Histone Methylation And Its Impact On Inflammatory Transcription In Endotoximia

Endotoxin, also called lipopolysaccharides, which is released into circulatory system by gram-negative bacteria from blood, can cause endotoxemia. The LPS in the circulation can broadly affect a variety of tissues and organs in the body, wherein neutrophils, macrophages and endothelial cells, which are main effector cells can produce large amounts of cytokines, such as tomor necrosis factor a(TNFa), interleukin6(IL-6), interleukin1(IL-1), interferon(INF), nitric oxide(NO), platelet activating factor(PAF),etc. as is the term "cytokine storm", cause the uncontrolled inflammation cascade reaction, eventually cause endotoxin shock and tissue injury, and even multiple organ dysfunction syndromes if the injury is seriously developed. Currently, the treatment of endotoximia mainly include antibiotics, anti-endotoxin antibodies, inflammatory mediators antagonists, traditional Chinese medicine and blood purification methods.But what makes us regret is the effect is not significant or the feedback is too big. Therefore, the exploration and understanding of the exactly pathogenesis and intervention measures to endotoximia for the prevention and treatment of endotoxin shock and MODS has important therapeutic significance and practical value.TLR4play a key role in LPS-induced inflammatory response as recognized LPS receptor. The signaling pathway mediated by TLR4induced the changes in hundreds of gene expression when the TLR4was activated. The products of these genes expression not only included pro-inflammatory cytokine and anti-inflammatory cytokines, but also contained chemokines, anti-microbial protein, tissue repair factors, metabolic regulation factor and acquired immune control factor. The function of these products is different, so the products had different influence on the tissue damage and internal environment. For example, pro-inflammatory cytokines and anti-microbial protein is critical for protecting immune response, but as mentioned above, sustained or excessive production of pro-inflammatory cytokines may lead to a systemic inflammatory response and an acute septic shock might occur. On the other hand, the excessive production of anti-microbial protein plays an sligt role on the stabilities of the internal environment. The researchers found that the body’s tolerance could reduce the release of the inflammatory cytokine. So in order to avoid the infection of pathogenic bacteria, it was necessary to regulate the expression of the gene strictly. So it is necessary to regulate the generation of the proinflammatory cytokines excessive in order to avoid tissue damage. Furthermore, the body needs to generate the anti-microbial prtein continuously in order to prevent the invasion of microorganisms.However, these genes with different functions are mediated by the same receptor (TLR4) and the same signal transduction pathway. Also it occurs in the same type cells (macrophages), therefore it is possible for the regulation level in the downstream. It is call gene specific regulation.The specificity of gene regulation is mainly occured in the histone modification level. Histone modification can be influenced by environments, can become the signal mark of gene expression. Histone modification has many types, such as lysine methylation, acetylation, serine phosphorylation, ubiquitination and glycosylation. Those modifications can be affected by environment, and they can participate in many cell biology courses which include gene expression, mitosis, cell differentiation, chromosome inactivation and genetic imprinting. The enzymes that can regulate the histone modification include histone methyltransferase, histone demethylase, histone acetyltransferase, histone deacetylase and the protein complex related to the enzyme. Those enzyme and protein complex contribute to the change of histone modification and influence the chromation construction and participate in the transduction regulation. The regulation has two facts:1, change the interaction between histone and DNA through acetylation and deacetylation;2, formation a site with high affinity to recruit enzyme and protein complex which have the reconstruction chromatin locally.More and more convincing evidence have confirmed the gene specific regulation. Endotoxin tolerance is termed that after LPS pre-stimulated the macrophage or monocyte in vivo or in vitro, the reaction to LPS is reduced when stimulated by LPS again compared to the first stimulation, the cytokine (TNFa, IL-6, IL-1) release reduce; but the release of the molecular which is involved with antibiotic ability rise; and the release of the complement receptor type3and Fclll/ll receptor go up. At last we can infer that their expression is regulated by gene-specific mechanisms rather than signal specific mechanisms due to these two types of genes were expressed differently.In order to understand the specific mechanisms of regulation in different gene with specific functions, the TLR-induced genes are divided into two categories by the different function and regulation needs. The first category of genes is called as tolerizeable gene, which could not be re-induced when the toleranced macrophage is re-stimulated by LPS, while the second class of genes, non-tolerizeable gene could be re-induced. It was reported that tolerance gene contains a variety of cytokine which can lead to the inflammation, while the non-tolerance gene contained antimicrobial factor, pathogen recognition receptors, chemokines and other genes that can cause tissue damage indirectly. A furthermore study showed that the induce expression of these gene of two types required different histone modification patterns. When the normal macrophages were stimulated by LPS, the histone H4at the promoter region of these two types gene can both be acetylated, but only the non-tolerance gene can be re-acetylated at the promoter region again. The dynamic