Toll样受体3、9在皮肤中的作用机制和信号转导通路研究及药物的干预
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摘要
目的：研究TLR3、TLR9在皮肤天然免疫系统中的作用及调控机制，深入了解皮肤在天然免疫和获得性免疫中的桥梁作用，为预防和治疗皮肤免疫性疾病提供新的作用靶点。
方法：通过体外培养人角质形成细胞株HaCaT细胞和真皮成纤维细胞，应用免疫组化、real-time PCR、流式细胞术和Western blotting方法考察TLR3、TLR9在两种细胞上的调控特征，探讨TLRs在天然免疫和获得性免疫中的桥梁作用。在此基础上，研究地塞米松、霉酚酸和他克莫司对TLR3、TLR9及其信号转导通路的干预作用。探讨CpG-ODN的乙肝质粒DNA柔性脂质体经皮肤以TLR9为靶标进行免疫的可行性，并从离体角度研究经皮免疫的机制，证明TLR9及其信号通路在经皮免疫接种中的作用机制。
结果：确定了TLR3、TLR9在HaCaT和真皮成纤维细胞有表达，且可特异性的识别其相应的配体poly(I：C)和ODN2216，其信号转导通路是MyD88依赖性的，通过调控MyD88信号通路，可激活获得性免疫，使得Th1和Th2型细胞因子的分泌均有不同程度的增加，但是以诱导Th1型为主。Th1型细胞因子也可对TLR3、TLR9及其信号转导通路进行调控，形成天然免疫-获得性免疫调控网络。EGF可显著增强HaCaT细胞TLR3、TLR9的表达，调控其信号转导通路。免疫抑制剂霉酚酸和地塞米松对正常的HaCaT细胞的TLR3、TLR9受体及其信号通路无显著性影响，但对配体激活状态下HaCaT细胞表现出免疫抑制作用。他克莫司并未显示出对TLR3、TLR9及其信号转导通路的影响作用。构建了新的可表达CpG-ODN的乙肝质粒DNA柔性脂质体，证明了其经皮肤以TLR9为靶标进行免疫的可行性，并从离体角度研究经皮免疫的机制，研究了TLR9及其信号通路的作用机制，有助于更好的产生抗体和细胞免疫反应。
结论：研究了TLR3、TLR9在皮肤天然免疫系统中的作用及调控机制，探讨了免疫抑制剂和新型疫苗对该靶点作用的可行性，为该领域新的TLRs激动剂和拮抗剂的研制开发展现了广阔的前景，同时为经皮免疫提供了良好的技术支持和理论依据。
The immune system is complex defense system that evolving through ages. Itsbasic function is to recognize the foreign antigen and the upcoming signals of danger,and start the down-stream activation events to eliminate the foreign pathogen andmaintain the homeostasis in the body. The immune system of mammary animalsconsists of two parts—natural immune and acquired immune. Natural immune is alsocalled inherent immune, which is very old and possessed by all kinds of multi-cellularanimals and includes many kinds of different host defense mechanism. The inherentimmune has a quick action on the antigens, but with no memories. Like the skin, itacts as the first defense to our body. And the acquired immune only exists in vertebralanimals, which is an advanced defense system mediated by the antigen-specific Tcells and B cells. It has a slow action, but also with a memory to the antigens. Thenatural immune is the first step in an immune response. The pathogens are swallowedby the antigen presenting cells (APC). And then the antigens are degenerated andpresented in form of MHCⅡantigen-peptides to the T-cell to activate the immuneresponse. Meanwhile the immune response is also activated through the recognition ofpathogens and activation of the immune and inflammatory cells to eliminate theinvading microbes.
Toll like receptors (TLRs) family is a new kind of mammary pattern recognitionreceptors (PRRs), which mediate the recognition of the pathogen-associatedmolecular pattern (PAMPs), and play an important role in the natural immune system.The TLR3 is involved in the recognition of virus dsRNA, and TLR9 mainly presentedin the surface of APC, with a specific recognition of the CpG gene sequence in thebacterial genome. The recognition of microbes, APC, activation of T-cell and cellsthat trigger the acquired immune response were all linked together by the TLRs. Andthe TLRs are activated by binding with their ligands and then transmit the signalsdown-stream through the Toll/IL-1R pathway, which finally activates transcription factors such as NF-κB、JNK、P38、ERK. Then the expression of MHCⅠand MHCⅡmolecules and their costimulating factors are up-regulated, the ability of APCs aresimulated, and various kinds of acute inflammatory factors such as IL-1、IL-6、IL-12and TNF-αare induced. So the interaction between TLRs and their ligands is animportant bridge in the process of natural and acquired immune responses.
