Mechanism Of IFN_γ-STAT1Signaling Pathway In The Abgogation Of LPS Tolerance

Bacterial lipopolysaccharide (LPS), the major structural component of the outer wall of Gram-negative bacteria, is a potent activator of macrophages. Prior exposure of innate immune cells like monocytes/macrophages to minute amounts of endotoxin causes them to become refractory to subsequent endotoxin challenge, a phenomenon called "endotoxin tolerance"(ET). Clinically, this STATe is associated with monocytes/macrophages in sepsis patients where they contribute to"immunosuppression" and mortality. The studies of endotoxin-tolerant murine macrophages and human monocytes show that most of the genes down-regulated upon LPS re-stimulation were inflammatory cytokines and chemokines like TNFa, IL-6, IL-12, IL-lb, CCL3, CCL4and CXCL10Up-regulated genes were more varied, consisting of anti-inflammatory cytokines such as IL-10, TGF-β and IL-1; scavenging C-type lectin receptors such as MARCO, CLEC4a and CD64; negative regulators such as IRAK-M and a variety of anti-microbial genes. Besides monocytes/macrophages, ET affects other myeloid cells, like dendritic cells (DC) and neutrophils. Endotoxin-tolerant DC show suppressed IL-12, TNFa and IL-6expression, but enhanced IL-10expression and endocytosis.From the previous studies, We know that1) ET can be viewed as a negative feedback response;2)ET is a case of gene reprogramming and immunomodulation;3) the regulation of ET is multi-leveled, involving receptors, signalling molecules, negative regulators and post-transcriptional changes like chromatin remodeling and microRNA regulation. Integrating these regulatory components into a mechanistic framework will be essential to the molecular basis of ET.There are many documents about the mechanisms of ET, but studies about the mechanisms to abrogate endotoxin tolerance have not been elucidated.Therefore, we will focus on the following aspects in this study:Objectives:1. Establish a model for IFN-γ-mediated inhibition of endotoxin tolerance, the influence of STAT1to inflammatory factors upon LPS stimulation;2. Investigating the role of STAT1in endotoxin stimulation and IFN-γ-mediated inhibition of endotoxin tolerance;3. Revealing the mechanism of of Ifnγ-STAT1signaling pathway in the abrogation of LPS tolerance.Methods:1. Establish a model for IFN-γ-mediated inhibition of endotoxin tolerance, testing the change of inflammatory factors.1.1Detection of inflammatory factors in the process of IFN-γ-mediated inhibition of endotoxin tolerance.Establish a model for IFN-γ-mediated inhibition of endotoxin tolerance, the expression of various inflammatory factors were measured at mRNA level by reverse transcriptional PCR and protein level by enzyme-linked immune sorbent assay (ELISA).1.2Detection mRNA level of inflammatory factors by RT-PCR after IFN stimulates various cell lines.After IFN γ stimulates cell line RAW264.7and mice peritoneal primary macrophages, detection mRNA level of inflammatory factors were measured by reverse transcriptional PCR. 1.3Investigating the role of STAT1SiRNA with IFN γ stimulates various cell lines.Confirmation STAT1SiRNA in HEK293cell and RAW264.7cell line by reverse transcriptional PCR and Western Blot. Tested STAT1SiRNA were transfected into cell lines,then mRNA level of inflammatory factors was measured by RT-PCR after stimulation by IFN γ,2. Determination the interaction between STAT1and transcription factors in IFN-γ-mediated inhibition of endotoxin tolerance.2.1Identification the interaction between STAT1and transcription factors in RAW264.7after IFNγ stimulation.IFNγ(10ng/ml) stimulates RAW264.7cell line, divide into control and stimulation groups, immune precipitation with antibody of P50、P65、P300, protein STAT1was measured by Wsetern blotting.2.2Identification the interaction between STAT1and transcription factors in IFN-γ-mediated inhibition of endotoxin tolerance.Establish a test for IFN-γ-mediated inhibition of endotoxin tolerance, set up three Raw264.7groups:Control, tolerance and IFN-y pretreatment groups.immune precipitation with antibody of P50、P65、P300, protein STAT1was measured by Wsetern blotting.2.3Dural fluorescence reporter gene assary about STAT1expressing vectors and NF-κB-luc reporter plasmid.NF-κB-luc reporter plasmid and adaptor MyD88plasmid in the LPS/TLR4signaling pathway were transfected together with STAT1expressing vectors in HEK293cell, analyzed the activation of NF-κB.3. Extraction cytoplasmic protein and nuclear protein, detect transcription factors P65and P50protein level.3.1Detect P50protein level in cytoplasm and nucleus through Western Blot.IFN-y-mediated inhibition of endotoxin tolerance,stimulation cells in various time point, extraction cytoplasmic protein and nuclear protein, detect the expression level of protein P50in cytoplasm and nucleus. 3.2Detect P65protein level in cytoplasm and nucleus through Western Blot.IFN-γ-mediated inhibition of endotoxin tolerance, stimulation cells in various time point, extraction cytoplasmic protein and nuclear protein, detect the expression level of protein P65in cytoplasm and nucleus.Results:1. Establish a model for IFN-γ-mediated inhibition of endotoxin tolerance successfully.1.1Establish a model for IFN-y-mediated inhibition of endotoxin tolerance, detect inflammatory factor P65.We use Raw264.7cell line to show that IFN-γ can block the development of endotoxin tolerance. We set up three Raw264.7groups:Control, tolerance and IFN-γ pretreatment groups. They are tolerized with low doses of LPS for24h before being restimulated with a second high dose of LPS in indicated times, and mRNA level of IL-6produce in response to LPS rechallenge is measured by RT-PCR and the protein level of IL-6is measured by ELISA.1.2IL-6is up-regulated by RT-PCR