to microbial components has been largely attributed to Toll-like receptors (TLRs). As a kind of important pattern recognition receptors (PRRs), TLRs recognize pathogen-associated molecule patterns (PAMPs), leading to a variety of signaling events that initiate innate immunity and activate immune cells to produce proinflammatory cytokines and type I interferon (IFN). In addition, TLR signaling can induce the up-regulation of MHC II and co-stimulatory molecules on antigen-prsenting cells such as dendritic cells (DC), facilitates DC maturation, and instructs development of antigen-specific adaptive immunity, especially Th1 response. Various other signal pathways are involved in the tight regulation of TLR signaling to enhance or attenuate their activation, which maintains the immunological homeostasis.There is a substantial body of literature detailing the expression of Notch family members as well as Notch ligands in cells and tissues of the immune system, and these reports provide supportive evidence that endogenous Notch may mediate regulation of immune function. Activated Notch can regulate the differentiation of thymocytes into either CD4 or CD8 SP cells. Notch signaling may regulate apoptosis during thymocyte maturation by preventing death by neglect and/or negative selection in cells destined to die. Notch is essential for Th2 cell differentiation but can also influence a Th1 cell response. However, the role of Notch signaling in TLR-triggered innate immunity remains unclear. It's been reported that LPS stimulation could increase the expression of Notch1, enhancing the Notch activation. Then, we wonder whether or not Notch receptors in turn affect the TLR-triggered signal pathway to regulate innate immunity? The aim of this study is to focus on the effect of activated Notch signaling on TLR-triggered production of inflammatory cytokines in macrophages and explore its underlying mechanisms.Part I. Notch expression in TLR-triggered macrophagesWe examined the expression of different Notch receptors in resting macrophages (Raw264.7 cells and mouse primary peritoneal macrophages) as well as macrophages after stimulation with different TLR ligands (LPS, CpG ODN or poly I:C). Fluorescence quota PCR (Q-PCR) result showed that both Raw246.7 cells and mouse peritoneal macrophages express Notch1, 2 and 4, but not Notch3. 100ng/ml LPS, 0.33M CpG and 10 ng/ml poly I:C stimulation could obviously increase the expression level of Notch1 in the Raw246.7 cells and mouse peritoneal macrophages. The results indicated that there may exist cross-regulation between the Notch and TLR signaling pathways. Part II. Effect of Notch signal on the TLR-triggered cytokine production in macrophagesConstitutive expression of active intracellular domain of Notch (NICD) in targeted cells could also result in an"activated"Notch phenotype. We constructed vectors expressing constitutively active intracellular domain of Notch1 (NICD1) and Notch2 (NICD2), as well as an inactive mutant of NICD1 (N1-6MT), in which the 3'PEST domain of NICD1 has been removed and replaced with 6 myc tags. The vectors were then transfected into mouse peritoneal macrophages and Raw 264.7 cells.Firstly, in mouse peritoneal macrophages, Q-PCR and ELISA results showed that overexpression of NICD1 and NICD2 can obviously strengthen LPS-induced secretion of IL-10, but decrease the secretion of inflammatory cytokines including IL-6,IL-1β, TNF-α, and IFN-β. But overexpression of the inactive form of NICD1, N1-6MT, had an opposite effects on cytokine secretion to that of NICD1 and NICD2, indicating that Notch signaling may inhibit TLR-triggered production of inflammatory cytokines in macrophages, and that the PEST domain of NICD1 may play an important role in the regulation of TLR-triggered cytokine secretion by NICD1.Secondly, the transfected Raw246.7cells were selected by resistance to neomycin. The stably transfected cells were designated as Raw246.7-NICD1, Raw246.7-NICD2 and Raw246.7-N1-6MT, respectively. We observed increased secretion of IL-10 but decreased secretion of inflammatory cytokines ( IL-6,IL-1β,TNF-αand IFN-β) after LPS stimulation in Raw246.7-NICD1 cells. But stable overexpression of N1-6MT results in opposite effect on cytokine production after TLR ligand stimulation. These results are consistent with those observed in mouse peritoneal macrophages, confirming the effect of Notch signaling on TLR-triggered cytokine production.Thirdly, Raw264.7 cells and mouse peritoneal macrophages were treated with Notch inhibitor, GSI, or transfected with Notch1-specific si-RNA, and then the secretion of cytokines (IL-10, IL-6, IL-1β, TNF-αand IFN-β) trigged by different TLR ligands were observed. The results showed that blockade of Notch1 signaling with GSI or inhibiting Notch1 expression by specific si-RNA could decrease IL-10 secretion, but increase IL-6, IL-1β, TNF-αand IFN-βsecretion induced by TLR ligands. The results further indicate the inhibitory effect of Notch signaling on TLR-triggered inflammatory cytokine production.Part III. The mechanisms for the regulation of TLR-triggered cytokine production in macrophages by Notch signalFirstly, we observed the effect of Notch signaling activation on the TLR signaling pathways in the macrophages. Western Blot result showed that overexpression of NICD1 could obviously suppress LPS-induced phosphorylation of ERK1/2, JNK p38 and I-κBαin Raw264.7 cells and mouse peritoneal macrophages. Accordingly, overexpression of N1-6MT had the opposite effect to that of NICD1. PD 98059 is a selective noncompetitive inhibitor of the MAPK pathway (Erk1/2, p-p44/42 MAPK). Pretreatment of macrophages with PD 98059 obviously antagonized the effect of Notch1 on TLR-triggered cytokine production. Therefore, the results suggest that Notch1 possibly participates in the TLR signaling through MAPK pathway.Secondly, we used the luciferase reporter gene assay to examine the effect of Notch signal on NF-κB as well as Notch and TNF-α. The results revealed that overexpression of NICD1 and NICD2 could obviously inhibit the reporter gene activities of NF-κB and TNF-αwhich activated by TRAF6 and MyD88; but overexpression of N1-6MT had a reverse effect on NF-κB and TNF-αreport gene activities.Thirdly, we used co-immunoprecipitation to identify target proteins that Notch1 interacted with in the TLR signaling pathways. We found that Notch1 promoted LPS-mediated phosphorylation of ERK1/2. But we detected no positive interaction between ERK1/2 and Notch1. So, it's speculated that Notch1 may not directly interact with ERK members, and that Notch1 may regulate ERK1/2 phosphorylation in an indirect manner. In conclusion, here we demonstrate the negative regulation of TLR-triggered inflammatory cytokine production and signaling pathway in macrophages by Notch1/2 signaling. We showed that activation of Notch1/2 signaling could regulate TLR-stimulated secrection of cytokines through ERK1/2 and NF-κB pathway. There are two possible explanations which need to be addressed. On one hand, Notch activation promotes LPS-induced IL-10, however, suppresss IL-6,IL-1β,TNF-αand IFN-βsecretion through different pathways. On the other hands, Notch activation firstly promotes IL-10 section, and then IL-10 in trun suppresses secretion of inflammatory cytokine (IL-6,IL-1β,TNF-αand IFN-β). The data may help us to understand the mechanisms by which Notch signaling might be involved in the innate immunity and provide the experimental basis for looking for the approaches to the treatment of inflammation by regulating Notch signaling.