The skin is the first defense in the natural immune system, and plays animportant role in the prevention of foreign microbe infection. First, it acts as aphysical barrier to the pathogens. Second, the skin can secrete many kinds ofcytokines, chemotactic factors and antibiotic peptides to mediate the natural andacquired immune process, and facilitate the elimination of pathogens. But the specificunderlying mechanisms of these defense systems are still unclear. Virus dsRNA andbacterial CpG are the main pathogenic factors in the skin-related diseases, and theyare the exactly the ligands of TLR3、TLR9. So is there an expression of TLR3、TLR9in the skin under normal circumstances? If there's an expression of these factors,where do they come from, and how do they function? If there's no expression of thefactors by the skin, are they induced from foreign cells? And how do these factorsactivate the signaling pathway to trigger a immune response? There questions willelucidate the roles of TLR3、TLR9 in the skin natural immune system.
This study is to observe the expression patterns of TLR3、TLR9 in two kinds ofcells—epidermis keratinocyte HaCaT and dermis fibroblast, by means ofimmunohistochemistry, real-time PCR, flowcytometry, and western blotting. After thedetermination of the existence of the TLR3、TLR9, we investigate the regulationeffects of TLR3、TLR9 ligands—poly(I:C), ODN2216 and LPS on the expression ofTLR3、TLR9, and discuss their influences to the signaling pathway. Meanwhile, aninvestigation of the time-dose relation in the secretion of Th1 and Th2 cytokines iscarried out to serve as a theoretical basis for the role of TLR3、TLR9 in the skinnatural immune system. Then discuss the regulation role of cytokines to the naturalimmune system. After that, we investigate the effect of immunosuppressants such asFK506, MPA and Dex on the expression of TLR3、TLR9 receptors and discover newtargets for the drugs, providing a theoretical basis for the development of new immunosuppressants. Finally, investigate the possibility of transdermal immune inboth in vivo and in vitro studies, after the construction of Hepatitis B plasmid DNAliposome with the expression of CpG-ODN and discuss the expression of HBsAG indifferent stages in the immune response process and elucidate the effect of vacantliposome and vaccine liposome on the skin natural immune system.
The study consists of the following five parts:
Part One: The expression of TLR3、TLR9 in human HaCaT and fibroblasts.
1. Immunohistochemistry results showed that there are positive expressions of TLR3、TLR9 in the normal HaCaT cells and dermis fibroblasts.
2. Real-time PCR showed that there are expressions of TLR3、TLR9 in normal HaCaTcells and dermis fibroblasts and the expressions can be significantly enhanced byLPS、poly(I:C)、ODN2216, and acts in a ligand specific fashion.
3. FC results showed that there are significant positive expressions of TLR3、TLR9 innormal HaCaT cells and dermis fibroblasts (P＜0.05). Various simulating factors allexerts influence over the expression of the TLR3 in HaCaT cells, with poly (I: C) cansignificantly upregulate the coexpression of TLR3 in the surface and inside of thecells. ODN2216 has no significant effect on the expression of TLR3 in two cell types,but can significantly upreguiate the coexpression of TLR9. poly (I:C) has nosignificant effect on the TLR9 expression in two cell types. And LPS can significantlyupregulate the coexpression of TLR3、TLR9 in both cell types.
4. Western blotting results showed that there are expressions of TLR3、TLR9 in bothnormal HaCaT cells and dermis fibroblasts and they can be regulated by variousfactors. Poly (I: C) and LPS can significantly increase the expression of TLR3 in bothcells, while ODN2216 has no effect. And ODN2216 and LPS can significantlyincrease the expression of TLR9 in both cells, while poly (I: C) has no effect.
All the studies showed that there are TLRs expressions in HaCaT cells and dermisfibroblasts which will play key role in the natural immune system. After binding withthe ligands, the same kind of biological characteristics as previous studies showed up,which is indicative an important role of both cells in the natural immune response.
Part Two: The effect of activation of TLR3、TLR9 on the secretion of Th1/Th2cytokines in HaCaT cells and fibroblasts and the MyD88 signaling pathway.
1. 1. Real-time PCR results showed that there are expressions of IFN-γ, IL-4 andMyD88 mRNA in normal HaCaT cells and dermis fibroblasts. And after poly(I:C),ODN2216 and LPS treatments with the HaCaT cells, the impacts on the connectinMyD88 are the same. And after treatments with FB cells, poly (I:C) has moresignificant effect on MyD88, while LPS has more significant effect on the secretion ofIFN-γand IL-4.
2. 2. ELISA assays have elucidated the time-effect relation in the secretion of IFN-γand IL-4 in both cells after the treatment of poly(I:C), LPS and ODN2216. the resultsshowed that both cells can secret some IFN-γand IL-4 in normal status. And aftertreatment with poly(I:C), ODN2216 and LPS for 0、6、12、24、48h, the concentrationof IFN-γand IL-4 in the supernatant fluids are elevated, and there's a more significantincreasing trend for IFN-γ.