after IFNγ stimulates various cell lines.IFNγ stimulates cell line RAW264.7and mice peritoneal primary macrophages in different time point, detection mRNA level of inflammatory factors were measured by reverse transcriptional PCR. IL-6was significantly increased in stimulation group.1.3IL-6is down-regulated after STAT1is interfered by SiRNA.Identifying STAT1SiRNA in HEK293cell and RAW264.7cell line by RT-PCR and Western Blot, Tested STAT1SiRNA were transfected into RAW264.7,then mRNA level ofIL-6was measured by RT-PCR after stimulation in different time point by IFNγ. IL-6is down-regulated in stimulation group.2. Confirmation the interaction between STAT1and p65、P50、P300by immune precipitation (IP).2.1The combination between STAT1and p65、P50、P300is increased after stimulation with IFNy.RAW264.7cell line is stimulated by IFNγ, separating into control and stimulation groups, the interaction between STAT1and p65、P50is increased in stimulation group, that is measured by immune precipitation (IP) and Western Blot.2.2The combination between STAT1and p65、P50in IFN-γ-mediated inhibition of endotoxin tolerance is diversing.We set up three Raw264.7groups:Control, tolerance and IFN-γ pretreatment groups, stimulated in various time point, detect the interaction between STAT1and p65、P50. comparing with tolerance group, in IFN-γ pretreatment groups, the interaction between STAT1and p65is decreased, the interaction between STAT1and p50is increased, based on the results, we consider that STAT1interacts with P65maybe inhibit the transcriptional activity of P65.2.3STAT1interactes with P65and inhibites its luciferase reporter activity.After NF-κB-luc reporter plasmid and STAT1expressing vector were transfected into HEK293T cells together with adaptor molecule MyD88plasmid induced NF-κB luciferase activity was greatly inhibited by STAT1expressing vector transfection, compared with control transfection. These data indicate that STAT1can inhibit NF-κB activation and subsequent production of proinflammatory cytokines such as IL-6.3. Extraction cytoplasmic protein and nuclear protein, detect protein expression of P65and P50.3.1Analyzation the expression of endogenous p50by Western Blot in cytoplasm and nucleus.We use the model of IFN-γ mediated inhibition of endotoxin tolerance, extract cytoplasmic protein and nuclear protein, detect the protein level of P50in cytoplasm and nucleus through Western Blot. Experiment results show that the protein level of P50has no obvious change. thus we consider that P50may not be an important regulate role.3.2Analyzation the expression of endogenous p65by Western Blot in cytoplasm and nucleus.Based above experiment method, we detect the protein level of P65in cytoplasm and nucleus through Western Blot. Experiment results indicate that comparing with tolerance group, the cytoplasmic protein level of P65in IFN-γ pretreatment group is significant decreased, the nuclear protein level of P65in IFN-γ pretreatment group is increased in a certain extent.Conclusion:1. IFN-y can mediate inhibition of endotoxin tolerance, STAT1plays a key role in the induce of inflammatory cytokine IL-6.2. STAT1can interact with P65、P50, inhibit NF-κB luciferase reporter activity.3. Analyzation cytoplasmic protein and nuclear protein, We find that STAT1functions as a cytoplasmic attenuator of P65, attenuating the activity of transcription factor P65in the cytoplasm and prevent nuclear import of p65, influencing expression of proinflammatory cytokines. However after IFN-y pretreatment, STAT1is activated with phosphorylation, the inhibiting effect of p65is weakened or disappeared, p65can import into nuclear and activate transcription of inflammatory factor:IL-6etc, and the endotoxin tolerance is blocked by IFNγ-STAT1signaling pathway.However, the mechanism of IFNγ-STAT1signaling pathway in the abrogation of LPS tolerance is still not very perfect and considerate,we should need further research to reveal the reaction about transcription factorSTAT1and transcription factor P65.At the same time,the function of E3ligase about STAT1is now beening studied,we consider that STAT1may promote P65degradation via ubiquitination,the next plan,we want to research the role of the ubiquitin-proteasome STAT1.Innovation and significances:1. This study indicates that STAT1plays an important role in the mechanisms of IFN-γ abrogate the endotoxin tolerance. We first demonstrate a novel function of the transcription factor STAT1in the regulation of blocking endotoxin tolerance formation. reveal a new regulatory mechanism in IFN-γ-mediated inhibition of endotoxin tolerance.1) we find that STATl functions as a cytoplasmic attenuator of P65, attenuating the activity of transcription factor P65in the cytoplasm.2) After IFN-γ pretreatment, STAT1is activated with phosphorylation, the inhibiting effect of p65is weakened or disappeared, activate transcription of inflammatory factor:IL-6etc, and the endotoxin tolerance is blocked by IFNy-STAT1signaling pathway.At the same time,the function of E3ligase about STAT1is now beening studied,we consider that STAT1may promote P65degradation via ubiquitination.2. Although tolerance may be a protective mechanism to curb deleterious inflammation in acute settings, it also contributes to the delayed immunosuppression that is an important cause of mortality in patients with sepsis. Our findings provide mechanistic insights that can be used to modulate macrophage activation and cytokine production in settings of infection and inflammation. But the mechanism of IFNy-STAT1signal pathway abrogates lps tolerance is not very perfect and considerate. Some of the areas need to be addressed in future. And molecular mechanism and signal pathway of abrogating endotoxin tolerance should be futher clarified in the future.