level of histone acetylation located in the promoter region of the non-tolerance gene in the endotoxin tolerance macrophages group reflects the changes in the level of gene expression. For example, the histone acetylation level of the tolerated cells was maintained at a relatively high level compared with normal cells. The histone acetylation level increased when it was stimulated, even may be faster. Therefore, the histone acetylation state is directly related with transcriptional activity between the tolerance gene and non-tolerance gene. These results show that the inflammatory cells can regulate specific gene expression through histone modification.The realization of gene specific regulation make us introspecte the reason of some anti-inflammation medicine have small effect or big feedback. Because of the tissue damage is mainly caused by the excessively release of inflammation mediator in sustained inflammation response. The excessively release of inflammation mediator is related with signaling pathways activation, so scientists have always pay attention to how effectively regulate the signaling pathways, and the method is to inhibit the signaling pathway with the feedback is that the anti-microbial protein is also inhibited at the same time, so the susceptibility of patients rises. So, it is a great idea to research the mechanism that histone modification can participate in the gene specific regulation. The idea not only contributes to demonstrate the pathophysiology between endotoxin shock and inflammation, but also guides searching more effective, reasonable anti-inflammation medicine.The preliminary studies of our laboratory have found acidification of some histone isoelectric point in liver cells of mice with endotoxin shock, and most of these changes occurred after LPS stimulation for30min to3h, indicating that histone acidification may be related to the endotoxin. Many papers also showed that the acidification of the histone isoelectric point is mainly due to post-translational modification of histones, including methylation, acetylation, phosphorylation, ubiquitin, and so on. Histone modifications change chromatin structure, which has an important impact on downstream gene transcription and expression.Therefore, the main purpose of this study was to detect which histone modification could be affected in the sepsis, and explored the expression of inflammatory genes change relations with the histone modification signals. On this bais, the study was divided into the following parts:The first part of the study was designed to study the histone modifications following LPS treatment. Those histone modifications included mono/di/tri-methylation on the forth lysine of histone H3(H3K4me1/2/3) and the twentieth lysine of histone H4(H4K20me1/2/3) and di-methylation on the ninth (H3K9me2), twenty-seventh (H3K27me2), thirty-sixth (H3K36me2), seventy-ninth lysine (H3K79me2) on histone H3and acetylation on histone H3and H4.The secone part was designed to explore the role of histone modifications in endotoxemia. We mainly focused on the transcription regulation of H3K4me2and H3K9me2on IL-6and TNFα.The results were as followed:1. Western Blot analysis demonstrated that H3K4me2/3, H3K9me2/3, H3K27me2, H3K36me2, H3K79me2, H3acetylation and H4acetylation were enhanced with different degree compared with control group following LPS(100μg/L) stimulation at0.5h,1h,2h,3h,6h, and the modification type that enhanced most obviously was H3K4me2and H3K9me2/3; however, H3K4me1, H3K9pan methylation, H4K20me1/2had no obvious change.2. Because H3K4me2and H3K9me2/3enhanced after LPS stimulated RAW264.7cell0.5h, we choose the two kinds of modifications to conduct next experiments. We used antibodies specific for H3K4me2and H3K9me2to recruit DNA which binded with the specific modifications-H3K4me2and H3K9me2(ChIP experiment), then we detected the interaction of the two histone modifications and the promoter of IL-6and TNFα with fluorescence quantitative PCR. The results demonstrated that the interaction of H3K4me2and the promoter of IL-6and TNFα increased after the RAW264.7cell was stimulated0.5h, the increase rate is121.8times and5.46times respectively; and the interaction of H3K9me2/3and the promoter of IL-6and TNFα abated after LPS(100μg/L) stimulated RAW264.7cells.3. In order to further clarified if H3K4me2was involved in the gene transcriptional regulation of IL-6and TNFα, we used histone methyltransferase inhibitors-MTA (5’-Deoxy-tp-(methylthio) adenosine) to inhibited H3K4me2/3, then tested the effect of MTA to the mRNA of IL-6and TNFa. The immunofluorescence experiments and western blot both showed that H3K4me2/3modifications were inhibited by MTA; then we found that MTA could inhibite the level of IL-6and TNFa mRNA with a dose-dependent manner. When the MTA dose was0.25mmol/L, MTA had no effect to the mRNA level; when the MTA dose was0.5mmol/L, the level of IL-6mRNA decreased by47%, and TNFa by48%compared with LPS group; when the MTA dose is1mmol/L, the level of IL-6mRNA decreased by90%, and TNFa by78%.Conclusions:1. In the course of endotoximia, H3K4me2/3, H3K9me2/3, H3K27me2, H3K36me2, H3K79me2, H4acetylation dynamic changed with the time. The change of histone modification was different in different cells (RAW264.7cell and BMM cell), and the change of different types of histone modification was different in the same cell. H3K4me2, H3K4me3and H3K9me2/3enhance most after LPS stimulated cells.2. H3K4me2modification levels rose at the promoters of IL-6and TNF alpha gene after stimulated by LPS for half of an hour in RAW264.7cells. After we inhibited the transcription of IL-6and TNF alpha with histone methyltransferase inhibitor MTA, the transcription of IL-6and TNF alpha could be inhibited, this demonstrated that H3K4me2participated in the transcription of inflammatory factor. Whether H3K9me2/3participated in the regulation of inflammatory factor in endotoximia needed experiment to be verificated.