3. The western blotting assays have semi-quantitatively detected the changes inthe expression of MyD88 after different ligands treatments in both cells. The resultsshowed expression in both cells and the expression could be upregulated by theTLR3、TLR9 ligands.
The studies in this part confirmed that the interaction between ligands and TLR3、TLR9 are not only regulated by the expression of receptors but also through theactivation of acquired immune response and influence over the expression of MyD88which will regulate the secretion of cytokines, induce the TH1 immune reaction.Therefore, the expression of TLR3、TLR9 with specific drugs and both the secretionof cytokines and signaling pathway would be affected, which has important roles inthe skin immune system. These results also confirmed that TLR3、TLR9 play animportant role in the natural and acquired immune response. And we can make furtherresearches in an integrated fashion, and a new age of the immunological research isopened to us.
Part Three: The effects of IFN-γ、TNF-αand EGF on the TLR3、TLR9 and theirsignaling pathway in HaCaT cells and FB cells
1. Detected the expression level of TLR3、TLR9 and MyD88 mRNA by means ofreal-time PCR in HaCaT cells and FB cells after treatment with IFN-γ(50ng·mL~(-1))、TNF-α(100ng·mL~(-1))and EGF(10ng·mL~(-1))for 24h. The results showed an elevationin the TLR3、TLR9 and MyD88 mRNA levels in both two cells (EGF has. nosignificant effect on the expression of TLR3、TLR9 and the signaling pathway in FBcells), from 2~2-2~8 folds.
2. Detected the amount of the receptors on the surface and inside the HaCaT cells andFB cells with flow cytometry after treatment with IFN-γ(50ng·mL.(-1))、TNF-α(100ng·mL~(-1))and EGF(10ng·mL~(-1))for 24h. the results showed an elevation in theexpression of TLR3、TLR9 after treatment with IFN-γ、TNF-αand EGF. And afterremoving the effects of membrane-expression, there's no significant differences foundin the expression level of receptors inside the cells for TLR3 and significant elevationfor TLRg. In normal FB cells, except the EGF, IFN-γ、TNF-αboth can increase thelevel of receptors. And after removing the effects of membrane-expression, there'ssignificant increase found in the expression level of both TLR3、TLR9 inside thecells.
3. Detected the expression level of the TLR3、TLR9 with western blotting in HaCaTcells and FB cells after treatment with IFN-γ(50ng·mL~(-1))、TNF-α(100ng·mL~(-1))andEGF (10ng·mL~(-1))for 24h. The results showed that IFN-γ、TNF-αand EGF all cansignificantly increase the expression of MyD88 in normal HaCaT cells. And TNF-αcan significantly increase the expression of MyD88 in normal FB cells, while EGBhas no effect.
Studies in this part IFN-γ, TNF-αand EGF can affect TLRs and their signalingpathway in different fashions. These results provide us a new drug target fortransdermal immune therapy and give us a new way to the development of newvaccines to treat and prevent the immunological and inflammatory diseases.
Part Four : The regulation of immunosuppressants on the TLR3、TLR9 and theirsignaling pathway in HaCaT cells.
1. Real-time PCR showed that given single dose of DEX、FK506 and MPA didn'taffect the expression of TLR3、TLR9 and MyD88. But in the presence of poly (I:C),the expression of TLR3 and MyD88 increased significantly. After co-culture withpoly (I:C), DEX can significantly increase the expression of TLR3 and MyD88, whileFK506 has no significant effect. The expression of TLR9 will be significantlyelevated after co-culture of DEX and poly (I: C). There's significant elevation in theexpression of TLR9 and MyD88 in the presence of only ODN2216, while there'ssignificant elevation in the induced expression of TLR9 and MyD88 after Co-cultureof ODN2216 and DEX. MPA can also increase the expression level of TLR9, whileFK506 has no obvious effect. MPA can increase the expression of MyD88 afterco-culture with poly(I:C)、ODN2216.
2. FC results showed that given single dose of DEX、FK506 and MPA can notchange the expression pattern of TLR3、TLR9 on the surface and inside the HaCaTcells. The expression of TLR3 can be elevated in the presence of DEX and poly (I: C),while there's no effect of FK506 and MPA on the expression of TLR3. Single dose ofpoly (I: C)、ODN221, FK506 and MPA exerts no effect on the expression of TLR3inside the cells, but it will be significantly elevated in the presence of DEX and poly(I: C). ODN2216 has no effect on the expression of TLR3, but it will increase theexpression of TLR3 inside the cell in the presence of DEX. When given together withODN2216, DEX can significantly increase the expression of TLR9, and so does MPA.But FK506 exerts no effect over the expression of TLR9. The expression level insideand outside the cell will be elevated in the presence of DEX and poly (I: C).
3. Western blotting results showed that DEX、MPA can significantly increase theexpression of MyD88. And FK506 has no effect on the expression of TLR3、TLR9and their signaling pathway.
4. ELISA results showed that the secretion of IL-4 and IFN-γhas no difference aftersingle treatment with DEX、FK506 and MPA only (the MPA group has a significantdepression in 12h.). But in the presence of poly (I: C), DEX and MPA can significantly reduce the secretion of IL-4. And the secretion of IL-4 can also besignificantly suppressed in the presence of ODN2216 and DEX. MPA only exerts itssuppression at 24h while FK506 has significant suppression at both 12h and 24h.DEX、FK506 and MPA cannot suppress the secretion of IFN-γin the presence ofpoly(I: C). And only MPA has significant suppression both at 12h and 24h.
The results of the studies suggest that the above three kinds of drugs cansuppress the expression of TLR3、TLR9 in skin cells, and increase the secretion ofcytokines, reduce the expression of cytokines, therefore exert a anti-inflammatoryeffect through their effect over the MyD88 singling pathway.
Part Five: The design of TLR9-targeted HBsAg vaccine and study of themechanism of its transdermal immune response.
1. Preparation of the flexible liposome. Construct the eukaryon vector pVAX/HBS-CpG which can express the Pre-S2/S protein and CpG sequence at the sametime. The Hepatitis B DNA vaccine cation flexible liposome was successfullyconstructed with the material of biodegradable and skin compatible DPPC andsoftener of octadecylamine. Investigate the size, distribution, shape, deformation rateand release rate, and determine the in vivo immune effect. The liposome was preparedby retro-evaporation and high-pressure homogen. Then investigate various parameters.Immunohistochemical assays were carried out to identify the distribution of HBsAgand titration of the antibody in the skin after local dermal administration of thevaccine. The results showed that the flexible liposome are transparent suspension witha encapsulating rate of (64.5±7.84)％, the diameter is (110.5±17.7) nm. EM showeda round shape. And no significant difference observed after storage under 4℃for 6months. Immunohistochemistry results showed strong positive expression of HBsAgat day 2, 3, 5, 7, with a main distribution in the hair follicles and tubular gland.
2. The in vivo immune study showed that the IgG titration has no significantdifference with the use of flexible liposome. Transdermal administration of theflexible liposome can induce a titration of antibody 3-4 fold higher than conventionalliposome. And the flexible liposome has a higher penetrativity and immunity, with more effect afterlocal administration to prevent the hepatitis B.(p＞0.05).
3. Detect the effect of vacant liposome and HBsAg plasmid DNA liposome on theexpression of TLR3、TLR9 in HaCaT cells and FB cells by means of real-time PCR.The results showed that both cells has an increased expression of TLR3、TLR9, butthe expression levels are significantly different (p＜0.05). And there is no significantdifference in the receptor expression after treatment of vacant liposome and HBsAgplasmid DNA liposome (p＞0.05).
4. Detect the effect of vacant liposome and HBsAg plasmid DNA liposome on theexpression levels of TLR3、TLR9 inside and on the surface of HaCaT cells and FBcells by means of FC. MFI of the TLR3、TLR9 on the membrane of HaCaT cells hasincreased 32.1％and 34.7％respectively, but the expression levels are significantlydifferent (p＜0.05). And there is no significant difference in the receptor expressionafter treatment of vacant liposome and HBsAg plasmid DNA liposome (p＞0.05).
5. Investigate the effect of vacant liposome and HBsAg plasmid DNA liposome on theexpression levels of MyD88 in HaCaT cells and FB cells by means of westernblotting. The results showed that the expression levels of MyD88 increased at 4h aftervacant liposome and HBsAg plasmid DNA liposome treatment, without adose-dependant fashion, which indicates an alternative pathway other than MyD88 inthe signaling of TLR in the two kinds of cells.
6. Investigate the effect of vacant liposome and HBsAg plasmid DNA liposome on thetime-effect relation of the secretion of Th1 and Th2 cytokines (IFN-γ, and IL-4) inHaCaT cells and FB cells by means of ELISA. The results showed that there's a dose-and time-dependant increase after vacant liposome and HBsAg plasmid DNAliposome treatment, with a maximum effect at 48h and the expression level in HBsAgplasmid DNA liposome group has a significant elevation (p＜0.05).
The study in this part is to design HBsAg vaccine targeted in TLR9 and maketransdermal immune possible. It will enhance our knowledge about the underlyingmechanism, and provide a helpful reference to the designation and enhancement ofthe immune response. The results showed that the vaccine can increase the immunitythrough the increase of TLRs which provide a theoretical foundation for thedevelopment of transdermal immunological drugs in the future.
In conclusion, this study elucidates the expression pattern of TLR3、TLR9receptors in the skin in various ways, discusses the role and regulation mechanism intransdermal and immunosuppressant action, and provides a new target for thetreatment of inflammatory diseases. The discussion about the possibility of effectsexerted by different immunosuppressants on the target provides a new insight into thedevelopment of new TLR promoter and antagonist, and provides a technical supportand theoretical foundation for the development of transdermal immunological drugsin the future.
方法：通过体外培养人角质形成细胞株HaCaT细胞和真皮成纤维细胞，应用免疫组化、real-time PCR、流式细胞术和Western blotting方法考察TLR3、TLR9在两种细胞上的调控特征，探讨TLRs在天然免疫和获得性免疫中的桥梁作用。在此基础上，研究地塞米松、霉酚酸和他克莫司对TLR3、TLR9及其信号转导通路的干预作用。探讨CpG-ODN的乙肝质粒DNA柔性脂质体经皮肤以TLR9为靶标进行免疫的可行性，并从离体角度研究经皮免疫的机制，证明TLR9及其信号通路在经皮免疫接种中的作用机制。
结果：确定了TLR3、TLR9在HaCaT和真皮成纤维细胞有表达，且可特异性的识别其相应的配体poly(I：C)和ODN2216，其信号转导通路是MyD88依赖性的，通过调控MyD88信号通路，可激活获得性免疫，使得Th1和Th2型细胞因子的分泌均有不同程度的增加，但是以诱导Th1型为主。Th1型细胞因子也可对TLR3、TLR9及其信号转导通路进行调控，形成天然免疫-获得性免疫调控网络。EGF可显著增强HaCaT细胞TLR3、TLR9的表达，调控其信号转导通路。免疫抑制剂霉酚酸和地塞米松对正常的HaCaT细胞的TLR3、TLR9受体及其信号通路无显著性影响，但对配体激活状态下HaCaT细胞表现出免疫抑制作用。他克莫司并未显示出对TLR3、TLR9及其信号转导通路的影响作用。构建了新的可表达CpG-ODN的乙肝质粒DNA柔性脂质体，证明了其经皮肤以TLR9为靶标进行免疫的可行性，并从离体角度研究经皮免疫的机制，研究了TLR9及其信号通路的作用机制，有助于更好的产生抗体和细胞免疫反应。
结论：研究了TLR3、TLR9在皮肤天然免疫系统中的作用及调控机制，探讨了免疫抑制剂和新型疫苗对该靶点作用的可行性，为该领域新的TLRs激动剂和拮抗剂的研制开发展现了广阔的前景，同时为经皮免疫提供了良好的技术支持和理论依据。
The immune system is complex defense system that evolving through ages. Itsbasic function is to recognize the foreign antigen and the upcoming signals of danger,and start the down-stream activation events to eliminate the foreign pathogen andmaintain the homeostasis in the body. The immune system of mammary animalsconsists of two parts—natural immune and acquired immune. Natural immune is alsocalled inherent immune, which is very old and possessed by all kinds of multi-cellularanimals and includes many kinds of different host defense mechanism. The inherentimmune has a quick action on the antigens, but with no memories. Like the skin, itacts as the first defense to our body. And the acquired immune only exists in vertebralanimals, which is an advanced defense system mediated by the antigen-specific Tcells and B cells. It has a slow action, but also with a memory to the antigens. Thenatural immune is the first step in an immune response. The pathogens are swallowedby the antigen presenting cells (APC). And then the antigens are degenerated andpresented in form of MHCⅡantigen-peptides to the T-cell to activate the immuneresponse. Meanwhile the immune response is also activated through the recognition ofpathogens and activation of the immune and inflammatory cells to eliminate theinvading microbes.
Toll like receptors (TLRs) family is a new kind of mammary pattern recognitionreceptors (PRRs), which mediate the recognition of the pathogen-associatedmolecular pattern (PAMPs), and play an important role in the natural immune system.The TLR3 is involved in the recognition of virus dsRNA, and TLR9 mainly presentedin the surface of APC, with a specific recognition of the CpG gene sequence in thebacterial genome. The recognition of microbes, APC, activation of T-cell and cellsthat trigger the acquired immune response were all linked together by the TLRs. Andthe TLRs are activated by binding with their ligands and then transmit the signalsdown-stream through the Toll/IL-1R pathway, which finally activates transcription factors such as NF-κB、JNK、P38、ERK. Then the expression of MHCⅠand MHCⅡmolecules and their costimulating factors are up-regulated, the ability of APCs aresimulated, and various kinds of acute inflammatory factors such as IL-1、IL-6、IL-12and TNF-αare induced. So the interaction between TLRs and their ligands is animportant bridge in the process of natural and acquired immune responses.
The skin is the first defense in the natural immune system, and plays animportant role in the prevention of foreign microbe infection. First, it acts as aphysical barrier to the pathogens. Second, the skin can secrete many kinds ofcytokines, chemotactic factors and antibiotic peptides to mediate the natural andacquired immune process, and facilitate the elimination of pathogens. But the specificunderlying mechanisms of these defense systems are still unclear. Virus dsRNA andbacterial CpG are the main pathogenic factors in the skin-related diseases, and theyare the exactly the ligands of TLR3、TLR9. So is there an expression of TLR3、TLR9in the skin under normal circumstances? If there's an expression of these factors,where do they come from, and how do they function? If there's no expression of thefactors by the skin, are they induced from foreign cells? And how do these factorsactivate the signaling pathway to trigger a immune response? There questions willelucidate the roles of TLR3、TLR9 in the skin natural immune system.
This study is to observe the expression patterns of TLR3、TLR9 in two kinds ofcells—epidermis keratinocyte HaCaT and dermis fibroblast, by means ofimmunohistochemistry, real-time PCR, flowcytometry, and western blotting. After thedetermination of the existence of the TLR3、TLR9, we investigate the regulationeffects of TLR3、TLR9 ligands—poly(I:C), ODN2216 and LPS on the expression ofTLR3、TLR9, and discuss their influences to the signaling pathway. Meanwhile, aninvestigation of the time-dose relation in the secretion of Th1 and Th2 cytokines iscarried out to serve as a theoretical basis for the role of TLR3、TLR9 in the skinnatural immune system. Then discuss the regulation role of cytokines to the naturalimmune system. After that, we investigate the effect of immunosuppressants such asFK506, MPA and Dex on the expression of TLR3、TLR9 receptors and discover newtargets for the drugs, providing a theoretical basis for the development of new immunosuppressants. Finally, investigate the possibility of transdermal immune inboth in vivo and in vitro studies, after the construction of Hepatitis B plasmid DNAliposome with the expression of CpG-ODN and discuss the expression of HBsAG indifferent stages in the immune response process and elucidate the effect of vacantliposome and vaccine liposome on the skin natural immune system.
The study consists of the following five parts:
Part One: The expression of TLR3、TLR9 in human HaCaT and fibroblasts.
1. Immunohistochemistry results showed that there are positive expressions of TLR3、TLR9 in the normal HaCaT cells and dermis fibroblasts.
2. Real-time PCR showed that there are expressions of TLR3、TLR9 in normal HaCaTcells and dermis fibroblasts and the expressions can be significantly enhanced byLPS、poly(I:C)、ODN2216, and acts in a ligand specific fashion.
3. FC results showed that there are significant positive expressions of TLR3、TLR9 innormal HaCaT cells and dermis fibroblasts (P＜0.05). Various simulating factors allexerts influence over the expression of the TLR3 in HaCaT cells, with poly (I: C) cansignificantly upregulate the coexpression of TLR3 in the surface and inside of thecells. ODN2216 has no significant effect on the expression of TLR3 in two cell types,but can significantly upreguiate the coexpression of TLR9. poly (I:C) has nosignificant effect on the TLR9 expression in two cell types. And LPS can significantlyupregulate the coexpression of TLR3、TLR9 in both cell types.
4. Western blotting results showed that there are expressions of TLR3、TLR9 in bothnormal HaCaT cells and dermis fibroblasts and they can be regulated by variousfactors. Poly (I: C) and LPS can significantly increase the expression of TLR3 in bothcells, while ODN2216 has no effect. And ODN2216 and LPS can significantlyincrease the expression of TLR9 in both cells, while poly (I: C) has no effect.
All the studies showed that there are TLRs expressions in HaCaT cells and dermisfibroblasts which will play key role in the natural immune system. After binding withthe ligands, the same kind of biological characteristics as previous studies showed up,which is indicative an important role of both cells in the natural immune response.
Part Two: The effect of activation of TLR3、TLR9 on the secretion of Th1/Th2cytokines in HaCaT cells and fibroblasts and the MyD88 signaling pathway.
1. 1. Real-time PCR results showed that there are expressions of IFN-γ, IL-4 andMyD88 mRNA in normal HaCaT cells and dermis fibroblasts. And after poly(I:C),ODN2216 and LPS treatments with the HaCaT cells, the impacts on the connectinMyD88 are the same. And after treatments with FB cells, poly (I:C) has moresignificant effect on MyD88, while LPS has more significant effect on the secretion ofIFN-γand IL-4.
2. 2. ELISA assays have elucidated the time-effect relation in the secretion of IFN-γand IL-4 in both cells after the treatment of poly(I:C), LPS and ODN2216. the resultsshowed that both cells can secret some IFN-γand IL-4 in normal status. And aftertreatment with poly(I:C), ODN2216 and LPS for 0、6、12、24、48h, the concentrationof IFN-γand IL-4 in the supernatant fluids are elevated, and there's a more significantincreasing trend for IFN-γ.
3. The western blotting assays have semi-quantitatively detected the changes inthe expression of MyD88 after different ligands treatments in both cells. The resultsshowed expression in both cells and the expression could be upregulated by theTLR3、TLR9 ligands.
The studies in this part confirmed that the interaction between ligands and TLR3、TLR9 are not only regulated by the expression of receptors but also through theactivation of acquired immune response and influence over the expression of MyD88which will regulate the secretion of cytokines, induce the TH1 immune reaction.Therefore, the expression of TLR3、TLR9 with specific drugs and both the secretionof cytokines and signaling pathway would be affected, which has important roles inthe skin immune system. These results also confirmed that TLR3、TLR9 play animportant role in the natural and acquired immune response. And we can make furtherresearches in an integrated fashion, and a new age of the immunological research isopened to us.
Part Three: The effects of IFN-γ、TNF-αand EGF on the TLR3、TLR9 and theirsignaling pathway in HaCaT cells and FB cells
1. Detected the expression level of TLR3、TLR9 and MyD88 mRNA by means ofreal-time PCR in HaCaT cells and FB cells after treatment with IFN-γ(50ng·mL~(-1))、TNF-α(100ng·mL~(-1))and EGF(10ng·mL~(-1))for 24h. The results showed an elevationin the TLR3、TLR9 and MyD88 mRNA levels in both two cells (EGF has. nosignificant effect on the expression of TLR3、TLR9 and the signaling pathway in FBcells), from 2~2-2~8 folds.
2. Detected the amount of the receptors on the surface and inside the HaCaT cells andFB cells with flow cytometry after treatment with IFN-γ(50ng·mL.(-1))、TNF-α(100ng·mL~(-1))and EGF(10ng·mL~(-1))for 24h. the results showed an elevation in theexpression of TLR3、TLR9 after treatment with IFN-γ、TNF-αand EGF. And afterremoving the effects of membrane-expression, there's no significant differences foundin the expression level of receptors inside the cells for TLR3 and significant elevationfor TLRg. In normal FB cells, except the EGF, IFN-γ、TNF-αboth can increase thelevel of receptors. And after removing the effects of membrane-expression, there'ssignificant increase found in the expression level of both TLR3、TLR9 inside thecells.
3. Detected the expression level of the TLR3、TLR9 with western blotting in HaCaTcells and FB cells after treatment with IFN-γ(50ng·mL~(-1))、TNF-α(100ng·mL~(-1))andEGF (10ng·mL~(-1))for 24h. The results showed that IFN-γ、TNF-αand EGF all cansignificantly increase the expression of MyD88 in normal HaCaT cells. And TNF-αcan significantly increase the expression of MyD88 in normal FB cells, while EGBhas no effect.
Studies in this part IFN-γ, TNF-αand EGF can affect TLRs and their signalingpathway in different fashions. These results provide us a new drug target fortransdermal immune therapy and give us a new way to the development of newvaccines to treat and prevent the immunological and inflammatory diseases.
Part Four : The regulation of immunosuppressants on the TLR3、TLR9 and theirsignaling pathway in HaCaT cells.
1. Real-time PCR showed that given single dose of DEX、FK506 and MPA didn'taffect the expression of TLR3、TLR9 and MyD88. But in the presence of poly (I:C),the expression of TLR3 and MyD88 increased significantly. After co-culture withpoly (I:C), DEX can significantly increase the expression of TLR3 and MyD88, whileFK506 has no significant effect. The expression of TLR9 will be significantlyelevated after co-culture of DEX and poly (I: C). There's significant elevation in theexpression of TLR9 and MyD88 in the presence of only ODN2216, while there'ssignificant elevation in the induced expression of TLR9 and MyD88 after Co-cultureof ODN2216 and DEX. MPA can also increase the expression level of TLR9, whileFK506 has no obvious effect. MPA can increase the expression of MyD88 afterco-culture with poly(I:C)、ODN2216.
2. FC results showed that given single dose of DEX、FK506 and MPA can notchange the expression pattern of TLR3、TLR9 on the surface and inside the HaCaTcells. The expression of TLR3 can be elevated in the presence of DEX and poly (I: C),while there's no effect of FK506 and MPA on the expression of TLR3. Single dose ofpoly (I: C)、ODN221, FK506 and MPA exerts no effect on the expression of TLR3inside the cells, but it will be significantly elevated in the presence of DEX and poly(I: C). ODN2216 has no effect on the expression of TLR3, but it will increase theexpression of TLR3 inside the cell in the presence of DEX. When given together withODN2216, DEX can significantly increase the expression of TLR9, and so does MPA.But FK506 exerts no effect over the expression of TLR9. The expression level insideand outside the cell will be elevated in the presence of DEX and poly (I: C).
3. Western blotting results showed that DEX、MPA can significantly increase theexpression of MyD88. And FK506 has no effect on the expression of TLR3、TLR9and their signaling pathway.
4. ELISA results showed that the secretion of IL-4 and IFN-γhas no difference aftersingle treatment with DEX、FK506 and MPA only (the MPA group has a significantdepression in 12h.). But in the presence of poly (I: C), DEX and MPA can significantly reduce the secretion of IL-4. And the secretion of IL-4 can also besignificantly suppressed in the presence of ODN2216 and DEX. MPA only exerts itssuppression at 24h while FK506 has significant suppression at both 12h and 24h.DEX、FK506 and MPA cannot suppress the secretion of IFN-γin the presence ofpoly(I: C). And only MPA has significant suppression both at 12h and 24h.
The results of the studies suggest that the above three kinds of drugs cansuppress the expression of TLR3、TLR9 in skin cells, and increase the secretion ofcytokines, reduce the expression of cytokines, therefore exert a anti-inflammatoryeffect through their effect over the MyD88 singling pathway.
Part Five: The design of TLR9-targeted HBsAg vaccine and study of themechanism of its transdermal immune response.
1. Preparation of the flexible liposome. Construct the eukaryon vector pVAX/HBS-CpG which can express the Pre-S2/S protein and CpG sequence at the sametime. The Hepatitis B DNA vaccine cation flexible liposome was successfullyconstructed with the material of biodegradable and skin compatible DPPC andsoftener of octadecylamine. Investigate the size, distribution, shape, deformation rateand release rate, and determine the in vivo immune effect. The liposome was preparedby retro-evaporation and high-pressure homogen. Then investigate various parameters.Immunohistochemical assays were carried out to identify the distribution of HBsAgand titration of the antibody in the skin after local dermal administration of thevaccine. The results showed that the flexible liposome are transparent suspension witha encapsulating rate of (64.5±7.84)％, the diameter is (110.5±17.7) nm. EM showeda round shape. And no significant difference observed after storage under 4℃for 6months. Immunohistochemistry results showed strong positive expression of HBsAgat day 2, 3, 5, 7, with a main distribution in the hair follicles and tubular gland.
2. The in vivo immune study showed that the IgG titration has no significantdifference with the use of flexible liposome. Transdermal administration of theflexible liposome can induce a titration of antibody 3-4 fold higher than conventionalliposome. And the flexible liposome has a higher penetrativity and immunity, with more effect afterlocal administration to prevent the hepatitis B.(p＞0.05).
3. Detect the effect of vacant liposome and HBsAg plasmid DNA liposome on theexpression of TLR3、TLR9 in HaCaT cells and FB cells by means of real-time PCR.The results showed that both cells has an increased expression of TLR3、TLR9, butthe expression levels are significantly different (p＜0.05). And there is no significantdifference in the receptor expression after treatment of vacant liposome and HBsAgplasmid DNA liposome (p＞0.05).
4. Detect the effect of vacant liposome and HBsAg plasmid DNA liposome on theexpression levels of TLR3、TLR9 inside and on the surface of HaCaT cells and FBcells by means of FC. MFI of the TLR3、TLR9 on the membrane of HaCaT cells hasincreased 32.1％and 34.7％respectively, but the expression levels are significantlydifferent (p＜0.05). And there is no significant difference in the receptor expressionafter treatment of vacant liposome and HBsAg plasmid DNA liposome (p＞0.05).
5. Investigate the effect of vacant liposome and HBsAg plasmid DNA liposome on theexpression levels of MyD88 in HaCaT cells and FB cells by means of westernblotting. The results showed that the expression levels of MyD88 increased at 4h aftervacant liposome and HBsAg plasmid DNA liposome treatment, without adose-dependant fashion, which indicates an alternative pathway other than MyD88 inthe signaling of TLR in the two kinds of cells.
6. Investigate the effect of vacant liposome and HBsAg plasmid DNA liposome on thetime-effect relation of the secretion of Th1 and Th2 cytokines (IFN-γ, and IL-4) inHaCaT cells and FB cells by means of ELISA. The results showed that there's a dose-and time-dependant increase after vacant liposome and HBsAg plasmid DNAliposome treatment, with a maximum effect at 48h and the expression level in HBsAgplasmid DNA liposome group has a significant elevation (p＜0.05).
The study in this part is to design HBsAg vaccine targeted in TLR9 and maketransdermal immune possible. It will enhance our knowledge about the underlyingmechanism, and provide a helpful reference to the designation and enhancement ofthe immune response. The results showed that the vaccine can increase the immunitythrough the increase of TLRs which provide a theoretical foundation for thedevelopment of transdermal immunological drugs in the future.
In conclusion, this study elucidates the expression pattern of TLR3、TLR9receptors in the skin in various ways, discusses the role and regulation mechanism intransdermal and immunosuppressant action, and provides a new target for thetreatment of inflammatory diseases. The discussion about the possibility of effectsexerted by different immunosuppressants on the target provides a new insight into thedevelopment of new TLR promoter and antagonist, and provides a technical supportand theoretical foundation for the development of transdermal immunological drugsin the future.
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